pH-dependent hydrolysis of acetylcholine: Consequences for non-neuronal acetylcholine.

PubMed

Wessler, Ignaz; Michel-Schmidt, Rosmarie; Kirkpatrick, Charles James

2015-11-01

Acetylcholine is inactivated by acetylcholinesterase and butyrylcholinesterase and thereby its cellular signalling is stopped. One distinguishing difference between the neuronal and non-neuronal cholinergic system is the high expression level of the esterase activity within the former and a considerably lower level within the latter system. Thus, any situation which limits the activity of both esterases will affect the non-neuronal cholinergic system to a much greater extent than the neuronal one. Both esterases are pH-dependent with an optimum at pH above 7, whereas at pH values below 6 particularly the specific acetylcholinesterase is more or less inactive. Thus, acetylcholine is prevented from hydrolysis at such low pH values. The pH of the surface of the human skin is around 5 and therefore non-neuronal acetylcholine released from keratinocytes can be detected in a non-invasive manner. Several clinical conditions like metabolic acidosis, inflammation, fracture-related haematomas, cardiac ischemia and malignant tumours are associated with local or systemic pH values below 7. Thus, the present article describes some consequences of an impaired inactivation of extracellular non-neuronal acetylcholine. Copyright © 2015 Elsevier B.V. All rights reserved.

Purification and characterization of the tween-hydrolyzing esterase of Mycobacterium smegmatis.

PubMed Central

Tomioka, H

1983-01-01

An esterase hydrolyzing Tween 80 (polyoxyethylene sorbitan monooleate) was purified from sonicated cell lysates of Mycobacterium smegmatis ATCC 14468 by DEAE-cellulose, Sephadex G-150, phenyl Sepharose, and diethyl-(2-hydroxypropyl) aminoethyl column chromatography and by subsequent preparative polyacrylamide gel electrophoresis. The molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 41,000 by gel filtration on a Sephadex G-150 column. The esterase contained a single polypeptide. The esterase was stable to heat treatment at 100 degrees C and to a wide range of pH. The temperature and pH optima for the hydrolysis of Tween 80 were 50 degrees C and 8.3, respectively. The esterase had a narrow substrate specificity; it exhibited a high activity only on compounds having both polyoxyethylene and fatty acyl moieties, such as Tweens. Monoacylglyceride was hydrolyzed more slowly by this esterase and this enzyme exhibited a nonspecific esterase activity on p-nitrophenyl acyl esters, especially those having short chain acyl moieties. The Km and Vmax were 19.2 mM and 1,670 mumol/min per mg of protein for Tween 20, 6.6 mM and 278 mumol/min per mg of protein for Tween 80, and 0.25 mM and 196 mumol/min per mg of protein for p-nitrophenyl acetate, respectively. Observations of the effects of various chemical modifications on the activity of the esterase indicated that tyrosine, histidine, arginine, and methionine (with tryptophan) residues may be active amino acids which play important roles in the expression of Tween 80-hydrolyzing activity of the enzyme. PMID:6885719

Eco-friendly surface modification on polyester fabrics by esterase treatment

NASA Astrophysics Data System (ADS)

Wu, Jindan; Cai, Guoqiang; Liu, Jinqiang; Ge, Huayun; Wang, Jiping

2014-03-01

Currently, traditional alkali deweighting technology is widely used to improve the hydrophilicity of polyester fabrics. However, the wastewater and heavy chemicals in the effluent cause enormous damage to the environment. Esterase treatment, which is feasible in mild conditions with high selectivity, can provide a clean and efficient way for polyester modification. Under the optimum conditions, the polyester fabric hydrolysis process of esterase had a linear kinetics. X-ray photoelectron spectrometry (XPS) results showed that hydroxyl and carboxyl groups were produced only on the surface of modified fiber without changing the chemical composition of the bulk. These fibers exhibited much improved fabric wicking, as well as greatly improved oily stain removal performance. Compared to the harsh alkali hydrolysis, the enzyme treatment led to smaller weight loss and better fiber integrity. The esterase treatment technology is promising to produce higher-quality polyester textiles with an environmental friendly approach.

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

PubMed Central

Rodríguez, María Cecilia; Loaces, Inés; Amarelle, Vanesa; Senatore, Daniella; Iriarte, Andrés; Fabiano, Elena; Noya, Francisco

2015-01-01

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases. PMID:25973851

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes.

PubMed

Rodríguez, María Cecilia; Loaces, Inés; Amarelle, Vanesa; Senatore, Daniella; Iriarte, Andrés; Fabiano, Elena; Noya, Francisco

2015-01-01

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40 °C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.

Human butyrylcholinesterase components differ in aryl acylamidase activity.

PubMed

Montenegro, María F; María, T Moral-Naranjo; de la Cadena, María Páez; Campoy, Francisco J; Muñoz-Delgado, Encarnación; Vidal, Cecilio J

2008-04-01

Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.

Hydrolysis of rosmarinic acid from rosemary extract with esterases and Lactobacillus johnsonii in vitro and in a gastrointestinal model.

PubMed

Bel-Rhlid, Rachid; Crespy, Vanessa; Pagé-Zoerkler, Nicole; Nagy, Kornél; Raab, Thomas; Hansen, Carl-Erik

2009-09-09

Rosmarinic acid (RA) was identified as one of the main components of rosemary extracts and has been ascribed to a number of health benefits. Several studies suggested that after ingestion, RA is metabolized by gut microflora into caffeic acid and derivatives. However, only limited information on the microorganisms and enzymes involved in this biotransformation is available. In this study, we investigated the hydrolysis of RA from rosemary extract with enzymes and a probiotic bacterium Lactobacillus johnsonii NCC 533. Chlorogenate esterase from Aspergillus japonicus (0.02 U/mg) hydrolyzed 90% of RA (5 mg/mL) after 2 h at pH 7.0 and 40 degrees C. Complete hydrolysis of RA (5 mg/mL) was achieved with a preparation of L. johnsonii (25 mg/mL, 3.3 E9 cfu/g) after 2 h of incubation at pH 7.0 and 37 degrees C. No hydrolysis of RA was observed after the passage of rosemary extract through the gastrointestinal tract model (GI model). Thus, RA is hydrolyzed neither chemically under the conditions of the GI model (temperature, pH, and bile salts) nor by secreted enzymatic activity (lipase and pancreatic enzymes). The addition of L. johnsonii cells to rosemary extract in the GI model resulted in substantial hydrolysis of RA (up to 99%).

Influence of chronic oral intake of cannabis extract on oxidative and hydrolytic metabolism of xenobiotics in rat.

PubMed

Khanna, P; Gupta, M B; Gupta, G P; Sanwal, G G; Ali, B

1991-01-01

Dietary intake of petroleum ether extract of cannabis leaves by rats in doses of 158, 250 and 500 mg/kg in the first, second and third week, respectively, caused selective induction of hepatic microsomal carboxylesterases/amidases without affecting the renal hydrolytic activity. Acetanilide N-deacetylase, p-nitrophenylacetate (NPA) esterase and acetylsalicylic acid (ASA) esterase I and II (active at pH 5.5 and 7.4) were stimulated 125, 64, 82 and 60%, respectively, whereas the activities of procaine esterase and acetylaminofluorene (AAF) N-deacetylase remained unaltered. The hydrolysis of acetylcholine was also unchanged. Upon withdrawal of treatment microsomal hydrolytic activity receded to basal levels within 7 days. Curiously though, the two-fold induction of thiacetazone N-deacetylase (118%), a cytosolic hydrolase, remained largely undiminished (62%). An appraisal of the hepatic cytochrome P450 mediated oxidative metabolism revealed approximately three-fold induction of aromatic hydrocarbon hydroxylase (AHH) metabolizing benzo(a)pyrene whereas the N-demethylation of aminopyrene was unaffected. These activities were restored to normal when resin administration was discontinued.

Gene cloning and characterization of a novel esterase from activated sludge metagenome

PubMed Central

2009-01-01

A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS) of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109) and Yarrowia lipolytica CLIB122 (XP_504639), respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His125 and Asp216) and a LHYFRG conserved motif. The EstAS was overexpressed, purified and shown to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤ C8). This EstAS had optimal temperature and pH at 35°C and 9.0, respectively, by hydrolysis of p-NP hexanoate. It also exhibited the same level of stability over wide temperature and pH ranges and in the presence of metal ions or detergents. The high level of stability of esterase EstAS with its unique substrate specificities make itself highly useful for biotechnological applications. PMID:20028524

Intracellular esterase activity in living cells may distinguish between metastatic and tumor-free lymph nodes.

PubMed

Afrimzon, Elena; Deutsch, Assaf; Shafran, Yana; Zurgil, Naomi; Sandbank, Judith; Pappo, Itzhak; Deutsch, Mordechai

2008-01-01

One of the major clinical problems in breast cancer detection is the relatively high incidence of occult lymph node metastases undetectable by standard procedures. Since the ascertainment of breast cancer stage determines the following treatment, such a "hypo-diagnosis" leads to inadequate therapy, and hence is detrimental for the outcome and survival of the patients. The purpose of our study was to investigate functional metabolic characteristics of living cells derived from metastatic and tumor-free lymph nodes of breast cancer (BC) patients. Our methodology is based on the ability of living cells to hydrolyze fluorescein diacetate (FDA) by intracellular esterases and on the association of FDA hydrolysis rates with a specific cell status, both in physiological and pathological conditions. The present study demonstrates a significant difference in the ability to utilize FDA by lymph node cells derived from metastatic and tumor-free lymph nodes in general average, as well as in the metastatic and tumor-free lymph nodes of individual patients. Cells from metastatic lymph nodes had a higher capacity for FDA hydrolysis, and increased this activity after additional activation by autologous tumor tissue (tt). The association between increased FDA hydrolysis rate and activated T lymphocytes and antigen-presenting cells (APC) was shown. The results of the present study may contribute to predicting the risk of involvement of seemingly "tumor-free" axillary lymph nodes in occult metastatic processes, and to reducing false-negative results of axillary examination.

Characterization of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes from Carbohydrate Esterase family 1 of the carbohydrate-active enzymes (CAZy) database.

PubMed

Mäkelä, Miia R; Dilokpimol, Adiphol; Koskela, Salla M; Kuuskeri, Jaana; de Vries, Ronald P; Hildén, Kristiina

2018-04-26

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Thermus thermophilus as a Source of Thermostable Lipolytic Enzymes

PubMed Central

López-López, Olalla; Cerdán, María-Esperanza; González-Siso, María-Isabel

2015-01-01

Lipolytic enzymes, esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), catalyze the hydrolysis of ester bonds between alcohols and carboxylic acids, and its formation in organic media. At present, they represent about 20% of commercialized enzymes for industrial use. Lipolytic enzymes from thermophilic microorganisms are preferred for industrial use to their mesophilic counterparts, mainly due to higher thermostability and resistance to several denaturing agents. However, the production at an industrial scale from the native organisms is technically complicated and expensive. The thermophilic bacterium Thermus thermophilus (T. thermophilus) has high levels of lipolytic activity, and its whole genome has been sequenced. One esterase from the T. thermophilus strain HB27 has been widely characterized, both in its native form and in recombinant forms, being expressed in mesophilic microorganisms. Other putative lipases/esterases annotated in the T. thermophilus genome have been explored and will also be reviewed in this paper. PMID:27682117

Formation of ethyl ferulate by rice koji enzyme in sake and mirin mash conditions.

PubMed

Hashizume, Katsumi; Ito, Toshihiko; Ishizuka, Takahiro; Takeda, Naoki

2013-08-01

Formation mechanism of ethyl ferulate (EF) in sake and mirin mash conditions was investigated to understand EF level control in the manufacturing process. Rice koji formed EF from ferulic acid (FA) and ethanol and decomposed EF to FA. This did not occur in sake yeast and chemical esterification was rare. Esterification of FA and hydrolysis of EF by rice koji might be due to feruloyl esterase(s). The rice koji enzyme showed normal Michaelis-Menten kinetics for FA in ethyl esterification and for EF in hydrolysis, but not for ethanol in the esterification reaction. Substrate specificity of the rice koji enzyme for hydroxycinnamic acids suggested that the main enzyme involved might be similar to type A feruloyl esterase. We studied the rice koji enzyme properties, short-term digestion of steamed rice grains with exogenous ethanol and small scale mirin making with pH adjustment. Our results suggested differences in the esterification and hydrolysis properties of the enzyme, in particular, different pH dependencies and different behaviors under high ethanol conditions; these factors might cause the differing EF levels in sake and mirin mashes. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Screening of adjunct cultures and their application in ester formation in Camembert-type cheese.

PubMed

Hong, Q; Liu, X M; Hang, F; Zhao, J X; Zhang, H; Chen, W

2018-04-01

The ethanol content and esterase and alcohol acyltransferase activities are the limiting factors in the synthesis of ethyl esters in Camembert-type cheeses. This study aimed to investigate the effects of alcohol, esterase and alcohol acyltransferase activities on ethyl ester formation in Camembert-type cheeses. Five experimental cheeses were prepared with three adjunct cultures with different enzyme activities and two levels of ethanol content (400 or 800 μg/g). The cheeses were aged for 4 weeks and analysed weekly for basic physicochemical, textural, volatile and sensory properties. The results showed that both the enzyme activity and ethanol content were limiting factors in the synthesis of ethyl esters in the Camembert-type cheeses. Variation in the esterase synthesis activity was observed among lactic acid bacteria, and the starter culture Lactococcus lactis MA 14 LYO distinguished itself through its high acidifying and esterase hydrolysis abilities. The addition of CCFM 12, a lactic acid bacteria strain with high esterase and alcohol acyltransferase activity, along with 400 or 800 μg/g of ethanol, notably enhanced the generation of ethyl esters and the corresponding fruity flavour, without causing dramatic changes in the basic physicochemical indices and microbial profile. In addition, cohesiveness was influenced by the addition of 400 and 800 μg/g of ethanol, and more resilience with 800 μg/g of ethanol had been found. The results showed that the addition of CCFM12 with 400 and 800 μg/g of ethanol may be applied in the production of Camembert cheese to enhance its fruity flavour. Copyright © 2017 Elsevier Ltd. All rights reserved.

Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of “hunger hormone” ghrelin

NASA Astrophysics Data System (ADS)

Yao, Jianzhuang; Yuan, Yaxia; Zheng, Fang; Zhan, Chang-Guo

2016-02-01

Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway.

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars.

PubMed

Rahmani, Nanik; Kahar, Prihardi; Lisdiyanti, Puspita; Hermiati, Euis; Lee, Jaemin; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2018-02-23

The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.

Interactions between resin monomers and commercial composite resins with human saliva derived esterases.

PubMed

Jaffer, F; Finer, Y; Santerre, J P

2002-04-01

Cholesterol esterase (CE) and pseudocholinesterase (PCE) have been reported to degrade commercial and model composite resins containing bisphenylglycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) or the latter in combination with urethane modified BisGMA monomer systems. In addition, human saliva has been shown to contain esterase like activities similar to CE and PCE. Hence, it was the aim of the current study to determine to what extent human saliva could degrade two common commercial composite resins (Z250 from 3M Inc. and Spectrum TPH from L.D. Caulk) which contain the above monomer systems. Saliva samples from different volunteers were collected, processed, pooled, and freeze-dried. TEGDMA and BisGMA monomers were incubated with human saliva derived esterase activity (HSDEA) and their respective hydrolysis was monitored using high performance liquid chromatography (HPLC). Both monomers were completely hydrolyzed within 25 h by HSDEA. Photopolymerized composites were incubated with buffer or human saliva (pH 7.0 and 37 C) for 2, 8 and 16 days. The incubation solutions were analyzed using HPLC and mass spectrometry. Surface morphology characterization was carried out using scanning electron microscopy. Upon biodegradation, the Z250 composite yielded higher amounts of BisGMA and TEGDMA related products relative to the TPH composite. However, there were higher amounts of ethoxylated bis-phenol A released from the TPH material. In terms of total mass of products released, human saliva demonstrated a greater ability to degrade Z250. In summary, HSDEA has been shown to contain esterase activities that can readily catalyze the biodegradation of current commercial composite resins.

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

PubMed

Komiya, Dai; Hori, Akane; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Koseki, Takuya; Fushinobu, Shinya

2017-10-15

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis ( Al AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. Al AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that Al AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of A lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers. Copyright © 2017 American Society for Microbiology.

Förster resonance energy transfer studies of luminescent gold nanoparticles functionalized with ruthenium(II) and rhenium(I) complexes: modulation via esterase hydrolysis.

PubMed

Leung, Frankie Chi-Ming; Tam, Anthony Yiu-Yan; Au, Vonika Ka-Man; Li, Mei-Jin; Yam, Vivian Wing-Wah

2014-05-14

A number of ruthenium(II) and rhenium(I) bipyridine complexes functionalized with lipoic acid moieties have been synthesized and characterized. Functionalization of gold nanoparticles with these chromophoric ruthenium(II) and rhenium(I) complexes has resulted in interesting supramolecular assemblies with Förster resonance energy transfer (FRET) properties that could be modulated via esterase hydrolysis. The luminescence of the metal complex chromophores was turned on upon cleavage of the ester bond linkage by esterase to reduce the efficiency of FRET quenching. The prepared nanoassembly conjugates have been characterized by transmission electron microscopy (TEM), energy-dispersive X-ray analysis (EDX), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), UV-visible spectroscopy, and emission spectroscopy. The quenching mechanism has also been studied by transient absorption and time-resolved emission decay measurements. The FRET efficiencies were found to vary with the nature of the chromophores and the length of the spacer between the donor (transition metal complexes) and the acceptor (gold nanoparticles).

Hydrolysis of chicoric and caftaric acids with esterases and Lactobacillus johnsonii in Vitro and in a gastrointestinal model.

PubMed

Bel-Rhlid, Rachid; Pagé-Zoerkler, Nicole; Fumeaux, René; Ho-Dac, Thang; Chuat, Jean-Yves; Sauvageat, Jean Luc; Raab, Thomas

2012-09-12

Chicoric acid (ChA) and caftaric acid (CafA) were identified as bioactive components of chicory and have been ascribed a number of health benefits. This study investigated the hydrolysis of ChA and CafA with enzymes and a probiotic bacterium Lactobacillus johnsonii (La1). Esterase from Aspergillus japonicus (24 U/mg) hydrolyzed 100% of ChA (5 mM) and CafA (5 mM) after 3 h, at pH 7.0 and 37 °C. Under the same reaction conditions, 100% hydrolysis of ChA and CafA was achieved with a spray-dried preparation of La1. The addition of La1 (100 mg/mL, 3.3 E9 cfu/g) to CafA solution in a gastrointestinal model (GI model) resulted in 65% hydrolysis of CafA. This model simulates the physicochemical conditions of the human gastrointestinal tract. No hydrolysis of CafA was observed after passage through the GI model in the absence of La1. The results of this study support the hypothesis that ChA and CafA are degraded by gut microflora before absorption and metabolization.

Identification of a Novel Esterase from Marine Environmental Genomic DNA Libraries and Its Application in Production of Free All- trans-Astaxanthin.

PubMed

Lu, Ping; Gao, Xinwei; Dong, Hao; Liu, Zhen; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

2018-03-21

Astaxanthin is a pigment with various functions. Free astaxanthin is obtained mainly through saponification methods, which could result in many byproducts. Enzymatic methods using lipases have been used in a few cases, while there are no reports on the use of esterases for the production of free astaxanthin. Herein we present the screening and identification of a novel esterase (Est3-14) from a marine mud metagenomic library. Est3-14 is pH-sensitive and keeps good stability in alkaline buffers (residual activity 94%, pH 8.0, 4 °C, and 36 h). Meanwhile, Est3-14 keeps a good stability in the medium temperature condition (residual activity 56.7%, pH 8.0, 40 °C, and 84 h). Est3-14 displayed high hydrolysis activity to prepare free all- trans-astaxanthin in biphasic systems. Furthermore, under optimal conditions (0.5 mL ethanol, 6 mL 0.1 M Tris-HCl buffer, pH 8.0, 0.5% (w/v) H. pluvialis oil, 40 °C), the hydrolytic conversion ratio was 99.3% after 36 h.

Modulating lipophilicity of rohitukine via prodrug approach: Preparation, characterization, and in vitro enzymatic hydrolysis in biorelevant media.

PubMed

Kumar, Vikas; Bharate, Sonali S; Vishwakarma, Ram A

2016-09-20

Rohitukine is a medicinally important natural product which has inspired the discovery of two anticancer clinical candidates. Rohitukine is highly hydrophilic in nature which hampers its oral bioavailability. Thus, herein our objective was to improve the drug-like properties of rohitukine via prodrug-strategy. Various ester prodrugs were synthesized and studied for solubility, lipophilicity, chemical stability and enzymatic hydrolysis in plasma/esterase. All prodrugs displayed lower aqueous solubility and improved lipophilicity compared with rohitukine, which was in accordance with the criteria of compounds in drug-discovery. The stability of synthesized prodrugs was evaluated in buffers at different pH, SGF, SIF, rat plasma and in esterase enzyme. The rate of hydrolysis in all incubation media was dependent primarily on the acyl promoieties. Hexanoyl ester prodrug of rohitukine, 3d, was stable under chemical conditions; however it was completely hydrolyzed to rohitukine, in plasma and in esterase in 4h. Hexanoate ester 3d appeared to be the most promising prodrug as it remained intact at gastric/intestinal pH and was completely transformed to the parent compound in plasma as desired for an ideal prodrug. The data presented herein, will help in designing prodrugs with desired physicochemical properties in future in structurally similar chemotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

VvMJE1 of the grapevine (Vitis vinifera) VvMES methylesterase family encodes for methyl jasmonate esterase and has a role in stress response

USDA-ARS?s Scientific Manuscript database

The known members of the plant methyl esterase (MES) family catalyze hydrolysis of a C-O ester linkage of methyl esters of several phytohormones including indole-3-acetic acid, salicylic acid, and jasmonic acid. The genome of grapevine (Vitis vinifera) was found to contain 15 MES genes, designated V...

Cloning and biochemical characterization of a novel lipolytic gene from activated sludge metagenome, and its gene product

PubMed Central

2010-01-01

In this study, a putative esterase, designated EstMY, was isolated from an activated sludge metagenomic library. The lipolytic gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene estMY contained a 1,083 bp open reading frame (ORF) encoding a polypeptide of 360 amino acids with a molecular mass of 38 kDa. Sequence analysis indicated that it showed 71% and 52% amino acid identity to esterase/lipase from marine metagenome (ACL67845) and Burkholderia ubonensis Bu (ZP_02382719), respectively; and several conserved regions were identified, including the putative active site, GDSAG, a catalytic triad (Ser203, Asp301, and His327) and a HGGG conserved motif (starting from His133). The EstMY was determined to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (≤C8). This EstMY exhibited the highest activity at 35°C and pH 8.5 respectively, by hydrolysis of p-NP caprylate. It also exhibited the same level of activity over wide temperature and pH spectra and in the presence of metal ions or detergents. The high level of stability of esterase EstMY with unique substrate specificities makes it highly valuable for downstream biotechnological applications. PMID:21054894

Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes.

PubMed

Wei, Ren; Oeser, Thorsten; Zimmermann, Wolfgang

2014-01-01

Thermophilic actinomycetes produce enzymes capable of hydrolyzing synthetic polyesters such as polyethylene terephthalate (PET). In addition to carboxylesterases, which have hydrolytic activity predominantly against PET oligomers, esterases related to cutinases also hydrolyze synthetic polymers. The production of these enzymes by actinomycetes as well as their recombinant expression in heterologous hosts is described and their catalytic activity against polyester substrates is compared. Assays to analyze the enzymatic hydrolysis of synthetic polyesters are evaluated, and a kinetic model describing the enzymatic heterogeneous hydrolysis process is discussed. Structure-function and structure-stability relationships of actinomycete polyester hydrolases are compared based on molecular dynamics simulations and recently solved protein structures. In addition, recent progress in enhancing their activity and thermal stability by random or site-directed mutagenesis is presented. © 2014 Elsevier Inc. All rights reserved.

Oleuropein hydrolysis in natural green olives: Importance of the endogenous enzymes.

PubMed

Ramírez, Eva; Brenes, Manuel; García, Pedro; Medina, Eduardo; Romero, Concepción

2016-09-01

The bitter taste of olives is mainly caused by the phenolic compound named oleuropein and the mechanism of its hydrolysis during the processing of natural green olives was studied. First, a rapid chemical hydrolysis of oleuropein takes place at a high temperature of 40°C and at a low pH value of 2.8, but the chemical hydrolysis of the bitter compound is slow at the common range of pH for these olives (3.8-4.2). However, decarboxymethyl elenolic acid linked to hydroxytyrosol and hydroxytyrosol have been found in a high concentration during the elaboration of natural green olives. When olives were heated at 90°C for 10min before brining, these compounds are not formed. Hence, the debittering process in natural green olives is due to the activity of β-glucosidase and esterase during the first months of storage and then a slow chemical hydrolysis of oleuropein happens throughout storage time. Copyright © 2016 Elsevier Ltd. All rights reserved.

Studies on the metabolism of benoxinate by human pseudocholinesterase.

PubMed

Dubbels, R; Schloot, W

1983-01-01

The local anesthetic drug benoxinate (oxybuprocaine, Novesine) is hydrolyzed to 3-butoxy-4-aminobenzoic acid. A rapid and simple spectrophotometric method for benoxinate hydrolysis by human plasma was developed. Benoxinate is hydrolyzed enzymatically by an esterase present in the serum. Heat stability characteristics and apparent affinity values of the benoxinate metabolizing enzyme were in the same range compared to benzoylcholine chloride hydrolysis. Apparent Vmax-values differ by a mean factor of about 18 between the hydrolysis of both substrates. Considerable interindividual variability of benoxinate hydrolysis and inhibition of the enzymatic reaction by dibucaine and sodium fluoride has been observed. Furthermore, enzyme activity with benoxinate as substrate is positively correlated (P less than 0.001) with benzoylcholine chloride hydrolysis. Therefore, we assume that benoxinate is metabolized by human pseudocholinesterase (PCHE, E.C. 3.1.1.8) and that ocular side effects after benoxinate application may be caused by altered metabolism of this drug, depending on genetically determined variants of pseudocholinesterase.

Functional Characterization of a Robust Marine Microbial Esterase and Its Utilization in the Stereo-Selective Preparation of Ethyl (S)-3-Hydroxybutyrate.

PubMed

Wang, Yilong; Zhang, Yun; Hu, Yunfeng

2016-11-01

One novel microbial esterase PHE21 was cloned from the genome of Pseudomonas oryzihabitans HUP022 identified from the deep sea of the Western Pacific. PHE21 was heterologously expressed and functionally characterized to be a robust esterase which behaved high resistance to various metal ions, organic solvents, surfactants, and NaCl. Despite the fact that the two enantiomers of ethyl 3-hydroxybutyrate were hard to be enzymatically resolved before, we successfully resolved racemic ethyl 3-hydroxybutyrate through direct hydrolysis reactions and generated chiral ethyl (S)-3-hydroxybutyrate using esterase PHE21. After process optimization, the enantiomeric excess, the conversion rate, and the yield of desired product ethyl (S)-3-hydroxybutyrate could reach 99, 65, and 87 %, respectively. PHE21 is a novel marine microbial esterase with great potential in asymmetric synthesis as well as in other industries.

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases.

PubMed

Chahinian, Henri; Ali, Yassine Ben; Abousalham, Abdelkarim; Petry, Stefan; Mandrich, Luigi; Manco, Guiseppe; Canaan, Stephane; Sarda, Louis

2005-12-30

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.

Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

PubMed Central

Sobek, H; Görisch, H

1989-01-01

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported. PMID:2508625

Synthesis of regioselectively protected forms of cytidine based on enzyme-catalyzed deacetylation as the key step.

PubMed

Kuboki, A; Ishihara, T; Kobayashi, E; Ohta, H; Ishii, T; Inoue, A; Mitsuda, S; Miyazaki, T; Kajihara, Y; Sugai, T

2000-02-01

N4-Acetylcytidine (77%) and 2',3'-O, N4-triacetylcytidine (95%) were obtained from the hydrolysis of a common precursor, the peracetylated form of cytidine with Aspergillus niger lipase (Amano A) and Burkholderia cepacia esterase (SC esterase S), respectively, under very mild conditions. The experimental procedure for the conversion of triacetylcytidine to a corresponding phosphoramidite (82%), an intermediate for sugar nucleotide synthesis, is also elaborated.

Microbial Glucuronoyl Esterases: 10 Years after Discovery

PubMed Central

2016-01-01

A carbohydrate esterase called glucuronoyl esterase (GE) was discovered 10 years ago in a cellulolytic system of the wood-rotting fungus Schizophyllum commune. Genes coding for GEs were subsequently found in a number of microbial genomes, and a new family of carbohydrate esterases (CE15) has been established. The multidomain structures of GEs, together with their catalytic properties on artificial substrates and positive effect on enzymatic saccharification of plant biomass, led to the view that the esterases evolved for hydrolysis of the ester linkages between 4-O-methyl-d-glucuronic acid of plant glucuronoxylans and lignin alcohols, one of the crosslinks in the plant cell walls. This idea of the function of GEs is further supported by the effects of cloning of fungal GEs in plants and by very recently reported evidence for changes in the size of isolated lignin-carbohydrate complexes due to uronic acid de-esterification. These facts make GEs interesting candidates for biotechnological applications in plant biomass processing and genetic modification of plants. This article is a brief summary of current knowledge of these relatively recent and unexplored esterases. PMID:27694239

Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

NASA Astrophysics Data System (ADS)

Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

2014-07-01

We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

Crystal structure of a feruloyl esterase belonging to the tannase family: a disulfide bond near a catalytic triad.

PubMed

Suzuki, Kentaro; Hori, Akane; Kawamoto, Kazusa; Thangudu, Ratna Rajesh; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Yamada, Chihaya; Arakawa, Takatoshi; Wakagi, Takayoshi; Koseki, Takuya; Fushinobu, Shinya

2014-10-01

Feruloyl esterase (FAE) catalyzes the hydrolysis of the ferulic and diferulic acids present in plant cell wall polysaccharides, and tannase catalyzes the hydrolysis of tannins to release gallic acid. The fungal tannase family in the ESTHER database contains various enzymes, including FAEs and tannases. Despite the importance of FAEs and tannases in bioindustrial applications, three-dimensional structures of the fungal tannase family members have been unknown. Here, we determined the crystal structure of FAE B from Aspergillus oryzae (AoFaeB), which belongs to the fungal tannase family, at 1.5 Å resolution. AoFaeB consists of a catalytic α/β-hydrolase fold domain and a large lid domain, and the latter has a novel fold. To estimate probable binding models of substrates in AoFaeB, an automated docking analysis was performed. In the active site pocket of AoFaeB, residues responsible for the substrate specificity of the FAE activity were identified. The catalytic triad of AoFaeB comprises Ser203, Asp417, and His457, and the serine and histidine residues are directly connected by a disulfide bond of the neighboring cysteine residues, Cys202 and Cys458. This structural feature, the "CS-D-HC motif," is unprecedented in serine hydrolases. A mutational analysis indicated that the novel structural motif plays essential roles in the function of the active site. © 2014 Wiley Periodicals, Inc.

Secreted protein extract analyses present the plant pathogen Alternaria alternata as a suitable industrial enzyme toolbox.

PubMed

García-Calvo, L; Ullán, R V; Fernández-Aguado, M; García-Lino, A M; Balaña-Fouce, R; Barreiro, C

2018-04-15

Lignocellulosic plant biomass is the most abundant carbon source in the planet, which makes it a potential substrate for biorefinery. It consists of polysaccharides and other molecules with applications in pharmaceutical, food and feed, cosmetics, paper and textile industries. The exploitation of these resources requires the hydrolysis of the plant cell wall, which is a complex process. Aiming to discover novel fungal natural isolates with lignocellulolytic capacities, a screening for feruloyl esterase activity was performed in samples taken from different metal surfaces. An extracellular enzyme extract from the most promising candidate, the natural isolate Alternaria alternata PDA1, was analyzed. The feruloyl esterase activity of the enzyme extract was characterized, determining the pH and temperature optima (pH 5.0 and 55-60 °C, respectively), thermal stability and kinetic parameters, among others. Proteomic analyses derived from two-dimensional gels allowed the identification and classification of 97 protein spots from the extracellular proteome. Most of the identified proteins belonged to the carbohydrates metabolism group, particularly plant cell wall degradation. Enzymatic activities of the identified proteins (β-glucosidase, cellobiohydrolase, endoglucanase, β-xylosidase and xylanase) of the extract were also measured. These findings confirm A. alternata PDA1 as a promising lignocellulolytic enzyme producer. Although plant biomass is an abundant material that can be potentially utilized by several industries, the effective hydrolysis of the recalcitrant plant cell wall is not a straightforward process. As this hydrolysis occurs in nature relying almost solely on microbial enzymatic systems, it is reasonable to infer that further studies on lignocellulolytic enzymes will discover new sustainable industrial solutions. The results included in this paper provide a promising fungal candidate for biotechnological processes to obtain added value from plant byproducts and analogous substrates. Moreover, the proteomic analysis of the secretome of a natural isolate of Alternaria sp. grown in the presence of one of the most used vegetal substrates on the biofuels industry (sugar beet pulp) sheds light on the extracellular enzymatic machinery of this fungal plant pathogen, and can be potentially applied to developing new industrial enzymatic tools. This work is, to our knowledge, the first to analyze in depth the secreted enzyme extract of the plant pathogen Alternaria when grown on a lignocellulosic substrate, identifying its proteins by means of MALDI-TOF/TOF mass spectrometry and characterizing its feruloyl esterase, cellulase and xylanolytic activities. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

PubMed Central

2011-01-01

Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. Results A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

Detoxification of diphenyl ether herbicide lactofen by Bacillus sp. Za and enantioselective characteristics of an esterase gene lacE.

PubMed

Zhang, Jing; Lu, Luyao; Chen, Feng; Chen, Lingling; Yin, Jingang; Huang, Xing

2018-01-05

A bacterial strain Za capable of degrading diphenyl ether herbicide lactofen was isolated and identified as Bacillus sp. This strain could degrade 94.8% of 50mgL -1 lactofen after 4days of inoculation in flasks. It was revealed that lactofen was initially hydrolyzed to desethyl lactofen, which was further transformed to acifluorfen, followed by the reduction of the nitro group to yield aminoacifluorfen. The phytotoxicity of the transformed product aminoacifluorfen to maize was decreased significantly compared with the lactofen. A gene lacE, encoding an esterase responsible for lactofen hydrolysis to desethyl lactofen and acifluorfen continuously, was cloned from Bacillus sp. Za. The deduced amino acid belonging to the esterase family VII contained a typical Ser-His-Asp/Glu catalytic triad and the conserved motifs GXSXG. The purified recombinant protein LacE displayed maximal esterase activity at 40°C and pH 7.0. Additionally, LacE had broad substrate specificity and was capable of hydrolyzing p-nitrophenyl esters. The enantioselectivity of LacE during lactofen degradation was further studied, and the results indicated that the (S)-(+)-lactofen was degraded faster than the (R)-(-)-lactofen, which could illustrate the reported phenomenon that (S)-(+)-lactofen was preferentially degraded in soil and sediment. Copyright © 2017 Elsevier B.V. All rights reserved.

Hyperproduction of sebaceous cis-6-hexadecenoic acid by esterase-reduced mutant of Rhodococcus sp. strain.

PubMed

Araki, Hiroyuki; Hagihara, Hiroshi; Takigawa, Hirofumi; Kotani, Nobuharu; Tsujino, Yukiharu; Koike, Kenzo; Kawai, Shuji; Ozaki, Katsuya; Ito, Susumu

2007-10-01

cis-6-Hexadecenoic acid is a major component of human sebaceous lipids that is involved in skin self-sterilization and atopic dermatitis amelioration. It can be prepared by hydrolysis of isopropyl cis-6-hexadecenoate produced by resting cells of Rhodococcus sp. strain KSM-MT66. To devise an economical industrial-scale process for the production of this rare fatty acid, we optimized the conditions for growing rhodococcal cells. Mg(2+) and Fe(2+) ions are essential for the efficient production of isopropyl cis-6-hexadecenoate. To further increase the production of isopropyl cis-6-hexadecenoate, we created a mutant strain (T64) with reduced esterase activity by random mutagenesis using UV irradiation of MT66. Under an optimized condition, the mutant T64 produced more than 60 g l(-1) isopropyl cis-6-hexadecenoate in a 4-d cultivation, corresponding to about 52 g l(-1)cis-6-hexadecenoate.

Aerobic biological treatment of synthetic municipal wastewater in membrane-coupled bioreactors.

PubMed

Klatt, Christian G; LaPara, Timothy M

2003-05-05

Membrane-coupled bioreactors (MBRs) offer many benefits compared to conventional biological wastewater treatment systems; however, their performance characteristics are poorly understood. Laboratory-scale MBRs were used to study bacterial adaptations in physiology and community structure. MBRs were fed a mixture of starch, gelatin, and polyoxyethylene-sorbitan monooleate to simulate the polysaccharide, protein, and lipid components of municipal wastewater. Physiological adaptations were detected by measuring ectoenzyme activity while structural dynamics were studied by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments. As cell biomass accumulated in the MBRs, pollutant removal efficiency initially improved and then stabilized with respect to effluent concentrations of chemical oxygen demand, protein, and carbohydrate. Comparison of the MBR effluent to filtered reactor fluid indicated that a portion of the observed pollutant removal was due to filtration by the membrane rather than microbial activity. The rates of ectoenzyme-mediated polysaccharide (alpha-glucosidase) and protein (leucine aminopeptidase) hydrolysis became relatively constant once pollutant removal efficiency stabilized. However, the maximum rate of lipid hydrolysis (heptanoate esterase) concomitantly increased more than 10-fold. Similarly, alpha-glucosidase and leucine aminopeptidase ectoenzyme affinities were relatively constant, while the heptanoate esterase affinity increased more than 30-fold. Community analysis revealed that a substantial community shift occurred within the first 7 days of operation. A Flavobacterium-like bacterial population dominated the community (>50% of total band intensity) and continued to do so for the remainder of the experiment. Copyright 2003 Wiley Periodicals, Inc.

Structural Characterization and Reversal of the Natural Organophosphate Resistance of a D-Type Esterase, Saccharomyces cerevisiae S-Formylglutathione Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Legler,P.; Kumaran, D.; Swaminathan, S.

2008-01-01

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 Angstroms resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme 'acyl pocket'. The W197I substitution enhances ySFGH reactivity with paraoxon bymore » >1000-fold (kiW197I = 16 {+-} 2 mM-1 h-1), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 Angstroms); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a 'D-type' esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon (ki = 42 or 80 mM-1 h-1, respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.« less

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin[S

PubMed Central

Camacho-Ruiz, María de los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A.

2015-01-01

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. PMID:25748441

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

PubMed

Abney, Kristopher K; Ramos-Hunter, Susan J; Romaine, Ian M; Godwin, J Shawn; Sulikowski, Gary A; Weaver, Charles David

2018-04-21

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated Porcine Liver Esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1

PubMed Central

Weiss, Michael; Denger, Karin; Huhn, Thomas

2012-01-01

Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C4-SPC). Second, these SPCs are mineralized. 3-C4-SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C4-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria. PMID:23001656

Elevated expression of esterase and cytochrome P450 are related with lambda-cyhalothrin resistance and lead to cross resistance in Aphis glycines Matsumura.

PubMed

Xi, Jinghui; Pan, Yiou; Bi, Rui; Gao, Xiwu; Chen, Xuewei; Peng, Tianfei; Zhang, Min; Zhang, Hua; Hu, Xiaoyue; Shang, Qingli

2015-02-01

A resistant strain of the Aphis glycines Matsumura (CRR) has developed 76.67-fold resistance to lambda-cyhalothrin compared with the susceptible (CSS) strain. Synergists piperonyl butoxide (PBO), S,S,S-Tributyltrithiophosphate (DEF) and triphenyl phosphate (TPP) dramatically increased the toxicity of lambda-cyhalothrin to the resistant strain. Bioassay results indicated that the CRR strain had developed high levels of cross-resistance to chlorpyrifos (11.66-fold), acephate (8.20-fold), cypermethrin (53.24-fold), esfenvalerate (13.83-fold), cyfluthrin (9.64-fold), carbofuran (14.60-fold), methomyl (9.32-fold) and bifenthrin (4.81-fold), but did not have cross-resistance to chlorfenapyr, imidacloprid, diafenthiuron, abamectin. The transcriptional levels of CYP6A2-like, CYP6A14-like and cytochrome b-c1 complex subunit 9-like increased significantly in the resistant strain than that in the susceptible. Similar trend were observed in the transcripts and DNA copy number of CarE and E4 esterase. Overall, these results demonstrate that increased esterase hydrolysis activity, combined with elevated cytochrome P450 monooxygenase detoxicatication, plays an important role in the high levels of lambda-cyhalothrin resistance and can cause cross-resistance to other insecticides in the CRR strain. Copyright © 2014 Elsevier Inc. All rights reserved.

Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC.

PubMed

Dilokpimol, Adiphol; Mäkelä, Miia R; Mansouri, Sadegh; Belova, Olga; Waterstraat, Martin; Bunzel, Mirko; de Vries, Ronald P; Hildén, Kristiina S

2017-07-25

A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

DOE PAGES

Chen, Qi; Luan, Zheng -Jiao; Yu, Hui -Lei; ...

2015-10-31

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst 1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residuemore » Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. In conclusion, this work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.« less

Human serum cholinesterase from liver pathological samples exhibit highly elevated aryl acylamidase activity.

PubMed

Boopathy, Rathanam; Rajesh, Ramanna Valmiki; Darvesh, Sultan; Layer, Paul G

2007-05-01

Although aspartate aminotransferase (AST) and gamma-glutamyltransferase (gamma GT) enzymes are widely used as markers for liver disorders, the ubiquitous enzyme butyrylcholinesterase (BChE), synthesized in liver is also used as marker in the assessment of liver pathophysiology. This BChE enzyme in addition to its esterase activity has yet another enzymatic function designated as aryl acylamidase (AAA) activity. It is determined in in vitro based on the hydrolysis of the synthetic substrate o-nitroacetanilide. In the present study, human serum cholinesterase (BChE) activity was studied with respect to its AAA activity on the BChE protein (AAA(BChE)) in patients with liver disorders. AST and gamma GT values were taken into account in this study as known markers for liver disorders. Blood samples were grouped into 3 based on esterase activity associated with BChE protein. They are normal, low, and very low BChE activity but with markedly increased AST and gamma GT levels. These samples were tested for their respective AAA function. Association of AAA with BChE from samples was proved using BChE monoclonal antibody precipitation experiment. The absolute levels of AAA were increased as BChE activity decreased while deviating from normal samples and such deviation was directly proportional to the severity of the liver disorder. Differences between these groups became prominent after determining the ratios of AAA(BChE) to BChE activities. Samples showing very high AAA(BChE) to BChE ratio were also showing high to very high gamma GT values. These findings establish AAA(BChE) as an independently regulated enzymatic activity on BChE especially in liver disorders. Moreover, since neither the low esterase activity of BChE by itself nor increased levels of AST/gamma GT are sufficient pathological indicators, this pilot study merits replication with large sample numbers.

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin.

PubMed

Camacho-Ruiz, María de Los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A

2015-05-01

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Role of the interfacial binding domain in the oxidative susceptibility of lecithin:cholesterol acyltransferase.

PubMed Central

Wang, Kewei; Subbaiah, Papasani V

2002-01-01

We had previously shown that the cholesterol esterification activity of lecithin:cholesterol acyltransferase (LCAT) is destroyed by oxidation, but still it retains the ability to hydrolyse water-soluble substrates. This suggested that the inactivation of the enzyme is not due to its catalytic function, but due to a loss of its hydrophobic binding. Since recent studies have shown that a tryptophan residue in the putative interfacial domain (Trp(61)) is critical for the activity, we determined the possible role of this residue in the oxidative susceptibility and substrate specificity of LCAT by site-directed mutagenesis. Deletion of Trp(61) resulted in a 56% loss of cholesterol esterification (LCAT) activity, but the phospholipase A(2) (PLA(2)) and the esterase activities of the enzyme were stimulated slightly. Replacing Trp(61) with another aromatic residue [Trp(61)-->Tyr (W61Y)] resulted in an increase in all activities (14-157%), whereas replacing it with an aliphatic residue [Trp(61)-->Gly (W61G)] caused a dramatic loss of LCAT (-90%) and PLA(2) (-82%) activities, but not the esterase activity (-5%). W61Y was the most sensitive to oxidation, whereas W61G was the most resistant, with respect to the LCAT and PLA(2) activities. However, the activities which do not involve interfacial binding, namely the esterase activity and the transesterification of short-chain phospholipids, were more resistant to oxidation in all LCATs, indicating a selective loss of the interfacial binding by oxidation. Furthermore, replacing the two cysteines (Cys(31) and Cys(184)) in the Trp(61) deletion mutant caused additional resistance of the enzyme to oxidizing agents, showing that both domains of the enzyme contribute independently to its oxidative susceptibility. Since the hydrolysis of truncated phospholipids, generated during the oxidation of low-density lipoproteins, does not require the interfacial-binding domain, our results suggest that LCAT may take part in the detoxification of these compounds even after the loss of its cholesterol esterification function. PMID:11966470

Presence and inter-individual variability of carboxylesterases (CES1 and CES2) in human lung.

PubMed

Gabriele, Morena; Puccini, Paola; Lucchi, Marco; Vizziello, Anna; Gervasi, Pier Giovanni; Longo, Vincenzo

2018-04-01

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (V max ) for pNPA and 4-MUA, unlike FD, were double in microsomes than in cytosol. In these cellular fractions, the V max of pNPA, as CES1 marker, were much greater (30-50-fold) than those of FD, as a specific CES2 marker. Conversely, the K m values were comparable suggesting the involvement of the same enzymes. Inhibition studies revealed that the FD hydrolysis was inhibited by bis-p-nitrophenylphosphate, phenylmethanesulfonyl fluoride, and loperamide (specific for CES2), whereas the pNPA and 4-MUA hydrolysis inhibition was limited. Inhibitors selective for other esterases missed having any effect on above-mentioned activities. In cytosol and microsomes of 20 lung samples, inter-individual variations were found for the hydrolysis of pNPA (2.5-5-fold), FD or 4-MUA (8-15-fold). Similar variations were also observed in CES1 and CES2 gene expression, although determined in a small number (n = 9) of lung samples. The identification of CES1 and CES2 and their variability in human lungs are important for drug metabolism and design of prodrugs which need to be activated in this organ. Copyright © 2018 Elsevier Inc. All rights reserved.

Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate.

PubMed

Masson, Patrick; Froment, Marie-Thérèse; Darvesh, Sultan; Schopfer, Lawrence M; Lockridge, Oksana

2007-08-01

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.

Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

PubMed Central

Rao, Lang; Zhao, Xiubo; Pan, Fang; Li, Yin; Xue, Yanfen; Ma, Yanhe; Lu, Jian R.

2009-01-01

Background Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity. PMID:19759821

An increase in pectin methyl esterase activity accompanies dormancy breakage and germination of yellow cedar seeds.

PubMed

Ren, C; Kermode, A R

2000-09-01

Pectin methyl esterase (PME) (EC 3.1.1.11) catalyzes the hydrolysis of methylester groups of cell wall pectins. We investigated the role of this enzyme in dormancy termination and germination of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds. PME activity was not detected in dormant seeds of yellow cedar but was induced and gradually increased during moist chilling; high activity coincided with dormancy breakage and germination. PME activity was positively correlated to the degree of dormancy breakage of yellow cedar seeds. The enzyme produced in different seed parts and in seeds at different times during moist chilling, germination, and early post-germinative growth consisted of two isoforms, both basic with isoelectric points of 8.7 and 8.9 and the same molecular mass of 62 kD. The pH optimum for the enzyme was between 7.4 and 8.4. In intact yellow cedar seeds, activities of the two basic isoforms of PME that were induced in embryos and in megagametophytes following dormancy breakage were significantly suppressed by abscisic acid. Gibberellic acid had a stimulatory effect on the activities of these isoforms in embryos and megagametophytes of intact seeds at the germinative stage. We hypothesize that PME plays a role in weakening of the megagametophyte, allowing radicle emergence and the completion of germination.

Carboxylesterases 1 and 2 hydrolyze phospho-nonsteroidal anti-inflammatory drugs: relevance to their pharmacological activity.

PubMed

Wong, Chi C; Cheng, Ka-Wing; Xie, Gang; Zhou, Dingying; Zhu, Cai-Hua; Constantinides, Panayiotis P; Rigas, Basil

2012-02-01

Phospho-nonsteroidal anti-inflammatory drugs (phospho-NSAIDs) are novel NSAID derivatives with improved anticancer activity and reduced side effects in preclinical models. Here, we studied the metabolism of phospho-NSAIDs by carboxylesterases and assessed the impact of carboxylesterases on the anticancer activity of phospho-NSAIDs in vitro and in vivo. The expression of human liver carboxylesterase (CES1) and intestinal carboxylesterase (CES2) in human embryonic kidney 293 cells resulted in the rapid intracellular hydrolysis of phospho-NSAIDs. Kinetic analysis revealed that CES1 is more active in the hydrolysis of phospho-sulindac, phospho-ibuprofen, phospho-naproxen, phospho-indomethacin, and phospho-tyrosol-indomethacin that possessed a bulky acyl moiety, whereas the phospho-aspirins are preferentially hydrolyzed by CES2. Carboxylesterase expression leads to a significant attenuation of the in vitro cytotoxicity of phospho-NSAIDs, suggesting that the integrity of the drug is critical for anticancer activity. Benzil and bis-p-nitrophenyl phosphate (BNPP), two carboxylesterase inhibitors, abrogated the effect of carboxylesterases and resensitized carboxylesterase-expressing cells to the potent cytotoxic effects of phospho-NSAIDs. In mice, coadministration of phospho-sulindac and BNPP partially protected the former from esterase-mediated hydrolysis, and this combination more effectively inhibited the growth of AGS human gastric xenografts in nude mice (57%) compared with phospho-sulindac alone (28%) (p = 0.037). Our results show that carboxylesterase mediates that metabolic inactivation of phospho-NSAIDs, and the inhibition of carboxylesterases improves the efficacy of phospho-NSAIDs in vitro and in vivo.

Carboxylesterases 1 and 2 Hydrolyze Phospho-Nonsteroidal Anti-Inflammatory Drugs: Relevance to Their Pharmacological Activity

PubMed Central

Wong, Chi C.; Cheng, Ka-Wing; Xie, Gang; Zhou, Dingying; Zhu, Cai-Hua; Constantinides, Panayiotis P.

2012-01-01

Phospho-nonsteroidal anti-inflammatory drugs (phospho-NSAIDs) are novel NSAID derivatives with improved anticancer activity and reduced side effects in preclinical models. Here, we studied the metabolism of phospho-NSAIDs by carboxylesterases and assessed the impact of carboxylesterases on the anticancer activity of phospho-NSAIDs in vitro and in vivo. The expression of human liver carboxylesterase (CES1) and intestinal carboxylesterase (CES2) in human embryonic kidney 293 cells resulted in the rapid intracellular hydrolysis of phospho-NSAIDs. Kinetic analysis revealed that CES1 is more active in the hydrolysis of phospho-sulindac, phospho-ibuprofen, phospho-naproxen, phospho-indomethacin, and phospho-tyrosol-indomethacin that possessed a bulky acyl moiety, whereas the phospho-aspirins are preferentially hydrolyzed by CES2. Carboxylesterase expression leads to a significant attenuation of the in vitro cytotoxicity of phospho-NSAIDs, suggesting that the integrity of the drug is critical for anticancer activity. Benzil and bis-p-nitrophenyl phosphate (BNPP), two carboxylesterase inhibitors, abrogated the effect of carboxylesterases and resensitized carboxylesterase-expressing cells to the potent cytotoxic effects of phospho-NSAIDs. In mice, coadministration of phospho-sulindac and BNPP partially protected the former from esterase-mediated hydrolysis, and this combination more effectively inhibited the growth of AGS human gastric xenografts in nude mice (57%) compared with phospho-sulindac alone (28%) (p = 0.037). Our results show that carboxylesterase mediates that metabolic inactivation of phospho-NSAIDs, and the inhibition of carboxylesterases improves the efficacy of phospho-NSAIDs in vitro and in vivo. PMID:22085648

Evaluation of liver and brain esterases in the spotted gar fish (Lepisosteus oculatus) as biomarkers of effect in the lower Mississippi River basin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, T.L.; Obih, P.O.; Jaiswal, R.

1997-05-01

The responses of various xenobiotic metabolizing enzymes in fish models are rapidly evolving as important biomarkers for monitoring unacceptable levels of environmental contaminants. Ethoxyresorufin O-deethylase, a specific cytochrome P450-dependent monooxygenase, is often used as an indicator of polycyclic aromatic hydrocarbon pollution. Another class of enzymes which are potential biomarkers are the B-type esterases. These enzymes are sensitive to inhibition by organophosphates, and include the cholinesterases (ChE) and carboxylesterases. ChEs are further subdivided into acetylcholinesterase and butyryl cholinesterase. Among fish, AChE is predominantly localized in the brain and muscle, whereas, BuChE activity is found mainly in liver and plasma. The precisemore » physiological role of BuChE is unknown, although it has been regarded as a marker enzyme for glial or supportive cells or other non-neuronal elements. Inhibition of ChE activity has often been associated with exposure to organophosphate and carbamate insecticides and other neurotoxic xenobiotics. Chemicals other than carbarnates and organophosphates that are environmental contaminants can also affect the activity of ChEs. Carboxylesterases represent a heterogenous group of isozymes that can catalyze the hydrolysis of a wide range of xenobiotic esters, amides and thioesters. For most CaE, their natural substrates are unknown, therefore, their physiological functions remain to be elucidated. These enzymes (CaE) occur widely in most tissues and are generally found in high levels in the liver. The purpose of this research was to evaluate the liver and brain esterases in the spotted gar fish as biomarkers of effect to multiple contaminants in the lower Mississippi River basin. 15 refs., 3 figs., 2 tabs.« less

Microbial degradation of polyurethane, polyester polyurethanes and polyether polyurethanes.

PubMed

Nakajima-Kambe, T; Shigeno-Akutsu, Y; Nomura, N; Onuma, F; Nakahara, T

1999-02-01

Polyurethane (PUR) is a polymer derived from the condensation of polyisocyanate and polyol and it is widely used as a base material in various industries. PUR, in particular, polyester PUR, is known to be vulnerable to microbial attack. Recently, environmental pollution by plastic wastes has become a serious issue and polyester PUR had attracted attention because of its biodegradability. There are many reports on the degradation of polyester PUR by microorganisms, especially by fungi. Microbial degradation of polyester PUR is thought to be mainly due to the hydrolysis of ester bonds by esterases. Recently, polyester-PUR-degrading enzymes have been purified and their characteristics reported. Among them, a solid-polyester-PUR-degrading enzyme (PUR esterase) derived from Comamonas acidovorans TB-35 had unique characteristics. This enzyme has a hydrophobic PUR-surface-binding domain and a catalytic domain, and the surface-binding domain was considered as being essential for PUR degradation. This hydrophobic surface-binding domain is also observed in other solid-polyester-degrading enzymes such as poly(hydroxyalkanoate) (PHA) depolymerases. There was no significant homology between the amino acid sequence of PUR esterase and that of PHA depolymerases, except in the hydrophobic surface-binding region. Thus, PUR esterase and PHA depolymerase are probably different in terms of their evolutionary origin and it is possible that PUR esterases come to be classified as a new solid-polyester-degrading enzyme family.

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.

PubMed

Chen, Qi; Luan, Zheng-Jiao; Yu, Hui-Lei; Cheng, Xiaolin; Xu, Jian-He

2015-11-01

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA.

PubMed

Fu, Jing; Sadgrove, Matthew; Marson, Lesley; Jay, Michael

2016-08-01

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [(14)C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CES-specific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

The role of negatively charged lipids in lysosomal phospholipase A2 function

PubMed Central

Abe, Akira; Shayman, James A.

2009-01-01

Lysosomal phospholipase A2 (LPLA2) is characterized by increased activity toward zwitterionic phospholipid liposomes containing negatively charged lipids under acidic conditions. The effect of anionic lipids on LPLA2 activity was investigated. Mouse LPLA2 activity was assayed as C2-ceramide transacylation. Sulfatide incorporated into liposomes enhanced LPLA2 activity under acidic conditions and was weakened by NaCl or increased pH. Amiodarone, a cationic amphiphilic drug, reduced LPLA2 activity. LPLA2 exhibited esterase activity when p-nitro-phenylbutyrate (pNPB) was used as a substrate. Unlike the phospholipase A2 activity, the esterase activity was detected over wide pH range and not inhibited by NaCl or amiodarone. Presteady-state kinetics using pNPB were consistent with the formation of an acyl-enzyme intermediate. C2-ceramide was an acceptor for the acyl group of the acyl-enzyme but was not available as the acyl group acceptor when dispersed in liposomes containing amiodarone. Cosedimentation of LPLA2 with liposomes was enhanced in the presence of sulfatide and was reduced by raising NaCl, amiodarone, or pH in the reaction mixture. LPLA2 adsorption to negatively charged lipid membrane surfaces through an electrostatic attraction, therefore, enhances LPLA2 enzyme activity toward insoluble substrates. Thus, anionic lipids present within lipid membranes enhance the rate of phospholipid hydrolysis by LPLA2 at lipid-water interfaces.—Abe, A., and J. A. Shayman. The role of negatively charged lipids in lysosomal phospholipase A2 function. PMID:19321879

ESTERASES OF THE POLYMORPHONUCLEAR LEUKOCYTE CAPABLE OF HYDROLYZING ACETYL DL-PHENYL-ALANINE β-NAPHTHYL ESTER

PubMed Central

Becker, Elmer L.; Ward, Peter A.

1969-01-01

Previous published work has led to the hypothesis that the activatable esterase of chemotaxis is a serine esterase of the rabbit polymorphonuclear leukocyte existing in an inert, phosphonate insusceptible form, which after activation is capable of hydrolyzing aromatic amino acid esters and being inhibited by phosphonates. In the present study, directed to the testing of this hypothesis, we have shown that rabbit peritoneal polymorphonuclear leukocytes contain three esterases capable of hydrolyzing the aromatic amino acid ester, acetyl DL-phenylalanine β-naphthyl ester. Two of these esterases, esterase 1 and esterase 2, are inhibited by various p-nitrophenyl ethyl phosphonate esters. The inhibition of each esterase is irreversible and progressive with time. When the logarithm of the esterase activity remaining after cell and inhibitor have been in contact for a constant time is plotted against the concentration of inhibitor, a straight line results. These results support the conclusion that both esterases are serine esterases. The third esterase, esterase 3, differs from the other two by its inability to be inactivated by any of the phosphonates no matter how high the concentration of phosphonate or prolonged the period of incubation of cell with phosphonate. The activity of esterase 1 is at least 10,000 times more easily inhibited by phosphonates than is that of esterase 2; incubating rabbit polymorphonuclear leukocytes for 15 min at 27°C with 10–9–10–8 M concentrations of various phosphonates inactivates esterase 1, but it required 10–6–10–4 M concentrations of the same phosphonates to inhibit esterase 2. The inhibition profiles of esterase 1 are distinctly different from those of esterase 2 when the two esterases are tested with the phenylalkylphosphonates, chloroalkylphosphonates, and alkylphosphonates. The inhibition profile of esterase 1 is essentially the same as that of the activatable esterase of chemotaxis obtained previously when the same three homologous series of phosphonates were tested for their ability to protect against deactivation by the chemotactic factor or give chemotactic-dependent inhibition. It is tentatively concluded that esterase 1 of the rabbit peritoneal neutrophil is the activated form of the activatable esterase of chemotaxis. PMID:5812915

Improved biomass degradation using fungal glucuronoyl-esterases-hydrolysis of natural corn fiber substrate.

PubMed

d'Errico, Clotilde; Börjesson, Johan; Ding, Hanshu; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

2016-02-10

Lignin-carbohydrate complexes (LCCs) are in part responsible for the recalcitrance of lignocellulosics in relation to industrial utilization of biomass for biofuels. Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 have been proposed to be able to degrade ester LCCs between glucuronic acids in xylans and lignin alcohols. By means of synthesized complex LCC model substrates we provide kinetic data suggesting a preference of fungal GEs for esters of bulky arylalkyl alcohols such as ester LCCs. Furthermore, using natural corn fiber substrate we report the first examples of improved degradation of lignocellulosic biomass by the use of GEs. Improved C5 sugar, glucose and glucuronic acid release was observed when heat pretreated corn fiber was incubated in the presence of GEs from Cerrena unicolor and Trichoderma reesei on top of different commercial cellulase/hemicellulase preparations. These results emphasize the potential of GEs for delignification of biomass thereby improving the overall yield of fermentable sugars for biofuel production. Copyright © 2015 Elsevier B.V. All rights reserved.

[Cloning, expression and characterization of a novel esterase from marine sediment microbial metagenomic library].

PubMed

Xu, Shiqing; Hu, Yongfei; Yuan, Aihua; Zhu, Baoli

2010-07-01

To clone, express and characterize a novel esterase from marine sediment microbial metagenomic library. Using esterase segregation agar containing tributyrin, we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library. This fosmid was partially digested with Sau3A I to construct the sublibrary, from which the esterase positive subclone pFLS10 was obtained. The full length of the esterase gene was amplified and cloned into the expressing vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 cells. We analyse the enzyme activity and study the characterization of the esterase after its expression and purification. An ORF (Open Reading Frame) of 924 bp was identified from the subclone pFLS10. Sequence analysis indicated that it showed 71% amino acid identity to esterase (ADA70030) from a marine sediment metagenomic library. The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate (C4). The optimum temperature of the esterase was 20 degrees C, the optimum pH was 7.5. The esterase in this study had good thermostability at 20 degrees C and good pH stability at pH8 -10. Significant increase in lipolytic activity was observed with addition of K+ and Mg2+, while decrease with Mn2+ etc. We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library. The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions, which laid a basis for industrial application.

Expression in Escherichia coli, refolding and crystallization of Aspergillus niger feruloyl esterase A using a serial factorial approach.

PubMed

Benoit, Isabelle; Coutard, Bruno; Oubelaid, Rachid; Asther, Marcel; Bignon, Christophe

2007-09-01

Hydrolysis of plant biomass is achieved by the combined action of enzymes secreted by microorganisms and directed against the backbone and the side chains of plant cell wall polysaccharides. Among side chains degrading enzymes, the feruloyl esterase A (FAEA) specifically removes feruloyl residues. Thus, FAEA has potential applications in a wide range of industrial processes such as paper bleaching or bio-ethanol production. To gain insight into FAEA hydrolysis activity, we solved its crystal structure. In this paper, we report how the use of four consecutive factorial approaches (two incomplete factorials, one sparse matrix, and one full factorial) allowed expressing in Escherichia coli, refolding and then crystallizing Aspergillus niger FAEA in 6 weeks. Culture conditions providing the highest expression level were determined using an incomplete factorial approach made of 12 combinations of four E. coli strains, three culture media and three temperatures (full factorial: 36 combinations). Aspergillus niger FAEA was expressed in the form of inclusion bodies. These were dissolved using a chaotropic agent, and the protein was purified by affinity chromatography on Ni column under denaturing conditions. A suitable buffer for refolding the protein eluted from the Ni column was found using a second incomplete factorial approach made of 96 buffers (full factorial: 3840 combinations). After refolding, the enzyme was further purified by gel filtration, and then crystallized following a standard protocol: initial crystallization conditions were found using commercial crystallization screens based on a sparse matrix. Crystals were then optimized using a full factorial screen.

Biodegradation of composite resin with ester linkages: identifying human salivary enzyme activity with a potential role in the esterolytic process.

PubMed

Cai, Kuihua; Delaviz, Yasaman; Banh, Michael; Guo, Yi; Santerre, J Paul

2014-08-01

The ester linkages contained within dental resin monomers (such as Bisphenol A-glycidylmethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA)) are susceptible to hydrolytic degradation by salivary esterases, however very little is known about the specific esterase activities implicated in this process. The objective of this work was to isolate and identify the dominant proteins from saliva that are associated with the esterase activities shown to be involved in the degradation of BisGMA. Human whole saliva was collected and processed prior to separation in a HiPrep 16/60 Sephacryl S-200 HR column. The fraction with the highest esterase activity was further separated by an anion exchange column (Mono-Q (10/100G)). Isolated fractions were then separated by gel electrophoresis, and compared to a common bench marker esterase, cholesterol esterase (CE), and commercial albumin which has been reported to express esterase activity. Proteins suspected of containing esterase activity were analyzed by Mass Spectroscopy (MS). Commercially available proteins, similar to the salivary esterase proteins identified by MS, were used to replicate the enzymatic complexes and confirm their degradation activity with respect to BisGMA. MS data suggested that the enzyme fraction with the highest esterase activity was contained among a group of proteins consisting of albumin, Zn-α2-glycoprotein, α-amylase, TALDO1 protein, transferrin, lipocalin2, and prolactin-induced protein. Studies concluded that the main esterase bands on the gels in each fraction did not overlap with CE activity, and that albumin activity emerged as a lead candidate with significant esterase activity relative to BisGMA degradation, particularly when it formed a complex with Zn-α2-glycoprotein, under slightly basic conditions. These enzyme complexes can be used as a physiologically relevant formulation to test the biostability of composite resins. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

PubMed

Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

2016-08-01

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei. Copyright © 2016 Elsevier Inc. All rights reserved.

Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure.

PubMed

Nigg, H N; Knaak, J B

2000-01-01

The organophosphorus pesticides of this review were discovered in 1936 during the search for a replacement for nicotine for cockroach control. The basic biochemical characteristics of RBC AChE and BChE were determined in the 1940s. The mechanism of inhibition of both enzymes and other serine esterases was known in the 1940s and, in general, defined in the 1950s. In 1949, the death of a parathion mixer-loader dictated blood enzyme monitoring to prevent acute illness from organophosphorus pesticide intoxication. However, many of the chemical and biochemical steps for serine enzyme inhibition by OP compounds remain unknown today. The possible mechanisms of this inhibition are presented kinetically beginning with simple (by comparison) Michaelis-Menten substrate enzyme interaction kinetics. As complicated as the inhibition kinetics appear here, PBPK model kinetics will be more complex. The determination of inter- and intraindividual variation in RBC ChE and BChE was recognized early as critical knowledge for a blood esterase monitoring program. Because of the relatively constant production of RBCs, variation in RBC AChE was determined by about 1970. The source of plasma (or serum) BChE was shown to be the liver in the 1960s with the change in BChE phenotype to the donor in liver transplant patients. BChE activity was more variable than RBC AChE, and only in the 1990s have BChE individual variation questions been answered. We have reviewed the chemistry, metabolism, and toxicity of organophosphorus insecticides along with their inhibitory action toward tissue acetyl- and butyrylcholinesterases. On the basis of the review, a monitoring program for individuals mixing-loading and applying OP pesticides for commercial applicators was recommended. Approximately 41 OPs are currently registered for use by USEPA in the United States. Under agricultural working conditions, OPs primarily are absorbed through the skin. Liver P-450 isozymes catalyze the desulfurization of phosphorothioates and phosphorodithioates (e.g., parathion and azinphosmethyl, respectively) to the more toxic oxons (P = O(S to O)). In some cases, P-450 isozymes catalyze the oxidative cleavage of P-O-aryl bonds (e.g., parathion, methyl parathion, fenitrothion, and diazinon) to form inactive water-soluble alkyl phosphates and aryl leaving groups that are readily conjugated with glucuronic or sulfuric acids and excreted. In addition to the P-450 isozymes, mammalian tissues contain ('A' and 'B') esterases capable of reacting with OPs to produce hydrolysis products or phosphorylated enzymes. 'A'-esterases hydrolyze OPs (i.e., oxons), while 'B'-esterases with serine at the active center are inhibited by OPs. OPs possessing carboxylesters, such as malathion and isofenphos, are hydrolyzed by the direct action of 'B'-esterases (i.e., carboxylesterase, CaE). Metabolic pathways shown for isofenphos, parathion, and malathion define the order in which these reactions occur, while Michaelis-Menten kinetics define reaction parameters (Vmax, K(m)) for the enzymes and substrates involved, and rates of inhibition of 'B'-esterases (kis, bimolecular rate constants) by OPs and their oxons. OPs exert their insecticidal action by their ability to inhibit AChE at the cholinergic synapse, resulting in the accumulation of acetylcholine. The extent to which AChE or other 'B'-esterases are inhibited in workers is dependent upon the rate the OP pesticide is activated (i.e., oxon formation), metabolized to nontoxic products by tissue enzymes, its affinity for AChE and other 'B'-esterases, and esterase concentrations in tissues. Rapid recovery of OP BChE inhibition may be related to reactivation of inhibited forms. AChE, BChE, and CaE appear to function in vivo as scavengers, protecting workers against the inhibition of AChE at synapses. Species sensitivity to OPs varies widely and results in part from binding affinities (Ka) and rates of phosphorylation (kp) rather than rates of activation and detoxif

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

PubMed

Sornlake, Warasirin; Rattanaphanjak, Phatcharamon; Champreda, Verawat; Eurwilaichitr, Lily; Kittisenachai, Suthathip; Roytrakul, Sittiruk; Fujii, Tatsuya; Inoue, Hiroyuki

2017-07-01

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and β-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external β-xylosidase.

Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases

PubMed Central

2014-01-01

Background Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Results Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. Conclusion The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw. PMID:24766728

Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases.

PubMed

Badhan, Ajay; Wang, Yuxi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim

2014-04-26

Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

PubMed

Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

2011-09-01

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.

2007-05-15

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrinmore » are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are {approx} 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts ({approx} 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected. Together, these results demonstrate that extrahepatic esterolytic metabolism of specific pyrethroids may be significant. Moreover, hepatic cytosolic and microsomal hydrolytic metabolism should each be considered during the development of pharmacokinetic models that predict the disposition of pyrethroids and other esterified compounds.« less

Evaluation of a Hypocrea jecorina enzyme preparation for hydrolysis of Tifton 85 bermudagrass.

PubMed

Ximenes, E A; Brandon, S K; Doran-Peterson, J

2008-03-01

Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.

Evaluation of a Hypocrea jecorina Enzyme Preparation for Hydrolysis of Tifton 85 Bermudagrass

NASA Astrophysics Data System (ADS)

Ximenes, E. A.; Brandon, S. K.; Doran-Peterson, J.

Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.

[An activity of nonspecific esterases in homogenates of Lymnaea stagnalis and Lymnaea tumida snails (Gastropoda: Pulmonata) infected by trematode cercariae Echinoparyphium aconiatum and Moliniella anceps (Echinostomatidae)].

PubMed

Vorontsova, Ia L; Iurlova, N I; Vodianitskaia, S N; Glupov, V V

2008-01-01

The comparative analysis of esterase changes in homogenates of the snails Lymnaea stagnalis and L. tumida bodies was carried out. Juvenile snails with shell size 2 mm, 3-4 mm, 5-6 mm and 7-8 mm were exposed to cercariae of the trematodes Echinoparyphium aconiatum and/or Moliniella anceps. The esterase activity was detected spectrofotometrically. The highest level of esterase activity in noninfected L. stagnalis was registered in snails with shell size 3-4 mm. The invasion of snails by trematode cercariae results in a change of esterase activity in the tissues of infected snails. The activity of easterases was increased in the infected L. stagnalis snails with shell size 5-8 mm at 2 days post invasion in comparison with control. The decrease of esterase activity in tissues of infected snails L. stagnalis (3-4 mm) and L. tumida (4 mm) was observed at 26 days post invasion by E. aconiatum only. The host size and parasite species was influenced on esterase activity in the snails.

Site-specific hydrolysis of chlorogenic acids by selected Lactobacillus species.

PubMed

Aguirre Santos, Elsa Anaheim; Schieber, Andreas; Weber, Fabian

2018-07-01

Hydroxycinnamic acids are a major group of phenolic compounds widely distributed in plants. Among them, chlorogenic acids and caffeic acid have been in the focus of interest due to their impact on food quality and their putative health benefits. Numerous microorganisms like lactic acid bacteria are able to hydrolyze chlorogenic acids by cinnamoyl esterase enzymes. Data on the specificity of theses enzymes regarding the cleavage of distinct isomers of mono- or dichlorogenic acids is lacking. Lactobacillus reuteri, Lactobacillus helveticus, and Lactobacillus fermentum were screened for their ability to hydrolyze chlorogenic acid isomers in culture medium. Concentrations of chlorogenic acids and the released caffeic acid were determined by UHPLC-ESI-MS. The highest hydrolysis rate (100%) was observed for the hydrolysis of 5-CQA by Lactobacillus helveticus. A so far unknown metabolic pathway for the cleavage of 4-CQA is proposed including isomerization to 5-CQA and 3-CQA followed by hydrolysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of a cold-active esterase from Lactobacillus plantarum suitable for food fermentations.

PubMed

Esteban-Torres, María; Mancheño, José Miguel; de las Rivas, Blanca; Muñoz, Rosario

2014-06-04

Lactobacillus plantarum is a lactic acid bacteria that can be found in numerous fermented foods. Esterases from L. plantarum exert a fundamental role in food aroma. In the present study, the gene lp_2631 encoding a putative esterase was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_2631 protein has been biochemically characterized. Lp_2631 exhibited optimal esterase activity at 20 °C and more than 90% of maximal activity at 5 °C, being the first cold-active esterase described in a lactic acid bacteria. Lp_2631 exhibited 40% of its maximal activity after 2 h of incubation at 65 °C. Lp_2631 also showed marked activity in the presence of compounds commonly found in food fermentations, such as NaCl, ethanol, or lactic acid. The results suggest that Lp_2631 might be a useful esterase to be used in food fermentations.

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.

PubMed

Rejón, Juan D; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J

2012-10-01

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes.

PubMed

Tchigvintsev, Anatoli; Tran, Hai; Popovic, Ana; Kovacic, Filip; Brown, Greg; Flick, Robert; Hajighasemi, Mahbod; Egorova, Olga; Somody, Joseph C; Tchigvintsev, Dmitri; Khusnutdinova, Anna; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Savchenko, Alexei; Golyshin, Peter N; Jaeger, Karl-Erich; Yakunin, Alexander F

2015-03-01

Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models.

PubMed

Tokudome, Yoshihiro; Katayanagi, Mishina; Hashimoto, Fumie

2015-06-01

Reconstructed human epidermal culture skin models have been developed for cosmetic and pharmaceutical research. This study evaluated the total and carboxyl esterase activities (i.e., Km and Vmax , respectively) and localization in two reconstructed human epidermal culture skin models (LabCyte EPI-MODEL [Japan Tissue Engineering] and EpiDerm [MatTek/Kurabo]). The usefulness of the reconstruction cultured epidermis was also verified by comparison with human and rat epidermis. Homogenized epidermal samples were fractioned by centrifugation. p-nitrophenyl acetate and 4-methylumbelliferyl acetate were used as substrates of total esterase and carboxyl esterase, respectively. Total and carboxyl esterase activities were present in the reconstructed human epidermal culture skin models and were localized in the cytosol. Moreover, the activities and localization were the same as those in human and rat epidermis. LabCyte EPI-MODEL and EpiDerm are potentially useful for esterase activity prediction in human epidermis.

Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models

PubMed Central

Katayanagi, Mishina; Hashimoto, Fumie

2015-01-01

Background Reconstructed human epidermal culture skin models have been developed for cosmetic and pharmaceutical research. Objective This study evaluated the total and carboxyl esterase activities (i.e., Km and Vmax, respectively) and localization in two reconstructed human epidermal culture skin models (LabCyte EPI-MODEL [Japan Tissue Engineering] and EpiDerm [MatTek/Kurabo]). The usefulness of the reconstruction cultured epidermis was also verified by comparison with human and rat epidermis. Methods Homogenized epidermal samples were fractioned by centrifugation. p-nitrophenyl acetate and 4-methylumbelliferyl acetate were used as substrates of total esterase and carboxyl esterase, respectively. Results Total and carboxyl esterase activities were present in the reconstructed human epidermal culture skin models and were localized in the cytosol. Moreover, the activities and localization were the same as those in human and rat epidermis. Conclusion LabCyte EPI-MODEL and EpiDerm are potentially useful for esterase activity prediction in human epidermis. PMID:26082583

The esterase and depsidase activities of tannase

PubMed Central

Haslam, E.; Stangroom, J. E.

1966-01-01

The esterase and depsidase activities of tannase have been examined by kinetic methods. Although the esterase/depsidase ratio of tannase may be varied by cultural methods and isolation procedures, evidence has been obtained to show that tannase, esterase and depsidase are enzymes with low specificities capable of hydrolysing both esters and depsides of gallic acid. PMID:5965343

Simultaneous pretreatment and enzymatic hydrolysis of forage biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henk, L.; Linden, J.C.

1993-12-31

Sweet sorghum is an attractive fermentation feedstock because as much as 40% of the dry weight consists of readily femented sugars such as sucrose, glucose and frutose. Cellulose and hemicellulose comprise another 50%. However, if this material is to be used a year-round feedstock for ethanol production, a stable method of storage must be developed to maintain the sugar content. A modified version of the traditional ensiling process is made effective by the addition of cellulolytic/hemicellulolytic enzymes and lactic acid bacteria to freshly chopped sweet sorghum prior to the production of silage. In situ hydrolysis of cellulose and hemicellulose occursmore » concurrently with the acidic ensiling fementation. By hydolyzing the acetyl groups using acetyl xylan esterase and 3-0-methyl glucuronyl side chains using pectinase from hemicellulose, cellulose becomes accessible to hydrolysis by cellulase, both during in situ ensiling with enzymes and in the simultaneous saccharification and fermentation (SSF) to ethanol.« less

Chemotactic activity from rabbit peritoneal neutrophils. Lack of identity with N-acetyl-DL-phenylalanine beta-napthyl esterase.

PubMed

Tsung, P K; Showell, H J; Kegeles, S W; Becker, E L

1976-08-12

The chemotactic and N-acetyl-DL-phenylalanine beta-naphthyl esterase activities of rabbit peritoneal neutrophils are separable from each other by both DEAE cellulose and Sephadex G-100 column chromatography. Partially purified esterase obtained from DEAE-cellulose chromatography had molecular weight of 70 000. However, the partially purified fraction contained chemotactic activities with major activity in molecular weight of 28000 and minor activities in the molecular weights of 45000, 21900, 14500 and 10500. Esterase activity is inhibited by 10(-7) M p-nitrophenylethyl-5-chloropentylphosphonate but chemotactic activity is not.

Discovery of Polyesterases from Moss-Associated Microorganisms.

PubMed

Müller, Christina Andrea; Perz, Veronika; Provasnek, Christoph; Quartinello, Felice; Guebitz, Georg M; Berg, Gabriele

2017-02-15

The growing pollution of the environment with plastic debris is a global threat which urgently requires biotechnological solutions. Enzymatic recycling not only prevents pollution but also would allow recovery of valuable building blocks. Therefore, we explored the existence of microbial polyesterases in microbial communities associated with the Sphagnum magellanicum moss, a key species within unexploited bog ecosystems. This resulted in the identification of six novel esterases, which were isolated, cloned, and heterologously expressed in Escherichia coli The esterases were found to hydrolyze the copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT) and the oligomeric model substrate bis[4-(benzoyloxy)butyl] terephthalate (BaBTaBBa). Two promising polyesterase candidates, EstB3 and EstC7, which clustered in family VIII of bacterial lipolytic enzymes, were purified and characterized using the soluble esterase substrate p-nitrophenyl butyrate (K m values of 46.5 and 3.4 μM, temperature optima of 48°C and 50°C, and pH optima of 7.0 and 8.5, respectively). In particular, EstC7 showed outstanding activity and a strong preference for hydrolysis of the aromatic ester bond in PBAT. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems as a source for novel hydrolytic enzymes hydrolyzing polymeric compounds. In this study, we describe the discovery and analysis of new enzymes from microbial communities associated with plants (moss). The recovered enzymes show the ability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate-co-butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be used as catalysts for selective recovery of valuable building blocks from this material. Copyright © 2017 American Society for Microbiology.

Discovery of Polyesterases from Moss-Associated Microorganisms

PubMed Central

Perz, Veronika; Provasnek, Christoph; Quartinello, Felice; Guebitz, Georg M.; Berg, Gabriele

2016-01-01

ABSTRACT The growing pollution of the environment with plastic debris is a global threat which urgently requires biotechnological solutions. Enzymatic recycling not only prevents pollution but also would allow recovery of valuable building blocks. Therefore, we explored the existence of microbial polyesterases in microbial communities associated with the Sphagnum magellanicum moss, a key species within unexploited bog ecosystems. This resulted in the identification of six novel esterases, which were isolated, cloned, and heterologously expressed in Escherichia coli. The esterases were found to hydrolyze the copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT) and the oligomeric model substrate bis[4-(benzoyloxy)butyl] terephthalate (BaBTaBBa). Two promising polyesterase candidates, EstB3 and EstC7, which clustered in family VIII of bacterial lipolytic enzymes, were purified and characterized using the soluble esterase substrate p-nitrophenyl butyrate (Km values of 46.5 and 3.4 μM, temperature optima of 48°C and 50°C, and pH optima of 7.0 and 8.5, respectively). In particular, EstC7 showed outstanding activity and a strong preference for hydrolysis of the aromatic ester bond in PBAT. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems as a source for novel hydrolytic enzymes hydrolyzing polymeric compounds. IMPORTANCE In this study, we describe the discovery and analysis of new enzymes from microbial communities associated with plants (moss). The recovered enzymes show the ability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate-co-butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be used as catalysts for selective recovery of valuable building blocks from this material. PMID:27940546

Targeting Transient Receptor Potential Vanilloid 1 (TRPV1) Channel Softly: The Discovery of Passerini Adducts as a Topical Treatment for Inflammatory Skin Disorders.

PubMed

Serafini, Marta; Griglio, Alessia; Aprile, Silvio; Seiti, Fabio; Travelli, Cristina; Pattarino, Franco; Grosa, Giorgio; Sorba, Giovanni; Genazzani, Armando A; Gonzalez-Rodriguez, Sara; Butron, Laura; Devesa, Isabel; Fernandez-Carvajal, Asia; Pirali, Tracey; Ferrer-Montiel, Antonio

2018-05-24

Despite being an old molecule, capsaicin is still a hot topic in the scientific community, and the development of new capsaicinoids is a promising pharmacological approach in the management of skin disorders related to inflammation and pruritus. Here we report the synthesis and the evaluation of capsaicin soft drugs that undergo deactivation by the hydrolyzing activity of skin esterases. The implanting of an ester group in the lipophilic moiety of capsaicinoids by the Passerini multicomponent reaction affords both agonists and antagonists that retain transient receptor potential vanilloid 1 channel (TRPV1) modulating activity and, at the same time, are susceptible to hydrolysis. The most promising antagonist identified shows in vivo anti-nociceptive activity on pruritus and hyperalgesia without producing hyperthermia, thus validating it as novel treatment for dermatological conditions that implicate TRPV1 channel dysfunction.

Esterase in Imported Fire Ants, Solenopsis invicta and S. richteri (Hymenoptera: Formicidae): Activity, Kinetics and Variation

PubMed Central

Chen, J.; Rashid, T.; Feng, G.

2014-01-01

Solenopsis invicta and Solenopsis richteri are two closely related invasive ants native to South America. Despite their similarity in biology and behavior, S. invicta is a more successful invasive species. Toxic tolerance has been found to be important to the success of some invasive species. Esterases play a crucial role in toxic tolerance of insects. Hence, we hypothesized that the more invasive S. invicta would have a higher esterase activity than S. richteri. Esterase activities were measured for workers and male and female alates of both ant species using α-naphthyl acetate and β-naphthyl acetate as substrates. Esterase activities in S. invicta were always significantly higher than those in S. richteri supporting our hypothesis. In S. invicta, male alates had the highest esterase activities followed by workers then female alates for both substrates. In S. richetri, for α-naphthyl acetate, male alates had the highest activity followed by female alates then workers, while for β-naphthyl acetate, female alates had the highest activity followed by male alates then workers. For workers, S. richteri showed significantly higher levels of variation about the mean esterase activity than S. invicta. However, S. invicta showed significantly higher levels of variation in both female and male alates. PMID:25408118

Two-stage statistical medium optimization for augmented cellulase production via solid-state fermentation by newly isolated Aspergillus niger HN-1 and application of crude cellulase consortium in hydrolysis of rice straw.

PubMed

Sandhu, Simranjeet Kaur; Oberoi, Harinder Singh; Babbar, Neha; Miglani, Kanupriya; Chadha, Bhupinder Singh; Nanda, Dhiraj Kumar

2013-12-26

Cellulolytic enzyme production by newly isolated Aspergillus niger HN-1 was statistically optimized using Plackett-Burman and central composite design (CCD). Optimum concentrations of 2, 0.40, 0.01, and 0.60 g L (-1) for KH2PO4, urea, trace elements solution, and CaCl2·2H2O, respectively, were suggested by Design-Expert software. The two-stage optimization process led to a 3- and 2-fold increases in the filter paper cellulase (FP) and β-glucosidase activities, respectively. FP, β-glucosidase, endoglucanase, exopolygalaturonase, cellobiohydrolase, xylanase, α-l-arabinofuranosidase, β-xylosidase, and xylan esterase activities of 36.7 ± 1.54 FPU gds(-1), 252.3 ± 7.4 IU gds(-1), 416.3 ± 22.8 IU gds(-1), 111.2 ± 5.4 IU gds(-1), 8.9 ± 0.50 IU gds(-1), 2593.5 ± 78.9 IU gds(-1), 79.4 ± 4.3 IU gds(-1), 180.8 ± 9.3 IU gds(-1), and 288.7 ± 11.8 IU gds(-1), respectively, were obtained through solid-state fermentation during the validation studies. Hydrolysis of alkali-treated rice straw with crude cellulases resulted in about 84% glucan to glucose, 89% xylan to xylose, and 91% arabinan to arabinose conversions, indicating potential for biomass hydrolysis by the crude cellulase consortium obtained in this study.

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

PubMed Central

Rejón, Juan D.; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J.

2012-01-01

Background and Aims A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. Methods The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Key Results Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. Conclusions In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma. PMID:22922586

In situ localization of the genetic locus encoding the lysosomal acid lipase/cholesteryl esterase (LIPA) deficient in wolman disease to chromosome 10q23. 2-q23. 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, R.A.; Rao, N.; Byrum, R.S.

1993-01-01

Human acid lipase/cholesteryl esterase (EC 3.1.1.13) is a 46-kDa glycoprotein required for the lysosomal hydrolysis of cholesteryl esters and triglycerides that cells acquire through the receptor-mediated endocytosis of low-density lipoproteins. This activity is essential in the provision of free cholesterol for cell metabolism as well as for the feedback signal that modulates endogenous cellular cholesterol production. The extremely low level of lysosomal acid lipase in patients afflicted with the hereditary, allelic lysosomal storage disorders Woman disease (WD) and cholesteryl ester storage disease (CESD) (MIM Number 278000 (6)) is associated with the massive intralysosomal lipid storage and derangements in the regulationmore » of cellular cholesterol production (10). Both WD and CESD cells lack a specific acid lipase isoenzyme and it is thought that the different mutations associated with WD and CESD are in the structural gene for this isoenzyme, LIPA. Analysis of the activity of the acid lipase isoenzyme in cell extracts from human-Chinese hamster somatic cell hybrids (4, 11) demonstrated the concordant segregation of the gene locus for lysosomal acid lipase with the glutamate oxaloacetate transaminase-1 (GOT1) enzyme marker for human chromosome 10 which was subsequently localized to 10q24.1 q25.1 (8). 11 refs., 1 figs.« less

Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

PubMed

Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

2016-01-01

Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic efficiencies towards target reactions.

Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model

PubMed Central

Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

2016-01-01

Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic efficiencies towards target reactions. PMID:27243223

Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases.

PubMed

Hajighasemi, Mahbod; Nocek, Boguslaw P; Tchigvintsev, Anatoli; Brown, Greg; Flick, Robert; Xu, Xiaohui; Cui, Hong; Hai, Tran; Joachimiak, Andrzej; Golyshin, Peter N; Savchenko, Alexei; Edwards, Elizabeth A; Yakunin, Alexander F

2016-06-13

Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.

A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil.

PubMed

Hu, Xiao Ping; Heath, Caroline; Taylor, Mark Paul; Tuffin, Marla; Cowan, Don

2012-01-01

A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic "families". The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T(opt) at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure-function relationships of esterase activity.

Determination of physicochemical properties and degradation kinetics of triamcinolone acetonide palmitate in vitro.

PubMed

Peng, Cuilian; Liu, Cong; Tang, Xing

2010-12-01

Triamcinolone acetonide palmitate (TAP) is a lipophilic prodrug of triamcinolone acetonide (TAA) to improve the insoluble TAA physicochemical properties for the preparation of emulsions. This investigation has focused on the preformulation study of TAP, including its physicochemical properties and hydrolysis kinetics in vitro. The solubility of TAP in medium-chain triglyceride is about twice greater than that in soybean oil (long-chain triglyceride) (19.17 versus 9.55 mg/g) at 25°C, and in all investigated cases, lecithin (80, 160, and 240 mg/g) as solubilizer provided increased solubility of drugs in medium-chain triglyceride and long-chain triglyceride, whereas the maximum water solubility of TAP was 0.10 μg/mL. The partition coefficient (log P) of TAP was 5.79 irrespective of the pH conditions. The hydrolysis of TAP followed pseudo-first-order kinetics in aqueous solutions, and the stable pH range was from pH 5.0 to 9.0. The in vitro enzymolysis kinetics of TAP in rat plasma and liver homogenate was evaluated by measuring the decrease of TAP as well as the increase of TAA at 37°C for 96 hours. The results demonstrated that the TAP may be hydrolyzed mainly by rat plasma esterase and, to a minor extent, by liver esterase, and the hydrolysis half-life of TAP in 100% rat plasma was 17.53 ± 6.85 hours at pH 7.4. All these results indicated that TAP had successfully obtained higher lipid-soluble property for the preparation of intravenous emulsion and may be an effective prodrug for sustained release of TAA in vivo.

Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius.

PubMed

Pennacchio, Angela; Mandrich, Luigi; Manco, Giuseppe; Trincone, Antonio

2015-09-01

The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two α-D-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl β-D-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3',5'-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.

Design of enzyme-mediated controlled release systems based on silica mesoporous supports capped with ester-glycol groups.

PubMed

Agostini, Alessandro; Mondragón, Laura; Pascual, Lluis; Aznar, Elena; Coll, Carmen; Martínez-Máñez, Ramón; Sancenón, Félix; Soto, Juan; Marcos, M Dolores; Amorós, Pedro; Costero, Ana M; Parra, Margarita; Gil, Salvador

2012-10-16

An ethylene glycol-capped hybrid material for the controlled release of molecules in the presence of esterase enzyme has been prepared. The final organic-inorganic hybrid solid S1 was synthesized by a two-step procedure. In the first step, the pores of an inorganic MCM-41 support (in the form of nanoparticles) were loaded with [Ru(bipy)(3)]Cl(2) complex, and then, in the second step, the pore outlets were functionalized with ester glycol moieties that acted as molecular caps. In the absence of an enzyme, release of the complex from aqueous suspensions of S1 at pH 8.0 is inhibited due to the steric hindrance imposed by the bulky ester glycol moieties. Upon addition of esterase enzyme, delivery of the ruthenium complex was observed due to enzymatic hydrolysis of the ester bond in the anchored ester glycol derivative, inducing the release of oligo(ethylene glycol) fragments. Hydrolysis of the ester bond results in size reduction of the appended group, therefore allowing delivery of the entrapped cargo. The S1 nanoparticles were not toxic for cells, as demonstrated by cell viability assays with HeLa and MCF-7 cell lines, and were found to be associated with lysosomes, as shown by confocal microscopy. However, when S1 nanoparticles were filled with the cytotoxic drug camptothecin (S1-CPT), S1-CPT-treated cells undergo cell death as a result of S1-CPT cell internalization and subsequent cellular enzyme-mediated hydrolysis and aperture of the molecular gate that induced the release of the camptothecin cargo. These findings point to a possible therapeutic application of these nanoparticles.

Novel choline esterase based sensor for monitoring of organophosphorus pollutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkins, E.S.; Ghindilis, A.L.; Atanasov, P.

1996-12-31

Organophosphorus compounds are significant major environmental pollutants due to their intensive use as pesticides. The modern techniques based on inhibition of choline esterase enzyme activity are discussed. Potentiometric electrodes based on detection of choline esterase inhibition by analytes has been developed. The detection of choline esterase activity is based on the novel principle of molecular transduction. Immobilized peroxidase acting as the molecular transducer, catalyzes the electroreduction of hydrogen peroxide by direct (mediatorless) electron transfer. The sensing element consists of a carbon based electrode containing an assembly of co-immobilized enzymes: choline esterase, choline oxidase and peroxidase.

Esterase reactions in acute myelomonocytic leukemia.

PubMed

Kass, L

1977-05-01

Specific and nonspecific esterase reactions of bone marrow cells from 14 patients with untreated acute myelomonocytic leukemia and six patients with acute histiomonocytic leukemia were examined. The technic for esterase determination permitted simultaneous visualization of both esterases on the same glass coverslip containing the marrow cells. In cases of acute histiomonocytic leukemia, monocytes, monocytoid hemohistioblasts and undifferentiated blasts stained intensely positive for nonspecific esterase, using alpha-naphthyl acetate as the substrate. No evidence of specific esterase activity using naphthol ASD-chloroacetate as the substrate and fast blue BBN as the dye coupler was apparent in these cells. In all of the cases of acute myelomonocytic leukemia, both specific and nonspecific esterases were visualized within monocytes, monocytoid cells, and granulocytic cells that had monocytoid-type nuclei. Nonspecific esterase activity was not observed in polymorphonuclear leukocytes in cases of myelomonocytic leukemia. The results support a current viewpoint that acute myelomonocytic leukemia may be a variant of acute myeloblastic leukemia, and that cytochemically, many of the leukemic cells in myelomonocytic leukemia share properties of both granulocytes and monocytes.

Extracellular fluid proteins of goldfish brain: evidence for the presence of proteases and esterases.

PubMed

Shashoua, V E; Holmquist, B

1986-09-01

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.

Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp.

PubMed

Oleas, Gabriela; Callegari, Eduardo; Sepúlveda, Romina; Eyzaguirre, Jaime

2017-04-18

The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10). Copyright © 2017 Elsevier Ltd. All rights reserved.

Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp

PubMed Central

Oleas, Gabriela; Callegari, Eduardo; Sepúlveda, Romina; Eyzaguirre, Jaime

2017-01-01

The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10). PMID:28342968

Effect of halogenated benzenes on acetanilide esterase, acetanilide hydroxylase and procaine esterase in rats.

PubMed

Carlson, G P; Dziezak, J D; Johnson, K M

1979-07-01

1,2,4-Trichlorobenzene, 1,3,5-trichlorobenzene, hexachlorobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene and hexabromobenzene were compared for their abilities to induce acetanilide esterase, acentailide hydroxylase and procaine esterase. Except for hexabromobenzene all induced acetanilide esterase whereas the hydroxylation of acetanilide was seen only with the fully halogenated benzenes and with 1,3,5-tribromobenzene. Hepatic procaine esterase activity was increased by the three chlorinated benzenes and 1,2,4-tribromobenzene.

Effect of rivastigmine on plasma butyrylcholine esterase activity and plasma ghrelin levels in patients with dementia in Alzheimer's disease.

PubMed

Kuroda, Atsushi; Setoguchi, Manabu; Uchino, Yasushi; Nagata, Kazuya; Hokonohara, Daisuke

2018-02-14

Alzheimer's disease causes loss of appetite, resulting in bodyweight reduction. This, in turn, causes progression of cognitive dysfunction and physical complications that hasten death. Earlier care for loss of appetite is essential in Alzheimer's disease management. Rivastigmine is a therapeutic agent for Alzheimer's disease that has dual inhibition effects on acetylcholine esterase and butyrylcholine esterase. Butyrylcholine esterase is known to degrade the gastric hormone, ghrelin, which regulates appetite; therefore, we considered that rivastigmine might have an effect on appetite. The present study aimed to evaluate the hypothesis that rivastigmine improves appetite in Alzheimer's disease patients. Rivastigmine was given to mild-to-moderate Alzheimer's disease patients for 16 weeks. We evaluated the effects of rivastigmine on food intake, bodyweight, motivation (estimated by the vitality index), cognition function (estimated by the Hasegawa Dementia Scale-Revised), plasma butyrylcholine esterase activity, active ghrelin and inactive ghrelin. Plasma butyrylcholine esterase activity significantly decreased over time (percent change: -18.9 ± 27.0%, P < 0.05 at week 8; percent change: -33.4 ± 45.4%, P < 0.05 at week 16). Negative correlations were detected between percent changes in butyrylcholine esterase activity and active ghrelin (r s  = -0.62, P = 0.033) or active/inactive ghrelin ratio (r s  = -0.73, P = 0.007). Furthermore, motivation (including appetite) improved significantly (percent change: 17.9 ± 18.6%, P < 0.05 at week 16). The present study suggests that rivastigmine might improve appetite in mild-to-moderate Alzheimer's disease patients by suppressing degradation of plasma active ghrelin through the inhibition of plasma butyrylcholine esterase. Geriatr Gerontol Int 2018; ••: ••-••. © 2018 Japan Geriatrics Society.

EstA from Arthrobacter nitroguajacolicus Rü61a, a thermo- and solvent-tolerant carboxylesterase related to class C beta-lactamases.

PubMed

Schütte, Marcus; Fetzner, Susanne

2007-03-01

The estA gene encoding a novel cytoplasmic carboxylesterase from Arthrobacter nitroguajacolicus Rü61a was expressed in Escherichia coli. Sequence analysis and secondary structure predictions suggested that EstA belongs to the family VIII esterases, which are related to class C beta-lactamases. The S-x-x-K motif that in beta-lactamases contains the catalytic nucleophile, and a putative active-site tyrosine residue are conserved in EstA. The native molecular mass of hexahistidine-tagged (His6) EstA, purified by metal chelate affinity chromatography, was estimated to be 95 kDa by gel filtration, whereas the His6EstA peptide has a calculated molecular mass of 42.1 kDa. The enzyme catalyzes the hydrolysis of short-chain phenylacyl esters and triglycerides, and shows weak activity toward 2-hydroxy- and 2-nitroacetanilide. Its catalytic activity was inhibited by the serine-specific effector phenylmethylsulfonyl fluoride, and by Cd2+ and Hg2+ ions. Maximum activity of His6EstA was observed at a pH of 9.5 and a temperature of 50 degrees C to 60 degrees C. The enzyme was fairly thermostable. After 19 days at 50 degrees C and after 24 hours at 60 degrees C, its residual relative esterase activity toward phenylacetate was still 53% and 30%, respectively. Exposure of His6EstA to buffer-solvent mixtures showed that the enzyme was inactivated by several high log P (hydrophobic) solvents, whereas it showed remarkable stability and activity in up to 30% (by volume) of polar (low log P) organic solvents such as dimethylsulfoxide, methanol, acetonitrile, acetone, and propanol.

Arylesterase activities in the plasma of rats, rabbits and humans on low- and high-cholesterol diets.

PubMed

Beynen, A C; Weinans, G J; Katan, M B

1984-01-01

Arylesterase activities were measured with beta-naphthylpropionate and/or alpha-naphthylacetate as substrate in the plasma of rats, rabbits and humans on low- and high-cholesterol diets. The plasma esterase activities measured with alpha-naphthylacetate were similar in rats, rabbits and humans. With beta-naphthylpropionate as a substrate, rabbits were found to have a markedly higher esterase activity than rats and humans. Basal plasma esterase activity was significantly higher in an inbred rat strain which is hyporesponsive to dietary cholesterol than in a hyperresponsive strain. In rats, but not in humans and rabbits, plasma esterase activity was significantly increased by a high-cholesterol diet. In individual humans and random-bred rabbits and rats there was no association between initial plasma total esterase activity and the subsequent plasma cholesterol response to cholesterol feeding. We suggest that arylesterases are associated with cholesterol metabolism and with the response to dietary cholesterol in rats; evidence for such a role in rabbits and humans is, however, inconclusive.

The search of the target of promotion: Phenylbenzoate esterase activities in hen peripheral nerve

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moretto, A.; Nicolli, A.; Lotti, M.

2007-03-15

Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crudemore » homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC{sub 50} of about 0.08 mM (20 min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds.« less

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

PubMed Central

Schreiber, Renate; Taschler, Ulrike; Wolinski, Heimo; Seper, Andrea; Tamegger, Stefanie N.; Graf, Maria; Kohlwein, Sepp D.; Haemmerle, Guenter; Zimmermann, Robert; Zechner, Rudolf; Lass, Achim

2009-01-01

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with β-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl β-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism. PMID:19723663

Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

PubMed

Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon; Lee, ChangWoo

2016-03-01

An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures.

Repeated Administration of a Mutant Cocaine Esterase: Effects on Plasma Cocaine Levels, Cocaine-Induced Cardiovascular Activity, and Immune Responses in Rhesus Monkeys

PubMed Central

Collins, Gregory T.; Brim, Remy L.; Noon, Kathleen R.; Narasimhan, Diwahar; Lukacs, Nicholas W.; Sunahara, Roger K.; Woods, James H.

2012-01-01

Previous studies have demonstrated the capacity of a long-acting mutant form of a naturally occurring bacterial double mutant cocaine esterase (DM CocE) to antagonize the reinforcing, discriminative, convulsant, and lethal effects of cocaine in rodents and reverse the increases in mean arterial pressure (MAP) and heart rate (HR) produced by cocaine in rhesus monkeys. This study was aimed at characterizing the immunologic responses to repeated dosing with DM CocE and determining whether the development of anti-CocE antibodies altered the capacity of DM CocE to reduce plasma cocaine levels and ameliorate the cardiovascular effects of cocaine in rhesus monkeys. Under control conditions, intravenous administration of cocaine (3 mg/kg) resulted in a rapid increase in the plasma concentration of cocaine (n = 2) and long-lasting increases in MAP and HR (n = 3). Administration of DM CocE (0.32 mg/kg i.v.) 10 min after cocaine resulted in a rapid hydrolysis of cocaine with plasma levels below detection limits within 5 to 8 min. Elevations in MAP and HR were significantly reduced within 25 and 50 min of DM CocE administration, respectively. Although slight (10-fold) increases in anti-CocE antibodies were observed after the fourth administration of DM CocE, these antibodies did not alter the capacity of DM CocE to reduce plasma cocaine levels or ameliorate cocaine's cardiovascular effects. Anti-CocE titers were transient and generally dissipated within 8 weeks. Together, these results suggest that highly efficient cocaine esterases, such as DM CocE, may provide a novel and effective therapeutic for the treatment of acute cocaine intoxication in humans. PMID:22518021

Distinct Effect of Benzalkonium Chloride on the Esterase and Aryl Acylamidase Activities of Butyrylcholinesterase.

PubMed

Jaganathan; Boopathy

2000-08-01

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) from vertebrates, other than their predominant acylcholine hydrolase (esterase) activity, display a genuine aryl acylamidase activity (AAA) capable of hydrolyzing the synthetic substrate o-nitroacetanilide to o-nitroaniline. This AAA activity is strongly inhibited by classical cholinesterase (ChE) inhibitors. In the present study, benzalkonium chloride (BAC), a cationic detergent widely used as a preservative in pharmaceutical preparations, has been shown to distinctly modulate the esterase and AAA activities of BChEs. The detergent BAC was able to inhibit the esterase activity of human serum and horse serum BChEs and AChEs from electric eel and human erythrocyte. The remarkable property of BAC was its ability to profoundly activate the AAA activity of human serum and horse serum BChEs but not the AAA activity of AChEs. Thus BAC seem to preferentially activate the AAA activity of BChEs alone. Results of the study using the ChE active site-specific inhibitor diisopropyl phosphorofluoridate indicated that BAC binds to the active site of ChEs. Furthermore, studies using a structural homolog of BAC indicated that the alkyl group of BAC is essential not only for its interaction with ChEs but also for its distinct effect on the esterase and AAA activities of BChEs. This is the first report of a compound that inhibits the esterase activity, while simultaneously activating the AAA activity, of BChEs. Copyright 2000 Academic Press.

Esterase inhibition by synergists in the western flower thrips Frankliniella occidentalis.

PubMed

López-Soler, Neus; Cervera, Amelia; Quinto, Vicente; Abellán, Jaime; Bielza, Pablo; Martínez-Pardo, Rafael; Garcerá, Maria Dolores

2011-12-01

Western flower thrips (WFT), Frankliniella occidentalis (Pergande), is among the most important crop pests in the south-eastern region of Spain. Its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. Use of synergists to inhibit the enzymes involved in insecticide detoxification is widely used to determine their responsibility for insecticide resistance. However, they do not always act as intended or expected, and caution must be exercised when interpreting synergist results. Laboratory-selected strains of WFT were used to analyse the effects of the synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and methiocarb on total esterase activity. Significant differences were found, indicating esterase activity inhibition by DEF, a lower effect for methiocarb and a small inhibition of the activity by PBO. Esterase isoenzyme inhibition by these compounds showed a similar result; this assay revealed an extreme sensitivity of Triplet A (resistance-associated esterases) to DEF. In an in vivo assay carried out with these compounds at different incubation times, only DEF caused posterior in vitro esterase activity inhibition, with a maximum effect 1 h after treatment. In this work, only DEF shows true synergistic inhibition of WFT esterases. Copyright © 2011 Society of Chemical Industry.

Cell-Bound Lipase and Esterase of Brevibacterium linens

PubMed Central

Sørhaug, Terje; Ordal, Z. John

1974-01-01

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity. PMID:4824883

Some Properties of Extracellular Acetylxylan Esterase Produced by the Yeast Rhodotorula mucilaginosa†

PubMed Central

Lee, Hung; To, Rebecca J. B.; Latta, Roger K.; Biely, Peter; Schneider, Henry

1987-01-01

The red yeast Rhodotorula mucilaginosa produced an esterase that accumulated in the culture supernatant on induction with triacetin. The enzyme was specific for substrates bearing an O-acetyl group, but was relatively nonspecific for the rest of the molecule, which could consist of a phenol, a monosaccharide, a polysaccharide, or an aliphatic alcohol. The esterase was more active against acetylxylan and glucose β-d-pentaacetate than were a number of esterases from plant and animal sources, when activities on 4-nitrophenyl acetate were compared. The enzyme exhibited Michaelis-Menten kinetics and was active over a broad pH range (5.5 to 9.2), with an optimum between pH 8 and 10. In addition, the enzyme retained its activity for 2 h at 55°C. The yeast that produced the enzyme did not produce xylanase and, hence, is of interest for the production of acetylxylan esterase that is free of xylanolytic activity. PMID:16347498

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01.

PubMed

Divakar, K; Suryia Prabha, M; Nandhinidevi, G; Gautam, P

2017-04-21

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking-Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820 × 10 3 U/L and extracellular protease activity of 172 × 10 3 U/L were obtained at the 16th hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.

Esterase isoenzymes and insecticide resistance in Frankliniella occidentalis populations from the south-east region of Spain.

PubMed

López-Soler, Neus; Cervera, Amelia; Moores, Graham D; Martínez-Pardo, Rafael; Garcerá, M Dolores

2008-12-01

Frankliniella occidentalis (Pergande) is among the most important crop pests in the south-east region of Spain; its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. To this end, F. occidentalis populations, collected from the field at different locations in south-east Spain, were studied in terms of total esterase activity and esterase isoenzyme pattern. Individual thrips extracts were analysed by native polyacrylamide gel electrophoresis (PAGE) and stained for esterase activity with the model substrate alpha-naphthyl acetate. Significant correlations were found between resistance to the insecticides acrinathrin and methiocarb and the presence of a group of three intensely stained bands, named Triplet A. For each individual thrips extract, total esterase activity towards the substrates alpha-naphthyl acetate and alpha-naphthyl butyrate was also measured in a microplate reader. Insects possessing Triplet A showed a significantly higher alpha-naphthyl acetate specific activity and alpha-naphthyl acetate/alpha-naphthyl butyrate activity ratio. This observation allowed a reliable classification of susceptible or resistant insects either by PAGE analysis or by total esterase activity determination. The PAGE and microplate assays described can be used as a monitoring technique for detecting acrinathrin- and methiocarb-resistant individuals among F. occidentalis field populations.

Comparative Study of Esterase and Hemolytic Activities in Clinically Important Candida Species, Isolated From Oral Cavity of Diabetic and Non-diabetic Individuals.

PubMed

Fatahinia, Mahnaz; Poormohamadi, Farzad; Zarei Mahmoudabadi, Ali

2015-03-01

Diabetes mellitus as a chronic metabolic disease occurs in patients with partial or complete deficiency of insulin secretion or disorder in action of insulin on tissue. The disease is known to provide conditions for overgrowth of Candida species. Candida spp. cause candidiasis by many virulence factors such as esterase, hemolysin and phospholipase. This study aimed to compare esterase and hemolytic activity in various Candida species isolated from oral cavity of diabetic and non-diabetic individuals. Swab samples were taken from 95 patients with diabetes (35 men and 60 women) and 95 normal persons (42 men and 53 women) and cultured on Sabouraud dextrose agar. Identification of isolated yeasts was performed by germ tube test, morphology on CHROMagar Candida medium, corn meal agar and ability to grow at 45°C. Hemolysin activity was evaluated using blood plate assay and esterase activity was determined using the Tween 80 opacity test. Different Candida species were isolated from 57 (60%) diabetic and 24 (25%) non-diabetic individuals. Esterase activity was detected in all Candida isolates. Only 21.6% of C. albicans from patients with diabetes had esterase activity as + 3, while it ranged from + 1 to + 2 in others. Hemolytic activity was determined in C. albicans, C. dubliniensis, C. glabrata and C. krusei as 0.79, 0.58, 0.66 and 0.74, respectively. Hemolytic activity was significantly different in the two groups of diabetics and non-diabetics. Oral carriage of C. albicans in the diabetic group (n = 42; 66.7%) was significantly greater than the control group (n = 16; 57.1%). Esterase activity of C. albicans in diabetic group was higher than non-diabetic group. Although C. albicans remains the most frequently pathogenic yeast for human, but other species are increasing.

A bacterial cocaine esterase protects against cocaine-induced epileptogenic activity and lethality.

PubMed

Jutkiewicz, Emily M; Baladi, Michelle G; Cooper, Ziva D; Narasimhan, Diwahar; Sunahara, Roger K; Woods, James H

2009-09-01

Cocaine toxicity results in cardiovascular complications, seizures, and death and accounts for approximately 20% of drug-related emergency department visits every year. Presently, there are no treatments to eliminate the toxic effects of cocaine. The present study hypothesizes that a bacterial cocaine esterase with high catalytic efficiency would provide rapid and robust protection from cocaine-induced convulsions, epileptogenic activity, and lethality. Cocaine-induced paroxysmal activity and convulsions were evaluated in rats surgically implanted with radiotelemetry devices (N=6 per treatment group). Cocaine esterase was administered 1 minute after a lethal dose of cocaine or after cocaine-induced convulsions to determine the ability of the enzyme to prevent or reverse, respectively, the effects of cocaine. The cocaine esterase prevented all cocaine-induced electroencephalographic changes and lethality. This effect was specific for cocaine because the esterase did not prevent convulsions and death induced by a cocaine analog, (-)-2beta-carbomethoxy-3beta-phenyltropane. The esterase prevented lethality even after cocaine-induced convulsions occurred. In contrast, the short-acting benzodiazepine, midazolam, prevented cocaine-induced convulsions but not the lethal effects of cocaine. The data showed that cocaine esterase successfully degraded circulating cocaine to prevent lethality and that cocaine-induced convulsions alone are not responsible for the lethal effects of cocaine in this model. Therefore, further investigation into the use of cocaine esterase for treating cocaine overdose and its toxic effects is warranted.

Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase.

PubMed

Steinbrecher, U P; Pritchard, P H

1989-03-01

Degradation of phosphatidylcholine to lysophosphatidylcholine occurs during oxidative modification of low density lipoproteins (LDL). In this study, we have shown that this phospholipid hydrolysis is brought about by an LDL-associated phospholipase A2 that can hydrolyze oxidized but not intact LDL phosphatidylcholine. The chemical nature of the oxidized phospholipids that can act as substrates for this enzyme was not fully characterized, but we hypothesized that the specificity of the enzyme for oxidized LDL phosphatidylcholine might be explained by fragmentation of polyunsaturated sn-2 fatty acyl groups in LDL phosphatidylcholine during oxidation. To facilitate characterization of this enzyme, we therefore selected a fluorescent phosphatidylcholine substrate that had a short-chain, polar residue in the sn-2 position: 1-palmitoyl 2-(6-[7-nitrobenzoxadiazolyl]amino) caproyl phosphatidylcholine, (C6NBD PC). This substrate was efficiently hydrolyzed by LDL, but the dodecanoyl analogue of C6NBD PC, which differed only in that a 12-carbon rather than a 6-carbon acyl derivative was present in the sn-2 position, was not hydrolyzed. The phospholipase activity was heat-stable, calcium-independent, and was inhibited by the serine esterase inhibitors phenylmethylsulfonyl-fluoride and diisopropylfluorophosphate, but was resistant to p-bromophenacylbromide and dithiobisnitrobenzoic acid. The phospholipid hydrolysis could not be attributed to the action of lecithin:cholesterol acyltransferase or lipoprotein lipase. Nearly all of the activity in EDTA-anticoagulated normal plasma was physically associated with apoB-containing lipoproteins, but this apoprotein was not essential as enzyme activity was present in plasma from abetalipoproteinemic patients. These properties are very similar to those recently reported for human plasma platelet-activating factor (PAF) acetylhydrolase. In the present study, we found that acylhydrolase activity against C6NBD PC, PAF, and oxidized phosphatidylcholine copurfied through gel filtration and ion-exchange chromatography. Substrate competition was demonstrated between C6NBD PC, PAF, and oxidized 2-arachidonyl phosphatidylcholine, suggesting that a single enzyme was active against all three substrates. The enzyme had an apparent molecular weight of 40,000-45,000 by high pressure gel exclusion chromatography. Inhibition of this activity with disopropyfluorophosphate prior to oxidative modification of LDL prevented phospholipid hydrolysis but did not affect the production of thiobarbituric acid reactive compounds or the change in electrophoretic mobility. In addition, this inhibition of phospholipase did not prevent the rapid degradati

The hydrolytic activity of esterases in the yeast Saccharomyces cerevisiae is strain dependent.

PubMed

Kwolek-Mirek, Magdalena; Bednarska, Sabina; Zadrąg-Tęcza, Renata; Bartosz, Grzegorz

2011-11-01

Ester precursors of fluorogenic or chromogenic probes are often employed in studies of yeast cell biology. This study was aimed at a comparison of the ability of several commonly used laboratory wild-type Saccharomyces cerevisiae strains to hydrolyse the following model esters: fluorescein diacetate, 2-naphthyl acetate, PNPA (p-nitrophenyl acetate) and AMQI (7-acetoxy-1-methylquinolinum iodide). In all the strains, the esterase activity was localized mainly to the cytosol. Considerable differences in esterase activity were observed between various wild-type laboratory yeast strains. The phase of growth also contributed to the variation in esterase activity of the yeast. This diversity implies the need for caution in using intracellularly hydrolysed probes for a comparison of yeast strains with various genetic backgrounds.

The secreted esterase of Propionibacterium freudenreichii has a major role in cheese lipolysis.

PubMed

Abeijón Mukdsi, María Claudia; Falentin, Hélène; Maillard, Marie-Bernadette; Chuat, Victoria; Medina, Roxana Beatriz; Parayre, Sandrine; Thierry, Anne

2014-01-01

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1(T) for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.

The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

PubMed Central

Abeijón Mukdsi, María Claudia; Falentin, Hélène; Maillard, Marie-Bernadette; Chuat, Victoria; Medina, Roxana Beatriz; Parayre, Sandrine

2014-01-01

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis. PMID:24242250

Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils.

PubMed

Yamamoto-Tamura, Kimiko; Hiradate, Syuntaro; Watanabe, Takashi; Koitabashi, Motoo; Sameshima-Yamashita, Yuka; Yarimizu, Tohru; Kitamoto, Hiroko

2015-01-01

The relationship between degradation speed of soil-buried biodegradable polyester film in a farmland and the characteristics of the predominant polyester-degrading soil microorganisms and enzymes were investigated to determine the BP-degrading ability of cultivated soils through characterization of the basal microbial activities and their transition in soils during BP film degradation. Degradation of poly(butylene succinate-co-adipate) (PBSA) film was evaluated in soil samples from different cultivated fields in Japan for 4 weeks. Both the degradation speed of the PBSA film and the esterase activity were found to be correlated with the ratio of colonies that produced clear zone on fungal minimum medium-agarose plate with emulsified PBSA to the total number colonies counted. Time-dependent change in viable counts of the PBSA-degrading fungi and esterase activities were monitored in soils where buried films showed the most and the least degree of degradation. During the degradation of PBSA film, the viable counts of the PBSA-degrading fungi and the esterase activities in soils, which adhered to the PBSA film, increased with time. The soil, where the film was degraded the fastest, recorded large PBSA-degrading fungal population and showed high esterase activity compared with the other soil samples throughout the incubation period. Meanwhile, esterase activity and viable counts of PBSA-degrading fungi were found to be stable in soils without PBSA film. These results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is. This could be due to the rapid accumulation of secreted esterases in these soils.

A Bacterial Cocaine Esterase Protects Against Cocaine-Induced Epileptogenic Activity and Lethality

PubMed Central

Jutkiewicz, Emily M.; Baladi, Michelle G.; Cooper, Ziva D.; Narasimhan, Diwahar; Sunahara, Roger K.; Woods, James H.

2012-01-01

Study objective Cocaine toxicity results in cardiovascular complications, seizures, and death and accounts for approximately 20% of drug-related emergency department visits every year. Presently, there are no treatments to eliminate the toxic effects of cocaine. The present study hypothesizes that a bacterial cocaine esterase with high catalytic efficiency would provide rapid and robust protection from cocaine-induced convulsions, epileptogenic activity, and lethality. Methods Cocaine-induced paroxysmal activity and convulsions were evaluated in rats surgically implanted with radiotelemetry devices (N=6 per treatment group). Cocaine esterase was administered 1 minute after a lethal dose of cocaine or after cocaine-induced convulsions to determine the ability of the enzyme to prevent or reverse, respectively, the effects of cocaine. Results The cocaine esterase prevented all cocaine-induced electroencephalographic changes and lethality. This effect was specific for cocaine because the esterase did not prevent convulsions and death induced by a cocaine analog, (−)-2β-carbomethoxy-3β-phenyltropane. The esterase prevented lethality even after cocaine-induced convulsions occurred. In contrast, the short-acting benzodiazepine, midazolam, prevented cocaine-induced convulsions but not the lethal effects of cocaine. Conclusion The data showed that cocaine esterase successfully degraded circulating cocaine to prevent lethality and that cocaine-induced convulsions alone are not responsible for the lethal effects of cocaine in this model. Therefore, further investigation into the use of cocaine esterase for treating cocaine overdose and its toxic effects is warranted. PMID:19013687

Evidence for a blockwise distribution of acetyl groups onto homogalacturonans from a commercial sugar beet (Beta vulgaris) pectin.

PubMed

Ralet, Marie-Christine; Crépeau, Marie-Jeanne; Bonnin, Estelle

2008-06-01

Commercial acid-extracted sugar beet pectin was extensively hydrolysed using an endo-polygalacturonase (AnPGI from Aspergillus niger or AnPGII from A. niger or FmPG from Fusarium moniliforme) in combination with Aspergillus aculeatus pectin methyl-esterase (AaPME). The homogalacturonan-derived oligogalacturonates released were quantified by high-performance anion-exchange chromatography and their structure determined by mass spectrometry. The different endo-polygalacturonases exhibited variable tolerance towards acetyl groups. AnPGI was the most active and FmPG the less. A hypothetical homogalacturonan was constructed using the AnPGI-recovered oligogalacturonates as building blocks and the validity of the model was checked taking into account FmPG observed requirements and hydrolysis products. A blockwise repartition of the acetyl groups onto sugar beet pectin homogalacturonan is proposed.

Non-specific esterases in the gustatory epithelia of man and dog.

PubMed

Rakhawy, M T

1976-01-01

(1) Simple esterase activity has been demonstrated in the gustatory epithelium of man and dog by the simultaneous coupling azo dye technique using alpha-naphthol and naphthol As acetate. Unfixed cryostat and fixed paraffin sections were used. (2) A peculiar pattern of simple esterase activity was encountered in which--contrary to what was to be expected--the taste bud-carrying papillae showed a very poor reaction while there was a gradual increase in the enzyme intensity as the epithelium was traced away from these papillae. (3) It seems that among the reported differences between simple esterases and cholinesterases is this differential activity in relation to the gemmal system. (4) A peculiar difference in the enzyme activity was reported between the unfixed cryostat and the fixed paraffin sections in the human material.

Molecular Self-Assembly Strategy for Generating Catalytic Hybrid Polypeptides

PubMed Central

Ikezoe, Yasuhiro; Pike, Douglas H.; Nanda, Vikas; Matsui, Hiroshi

2016-01-01

Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aβ. When the fused CP4-Aβ construct assembled into antiparallel β-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aβ assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality. PMID:27116246

Molecular self-assembly strategy for generating catalytic hybrid polypeptides

DOE PAGES

Maeda, Yoshiaki; Fang, Justin; Ikezoe, Yasuhiro; ...

2016-04-26

Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aβ. When the fused CP4-Aβ construct assembled into antiparallel β- sheets and amyloidmore » fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. Furthermore, the enhanced catalytic activity of CP4-Aβ assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality.« less

Characterization and function of Mycobacterium tuberculosis H37Rv Lipase Rv1076 (LipU).

PubMed

Li, Chunyan; Li, Qiming; Zhang, Yuan; Gong, Zhen; Ren, Sai; Li, Ping; Xie, Jianping

2017-03-01

Lipids and lipases/esterases are essential for Mycobacterium tuberculosis (Mtb) survival and persistence, even virulence. Mycobacterium tuberculosis H37Rv Rv1076 (LipU), a member of lipase family, is homologous to the human Hormone Sensitive Lipase (HSL) based on the presence of conserved motif 'GXSXG'. To define the enzymatic characteristics of rv1076, the gene was cloned, and expressed in Escherichia coli. The protein was purified for enzymatic characterization. LipU showed high specific activity for the hydrolysis of short carbon chain substrates with optimal activity at 40°C/pH 8.0 and stability at low temperature and near-neutral pH. The specific activity, Km and Vmax of LipU was calculated to 176.7U/mg, 1.73μM and 62.24μM/min respectively. Ionic detergents can inhibit its activity. The active-site residues of LipU were determined to be Ser140, Asp244 and His269 by site-directed mutagenesis. The upregulation of Mycobacterium tuberculosis rv1076 under nutritive stress implicates a role in starvation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

DOE Office of Scientific and Technical Information (OSTI.GOV)

Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newlymore » modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.« less

Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence TimeS⃞

PubMed Central

Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.; Nichols, Joseph; Edwald, Elin; Chan, Jimmy; Ko, Mei-Chuan; Woods, James H.; Tesmer, John J. G.

2011-01-01

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37°C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37°C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo. PMID:21890748

Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response▿ †

PubMed Central

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J.

2006-01-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway. PMID:16980424

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response.

PubMed

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J

2006-11-01

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

Selective enzymatic hydrolysis of chlorogenic acid lactones in a model system and in a coffee extract. Application to reduction of coffee bitterness.

PubMed

Kraehenbuehl, Karin; Page-Zoerkler, Nicole; Mauroux, Olivier; Gartenmann, Karin; Blank, Imre; Bel-Rhlid, Rachid

2017-03-01

Chlorogenic acid lactones have been identified as key contributors to coffee bitterness. These compounds are formed during roasting by dehydration and cyclization of their precursors, the chlorogenic acids (CGAs). In the present study, we investigated an approach to decompose these lactones in a selective way without affecting the positive coffee attributes developed during roasting. A model system composed of (3-caffeoylquinic acid lactone (3-CQAL), 4- caffeoyl quinic acid lactone (4-CQAL), and 4-feruloylquinic acid lactone (4-FQAL)) was used for the screening of enzymes before treatment of the coffee extracts. Hog liver esterase (HLE) hydrolyzed chlorogenic acid lactones (CQALs, FQALs) selectively, while chlorogenate esterase hydrolyzed all chlorogenic acids (CQAs, FQAs) and their corresponding lactones (CQALs, FQALs) in a non-selective way. Enzymatically treated coffee samples were evaluated for their bitterness by a trained sensory panel and were found significantly less bitter than the untreated samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC 533.

PubMed

Bel-Rhlid, Rachid; Thapa, Dinesh; Kraehenbuehl, Karin; Hansen, Carl Erik; Fischer, Lutz

2013-01-01

The potential of Lactobacillus johnsonii NCC 533 to metabolize chlorogenic acids from green coffee extract was investigated. Two enzymes, an esterase and a hydroxycinnamate decarboxylase (HCD), were involved in this biotransformation. The complete hydrolysis of 5-caffeoylquinic acid (5-CQA) into caffeic acid (CA) by L. johnsonii esterase occurred during the first 16 h of reaction time. No dihydrocaffeic acid was identified in the reaction mixture. The decarboxylation of CA into 4-vinylcatechol (4-VC) started only when the maximum concentration of CA was reached (10 μmol/ml). CA was completely transformed into 4-VC after 48 h of incubation. No 4-vinylphenol or other derivatives could be identified in the reaction media. In this study we demonstrate the capability of L. johnsonii to transform chlorogenic acids from green coffee extract into 4-VC in two steps one pot reaction. Thus, the enzymatic potential of certain lactobacilli might be explored to generate flavor compounds from plant polyphenols.

Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors

PubMed Central

Bam, Rujuta A.; Willkom, Madeleine; Frey, Christian R.; Tsai, Luong; Stray, Kirsten M.; Yant, Stephen R.; Cihlar, Tomas

2015-01-01

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4+ T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF. PMID:26503655

Enzymatic characterization of insecticide resistance mechanisms in field populations of Malaysian Culex quinquefasciatus say (Diptera: Culicidae).

PubMed

Low, Van Lun; Chen, Chee Dhang; Lee, Han Lim; Tan, Tiong Kai; Chen, Chin Fong; Leong, Cherng Shii; Lim, Yvonne Ai Lian; Lim, Phaik Eem; Norma-Rashid, Yusoff; Sofian-Azirun, Mohd

2013-01-01

There has been no comprehensive study on biochemical characterization of insecticide resistance mechanisms in field populations of Malaysian Culex quinquefasciatus. To fill this void in the literature, a nationwide investigation was performed to quantify the enzyme activities, thereby attempting to characterize the potential resistance mechanisms in Cx. quinquefasciatus in residential areas in Malaysia. Culex quinquefasciatus from 14 residential areas across 13 states and one federal territory were subjected to esterases, mixed function oxidases, glutathione-S-transferase and insensitive acetylcholinesterase assays. Enzyme assays revealed that α-esterases and β-esterases were elevated in 13 populations and 12 populations, respectively. Nine populations demonstrated elevated levels of mixed function oxidases and glutathione-S-transferase. Acetylcholinesterase was insensitive to propoxur in all 14 populations. Activity of α-esterases associated with malathion resistance was found in the present study. In addition, an association between the activity of α-esterases and β-esterases was also demonstrated. The present study has characterized the potential biochemical mechanisms in contributing towards insecticide resistance in Cx. quinquefasciatus field populations in Malaysia. Identification of mechanisms underlying the insecticide resistance will be beneficial in developing effective mosquito control programs in Malaysia.

Biochemical Characterization and Relative Expression Levels of Multiple Carbohydrate Esterases of the Xylanolytic Rumen Bacterium Prevotella ruminicola 23 Grown on an Ester-Enriched Substrate ▿ †

PubMed Central

Kabel, Mirjam A.; Yeoman, Carl J.; Han, Yejun; Dodd, Dylan; Abbas, Charles A.; de Bont, Jan A. M.; Morrison, Mark; Cann, Isaac K. O.; Mackie, Roderick I.

2011-01-01

We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOSFA,Ac) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOSFA,Ac, a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose. PMID:21742923

Conifer flavonoid compounds inhibit detoxification enzymes and synergize insecticides.

PubMed

Wang, Zhiling; Zhao, Zhong; Cheng, Xiaofei; Liu, Suqi; Wei, Qin; Scott, Ian M

2016-02-01

Detoxification by glutathione S-transferases (GSTs) and esterases are important mechanisms associated with insecticide resistance. Discovery of novel GST and esterase inhibitors from phytochemicals could provide potential new insecticide synergists. Conifer tree species contain flavonoids, such as taxifolin, that inhibit in vitro GST activity. The objectives were to test the relative effectiveness of taxifolin as an enzyme inhibitor and as an insecticide synergist in combination with the organophosphorous insecticide, Guthion (50% azinphos-methyl), and the botanical insecticide, pyrethrum, using an insecticide-resistant Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) strain. Both taxifolin and its isomer, quercetin, increased the mortality of 1(st) instar CPB larvae after 48h when combined with Guthion, but not pyrethrum. Taxifolin had greater in vitro esterase inhibition compared with the commonly used esterase inhibitor, S, S, S-tributyl phosphorotrithioate (DEF). An in vivo esterase and GST inhibition effect after ingestion of taxifolin was measured, however DEF caused a greater suppression of esterase activity. This study demonstrated that flavonoid compounds have both in vitro and in vivo esterase inhibition, which is likely responsible for the insecticide synergism observed in insecticide-resistant CPB. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

Crystal structure of tannase from Lactobacillus plantarum.

PubMed

Ren, Bin; Wu, Mingbo; Wang, Qin; Peng, Xiaohong; Wen, Hua; McKinstry, William J; Chen, Qianming

2013-08-09

Tannins are water-soluble polyphenolic compounds in plants. Hydrolyzable tannins are derivatives of gallic acid (3,4,5-trihydroxybenzoic acid) or its meta-depsidic forms that are esterified to polyol, catechin, or triterpenoid units. Tannases are a family of esterases that catalyze the hydrolysis of the galloyl ester bond in hydrolyzable tannins to release gallic acid. The enzymes have found wide applications in food, feed, beverage, pharmaceutical, and chemical industries since their discovery more than a century ago, although little is known about them at the molecular level, including the details of the catalytic and substrate binding sites. Here, we report the first three-dimensional structure of a tannase from Lactobacillus plantarum. The enzyme displays an α/β structure, featured by a large cap domain inserted into the classical serine hydrolase fold. A catalytic triad was identified in the structure, which is composed of Ser163, His451, and Asp419. During the binding of gallic acid, the carboxyl group of the molecule forges hydrogen-bonding interactions with the catalytic triad of the enzyme while the three hydroxyl groups make contacts with Asp421, Lys343, and Glu357 to form another hydrogen-bonding network. Mutagenesis studies demonstrated that these residues are indispensable for the activity of the enzyme. Structural studies of the enzyme in complex with a number of substrates indicated that the interactions at the galloyl binding site are the determinant force for the binding of substrates. The single galloyl binding site is responsible for the esterase and depsidase activities of the enzyme. Copyright © 2013 Elsevier Ltd. All rights reserved.

Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate.

PubMed

Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J

2015-01-27

Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05-1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.

Monitoring Lipase/Esterase Activity by Stopped Flow in a Sequential Injection Analysis System Using p-Nitrophenyl Butyrate

PubMed Central

Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J.

2015-01-01

Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05–1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed. PMID:25633600

Modulated growth of nanoparticles. Application for sensing nerve gases.

PubMed

Virel, Ana; Saa, Laura; Pavlov, Valeri

2009-01-01

Hydrolysis of acetylthiocholine mediated by acetylcholine esterase yields the thiol-bearing compound thiocholine. At trace concentrations, thiocholine modulates the growth of Au-Ag nanoparticles on seeding gold nanoparticles in the presence of ascorbic acid. Inhibition of the enzyme by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) or by diethyl p-nitrophenyl phosphate (paraoxon) produces lower yields of thiocholine, promoting the catalytic growth of Au-Ag nanoparticles. Here, we describe the development of a simple and sensitive colorimetric assay for the detection of AChE inhibitors.

Crystal Structure and Functional Characterization of an Esterase (EaEST) from Exiguobacterium antarcticum.

PubMed

Lee, Chang Woo; Kwon, Sena; Park, Sun-Ha; Kim, Boo-Young; Yoo, Wanki; Ryu, Bum Han; Kim, Han-Woo; Shin, Seung Chul; Kim, Sunghwan; Park, Hyun; Kim, T Doohun; Lee, Jun Hyuck

2017-01-01

A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 Å, showed that the enzyme has a canonical α/β hydrolase fold with an α-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (≤C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80°C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications.

Bound Phenolics of Quinoa Seeds Released by Acid, Alkaline, and Enzymatic Treatments and Their Antioxidant and α-Glucosidase and Pancreatic Lipase Inhibitory Effects.

PubMed

Tang, Yao; Zhang, Bing; Li, Xihong; Chen, Peter X; Zhang, Hua; Liu, Ronghua; Tsao, Rong

2016-03-02

Unextractable phenolics from plant foods and their role in health benefits have become increasingly important. Meal residues of three quinoa seeds free of fat and extractable phenolics were subjected to acid, alkaline, and enzymatic hydrolyses. The total and individual phenolic compounds released were analyzed, and 19 phenolics, predominantly phenolic acids and several flavonoids, were identified. The concentration of bound phenolics was highest in black quinoa followed by red and white, regardless of the hydrolysis method. Higher phenolic contents also showed stronger antioxidant activities and inhibition of α-glucosidase and pancreatic lipase activities. Carbohydrases, that is, pectinase, xylanase and feruloyl esterase, which effectively liberated bound phenolics are known to be secreted by colonic bacteria, suggesting potential antioxidant and anti-inflammatory effects by these compounds in the large intestine during colonic fermentation. These results can also be applied to treat foods high in bound phenolics to enhance bioaccessibility.

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

PubMed

Dias, Albino A; Freitas, Gil S; Marques, Guilhermina S M; Sampaio, Ana; Fraga, Irene S; Rodrigues, Miguel A M; Evtuguin, Dmitry V; Bezerra, Rui M F

2010-08-01

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities. (c) 2010 Elsevier Ltd. All rights reserved.

In vitro enzymic hydrolysis of chlorogenic acids in coffee.

PubMed

da Encarnação, Joana Amarante; Farrell, Tracy L; Ryder, Alexandra; Kraut, Nicolai U; Williamson, Gary

2015-02-01

Coffee is rich in quinic acid esters of phenolic acids (chlorogenic acids) but also contains some free phenolic acids. A proportion of phenolic acids appear in the blood rapidly after coffee consumption due to absorption in the small intestine. We investigated in vitro whether this appearance could potentially be derived from free phenolic acids in instant coffee or from hydrolysis of chlorogenic acids by pancreatic or brush border enzymes. We quantified six free phenolic acids in instant coffees using HPLC-DAD-mass spectrometry. The highest was caffeic acid, but all were present at low levels compared to the chlorogenic acids. Roasting and decaffeination significantly reduced free phenolic acid content. We estimated, using pharmacokinetic modelling with previously published data, that the contribution of these compounds to small intestinal absorption is minimal. Hydrolysis of certain chlorogenic acids was observed with human-differentiated Caco-2 cell monolayers and with porcine pancreatin, which showed maximal rates on 3- and 5-O-caffeoylquinic acids, respectively. The amounts of certain free phenolic acids in coffee could only minimally account for small intestinal absorption based on modelling. The hydrolysis of caffeoylquinic, but not feruloylquinic acids, by enterocyte and pancreatic esterases is potentially a contributing mechanism to small intestinal absorption. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cholesterol esterase inhibitory activity of bioactives from leaves of Mangifera indica L

PubMed Central

Gururaja, G. M.; Mundkinajeddu, Deepak; Dethe, Shekhar M.; Sangli, Gopala K.; Abhilash, K.; Agarwal, Amit

2015-01-01

Background: In the earlier studies, methanolic extract of Mangifera indica L leaf was exhibited hypocholesterol activity. However, the bioactive compounds responsible for the same are not reported so far. Objective: To isolate the bioactive compounds with hypocholesterol activity from the leaf extract using cholesterol esterase inhibition assay which can be used for the standardization of extract. Materials and Methods: The leaf methanolic extract of M. indica (Sindoora variety) was partitioned with ethyl acetate and chromatographed on silica gel to yield twelve fractions and the activity was monitored by using cholesterol esterase inhibition assay. Active fractions were re-chromatographed to yield individual compounds. Results and Discussion: A major compound mangiferin present in the extract was screened along with other varieties of mango leaves for cholesterol esterase inhibition assay. However, the result indicates that compounds other than mangiferin may be active in the extract. Invitro pancreatic cholesterol esterase inhibition assay was used for bioactivity guided fractionation (BAGF) to yield bioactive compound for standardization of extract. Bioactivity guided fractionation afford the active fraction containing 3b-taraxerol with an IC50 value of 0.86μg/ml. Conclusion: This study demonstrates that M. indica methanol extract of leaf have significant hypocholesterol activity which is standardized with 3b-taraxerol, a standardized extract for hypocholesterol activity resulted in development of dietary supplement from leaves of Mangifera indica. PMID:26692750

Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin.

PubMed

A L Rocha, Vanessa; N Maeda, Roberto; Pereira, Nei; F Kern, Marcelo; Elias, Luisa; Simister, Rachael; Steele-King, Clare; Gómez, Leonardo D; McQueen-Mason, Simon J

2016-03-01

This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS-MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono-oxygenases (AA9), carbohydrate-binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two-fold the hydrolysis efficiency of a commercial cellulase cocktail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327-336, 2016. © 2016 American Institute of Chemical Engineers.

Mycoplasma hyopneumoniae p65 Surface Lipoprotein Is a Lipolytic Enzyme with a Preference for Shorter-Chain Fatty Acids

PubMed Central

Schmidt, Jono A.; Browning, Glenn F.; Markham, Philip F.

2004-01-01

Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39°C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma. PMID:15317784

A p-coumaroyl esterase from Rhizoctonia solani with a pronounced chlorogenic acid esterase activity.

PubMed

Nieter, Annabel; Kelle, Sebastian; Linke, Diana; Berger, Ralf G

2017-07-25

Extracellular esterase activity was detected in submerged cultures of Rhizoctonia solani grown in the presence of sugar beet pectin or Tween 80. Putative type B feruloyl esterase (FAE) coding sequences found in the genome data of the basidiomycete were heterologously expressed in Pichia pastoris. Recombinant enzyme production on the 5-L bioreactor scale (Rs pCAE: 3245UL -1 ) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant Rs pCAE was classified as a p-coumaroyl esterase (pCAE) with a pronounced chlorogenic acid esterase side activity. The Rs pCAE was also active on methyl cinnamate, caffeate and ferulate and on feruloylated saccharides. The unprecedented substrate profile of Rs pCAE together with the lack of sequence similarity to known FAEs or pCAEs suggested that the Rs pCAE represents a new type of enzyme. Hydroxycinnamic acids were released from agro-industrial side-streams, such as destarched wheat bran (DSWB), sugar beet pectin (SBP) and coffee pulp (CP). Overnight incubation of coffee pulp with the Rs pCAE resulted in the efficient release of p-coumaric (100%), caffeic (100%) and ferulic acid (85%) indicating possible applications for the valorization of food processing wastes and for the enhanced degradation of lignified biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of a Feruloyl Esterase from Lactobacillus plantarum

PubMed Central

Esteban-Torres, María; Reverón, Inés; Mancheño, José Miguel; de las Rivas, Blanca

2013-01-01

Lactobacillus plantarum is frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant. L. plantarum WCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methyl p-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl p-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present among L. plantarum strains. The present study constitutes the description of feruloyl esterase activity in L. plantarum and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species. PMID:23793626

Characterization of a feruloyl esterase from Lactobacillus plantarum.

PubMed

Esteban-Torres, María; Reverón, Inés; Mancheño, José Miguel; de Las Rivas, Blanca; Muñoz, Rosario

2013-09-01

Lactobacillus plantarum is frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant. L. plantarum WCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methyl p-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl p-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present among L. plantarum strains. The present study constitutes the description of feruloyl esterase activity in L. plantarum and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.

Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

PubMed Central

Fukuda, Kiyoshi; Yamamoto, Nagi; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

1998-01-01

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash. PMID:9758847

Mechanism-based inhibition of HsaD: a C-C bond hydrolase essential for survival of Mycobacterium tuberculosis in macrophage.

PubMed

Ryan, Ali; Keany, Sebastian; Eleftheriadou, Olga; Ballet, Romain; Cheng, Hung-Yuan; Sim, Edith

2014-01-01

Mycobacterium tuberculosis remains the leading cause of death by a bacterial pathogen worldwide. Increasing prevalence of multidrug-resistant organisms means prioritizing identification of targets for antituberculars. 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD), part of the cholesterol metabolism operon, is vital for survival within macrophage. The C-C bond hydrolase, HsaD, has a serine protease-like catalytic triad. We tested a range of serine protease and esterase inhibitors for their effects on HsaD activity. As well as providing a potential starting point for drug development, the data provides evidence for the mechanism of C-C bond hydrolysis. This screen also provides a route to initiate development of fragment-based inhibitors. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

PubMed

Martin del Campo, Martha; Camacho, Rosa M; Mateos-Díaz, Juan C; Müller-Santos, Marcelo; Córdova, Jesus; Rodríguez, Jorge A

2015-11-01

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

PubMed

Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

2015-07-01

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

Disposition and biotransformation of the acetylenic retinoid tazarotene in humans.

PubMed

Attar, Mayssa; Yu, Dale; Ni, Jinsong; Yu, Zhiling; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

2005-10-01

Oral tazarotene, an acetylenic retinoid, is in clinical development for the treatment of psoriasis. The disposition and biotransformation of tazarotene were investigated in six healthy male volunteers, following a single oral administration of a 6 mg (100 microCi) dose of [14C]tazarotene, in a gelatin capsule. Blood levels of radioactivity peaked 2 h postdose and then rapidly declined. Total recovery of radioactivity was 89.2+/-8.0% of the administered dose, with 26.1+/-4.2% in urine and 63.0+/-7.0% in feces, within 7 days of dosing. Only tazarotenic acid, the principle active metabolite formed via esterase hydrolysis of tazarotene, was detected in blood. One major urinary oxidative metabolite, tazarotenic acid sulfoxide, accounted for 19.2+/-3.0% of the dose. The majority of radioactivity recovered in the feces was attributed to tazarotenic acid representing 46.9+/-9.9% of the dose and only 5.82+/-3.84% of dose was excreted as unchanged tazarotene. Thus following oral administration, tazarotene was rapidly absorbed and underwent extensive hydrolysis to tazarotenic acid, the major circulating species in the blood that was then excreted unchanged in feces. A smaller fraction of tazarotenic acid was further metabolized to an inactive sulfoxide that was excreted in the urine. Copyright (c) 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Identification of a type-D feruloyl esterase from Neurospora crassa.

PubMed

Crepin, V F; Faulds, C B; Connerton, I F

2004-02-01

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.

Three feruloyl esterases in Cellulosilyticum ruminicola H1 act synergistically to hydrolyze esterified polysaccharides.

PubMed

Li, Jiabao; Cai, Shichun; Luo, Yuanming; Dong, Xiuzhu

2011-09-01

Feruloyl esterases (Faes) constitute a subclass of carboxyl esterases that specifically hydrolyze the ester linkages between ferulate and polysaccharides in plant cell walls. Until now, the described microbial Faes were mainly from fungi. In this study, we report that Cellulosilyticum ruminicola H1, a previously described fibrolytic rumen bacterium, possesses three different active feruloyl esterases, FaeI, FaeII, and FaeIII. Phylogenetic analysis classified the described bacterial Faes into two types, FaeI and FaeII in type I and FaeIII in type II. Substrate specificity assays indicated that FaeI is more active against the ester bonds in natural hemicelluloses and FaeIII preferentially attacks the ferulate esters with a small moiety, such as methyl groups, while FaeII is active on both types of substrates. Among the three feruloyl esterase genes, faeI was the only one induced significantly by xylose and xylan, while pectin appeared to moderately induce the three genes during the late log phase to stationary phase. Western blot analysis determined that FaeI and FaeIII were secreted and cytoplasmic proteins, respectively, whereas FaeII seemed to be cell associated. The addition of FaeI and FaeII but not FaeIII enhanced the activity of a xylanase on maize cob, suggesting a synergy of the former two with xylanase. Hence, we propose that the three feruloyl esterases work in concert to hydrolyze ferulate esters in natural hemicelluloses.

Changes in activity of non-specific esterases in cadmium treated Lymantria dispar larvae.

PubMed

Vlahović, Milena; Mataruga, Vesna Perić; Ilijin, Larisa; Mrdaković, Marija; Mirčić, Dejan; Todorović, Dajana; Lazarević, Jelica

2012-03-01

Many biochemical, physiological and histological criteria have been used as indicators of exposures and effects of the contaminants. These changes can indicate the response of an organism to a specific environmental stressor. In the present paper, the effect of the acute and chronic exposure to cadmium as well as recovery from two cadmium concentrations (10 and 30 μgCd/g dry food) on gypsy moth (Lymantria dispar) midgut esterases was investigated. The influence of cadmium on trait plasticity was also examined. Esterases showed great sensitivity to low metal concentrations during acute and chronic treatments. Their activities during short-term exposure and after recovery significantly depended on cadmium concentrations. The esterases had greater index of plasticity during chronic treatments with 10 and 30 μgCd/dry food. Five esterase isoforms between 64 and 250 kDa were detected. Isoforms of esterases exposed to any of the two cadmium effects differed among several egg-masses. Isozymes were distinguished in one egg-mass during different cadmium treatments. We conclude that these enzymes could be considered potential and sensitive non-selective biomarkers for the presence of cadmium in food.

A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution.

PubMed

Lülsdorf, Nina; Vojcic, Ljubica; Hellmuth, Hendrik; Weber, Thomas T; Mußmann, Nina; Martinez, Ronny; Schwaneberg, Ulrich

2015-06-01

Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, especially for complex aromatic substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing phenyl benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compound 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to standard deviation (5 %), linear detection range (15 to 250 μM), lower detection limit (15 μM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temperatures. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temperature (WT 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).

Biochemical evaluation of interactions between synergistic molecules and phase I enzymes involved in insecticide resistance in B- and Q-type Bemisia tabaci (Hemiptera: Aleyrodidae).

PubMed

Panini, Michela; Tozzi, Francesco; Zimmer, Christoph T; Bass, Chris; Field, Linda; Borzatta, Valerio; Mazzoni, Emanuele; Moores, Graham

2017-09-01

Metabolic resistance is an important consideration in the whitefly Bemisia tabaci, where an esterase-based mechanism has been attributed to pyrethroid resistance and over-expression of the cytochrome P450, CYP6CM1, has been correlated to resistance to imidacloprid and other neonicotinoids. In vitro interactions between putative synergists and CYP6CM1, B and Q-type esterases were investigated, and structure-activity relationship analyses allowed the identification of chemical structures capable of acting as inhibitors of esterase and oxidase activities. Specifically, methylenedioxyphenyl (MDP) moieties with a polyether chain were preferable for optimum inhibition of B-type esterase, whilst corresponding dihydrobenzofuran structures were potent for the Q-esterase variation. Potent inhibition of CYP6CM1 resulted from structures which contained an alkynyl chain with a terminal methyl group. Synergist candidates could be considered for field control of B. tabaci, especially to abrogate neonicotinoid resistance. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål).

PubMed

Vontas, J G; Small, G J; Hemingway, J

2000-12-01

Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3-7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1-15-fold more Nl-EST1 mRNA in individual insects and 5-11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8-10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear.

Taraxinic acid, a hydrolysate of sesquiterpene lactone glycoside from the Taraxacum coreanum NAKAI, induces the differentiation of human acute promyelocytic leukemia HL-60 cells.

PubMed

Choi, Jung-Hye; Shin, Kyung-Min; Kim, Na-Young; Hong, Jung-Pyo; Lee, Yong Sup; Kim, Hyoung Ja; Park, Hee-Juhn; Lee, Kyung-Tae

2002-11-01

The present work was performed to elucidate the active moiety of a sesquiterpene lactone, taraxinic acid-1'-O-beta-D-glucopyranoside (1). from Taraxacum coreanum NAKAI on the cytotoxicity of various cancer cells. Based on enzymatic hydrolysis and MTT assay, the active moiety should be attributed to the aglycone taraxinic acid (1a). rather than the glycoside (1). Taraxinic acid exhibited potent antiproliferative activity against human leukemia-derived HL-60. In addition, this compound was found to be a potent inducer of HL-60 cell differentiation as assessed by a nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD 14 and CD 66 b surface antigens. These results suggest that taraxinic acid induces the differentiation of human leukemia cells to monocyte/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during taraxinic acid-dependent HL-60 cell differentiation, whereas p21(CIP1) and p27(KIP1) were up-regulated. Taken together, our results suggest that taraxinic acid may have potential as a therapeutic agent in human leukemia.

Enzymatic production of ethanol from cellulose using soluble cellulose acetate as an intermediate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Downing, K.M.; Ho, C.S.; Zabriskie, D.W.

1987-01-01

A two-stage process for the enzymatic conversion of cellulose to ethanol is proposed as an alternative to currently incomplete and relatively slow enzymatic conversion processes employing natural insoluble cellulose. This alternative approach is designed to promote faster and more complete conversion of cellulose to fermentable sugars through the use of a homogeneous enzymatic hydrolysis reaction. Cellulose is chemically dissolved in the first stage to form water-soluble cellulose acetate (WSCA). The WSCA is then converted to ethanol in a simultaneous saccharification-fermentation with Pestalotiopsis westerdijkii enzymes (containing cellulolytic and acetyl esterase components) and yeast.

Enzyme-catalyzed hydrolysis of dentin adhesives containing a new urethane-based trimethacrylate monomer

PubMed Central

Park, Jong-Gu; Ye, Qiang; Topp, Elizabeth M.; Spencer, Paulette

2009-01-01

A new trimethacrylate monomer with urethane-linked groups, 1,1,1-tri-[4-(methacryloxyethylamino-carbonyloxy)-phenyl]ethane (MPE), was synthesized, characterized, and used as a co-monomer in dentin adhesives. Dentin adhesives containing 2-hydroxyethyl methacrylate (HEMA, 45% w/w) and 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane (BisGMA, 30% w/w) in addition to MPE (25% w/w) were formulated with H2O at 0 (MPE0), 8 (MPE8) and 16 wt % water (MPE16) to simulate the wet demineralized dentin matrix and compared with controls [HEMA/BisGMA, 45/55 w/w, at 0 (C0), 8 (C8) and 16 wt% water (C16)]. The new adhesive showed a degree of double bond conversion and mechanical properties comparable with control, with good penetration into the dentin surface and a uniform adhesive/dentin interface. On exposure to porcine liver esterase, the net cumulative methacrylic acid (MAA) release from the new adhesives was dramatically (P < 0.05) decreased relative to the control, suggesting that the new monomer improves esterase resistance. PMID:19582843

Interaction of Triton X-100 with acyl pocket of butyrylcholinesterase: effect on esterase activity and inhibitor sensitivity of the enzyme.

PubMed

Jaganathan, L; Boopathy, R

1998-06-01

The effect of non-ionic detergents like Triton X-100, Lubrol PX, Brij 35 and Tween 80 on the esterase activity and inhibitor sensitivity of human serum butyrylcholinesterase (BuChE) were studied. The results showed that though BuChE is not a detergent dependent enzyme, the esterase activity and inhibitor sensitivity of it can be modulated by the presence of detergents. All the detergents caused a marginal activation of the esterase activity. The presence of Lubrol PX, Brij 35 or Tween 80 did not affect the 50% molar inhibition concentration (IC50) of the inhibitors tested. But in the presence of Triton X-100 the IC50 values were increased for neostigmine, eserine and tetraisopropylpyrophosphoramide (acylation site interacting inhibitors), whereas for inhibitors like ethopropazine, imipramine and procainamide (choline binding pocket specific inhibitors) the IC50 values were unaltered. In addition, in the presence of Triton X-100 the bimolecular reaction constant for phosphorylation reaction (ki) of BuChE for the acyl pocket specific tetraisopropylpyrophosphoramide was reduced. Triton X-100 partially protected BuChE against this tetraisopropylpyrophosphoramide inactivation. These results indicate that Triton X-100 by interacting with the acyl pocket hydrophobic region is able to activate the esterase activity of BuChE. Further it reduces the capacity of the enzyme to react with inhibitors that inactivate it by interacting with the serine residue of the acylation site.

Comparative evaluation of lignocellulolytic activities of filamentous cultures of monocentric and polycentric anaerobic fungi.

PubMed

Dagar, Sumit Singh; Kumar, Sanjay; Mudgil, Priti; Puniya, Anil Kumar

2018-04-01

Sixteen strains of monocentric and polycentric anaerobic fungi were evaluated for cellulase, xylanase and esterase activities. Though strain level variations were observed among all genera, Neocallimastix and Orpinomyces strains exhibited the highest lignocellulolytic activities. The esterase activities of monocentric group of anaerobic fungi were better than the polycentric group. Copyright © 2018 Elsevier Ltd. All rights reserved.

A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids

USDA-ARS?s Scientific Manuscript database

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone releas...

Nebulized C1-Esterase Inhibitor does not Reduce Pulmonary Complement Activation in Rats with Severe Streptococcus Pneumoniae Pneumonia.

PubMed

de Beer, Friso; Lagrand, Wim; Glas, Gerie J; Beurskens, Charlotte J P; van Mierlo, Gerard; Wouters, Diana; Zeerleder, Sacha; Roelofs, Joris J T H; Juffermans, Nicole P; Horn, Janneke; Schultz, Marcus J

2016-12-01

Complement activation plays an important role in the pathogenesis of pneumonia. We hypothesized that inhibition of the complement system in the lungs by repeated treatment with nebulized plasma-derived human C1-esterase inhibitor reduces pulmonary complement activation and subsequently attenuates lung injury and lung inflammation. This was investigated in a rat model of severe Streptococcus pneumoniae pneumonia. Rats were intra-tracheally challenged with S. pneumoniae to induce pneumonia. Nebulized C1-esterase inhibitor or saline (control animals) was repeatedly administered to rats, 30 min before induction of pneumonia and every 6 h thereafter. Rats were sacrificed 20 or 40 h after inoculation with bacteria. Brochoalveolar lavage fluid and lung tissue were obtained for measuring levels of complement activation (C4b/c), lung injury and inflammation. Induction of pneumonia was associated with pulmonary complement activation (C4b/c at 20 h 1.24 % [0.56-2.59] and at 40 h 2.08 % [0.98-5.12], compared to 0.50 % [0.07-0.59] and 0.03 % [0.03-0.03] in the healthy control animals). The functional fraction of C1-INH was detectable in BALF, but no effect was found on pulmonary complement activation (C4b/c at 20 h 0.73 % [0.16-1.93] and at 40 h 2.38 % [0.54-4.19]). Twenty hours after inoculation, nebulized C1-esterase inhibitor treatment reduced total histology score, but this effect was no longer seen at 40 h. Nebulized C1-esterase inhibitor did not affect other markers of lung injury or lung inflammation. In this negative experimental animal study, severe S. pneumoniae pneumonia in rats is associated with pulmonary complement activation. Repeated treatment with nebulized C1-esterase inhibitor, although successfully delivered to the lungs, does not affect pulmonary complement activation, lung inflammation or lung injury.

Production of Null Mutants in the Major Intestinal Esterase Gene (Ges-1) of the Nematode Caenorhabditis Elegans

PubMed Central

McGhee, J. D.; Birchall, J. C.; Chung, M. A.; Cottrell, D. A.; Edgar, L. G.; Svendsen, P. C.; Ferrari, D. C.

1990-01-01

The ges-1 gene of the nematode Caenorhabditis elegans codes for a nonspecific carboxylesterase that is expressed only in the intestinal lineage. This esterase has turned out to be a convenient biochemical marker for lineage-specific differentiation. In the present paper, we describe the production of several C. elegans strains that lack detectable activity of the ges-1 esterase. To isolate these ges-1 null strains, we first produced a strain of hermaphrodites in which the wild-type copy of the ges-1 gene was stably balanced over a previously isolated isoelectric focusing allele, ges-1(ca6); this parental strain was then mutagenized with EMS and isoelectric focusing gels were used to identify progeny populations that lacked either ges-1(+) or ges-1(ca6) esterase activity. This method is a straightforward and general approach to obtaining null mutations in any gene that has a biochemical or immunological assay. The ges-1 gene is not essential to worm survival, development or reproduction. Furthermore, lack of the ges-1 product has no obvious effect on the ability of worms (containing either normal or greatly reduced levels of acetylcholinesterases) to survive exposure to esterase inhibitors. The ges-1 gene product provides roughly half of the total esterase activity measured in crude extracts of L1 larvae or mixed worm populations. However, histochemical staining of individual ges-1(0) embryos shows that the ges-1 esterase is the first and essentially the only esterase to be produced during embryonic development, from the midproliferation phase up to at least the twofold stage of morphogenesis. These ges-1(0) strains now allow us to investigate the developmental control of the ges-1 gene by DNA-mediated transformation, in which the ges-1 gene acts as its own reporter. PMID:2379823

A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold.

PubMed

De Vitis, Valerio; Nakhnoukh, Cristina; Pinto, Andrea; Contente, Martina L; Barbiroli, Alberto; Milani, Mario; Bolognesi, Martino; Molinari, Francesco; Gourlay, Louise J; Romano, Diego

2018-03-01

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a "lid" domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG. Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 5O7G (apo-BCE) and 5OLU (glycerol-bound BCE). © 2017 Federation of European Biochemical Societies.

Biochemical characterisation of the esterase activities of wine lactic acid bacteria.

PubMed

Matthews, Angela; Grbin, Paul R; Jiranek, Vladimir

2007-11-01

Esters are an important group of volatile compounds that can contribute to wine flavour. Wine lactic acid bacteria (LAB) have been shown to produce esterases capable of hydrolysing ester substrates. This study aims to characterise the esterase activities of nine LAB strains under important wine conditions, namely, acidic conditions, low temperature (to 10 degrees C) and in the presence of ethanol (2-18% v/v). Esterase substrate specificity was also examined using seven different ester substrates. The bacteria were generally found to have a broad pH activity range, with the majority of strains showing maximum activity close to pH 6.0. Exceptions included an Oenococcus oeni strain that retained most activity even down to a pH of 4.0. Most strains exhibited highest activity across the range 30-40 degrees C. Increasing ethanol concentration stimulated activity in some of the strains. In particular, O. oeni showed an increase in activity up to a maximum ethanol concentration of around 16%. Generally, strains were found to have greater activity towards short-chained esters (C2-C8) compared to long-chained esters (C10-C18). Even though the optimal physicochemical conditions for enzyme activity differed from those found in wine, these findings are of potential importance to oenology because significant activities remained under wine-like conditions.

Juvenile hormone activity in Dysdercus cingulatus Fabr by juvenile hormone esterase inhibitor, OTFP.

PubMed

Elayidam, U Gayathri; Muraleedharan, D

2007-10-01

Application of juvenile hormone esterase inhibitor 3-octylthio-1,1,1- trifluropropan-2-one (OTFP) to 5th instar nymphs and virgin females of D. cingulatus revealed the profound role played by juvenile hormone esterase (JHE) in metamorphosis and reproduction. The ability of OTFP to cause delay and the formation of malformed nymphs, suggests that inhibition of JHE in vivo maintains a higher than normal hemolymph JH titer. It is obvious that OTFP does inhibit in vivo JHE activity in late instar nymphs. Further, the application of JHE inhibitor, OTFP to virgin females demonstrates that substituted trifluropropanones can indirectly stimulate egg development by inhibiting JHE activity in virgin females.

Inhibition of pectin methyl esterase activity by green tea catechins.

PubMed

Lewis, Kristin C; Selzer, Tzvia; Shahar, Chen; Udi, Yael; Tworowski, Dmitry; Sagi, Irit

2008-10-01

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.

The impact of hydroquinone on acetylcholine esterase and certain human carbonic anhydrase isoenzymes (hCA I, II, IX, and XII).

PubMed

Scozzafava, Andrea; Kalın, Pınar; Supuran, Claudiu T; Gülçin, İlhami; Alwasel, Saleh H

2015-12-01

Carbonic anhydrases (CAs) are widespread and the most studied members of a great family of metalloenzymes in higher vertebrates including humans. CAs were investigated for their inhibition of all of the catalytically active mammalian isozymes of the Zn(2+)-containing CA, (CA, EC 4.2.1.1). On the other hand, acetylcholinesterase (AChE. EC 3.1.1.7), a serine protease, is responsible for ACh hydrolysis and plays a fundamental role in impulse transmission by terminating the action of the neurotransmitter ACh at the cholinergic synapses and neuromuscular junction. In the present study, the inhibition effect of the hydroquinone (benzene-1,4-diol) on AChE activity was evaluated and effectively inhibited AChE with Ki of 1.22 nM. Also, hydroquinone strongly inhibited some human cytosolic CA isoenzymes (hCA I and II) and tumour-associated transmembrane isoforms (hCA IX, and XII), with Kis in the range between micromolar (415.81 μM) and nanomolar (706.79 nM). The best inhibition was observed in cytosolic CA II.

Characterization of a cold-adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

PubMed

Dong, Juan; Gasmalla, Mohammed A A; Zhao, Wei; Sun, Jingtao; Liu, Wenyu; Wang, Mingming; Han, Liang; Yang, Ruijin

2017-09-01

A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No. 1, Tianshan, People's Republic of China and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room temperature plasma method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30 ℃, pH 9.0 and 25 ℃, pH 8.5, respectively. EstTB11 was thermally more stable (50 ℃ for 1 H) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0 ℃ and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4 ℃. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation.

PubMed

Ramya, Shanivarsanthe Leelesh; Venkatesan, Thiruvengadam; Srinivasa Murthy, Kottilingam; Jalali, Sushil Kumar; Verghese, Abraham

2016-01-01

Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n=13) and adults (n=12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32μmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus - KC985225 and Pantoea agglomerans - KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26μmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus.

PubMed

Leis, Benedikt; Angelov, Angel; Mientus, Markus; Li, Haijuan; Pham, Vu T T; Lauinger, Benjamin; Bongen, Patrick; Pietruszka, Jörg; Gonçalves, Luís G; Santos, Helena; Liebl, Wolfgang

2015-01-01

Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

A critical examination of Escherichia coli esterase activity.

PubMed

Antonczak, Alicja K; Simova, Zuzana; Tippmann, Eric M

2009-10-16

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances.

A Critical Examination of Escherichia coli Esterase Activity*

PubMed Central

Antonczak, Alicja K.; Simova, Zuzana; Tippmann, Eric M.

2009-01-01

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances. PMID:19666472

Bacterial Cinnamoyl Esterase Activity Screening for the Production of a Novel Functional Food Product▿ †

PubMed Central

Guglielmetti, Simone; De Noni, Ivano; Caracciolo, Federica; Molinari, Francesco; Parini, Carlo; Mora, Diego

2008-01-01

Lactobacillus helveticus MIMLh5 was selected for its strong cinnamoyl esterase activity on chlorogenic acid and employed for the preparation of a food product containing a high concentration of free caffeic acid. The novel food product was demonstrated to display high total antioxidant power and potential probiotic properties. PMID:18165367

Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vose, Sarah C.; Center for Children's Environmental Health Research, School of Public Health, University of California, Berkeley, CA 94720; Holland, Nina T.

2007-10-01

Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC{sub 50} valuesmore » of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.« less

Novel function of lecithin-cholesterol acyltransferase. Hydrolysis of oxidized polar phospholipids generated during lipoprotein oxidation.

PubMed

Goyal, J; Wang, K; Liu, M; Subbaiah, P V

1997-06-27

Although the major function of lecithin-cholesterol acyltransferase (LCAT) is cholesterol esterification, our previous studies showed that it can also hydrolyze platelet-activating factor (PAF). Because of the structural similarities between PAF and the truncated phosphatidylcholines (polar PCs) generated during lipoprotein oxidation, we investigated the possibility that LCAT may also hydrolyze polar PCs to lyso-PC during the oxidation of plasma. PAF acetylhydrolase (PAF-AH), which is known to hydrolyze polar PCs in human plasma, was completely inhibited by 0.2 mM p-aminoethyl benzenesulfonyl fluoride (Pefabloc), a new serine esterase inhibitor, which had no effect on LCAT at this concentration. On the other hand, 1 mM diisopropylfluorophosphate (DFP) completely inhibited LCAT but had no effect on PAF-AH. Polar PC accumulation during the oxidation of plasma increased by 44% in the presence of 0.2 mM Pefabloc and by 30% in the presence of 1 mM DFP. The formation of lyso-PC was concomitantly inhibited by both of the inhibitors. The combination of the two inhibitors resulted in the maximum accumulation of polar PCs, suggesting that both PAF-AH and LCAT are involved in their breakdown. Oxidation of chicken plasma, which has no PAF-AH activity, also resulted in the formation of lyso-PC from the hydrolysis of polar PC, which was inhibited by DFP. Polar PCs, either isolated from oxidized plasma or by oxidation of labeled synthetic PCs, were hydrolyzed by purified LCAT, which had no detectable PAF-AH activity. These results demonstrate a novel function for LCAT in the detoxification of polar PCs generated during lipoprotein oxidation, especially when the PAF-AH is absent or inactivated.

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

PubMed

Nicolas, A; Egmond, M; Verrips, C T; de Vlieg, J; Longhi, S; Cambillau, C; Martinez, C

1996-01-16

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

Silica-bound copper(II)triazacyclononane as a phosphate esterase: effect of linker length and surface hydrophobicity.

PubMed

Bodsgard, Brett R; Clark, Robert W; Ehrbar, Anthony W; Burstyn, Judith N

2009-04-07

A series of silica-bound Cu(ii) triazacyclononane materials was prepared to study the effect of linker length and surface hydrophobicity on the hydrolysis of phosphate esters. The general synthetic approach for these heterogeneous reagents was rhodium-catalyzed hydrosilation between an alkenyl-modified triazacyclononane and hydride-modified silica followed by metallation with a Cu(ii) salt. Elemental analysis confirmed that organic functionalization of the silica gel was successful and provided an estimate of the surface concentration of triazacyclononane. EPR spectra were consistent with square pyramidal Cu(ii), indicating that Cu(ii) ions were bound to the immobilized macrocycles. The hydrolytic efficacies of these heterogeneous reagents were tested with bis(p-nitrophenyl)phosphate (BNPP) and diethyl 4-nitrophenyl phosphate (paraoxon). The agent that performed best was an octyl-linked, propanol-blocked material. This material had the most hydrophilic surface and the most accessible active site, achieving a rate maximum on par with the other materials, but in fewer cycles and without an induction period.

Nucleotide and amino acid variations of tannase gene from different Aspergillus strains.

PubMed

Borrego-Terrazas, J A; Lara-Victoriano, F; Flores-Gallegos, A C; Veana, F; Aguilar, C N; Rodríguez-Herrera, R

2014-08-01

Tannase is an enzyme that catalyses the hydrolysis of ester bonds present in tannins. Most of the scientific reports about this biocatalysis focus on aspects related to tannase production and its recovery; on the other hand, reports assessing the molecular aspects of the tannase gene or protein are scarce. In the present study, a tannase gene fragment from several Aspergillus strains isolated from the Mexican semidesert was sequenced and compared with tannase amino acid sequences reported in NCBI database using bioinformatics tools. The genetic relationship among the different tannase sequences was also determined. A conserved region of 7 amino acids was found with the conserved motif GXSXG common to esterases, in which the active-site serine residue is located. In addition, in Aspergillus niger strains GH1 and PSH, we found an extra codon in the tannase sequences encoding glycine. The tannase gene belonging to semidesert fungal strains followed a neutral evolution path with the formation of 10 haplotypes, of which A. niger GH1 and PSH haplotypes are the oldest.

Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

PubMed

Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

2016-01-01

This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

Production and characterization of a tributyrin esterase from Lactobacillus plantarum suitable for cheese lipolysis.

PubMed

Esteban-Torres, M; Mancheño, J M; de las Rivas, B; Muñoz, R

2014-11-01

Lactobacillus plantarum is a lactic acid bacterium that can be found during cheese ripening. Lipolysis of milk triacylglycerols to free fatty acids during cheese ripening has fundamental consequences on cheese flavor. In the present study, the gene lp_1760, encoding a putative esterase or lipase, was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_1760 protein was biochemically characterized. Lp_1760 hydrolyzed p-nitrophenyl esters of fatty acids from C2 to C16, with a preference for p-nitrophenyl butyrate. On triglycerides, Lp_1760 showed higher activity on tributyrin than on triacetin. Although optimal conditions for activity were 45°C and pH 7, Lp_1760 retains activity under conditions commonly found during cheese making and ripening. The Lp_1760 showed more than 50% activity at 5°C and exhibited thermal stability at high temperatures. Enzymatic activity was strongly inhibited by sodium dodecyl sulfate and phenylmethylsulfonyl fluoride. The Lp_1760 tributyrin esterase showed high activity in the presence of NaCl, lactic acid, and calcium chloride. The results suggest that Lp_1760 might be a useful tributyrin esterase to be used in cheese manufacturing. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The Activity of Cholinesterases in Diapausing and Flying Red Mason Bees Osmia bicornis (Megachilidae).

PubMed

Dmochowska-Slezak, Kamila; Zaobidna, Ewa; Domeracka, Joanna; Swiatkowska, Marta; Rusznica, Małgorzata; Zółtowska, Krystyna

2015-01-01

The red mason bee (Osmia bicornis) is a highly effective pollinator that is exposed to various xenobiotics. The organism's potential resistance to the toxic effects of xenobiotics can be determined based on cholinesterase activity. The activity of cholinesterases (ChEs) towards acetylcholine (ACh) and butyrylcholine (BCh) was determined in extracts of diapausing (between October and late March) and flying bees (May). In both males and females, enzyme activity was higher towards ACh than towards BCh. The ratio of ACh/BCh activity was determined in the range of 1.43 to 4.15 in diapausing females and 3.00 to 7.18 in diapausing males. No significant changes in ChE activity towards ACh were observed in females before December and in males before February. Enzyme activity towards ACh increased dynamically in the second half of March. Enzyme activity towards BCh remained stable in both sexes until mid-March, after which it increased significantly. Excluding mid-March, enzyme BCh activity was significantly higher in females than in males. The activity of carboxylesterase towards 4-p-nitrophenyl butyrate was determined in females to assess the involvement of non-specific esterases in the hydrolysis of choline esters. Carboxylesterase activity was low in comparison with cholinesterase activity, and it remained practically unchanged throughout diapause, suggesting that choline esters in female O. bicornis extracts were hydrolyzed mainly by acetylcholinesterases.

New insights on molecular interactions of organophosphorus pesticides with esterases.

PubMed

Mangas, Iris; Estevez, Jorge; Vilanova, Eugenio; França, Tanos Celmar Costa

2017-02-01

Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the organophosphorus toxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

PubMed

Du, Liangjun; Huo, Ying; Ge, Fanglan; Yu, Jiajun; Li, Wei; Cheng, Guiying; Yong, Bin; Zeng, Lihuang; Huang, Min

2010-12-01

CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

Purification and general properties of pectin methyl esterase from Curvularia inaequalis NRRL 13884 in solid state culture using orange peels as an inducer.

PubMed

Afifi, A F; Fawzi, E M; Foaad, M A

2002-01-01

Pectin methyl esterase (PME) [E.C.3. 1.1.11] production by Curvularia inaequalis (Shear) Boedijn NRRL 13884 was investigated using solid-state culture. The highest level of extracellular pectin methyl esterase was detected with orange peels as an inducing substrate and as a sole carbon source. The enzyme was partially purified using Sephadex G-100 and DEAE-Cellulose column chromatography. It was purified about 40 fold with optimum activity at pH 4.4 and 45 degrees C. The enzyme was activated by Co++, Mg++, Na+, whereas it was slightly activated in the presence of Cu++, K+, Mn++, Zn++. On the other hand Ag++, Ca++ and Hg++ inhibited the activity of the enzyme. The Km was calculated to be 0.52 mM.

Development of a bifunctional xylanase-cellulase chimera with enhanced activity on rice and barley straws using a modular xylanase and an endoglucanase procured from camel rumen metagenome.

PubMed

Khalili Ghadikolaei, Kamran; Akbari Noghabi, Kambiz; Shahbani Zahiri, Hossein

2017-09-01

The camel rumen metagenome is an untapped source of glycoside hydrolases. In this study, novel genes encoding for a modular xylanase (XylC) and a cellulase (CelC) were isolated from a camel rumen metagenome and expressed in Escherichia coli BL21 (DE3). XylC with xylanase (Xyn), CBM, and carbohydrate esterase (CE) domains was characterized as a β-1,4-endoxylanase with remarkable catalytic activity on oat-spelt xylan (K cat  = 2919 ± 57 s -1 ). The implication of XylC's modular structure in its high catalytic activity was analyzed by truncation and fusion construction with CelC. The resulting fusions including Cel-CBM, Cel-CBM-CE, and Xyn-CBM-Cel showed remarkable enhancement in CMCase activity with K cat values of 742 ± 12, 1289 ± 34.5, and 2799 ± 51 s -1 compared to CelC with a K cat of 422 ± 3.5 s -1 . It was also shown that the bifunctional Xyn-CBM-Cel with synergistic xylanase/cellulase activities was more efficient than XylC and CelC in hydrolysis of rice and barley straws.

Assessment of bermudagrass and bunch grasses as feedstock for conversion to ethanol.

PubMed

Anderson, William F; Dien, Bruce S; Brandon, Sarah K; Peterson, Joy Doran

2008-03-01

Research is needed to allow more efficient processing of lignocellulose from abundant plant biomass resources for production to fuel ethanol at lower costs. Potential dedicated feedstock species vary in degrees of recalcitrance to ethanol processing. The standard dilute acid hydrolysis pretreatment followed by simultaneous sacharification and fermentation (SSF) was performed on leaf and stem material from three grasses: giant reed (Arundo donax L.), napiergrass (Pennisetum purpureum Schumach.), and bermudagrass (Cynodon spp). In a separate study, napiergrass, and bermudagrass whole samples were pretreated with esterase and cellulose before fermentation. Conversion via SSF was greatest with two bermudagrass cultivars (140 and 122 mg g(-1) of biomass) followed by leaves of two napiergrass genotypes (107 and 97 mg g(-1)) and two giant reed clones (109 and 85 mg g(-1)). Variability existed among bermudagrass cultivars for conversion to ethanol after esterase and cellulase treatments, with Tifton 85 (289 mg g) and Coastcross II (284 mg g(-1)) being superior to Coastal (247 mg g(-1)) and Tifton 44 (245 mg g(-1)). Results suggest that ethanol yields vary significantly for feedstocks by species and within species and that genetic breeding for improved feedstocks should be possible.

Assessment of Bermudagrass and Bunch Grasses as Feedstock for Conversion to Ethanol

NASA Astrophysics Data System (ADS)

Anderson, William F.; Dien, Bruce S.; Brandon, Sarah K.; Peterson, Joy Doran

Research is needed to allow more efficient processing of lignocellulose from abundant plant biomass resources for production to fuel ethanol at lower costs. Potential dedicated feedstock species vary in degrees of recalcitrance to ethanol processing. The standard dilute acid hydrolysis pretreatment followed by simultaneous sacharification and fermentation (SSF) was performed on leaf and stem material from three grasses: giant reed (Arundo donax L.), napiergrass (Pennisetum purpureum Schumach.), and bermudagrass (Cynodon spp). In a separate study, napiergrass, and bermudagrass whole samples were pretreated with esterase and cellulose before fermentation. Conversion via SSF was greatest with two bermudagrass cultivars (140 and 122 mg g-1 of biomass) followed by leaves of two napiergrass genotypes (107 and 97 mg g-1) and two giant reed clones (109 and 85 mg g-1). Variability existed among bermudagrass cultivars for conversion to ethanol after esterase and cellulase treatments, with Tifton 85 (289 mg g) and Coastcross II (284 mg g-1) being superior to Coastal (247 mg g-1) and Tifton 44 (245 mg g-1). Results suggest that ethanol yields vary significantly for feedstocks by species and within species and that genetic breeding for improved feedstocks should be possible.

A novel cold-adapted esterase from Enterobacter cloacae: Characterization and improvement of its activity and thermostability via the site of Tyr193Cys.

PubMed

Gao, Haofeng; Li, Chanjuan; Bandikari, Ramesh; Liu, Ziduo; Hu, Nan; Yong, Qiang

2018-03-19

In industries lipolytic reactions occur in insensitive conditions such as high temperature thus novel stout esterases with unique properties are attracts to the industrial application. Protein engineering is the tool to obtain desirable characters of enzymes. A novel esterase gene was isolated from South China Sea and subjected to a random mutagenesis and site directed mutagenesis for higher activity and thermo-stability compared to wild type. A novel esterase showed the highest hydrolytic activity against p-nitrophenyl acetate (pNPA, C2) and the optimal activity at 40 °C and pH 8.5. It was a cold-adapted enzyme and retained approximately 40% of its maximum activity at 0 °C. A mutant, with higher activity and thermo-stability was obtained by random mutagenesis. Kinetic analysis indicated that the mutant Val29Ala/Tyr193Cys shown 43.5% decrease in K m , 2.6-fold increase in K cat , and 4.7-fold increase in K cat /K m relative to the wild type. Single mutants V29A and Y193C were constructed and their kinetic parameters were measured. The results showed that the values of K m , K cat , and K cat /K m of V29A were similar to those of the wild type while Y193C showed 52.7% decrease in K m , 2.7-fold increase in K cat , and 5.6-fold increase in K cat /K m compared with the wild type. The 3-D structure and docking analysis revealed that the replacement of Tyr by Cys could enlarge the binding pocket. Moreover Y193C also showed a better thermo-stability for the reason its higher hydrophobicity and retained 67% relative activity after incubation for 3 h at 50 °C. The superior quality of modified esterase suggested it has great potential application in extreme conditions and the mutational work recommended that important information for the study of esterase structure and function.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates.

PubMed

Ishida, K; Alviano, D S; Silva, B G; Guerra, C R; Costa, A S; Nucci, M; Alviano, C S; Rozental, S

2012-05-01

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes.

Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

PubMed Central

Ishida, K.; Alviano, D.S.; Silva, B.G.; Guerra, C.R.; Costa, A.S.; Nucci, M.; Alviano, C.S.; Rozental, S.

2012-01-01

Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes. PMID:22415116

Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots.

PubMed

Negrel, Jonathan; Javelle, Francine; Morandi, Dominique; Lucchi, Géraldine

2016-12-01

A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (K m  = 2 μM) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Glucuronoyl esterases are active on polymeric substrate, methyl esterified glucuronoxylan

USDA-ARS?s Scientific Manuscript database

Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic d...

Phenol esterase activity of porcine skin

USDA-ARS?s Scientific Manuscript database

The alkyl esters of plant-derived phenols may serve as slow-release sources for cutaneous delivery of antioxidants. The ability of skin esterases to hydrolyze phenolic esters was examined. Esters of tyrosol and hydroxytyrosol were prepared from decanoic and lipoic acids. Ferulic acid was esterified ...

Mass Spectrometry to Identify New Biomarkers of Nerve Agent Exposure

DTIC Science & Technology

2009-04-01

covalent bond with the active site serine in the consensus sequence GXSXG of esterases and proteases. However, the site of attachment to proteins...that have no active site serine has only recently been recognized as tyrosine. In last year’s report we provided mass spectrometry evidence that...PMID: 18502412 Lockridge O, Xue W, Gaydess A, Grigoryan H, Ding SJ, Schopfer LM, Hinrichs SH, Masson P. Pseudo- esterase activity of human albumin

Screening exogenous fibrolytic enzyme preparations for improved in vitro digestibility of bermudagrass haylage.

PubMed

Romero, J J; Zarate, M A; Arriola, K G; Gonzalez, C F; Silva-Sanchez, C; Staples, C R; Adesogan, A T

2015-04-01

Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 μg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to the least effective, the most effective EFE at increasing NDFD contained 10 times more endoglucanase III, 17 times more acetylxylan esterase with a cellulose-binding domain 1, 33 times more xylanase III, 25 times more β-xylosidase, and 7.7 times more polysaccharide monooxygenase with cellulose-binding domain 1 and 3 times more swollenin. The most effective EFE had a much greater quantity of fibrolytic enzymes and key proteins necessary for hemicellulose and lignocellulase deconstruction. This study identified several EFE that increased the NDFD and in vitro fermentation of 4-wk BH and revealed why some EFE are more effective than others. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

In vitro metabolism and drug-drug interaction potential of UTL-5g, a novel chemo- and radioprotective agent.

PubMed

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick; Li, Jing

2014-12-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute(-1)·mM(-1), respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

In Vitro Metabolism and Drug-Drug Interaction Potential of UTL-5g, a Novel Chemo- and Radioprotective Agent

PubMed Central

Wu, Jianmei; Shaw, Jiajiu; Dubaisi, Sarah; Valeriote, Frederick

2014-01-01

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute−1·mM−1, respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g. PMID:25249693

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

PubMed Central

Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

2014-01-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prestwich, G.D.; Streinz, L.

A series of mono, di-, and trihalogenated acetate analogs of Z11-16:Ac were prepared and examined for electrophysiological activity in antennae of males of the diamondback moth, Plutella xylostella. In addition, two potential affinity labels, a diazoacetate (Dza) and a trifluoromethyl ketone (Tfp), were evaluated for EAG activity. The Z11-16:Ac showed the highest activity in EAG assays, followed by the fluorinated acetates, but other haloacetates were essentially inactive. The effects of these analogs on the hydrolysis of (/sup 3/H)Z11-16:Ac to (/sup 3/H)Z11-16:OH by antennal esterases was also examined. The three fluorinated acetates showed the greatest activity as inhibitors in competition assays,more » with rank order F/sub 2/Ac > F/sub 3/Ac > FAc > AC > Cl/sub 2/Ac > ClAc > Dza > Br/sub 2/Ac > BrAc > Tfp > I > Cl/sub 3/Ac > Br/sub 3/Ac > OH. The relative polarities of the haloacetates, as determined by TLC mobility, are in the order mono- > di- > trihalo, but F, Cl, Br, and I all confer similar polarities within a substitution group. Thus, the steric size appears to be the predominant parameter affecting the interactions of the haloacetate analogs with both receptor and catabolic proteins in P. xylostella males.« less

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.

PubMed

de Vries, Ronald P; vanKuyk, Patricia A; Kester, Harry C M; Visser, Jaap

2002-04-15

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.

PubMed Central

de Vries, Ronald P; vanKuyk, Patricia A; Kester, Harry C M; Visser, Jaap

2002-01-01

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin. PMID:11931668

Evaluation of esterase and hemolysin activities of different Candida species isolated from vulvovaginitis cases in Lorestan Province, Iran

PubMed Central

Noori, Maryam; Dakhili, Mohammad; Sepahvand, Asghar; Davari, Nader

2017-01-01

Background and Purpose: Annually affecting millions of women, vulvovaginal candidiasis (VVC) is commonly described by signs and symptoms of vulvovaginal inflammation in the presence of Candida species. Today, the detection of the virulence factors plays a major role in the understanding of pathogenesis of candidiasis and helps produce new anticandidial drugs to improve its treatment efficiency. Herein, we aimed to evaluate the esterase and hemolysin activities of the vaginal isolates of Candida and their relationship with the presence of VVC. Materials and Methods: One-hundred vaginal clinical specimens were randomly collected during September-December 2016. The target population consisted of married women suspected of VVC who presented to health centers in Lorestan Province, Iran. In this study, the esterase activity and hemolysin production of Candida clinical isolates were evaluated using the Tween 80 opacity test and the plate assay, respectively. Results: The most frequent Candida species was C. albicans (66; 66%), followed by C. glabrata (11; 11%) and C. tropicalis (11; 11%). The highest esterase activity was found in C. krusei (75%), followed by C. albicans (68.2%) and C. glabrata (54.5%). The greater part of the positive esterase isolates had Pz 4+ scores. Among the Candida species, C. albicans (22.7%), C. glabrata (63.6%), and C. krusei (50%) were found to have the highest rates of alpha, beta, and gamma hemolysin production, respectively. The level of hemolytic activity in 51% of the Candida species was Pz 4+ scores. Conclusion: According to our results, the higher expression rates of both enzymes in C. albicans species relative to those of non-albicans Candidate species can partly reflect the role of the virulence factors involved in C. albicans pathogenicity. PMID:29707672

Evaluation of esterase and hemolysin activities of different Candida species isolated from vulvovaginitis cases in Lorestan Province, Iran.

PubMed

Noori, Maryam; Dakhili, Mohammad; Sepahvand, Asghar; Davari, Nader

2017-12-01

Annually affecting millions of women, vulvovaginal candidiasis (VVC) is commonly described by signs and symptoms of vulvovaginal inflammation in the presence of  Candida  species. Today, the detection of the virulence factors plays a major role in the understanding of pathogenesis of candidiasis and helps produce new anticandidial drugs to improve its treatment efficiency. Herein, we aimed to evaluate the esterase and hemolysin activities of the vaginal isolates of Candida and their relationship with the presence of VVC. One-hundred vaginal clinical specimens were randomly collected during September-December 2016. The target population consisted of married women suspected of VVC who presented to health centers in Lorestan Province, Iran. In this study, the esterase activity and hemolysin production of Candida clinical isolates were evaluated using the Tween 80 opacity test and the plate assay, respectively. The most frequent Candida species was C. albicans (66; 66%), followed by C. glabrata (11; 11%) and C. tropicalis (11; 11%). The highest esterase activity was found in C. krusei (75%), followed by C. albicans (68.2%) and C. glabrata (54.5%). The greater part of the positive esterase isolates had Pz 4+ scores. Among the Candida species, C. albicans (22.7 % ), C. glabrata (63.6%), and C. krusei (50%) were found to have the highest rates of alpha, beta, and gamma hemolysin production, respectively. The level of hemolytic activity in 51% of the Candida species was Pz 4+ scores. According to our results, the higher expression rates of both enzymes in C. albicans species relative to those of non-albicans Candidate species can partly reflect the role of the virulence factors involved in C. albicans pathogenicity.

Evidence of multiple/cross resistance to Bt and organophosphate insecticides in Puerto Rico population of the fall armyworm, Spodoptera frugiperda.

PubMed

Zhu, Yu Cheng; Blanco, Carlos A; Portilla, Maribel; Adamczyk, John; Luttrell, Randall; Huang, Fangneng

2015-07-01

Fall armyworm (FAW) is a damaging pest of many economic crops. Long-term use of chemical control prompted resistance development to many insecticide classes. Many populations were found to be significantly less susceptible to major Bt toxins expressed in transgenic crops. In this study, a FAW strain collected from Puerto Rico (PR) with 7717-fold Cry1F-resistance was examined to determine if it had also developed multiple/cross resistance to non-Bt insecticides. Dose response assays showed that the PR strain developed 19-fold resistance to acephate. Besides having a slightly smaller larval body weight and length, PR also evolved a deep (2.8%) molecular divergence in mitochondrial oxidase subunit II. Further examination of enzyme activities in the midgut of PR larvae exhibited substantial decreases of alkaline phosphatase (ALP), aminopeptidase (APN), 1-NA- and 2-NA-specific esterase, trypsin, and chymotrypsin activities, and significant increases of PNPA-specific esterase and glutathione S-transferase (GST) activities. When enzyme preparations from the whole larval body were examined, all three esterase, GST, trypsin, and chymotrypsin activities were significantly elevated in the PR strain, while ALP and APN activities were not significantly different from those of susceptible strain. Data indicated that multiple/cross resistances may have developed in the PR strain to both Bt toxins and conventional insecticides. Consistently reduced ALP provided evidence to support an ALP-mediated Bt resistance mechanism. Esterases and GSTs may be associated with acephate resistance through elevated metabolic detoxification. Further studies are needed to clarify whether and how esterases, GSTs, and other enzymes (such as P450s) are involved in cross resistance development to Bt and other insecticide classes. Published by Elsevier Inc.

KINETIC STUDY ON THE INHIBITION OF HEN BRAIN NEUROTOXIC ESTERASE BY MIPAFOX

EPA Science Inventory

A direct method of assaying neurotoxic esterase (NTE) activity, using 4-nitrophenyl valerate, has been described. The technique was used to determine the biomolecular rate (ki), phosphorylation (k2), and affinity (kd) constants for the reaction of hen brain microsomal NTE with mi...

Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

1999-10-01

The HL-60 differentiation-inducing compounds in bergamot fruits were isolated with column chromatography and identified as bergamottin, bergapten, and citropten by (1)H and (13)C NMR. Their HL-60 differentiation-inducing activity was measured by examining nitro blue tetrazolium (NBT) reducing, nonspecific acid esterase (NSE), specific esterase (SE), and phagocytic activities, and bergamottin showed the strongest activity among the coumarins isolated from bergamot fruits. The structure-activity relationship obtained from HL-60 differentiation assay suggests that hydrophobicity of furocoumarins is correlated with their activity.

The Lp_3561 and Lp_3562 Enzymes Support a Functional Divergence Process in the Lipase/Esterase Toolkit from Lactobacillus plantarum

PubMed Central

Esteban-Torres, María; Reverón, Inés; Santamaría, Laura; Mancheño, José M.; de las Rivas, Blanca; Muñoz, Rosario

2016-01-01

Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions. PMID:27486450

Electrochemical biosensor for carbofuran pesticide based on esterases from Eupenicillium shearii FREI-39 endophytic fungus.

PubMed

Grawe, Gregory Ferreira; de Oliveira, Tássia Regina; de Andrade Narciso, Esther; Moccelini, Sally Katiuce; Terezo, Ailton José; Soares, Marcos Antonio; Castilho, Marilza

2015-01-15

In this work, a biosensor was constructed by physical adsorption of the isolated endophytic fungus Eupenicillium shearii FREI-39 esterase on halloysite, using graphite powder, multi-walled carbon nanotubes and mineral oil for the determination of carbofuran pesticide by inhibition of the esterase using square-wave voltammetry (SWV). Specific esterase activities were determined each 2 days over a period of 15 days of growth in four different inoculation media. The highest specific activity was found on 6th day, with 33.08 U on PDA broth. The best performance of the proposed biosensor was obtained using 0.5 U esterase activity. The carbofuran concentration response was linear in the range from 5.0 to 100.0 µg L(-1) (r=0.9986) with detection and quantification limits of 1.69 µg L(-1) and 5.13 µg L(-1), respectively. A recovery study of carbofuran in spiked water samples showed values ranging from 103.8±6.7% to 106.7±9.7%. The biosensor showed good repeatability and reproducibility and remained stable for a period of 20 weeks. The determination of carbofuran in spiked water samples using the proposed biosensor was satisfactory when compared to the chromatographic reference method. The results showed no significant difference at the 95% confidence level with t-test statistics. The application of enzymes from endophytic fungi in constructing biosensors broadens the biotechnological importance of these microorganisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

PubMed

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J; Madrid, Susan M; Brinch-Pedersen, Henrik; Holm, Preben B; Scheller, Henrik V

2010-04-01

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig

2009-12-08

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and three fold relative to wild type. The grains were shriveled and had a 25-33% decrease in mass. Extensive analysis of the cell walls showed a 10-15% increase in arabinose to xylosemore » ratio, a 50% increase in the proportion of water extractable arabinoxylan, and a shift in the MW of the water extractable arabinoxylan from being mainly larger than 85 kD to being between 2 kD and 85 kD. Ferulic acid esterase expressing grains were also shriveled and the seed weight was decreased by 20-50%. No ferulic acid esterase activity could be detected in wild type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15-40% increase in water unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13 and 34%. In all the plants the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.« less

Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis.

PubMed

Kwon, Deok Ho; Kim, Ju Hyeon; Kim, Young Ho; Yoon, Kyong Sup; Clark, J Marshall; Lee, Si Hyeock

2014-06-01

Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance. Copyright © 2014 Elsevier Inc. All rights reserved.

Diazo compounds for the bioreversible esterification of proteins† †Electronic supplementary information (ESI) available: Experimental procedures, analytical data, and spectral data for novel compounds. See DOI: 10.1039/c4sc01768d Click here for additional data file.

PubMed Central

McGrath, Nicholas A.; Andersen, Kristen A.; Davis, Amy K. F.; Lomax, Jo E.

2015-01-01

A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups. This new mode of chemical modification could enable the key advantages of prodrugs to be translated from small-molecules to proteins. PMID:25544883

Monocyte esterase deficiency in malignant neoplasia.

PubMed Central

Markey, G M; McCormick, J A; Morris, T C; Alexander, H D; Nolan, L; Morgan, L M; Reynolds, M E; Edgar, S; Bell, A L; McCaigue, M D

1990-01-01

A survey of the incidence of monocyte esterase deficiency in 4000 inpatients (including 808 with malignant neoplastic disease) and 474 normal controls was performed using an automated esterase method. A highly significant excess of patients with malignant disease and the deficiency was evident when compared with normal controls or all other patients. Within the group of patients with malignant disease the demonstrable excess occurred in B chronic lymphocytic leukaemia, non-Hodgkin's and Hodgkin's lymphoma, and carcinoma of the gastrointestinal tract. There was also a significant excess of patients with the deficiency attending the renal unit, both among patients who had had renal transplants and those who had not. A familial incidence of monocyte esterase deficiency was found in 19 (35%) of first degree relatives of those patients in whom family studies were done. It is suggested that the reason for the increased prevalence of the anomaly in these disorders might be that the diminution of esterase activity has a role in their development. PMID:2341564

Effects of Model Salivary Esterases and MMP Inhibition on the Restoration's Marginal Integrity and Potential Degradative Contribution of Cariogenic Bacteria

NASA Astrophysics Data System (ADS)

Huang, Bo

Enzyme-catalyzed degradation of the restoration-tooth interface compromises interfacial integrity, thereby contributing to secondary caries, which is a major cause of resin-based restoration failure. It is hypothesized that in addition to salivary esterases, the cariogenic bacterium Streptococcus mutans has specific esterases that degrade the resin-dentin interface, releasing biodegradation by- products (BBPs) such as bis-hydroxy-propoxy-phenyl-propane (BisHPPP). In turn, BisHPPP affects S. mutans by stimulating the expression of esterases. Another hypothesis is that the biostability of the resin-dentin interface is affected by simulated salivary esterases, dentinal matrix metalloproteinase (MMP) inhibition, and restorative materials. To test the first hypothesis, putative esterase genes in S. mutans UA159 were identified, purified, and characterized. SMU_118c was identified as the dominant esterase in S. mutans UA159 and showed a similar hydrolytic activity profile to salivary esterases. BisHPPP upregulated expression of the SMU_118c gene and related protein in a concentration-dependent manner. This positive feedback process could accelerate the degradation of the restoration-tooth interface and lead to premature restoration failure. To test the second hypothesis, an in vitro model was established to evaluate the effects of salivary esterases, MMP inhibition and restorative materials on interfacial integrity. It was confirmed that interfacial integrity was compromised with time and was further deteriorated by simulated salivary esterases, as indicated by the greater depth of bacterial ingress and more bacterial biomass of biofilm along the interface. However, this process could be modulated by using different restorative materials and MMPs inhibition. This project elucidated the mechanistic interaction between oral bacteria and restorative materials and established a new, in vitro, and physiologically relevant model to assess the effect of material chemistry, properties, and application modes on bacterial penetration and biofilm formation. These findings offer the oral health community practical ways to reduce secondary caries by altering material composition and restorative procedures.

Genome-wide analysis of esterase-like genes in the striped rice stem borer, Chilo suppressalis.

PubMed

Wang, Baoju; Wang, Ying; Zhang, Yang; Han, Ping; Li, Fei; Han, Zhaojun

2015-06-01

The striped rice stem borer, Chilo suppressalis, a destructive pest of rice, has developed high levels of resistance to certain insecticides. Esterases are reported to be involved in insecticide resistance in several insects. Therefore, this study systematically analyzed esterase-like genes in C. suppressalis. Fifty-one esterase-like genes were identified in the draft genomic sequences of the species, and 20 cDNA sequences were derived which encoded full- or nearly full-length proteins. The putative esterase proteins derived from these full-length genes are overall highly diversified. However, key residues that are functionally important including the serine residue in the active site are conserved in 18 out of the 20 proteins. Phylogenetic analysis revealed that most of these genes have homologues in other lepidoptera insects. Genes CsuEst6, CsuEst10, CsuEst11, and CsuEst51 were induced by the insecticide triazophos, and genes CsuEst9, CsuEst11, CsuEst14, and CsuEst51 were induced by the insecticide chlorantraniliprole. Our results provide a foundation for future studies of insecticide resistance in C. suppressalis and for comparative research with esterase genes from other insect species.

New medium for detection of esterase and gelatinase activity.

PubMed

Pácová, Z; Kocur, M

1984-10-01

A new medium was developed for detecting esterase and gelatinase activities in aerobic and facultatively anaerobic bacteria. The new medium was tested with various strains of bacteria and the results showed agreement between the reactions in the new medium and those obtained by conventional techniques. The new medium is more economical and may be used for a rapid differentiation of Serratia, Aeromonas and Vibrio species from biochemically similar bacteria.

Survival of Burkholderia pseudomallei on Environmental Surfaces.

PubMed

Shams, Alicia M; Rose, Laura J; Hodges, Lisa; Arduino, Matthew J

2007-12-01

The survival of the biothreat agent Burkholderia pseudomallei on the surfaces of four materials was measured by culture and esterase activity analyses. The culture results demonstrated that this organism persisted for <24 h to <7 days depending on the material, bacterial isolate, and suspension medium. The persistence determined by analysis of esterase activity, as measured with a ScanRDI solid-phase cytometer, was always longer than the persistence determined by culture analysis.

Evaluation of secretome of highly efficient lignocellulolytic Penicillium sp. Dal 5 isolated from rhizosphere of conifers.

PubMed

Rai, Rohit; Kaur, Baljit; Singh, Surender; Di Falco, Macros; Tsang, Adrian; Chadha, B S

2016-09-01

Penicillium sp. (Dal 5) isolated from rhizosphere of conifers from Dalhousie (Himachal Pradesh, India) was found to be an efficient cellulolytic strain. The culture under shake flask on CWR (cellulose, wheat bran and rice straw) medium produced appreciably higher levels of endoglucanase (35.69U/ml), β-glucosidase (4.20U/ml), cellobiohydrolase (2.86U/ml), FPase (1.2U/ml) and xylanase (115U/ml) compared to other Penicillium strains reported in literature. The mass spectroscopy analysis of Penicillium sp. Dal 5 secretome identified 108 proteins constituting an array of CAZymes including glycosyl hydrolases (GH) belonging to 24 different families, polysaccharide lyases (PL), carbohydrate esterases (CE), lytic polysaccharide mono-oxygenases (LPMO) in addition to swollenin and a variety of carbohydrate binding modules (CBM) indicating an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. Further, the culture extract was evaluated for hydrolysis of alkali treated rice straw, wheat straw, bagasse and corn cob at 10% substrate loading rate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Spinosad resistance, esterase isoenzymes and temporal synergism in Frankliniella occidentalis (Pergande) in Australia.

PubMed

Herron, Grant A; Gunning, Robin V; Cottage, Emma L A; Borzatta, Valerio; Gobbi, Carlotta

2014-09-01

Spinosad has been widely used in Australia to control western flower thrips Frankliniella occidentalis (Pergande) but spinosad usefulness is now compromised by resistance. Here we studied a highly spinosad resistant strain of F. occidentalis to explore if esterases had a role in spinosad resistance. Enhanced esterase activity in pressured spinosad-resistant F. occidentalis was confirmed via PAGE electrophoresis and estimated to be approximately three times higher than that in a susceptible strain. Spinosad-esterase inhibition data in the resistant strain, showed a concentration effect with significant esterase-spinosad binding occurring at spinosad concentrations from 6.2× 10(-7) to 1.5× 10(-5) M. Similarly, a spinosad-piperonyl butoxide (PBO) inhibition curve showed a concentration effect, with significant esterase-PBO binding occurring in the resistant strain at PBO concentrations between 3.3× 10(-5) M and 8.4× 10(-4) M. No binding of esterase to spinosad or PBO occurred in the susceptible strain. Results of bioassays in which spinosad resistant F. occidentalis were sprayed with a 4h delayed release formulation of cyclodextrin-complexed spinosad with immediately available PBO demonstrated that spinosad resistance was significantly reduced from 577 to 72-fold. With further development the PBO synergism of spinosad using a delayed release formulation, similar to that used here, may provide effective control for spinosad resistant F. occidentalis. Temporal synergism of spinosad may prove to be effective tactic for the control of spinosad resistant F. occidentalis where the main resistance mechanism involved has been confirmed to be esterase based. Copyright © 2014 Elsevier Inc. All rights reserved.

Extracellular esterases of phylloplane yeast Pseudozyma antarctica induce defect on cuticle layer structure and water-holding ability of plant leaves.

PubMed

Ueda, Hirokazu; Mitsuhara, Ichiro; Tabata, Jun; Kugimiya, Soichi; Watanabe, Takashi; Suzuki, Ken; Yoshida, Shigenobu; Kitamoto, Hiroko

2015-08-01

Aerial plant surface (phylloplane) is a primary key habitat for many microorganisms but is generally recognized as limited in nutrient resources. Pseudozyma antarctica, a nonpathogenic yeast, is commonly isolated from plant surfaces and characterized as an esterase producer with fatty acid assimilation ability. In order to elucidate the biological functions of these esterases, culture filtrate with high esterase activity (crude enzyme) of P. antarctica was applied onto leaves of tomato and Arabidopsis. These leaves showed a wilty phenotype, which is typically associated with water deficiency. Furthermore, we confirmed that crude enzyme-treated detached leaves clearly lost their water-holding ability. In treated leaves of both plants, genes associated to abscisic acid (ABA; a plant stress hormone responding osmotic stress) were activated and accumulation of ABA was confirmed in tomato plants. Microscopic observation of treated leaf surfaces revealed that cuticle layer covering the aerial epidermis of leaves became thinner. A gas chromatography-mass spectrometry (GC-MS) analysis exhibited that fatty acids with 16 and 18 carbon chains were released in larger amounts from treated leaf surfaces, indicating that the crude enzyme has ability to degrade lipid components of cuticle layer. Among the three esterases detected in the crude enzyme, lipase A, lipase B, and P. antarctica esterase (PaE), an in vitro enzyme assay using para-nitrophenyl palmitate as substrate demonstrated that PaE was the most responsible for the degradation. These results suggest that PaE has a potential role in the extraction of fatty acids from plant surfaces, making them available for the growth of phylloplane yeasts.

Effect of coumarins on HL-60 cell differentiation.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

2000-01-01

Twenty-eight coumarins, including 7 furocoumarins, were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase and phagocytic activities. Esculetin, nordalbergin, 6,7-dihydroxy-4-methylcoumarin and imperatorin had strong activity among the coumarins examined. HL-60 cells treated with these coumarins differentiated into mature monocyte/macrophage. The structure-activity relationship established from the results revealed that 6,7-dihydroxy moiety had an important role in the induction of differentiation of HL-60.

Physiological and biochemical effect of neem and other Meliaceae plants secondary metabolites against Lepidopteran insects

PubMed Central

Senthil-Nathan, Sengottayan

2013-01-01

This review described the physiological and biochemical effects of various secondary metabolites from Meliaceae against major Lepidopteran insect pest including, Noctuidae and Pyralidae. The biochemical effect of major Meliaceae secondary metabolites were discussed more in this review. Several enzymes based on food materials have critical roles in nutritional indices (food utilization) of the insect pest population. Several research work has been referred and the effect of Meliaceae secondary metabolites on feeding parameters of insects by demonstrating food consumption, approximate digestibility of consumed food, efficiency of converting the ingested food to body substance, efficiency of converting digested food to body substance and consumption index was reviewed in detail. Further how the digestive enzymes including a-Amylases, α and β-glucosidases (EC 3.2.1.1), lipases (EC 3.1.1) Proteases, serine, cysteine, and aspartic proteinases affected by the Meliaceae secondary metabolites was reviewed. Further effect of Meliaceae secondary metabolites on detoxifying enzymes have been found to react against botanical insecticides including general esterases (EST), glutathione S-transferase (GST) and phosphatases was reviewed. Alkaline phosphatase (ALP, E.C.3.1.3.1) and acid phosphatase (ACP, E.C.3.1.3.2) are hydrolytic enzymes, which hydrolyze phosphomonoesters under alkaline or acid conditions, respectively. These enzymes were affected by the secondary metabolites treatment. The detailed mechanism of action was further explained in this review. Acethylcholine esterase (AChE) is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of neurotransmitter, acetylcholine, in the nervous system of various organisms. How the AChE activity was altered by the Meliaceae secondary metabolites reviewed in detail. PMID:24391591

Liposomal formulation of {alpha}-tocopheryl maleamide: In vitro and in vivo toxicological profile and anticancer effect against spontaneous breast carcinomas in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turanek, Jaroslav; Wang Xiufang; Knoetigova, Pavlina

2009-06-15

The vitamin E analogue {alpha}-tocopheryl succinate ({alpha}-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of {alpha}-tocopheryl maleamide ({alpha}-TAM), an esterase-resistant analogue of {alpha}-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of {alpha}-TAM towards cancer cells (MCF-7, B16F10) compared to {alpha}-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that {alpha}-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both {alpha}-TOS and {alpha}-TAM to solve the problem with cytotoxicity of free {alpha}-TAM (neurotoxicity and anaphylaxis), as well as the low solubilitymore » of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal {alpha}-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of {alpha}-TAM and {alpha}-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of {alpha}-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of {alpha}-TOS. Thus, the liposomal formulation of {alpha}-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.« less

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.

PubMed

Gouran, Hossein; Chakraborty, Sandeep; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

PubMed Central

Rao, Basuthkar J.; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction. PMID:25717364

Enzymes as Enhancers for the Biodegradation of Synthetic Polymers in Wastewater.

PubMed

Haernvall, Karolina; Zitzenbacher, Sabine; Biundo, Antonino; Yamamoto, Motonori; Schick, Michael Bernhard; Ribitsch, Doris; Guebitz, Georg M

2018-02-16

Synthetic polyesters are today the second-largest class of ingredients in household products and are entering wastewater treatment plants (WWTPs) after product utilization. One approach to improve polymer biodegradation in wastewater would be to complement current processes with polyester-hydrolyzing enzymes and their microbial producers. In this study, the hydrolysis of poly(oxyethylene terephthalate) polymer by hydrolases from wastewater microorganisms was investigated in vitro and under realistic WWTP conditions. An esterase and a cutinase from Pseudomonas pseudoalcaligenes and a lipase from Pseudomonas pelagia were heterologously expressed in Escherichia coli BL21-Gold(DE3) and were purified by a C-terminal His 6 tag. The hydrolases were proven to hydrolyze the polymer effectively, which is a prerequisite for further biodegradation. The hydrolases maintained high activity up to 50 % upon lowering the temperature from 28 to 15 °C to mimic WWTP conditions. The hydrolases were also not inhibited by the wastewater matrix. Polyester-hydrolyzing enzymes active under WWTP conditions and their microbial producers thus have the potential to improve biological treatment of wastewater rich in synthetic polymers. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vine Trimming Shoots as Substrate for Ferulic Acid Esterases Production.

PubMed

Pérez-Rodríguez, N; Outeiriño, D; Torrado Agrasar, A; Domínguez, J M

2017-02-01

Ferulic acid esterases (FAE) possess a large variety of biotechnological applications mainly based on their ability to release ferulic acid from lignocellulosic matrixes. The use of vine trimming shoots (VTS), an agricultural waste, as substrate for the generation of this kind of esterases represents an attractive alternative to change the consideration of VTS from residue to resource. Furthermore, xylanase, cellobiase, and cellulase activities were quantified. Six microorganisms were screened for FAE production by solid-state fermentation, and the effects of the additional supplementation and substrate size were also tested. Finally, the process was scaled-up to a horizontal bioreactor where the influence of aeration in enzymatic activities was evaluated. Thus, the optimal FAE activity (0.44 U/g dry VTS) was attained by Aspergillus terreus CECT 2808, in non-additional supplementation media, using the larger particles size of substrate (≤ 5 mm) and at a flow rate of 0.7 L/min.

Xylella fastidiosa esterase rather than hydroxynitrile lyase.

PubMed

Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

2015-03-02

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate.

PubMed

Morono, Yuki; Takano, Suguru; Miyanaga, Kazuhiko; Tanji, Yasunori; Unno, Hajime; Hori, Katsutoshi

2004-03-01

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

PubMed

Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

2015-03-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Novel ferulate esterase from Gram-positive lactic acid bacteria and analyses of the recombinant enzyme produced in E. coli

USDA-ARS?s Scientific Manuscript database

Using a plate containing ethyl ferulate as sole carbon source, various bacteria cultures were screened for ferulate esterase (FAE). Among a dozen of species showing positive FAE, one Lactobacillus fermentum strain NRRL 1932 demonstrated the strongest activity. Using a published sequence of ferulate ...

Novel feruloyl esterase from Lactobacillus fermentum NRRL B-1932 and analysis of the recombinant enzyme produced in Escherichia coli.

USDA-ARS?s Scientific Manuscript database

Using agar plates containing ethyl ferulate as the sole carbon source, 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity. Among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate, Lactobacillus fermentum NRRL B-1932 demonstrated the stronge...

Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome

NASA Astrophysics Data System (ADS)

Zhang, Hao; Li, Fuchao; Chen, Huaxin; Zhao, Jin; Yan, Jinfei; Jiang, Peng; Li, Ronggui; Zhu, Baoli

2015-07-01

Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. ScsEst01 was successfully co-expressed in Escherichia coli BL21 (DE3) with chaperones (dnaK-dnaJ-grpE) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2+. The enzyme was characterized using p -nitrophenol butyrate as a substrate. ScsEst01 had the highest lipolytic activity at 35°C and pH 8.0, indicative of a meso-thermophilic alkaline esterase. ScsEst01 was thermostable at 20°C. The lipolytic activity of scsEst01 was strongly increased by Fe2+, Mn2+ and 1% Tween 80 or Tween 20.

Effect of a mixture of iprobenfos and malathion on the development of malathion resistance in the mosquito Culex pipiens pallens Coq.

PubMed

Tao, Li-Ming; Yang, Jian-Zhong; Zhuang, Pei-Jun; Tang, Zhen-Hua

2006-01-01

A malathion-resistant (RM) strain of Culex pipiens pallens Coq was obtained by successively selecting a field population with malathion in the laboratory. The synergistic effect of iprobenfos on malathion toxicity and alpha-naphthyl acetate (alpha-NA) esterase assay revealed that malathion resistance in the RM strain was associated with increased alpha-NA esterase activity and the synergism was mainly due to the inhibition by iprobenfos of this activity. There was no difference in alpha-NA esterase activity between the larvae and female adults in the susceptible (S) strain, but the activity in the adults was 13-fold higher than in the larvae of the RM strain. To understand the effect of the application of a mixture of iprobenfos and malathion on the evolution of malathion resistance, an artificial strain (Syn) was generated by mixing the RM and S strains with 0.1 frequency of the malathion-resistant individuals. The offspring of the Syn strain were divided into two sub-strains, Rm and Rm+ibp, which were successively treated with, respectively, malathion alone and malathion + iprobenfos (1:2) at LC70. In the mixture, the fungicide iprobenfos acted as a synergist of malathion. After treatment for 10 generations, the resistance level to malathion was 317.4-fold for the Rm sub-strain, whereas for the Rm+ibp sub-strain it was only 38.9-fold, compared with the Syn strain. Similar results were obtained by measurement of alpha-NA esterase activity from both larvae and female adults. The alpha-NA esterase activities in larvae and female adults at F10 generation were 2.6- and 10.9-fold from the Rm+ibp sub-strain and 5.7- and 98.5-fold from the Rm sub-strain, respectively, compared with the Syn strain. The above results suggested that iprobenfos, although it cannot completely stop or prevent the onset of malathion resistance, could dramatically delay its evolution. Copyright 2005 Society of Chemical Industry.

The Fate of Bacterial Cocaine Esterase (CocE): An In Vivo Study of CocE-Mediated Cocaine Hydrolysis, CocE Pharmacokinetics, and CocE EliminationS⃞

PubMed Central

Brim, Remy L.; Noon, Kathleen R.; Collins, Gregory T.; Stein, Aron; Nichols, Joseph; Narasimhan, Diwa; Ko, Mei-Chuan; Woods, James H.

2012-01-01

Cocaine abuse and toxicity remain widespread problems in the United States. Currently cocaine toxicity is treated only symptomatically, because there is no Food and Drug Administration-approved pharmacotherapy for this indication. To address the unmet need, a stabilized mutant of bacterial cocaine esterase [T172R/G173Q-CocE (DM-CocE)], which hydrolyzes cocaine into inactive metabolites and has low immunogenic potential, has been developed and previously tested in animal models of cocaine toxicity. Here, we document the rapid cocaine hydrolysis by low doses of DM-CocE in vitro and in vivo, as well as the pharmacokinetics and distribution of the DM-CocE protein in rats. DM-CocE at 50.5 μg/kg effectively eliminated 4 mg/kg cocaine within 2 min in both male and female rats as measured by mass spectrometry. We expanded on these findings by using a pharmacologically relevant dose of DM-CocE (0.32 mg/kg) in rats and monkeys to hydrolyze convulsant doses of cocaine. DM-CocE reduced cocaine to below detection limits rapidly after injection; however, elimination of DM-CocE resulted in peripheral cocaine redistribution by 30 to 60 min. Elimination of DM-CocE was quantified by using [35S] labeling of the enzyme and was found to have a half-life of 2.1 h in rats. Minor urinary output of DM-CocE was also observed. Immunohistochemistry, Western blotting, and radiography all were used to elucidate the mechanism of DM-CocE elimination, rapid proteolysis, and recycling of amino acids into all tissues. This rapid elimination of DM-CocE is a desirable property of a therapeutic for cocaine toxicity and should reduce the likelihood of immunogenic or adverse reactions as DM-CocE moves toward clinical use. PMID:21990608

A Two-Step Bioconversion Process for Canolol Production from Rapeseed Meal Combining an Aspergillus niger Feruloyl Esterase and the Fungus Neolentinus lepideus

PubMed Central

Odinot, Elise; Fine, Frédéric; Sigoillot, Jean-Claude; Navarro, David; Laguna, Oscar; Bisotto, Alexandra; Peyronnet, Corinne; Lecomte, Jérôme; Faulds, Craig B.

2017-01-01

Rapeseed meal is a cheap and abundant raw material, particularly rich in phenolic compounds of biotechnological interest. In this study, we developed a two-step bioconversion process of naturally occurring sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid) from rapeseed meal into canolol by combining the complementary potentialities of two filamentous fungi, the micromycete Aspergillus niger and the basidiomycete Neolentinus lepideus. Canolol could display numerous industrial applications because of its high antioxidant, antimutagenic and anticarcinogenic properties. In the first step of the process, the use of the enzyme feruloyl esterase type-A (named AnFaeA) produced with the recombinant strain A. niger BRFM451 made it possible to release free sinapic acid from the raw meal by hydrolysing the conjugated forms of sinapic acid in the meal (mainly sinapine and glucopyranosyl sinapate). An amount of 39 nkat AnFaeA per gram of raw meal, at 55 °C and pH 5, led to the recovery of 6.6 to 7.4 mg of free sinapic acid per gram raw meal, which corresponded to a global hydrolysis yield of 68 to 76% and a 100% hydrolysis of sinapine. Then, the XAD2 adsorbent (a styrene and divinylbenzene copolymer resin), used at pH 4, enabled the efficient recovery of the released sinapic acid, and its concentration after elution with ethanol. In the second step, 3-day-old submerged cultures of the strain N. lepideus BRFM15 were supplied with the recovered sinapic acid as the substrate of bioconversion into canolol by a non-oxidative decarboxylation pathway. Canolol production reached 1.3 g/L with a molar yield of bioconversion of 80% and a productivity of 100 mg/L day. The same XAD2 resin, when used at pH 7, allowed the recovery and purification of canolol from the culture broth of N. lepideus. The two-step process used mild conditions compatible with green chemistry. PMID:29036919

Synthesis of fruity ethyl esters by acyl coenzyme A: alcohol acyltransferase and reverse esterase activities in Oenococcus oeni and Lactobacillus plantarum.

PubMed

Costello, P J; Siebert, T E; Solomon, M R; Bartowsky, E J

2013-03-01

To assess the abilities of commercial wine lactic acid bacteria (LAB) to synthesize potentially flavour active fatty acid ethyl esters and determine mechanisms involved in their production. Oenococcus oeni AWRI B551 produced significant levels of ethyl hexanoate and ethyl octanoate following growth in an ethanolic test medium, and ester formation generally increased with increasing pH (4.5 > 3.5), anaerobiosis and precursor supplementation. Cell-free extracts of commercial O. oeni strains and Lactobacillus plantarum AWRI B740 were also tested for ester-synthesizing capabilities in a phosphate buffer via: (i) acyl coenzyme A: alcohol acyltransferase (AcoAAAT) activity and (ii) reverse esterase activity. For both ester-synthesizing activities, strain-dependent variation was observed, with AcoAAAT activity generally greater than reverse esterase. Reverse esterase in O. oeni AWRI B551 also esterified 1-propanol to produce propyl octanoate, and deuterated substrates ([(2)H(6)]ethanol and [(2)H(15)]octanoic acid) to produce the fully deuterated ester, [(2)H(5)]ethyl [(2)H(15)]octanoate. Wine LAB exhibit ethyl ester-synthesizing capability and possess two different ester-synthesizing activities, one of which is associated with an acyl coenzyme A: alcohol acyltransferase. This study demonstrates that wine LAB exhibit enzyme activities that can augment the ethyl ester content of wine. This knowledge will facilitate greater control over the impacts of malolactic fermentation on the fruity sensory properties and quality of wine. © 2012 Australian Wine Research Institute © 2012 The Society for Applied Microbiology.

In-vivo measurements of regional acetylcholine esterase activity in degenerative dementia: comparison with blood flow and glucose metabolism.

PubMed

Herholz, K; Bauer, B; Wienhard, K; Kracht, L; Mielke, R; Lenz, M O; Strotmann, T; Heiss, W D

2000-01-01

Memory and attention are cognitive functions that depend heavily on the cholinergic system. Local activity of acetylcholine esterase (AChE) is an indicator of its integrity. Using a recently developed tracer for positron emission tomography (PET), C-11-labeled N-methyl-4-piperidyl-acetate (C11-MP4A), we measured regional AChE activity in 4 non-demented subjects, 4 patients with dementia of Alzheimer type (DAT) and 1 patient with senile dementia of Lewy body type (SDLT), and compared the findings with measurements of blood flow (CBF) and glucose metabolism (CMRGlc). Initial tracer extraction was closely related to CBF. AChE activity was reduced significantly in all brain regions in demented subjects, whereas reduction of CMRGlc and CBF was more limited to temporo-parietal association areas. AChE activity in SDLT was in the lower range of values in DAT. Our results indicate that, compared to non-demented controls, there is a global reduction of cortical AChE activity in dementia. Dementia, cholinergic system, acetylcholine esterase, positron emission tomography, cerebral blood flow, cerebral glucose metabolism.

Unexpected Diversity of Escherichia coli Sialate O-Acetyl Esterase NanS

PubMed Central

Rangel, Ariel; Steenbergen, Susan M.

2016-01-01

ABSTRACT The sialic acids (N-acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, where N-acetyl- or N-glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations to N-acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria with O-acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity of Escherichia coli NanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show that E. coli strain O157:H7 Stx prophage or prophage remnants invariably include paralogs of nanS often located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialate O-acetyl esterases, as shown by complementation of an E. coli strain K-12 nanS mutant and the unimpaired growth of an E. coli O157 nanS mutant on O-acetylated sialic acid. We further demonstrate that nanS homologs in Streptococcus spp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialate O-acetyl esterase. IMPORTANCE The sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show that nanS homologs exist in bacteria other than Escherichia coli, as well as part of toxigenic E. coli prophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies of nanS to include mucosal pathogens, prophage, and prophage remnants. This expansion of the nanS superfamily suggests important, although as-yet-unknown, functions in host-microbe interactions. PMID:27481927

A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition.

PubMed Central

Crepin, Valerie F; Faulds, Craig B; Connerton, Ian F

2003-01-01

Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall. The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties. We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris. The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no. AJ293029). The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (K(m) 0.048 mM, K (i) 2.5 mM and V(max) 8.2 units/mg against methyl 3,4-dihydroxycinnamate). The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action. PMID:12435269

An Inserted α/β Subdomain Shapes the Catalytic Pocket of Lactobacillus johnsonii Cinnamoyl Esterase

PubMed Central

Vu, Clara; Xu, Xiaohui; Cui, Hong; Molloy, Sara; Savchenko, Alexei; Yakunin, Alexander; Gonzalez, Claudio F.

2011-01-01

Background Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2. Methodology/Principal Findings We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser106, His225, and Asp197, while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank. Conclusions The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases. PMID:21876742

An inserted α/β subdomain shapes the catalytic pocket of Lactobacillus johnsonii cinnamoyl esterase.

PubMed

Lai, Kin-Kwan; Stogios, Peter J; Vu, Clara; Xu, Xiaohui; Cui, Hong; Molloy, Sara; Savchenko, Alexei; Yakunin, Alexander; Gonzalez, Claudio F

2011-01-01

Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2. We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser(106), His(225), and Asp(197), while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank. The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases.

Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

PubMed

Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2017-03-01

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096.

PubMed

Kumar, C Ganesh; Kamle, Avijeet; Kamal, Ahmed

2013-01-01

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE-1, AtFAE-2, and AtFAE-3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH-dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5-8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca(2+), K(+), and Mg(2+) stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k(cat) /K(m)) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF-MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE-1 belonged to type A while AtFAE-2 and AtFAE-3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers.

Effect of phorbol esters on the macrophage-mediated biodegradation of polyurethanes via protein kinase C activation and other pathways.

PubMed

McBane, Joanne Eileen; Santerre, J P; Labow, Rosalind

2009-01-01

It was previously found that re-seeding monocyte-derived macrophages (MDM) on polycarbonate-based polyurethanes (PCNUs) in the presence of the protein kinase C (PKC) activator phorbol myristate acetate (PMA) inhibited MDM-mediated degradation of PCNUs synthesized with 1,6-hexane diisocyanate (HDI), as well as esterase activity and monocyte-specific esterase (MSE) protein. However, no effect on the degradation of a 4,4'-methylene bisphenyl (MDI)-derived PCNU (MDI321) occurred. This finding suggested that oxidation, a process linked to the PKC pathway, was not activated in the same manner for all PCNUs. In the current study MDM were re-seeded onto the above PCNU surfaces with PMA, PKC-inactive 4alphaPMA and the PKC inhibitor bisindolylmaleimide I hydrochloride (BIM) for 48 h before assaying for PCNU degradation, esterase activity, MSE protein, DNA, cell viability and cell morphology. 4alphaPMA did not alter MDM-mediated HDI PCNU degradation but MDI321 degradation increased in this condition. BIM alone had no effect on any parameter; however, when BIM and PMA were added together, the PMA inhibition of biodegradation, esterase activity and MSE protein was partially reversed for MDM on HDI PCNUs only. Adding PMA to MDM on HDI PCNUs increased intercellular connections, whereas 4alphaPMA or BIM+PMA increased cell size. Although this study demonstrated a role for oxidation via a PKC-activated pathway in MDM-mediated PCNU degradation, phorbol esters appear to also activate non-PKC pathways that have roles in biodegradation. Moreover, the sensitivity to material surface chemistry in the MDM response to each PCNU dictates a multi-factorial degradative process involving alternate material specific oxidative and hydrolytic mechanisms.

Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune.

PubMed

Spániková, Silvia; Biely, Peter

2006-08-21

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.

Haloacetate analogs of pheromones: Effects on catabolism and electrophysiology inPlutella xylostella.

PubMed

Prestwich, G D; Streinz, L

1988-03-01

A series of mono-, di-, and trihalogenated acetate analogs of Zl 1-16: Ac were prepared and examined for electrophysiological activity in antennae of males of the diamondback moth,Plutella xylostella. In addition, two potential affinity labels, a diazoacetate (Dza) and a trifluoromethyl ketone (Tfp), were evaluated for EAG activity. The Z11-16∶Ac showed the highest activity in EAG assays, followed by the fluorinated acetates, but other halo-acetates were essentially inactive. The polar diazoacetate and the trifluoromethyl ketone were also very weak EAG stimulants. The effects of these analogs on the hydrolysis of [(3)H]Z11-16∶Ac to [(3)H]Z11-16∶OH by antennal esterases was also examined. The three fluorinated acetates showed the greatest activity as inhibitors in competition assays, with rank order F2Ac > F(3)Ac > FAc > Ac > Cl2Ac > ClAc > Dza > Br2Ac > BrAc > Tfp > I > Cl3Ac > Br3Ac > OH. The relative polarities of the haloacetates, as determined by TLC mobility, are in the order mono- > di- > trihalo, but F, Cl, Br, and I all confer similar polarities within a substitution group. Thus, the steric size appears to be the predominant parameter affecting the interactions of the haloacetate analogs with both receptor and catabolic proteins inP. xylostella males.

Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

PubMed

Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda

2014-06-01

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism.

PubMed

Parsons, Harriet T; Yasmin, Tayyaba; Fry, Stephen C

2011-12-15

L-Ascorbate catabolism involves reversible oxidation to DHA (dehydroascorbic acid), then irreversible oxidation or hydrolysis. The precursor-product relationships and the identity of several major DHA breakdown products remained unclear. In the presence of added H2O2, DHA underwent little hydrolysis to DKG (2,3-dioxo-L-gulonate). Instead, it yielded OxT (oxalyl L-threonate), cOxT (cyclic oxalyl L-threonate) and free oxalate (~6:1:1), essentially simultaneously, suggesting that all three product classes independently arose from one reactive intermediate, proposed to be cyclic-2,3-O-oxalyl-L-threonolactone. Only with plant apoplastic esterases present were the esters significant precursors of free oxalate. Without added H2O2, DHA was slowly hydrolysed to DKG. Downstream of DKG was a singly ionized dicarboxy compound (suggested to be 2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone), which reversibly de-lactonized to a dianionic carboxypentonate. Formation of these lactones and acid was minimized by the presence of residual unreacted ascorbate. In vivo, the putative 2-carboxy-L-pentonolactones were relatively stable. We propose that DHA is a branch-point in ascorbate catabolism, being either oxidized to oxalate and its esters or hydrolysed to DKG and downstream carboxypentonates. The oxidation/hydrolysis ratio is governed by reactive oxygen species status. In vivo, oxalyl esters are enzymatically hydrolysed, but the carboxypentonates are stable. The biological roles of these ascorbate metabolites invite future exploration.

Synergistic action of recombinant accessory hemicellulolytic and pectinolytic enzymes to Trichoderma reesei cellulase on rice straw degradation.

PubMed

Laothanachareon, Thanaporn; Bunterngsook, Benjarat; Suwannarangsee, Surisa; Eurwilaichitr, Lily; Champreda, Verawat

2015-12-01

Synergism between core cellulases and accessory hydrolytic/non-hydrolytic enzymes is the basis of efficient hydrolysis of lignocelluloses. In this study, the synergistic action of three recombinant accessory enzymes, namely GH62 α-l-arabinofuranosidase (ARA), CE8 pectin esterase (PET), and GH10 endo-1,4-beta-xylanase (XYL) from Aspergillus aculeatus expressed in Pichia pastoris to a commercial Trichoderma reesei cellulase (Accellerase® 1500; ACR) on hydrolysis of alkaline pretreated rice straw was studied using a mixture design approach. Applying the full cubic model, the optimal ratio of quaternary enzyme mixture was predicted to be ACR:ARA:PET:XYL of 0.171:0.079:0.100:0.150, which showed a glucose releasing efficiency of 0.173 gglc/FPU, higher than the binary ACR:XYL mixture (0.122 gglc/FPU) and ACR alone (0.081 gglc/FPU) leading to a 47.3% increase in glucose yield compared with that from ACR at the same cellulase dosage. The result demonstrates the varying degree of synergism of accessory enzymes to cellulases useful for developing tailor-made enzyme systems for bio-industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

PubMed Central

Wheelock, Craig E.; Eder, Kai J.; Werner, Inge; Huang, Huazhang; Jones, Paul D.; Brammell, Benjamin F.; Elskus, Adria A.; Hammock, Bruce D.

2006-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 μg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 μg/l), but not a low dose (1.2 μg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ∼30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish. PMID:16011852

Individual variability in esterase activity and CYP1A levels in Chinook salmon (Oncorhynchus tshawytscha) exposed to esfenvalerate and chlorpyrifos

USGS Publications Warehouse

Wheelock, C.E.; Eder, K.J.; Werner, I.; Huang, H.; Jones, P.D.; Brammell, B.F.; Elskus, A.A.; Hammock, B.D.

2005-01-01

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 ??g/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 ??g/l), but not a low dose (1.2 ??g/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ???30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish. ?? 2005 Elsevier B.V. All rights reserved.

Plasma B-esterase activities in European raptors.

PubMed

Roy, Claudie; Grolleau, Gérard; Chamoulaud, Serge; Rivière, Jean-Louis

2005-01-01

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and Strigidae families were characterized by a BChE contribution that dominated the total ChE activity, while in the species of the Falconidae family, AChE activity dominated. With the exception of the barn owl, CbE activity (eserine-insensitive alpha-naphthyl acetate esterase [alpha-NAE] activity) in all species was almost absent or very low. The values obtained in this study for ChE, AChE, and BChE activities and the AChE:BChE ratios for buzzard, kestrel, barn owl, and tawny owl provide a good estimate of the normal values in free-living individuals of these European species. They can be used as a baseline to evaluate the effect of anticholinesterase insecticides in the field.

Behavior of detoxifying enzymes of Aedes aegypti exposed to girgensohnine alkaloid analog and Cymbopogon flexuosus essential oil.

PubMed

Carreño Otero, Aurora L; Palacio-Cortés, Angela Maria; Navarro-Silva, Mario Antonio; Kouznetsov, Vladimir V; Duque L, Jonny E

2018-01-01

Because mosquito control depend on the use of commercial insecticides and resistance has been described in some of them, there is a need to explore new molecules no resistant. In vivo effects of girgensohnine analog 2-(3,4-dimethoxyphenyl)-2-(piperidin-1-yl)acetonitrile DPPA and Cymbopogon flexuosus essential oil CFEO, on the detoxifying enzymes acetylcholinesterase (AChE), glutathione-S-transferase (GST), nonspecific esterases (α- and β-), mixed function oxidases (MFO) and p-NPA esterases were evaluated on a Rockefeller (Rock) and wild Aedes aegypti population from Santander, Colombia (WSant). The action was tested after 24h of exposure at concentrations of 20.10, 35.18 and 70.35mgL -1 of DPPA and 18.45, 30.75 and 61.50mgL -1 of CFEO, respectively. It was found that AChE activity of Rock and WSant was not influenced by the evaluated concentration of DPPA and CFEO (p>0.05), while MFO activity was significantly affected by all CFEO concentrations in WSant (p<0.05). GST, α- and β-esterase activities were affected in Rock exposed at the highest CFEO concentration, this concentration also modified β-esterases activity of WSant. DPPA and CFEO sublethal doses induced inhibition of AChE activity on untreated larvae homogenate from 12 to 20% and 18 to 26%, respectively. For untreated adult homogenate, the inhibition activity raised up to 14 to 27% for DPPA and 26 to 34% for CFEO. Elevated levels of detoxifying enzymes, found when CFEO was evaluated, showed a larval sensitivity not observed by the pure compound suggesting that DPPA, contrary to CFEO, was not recognized, transformed or eliminated by the evaluated detoxifying enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

A case of hereditary angioneurotic oedema, successfully treated with ε-aminocaproic acid. Studies on C'1 esterase inhibitor, C'1 activation, plasminogen level and histamine metabolism

PubMed Central

Lundh, B.; Laurell, Anna-Brita; Wetterqvist, H.; White, T.; Granerus, G.

1968-01-01

A patient with clinical and laboratory findings characteristic of hereditary angioneurotic oedema was investigated. The patient was observed for a period of 5 weeks, during which he had four attacks. ε-Aminocaproic acid (EACA) was then given continuously for 5 months, during which time the patient had no attacks. Attacks reappeared on withdrawal of EACA. Trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCA®) was found to be equally effective in later therapeutic trials. C'1 esterase inhibitor was found in low concentration in defibrinated plasma also during attacks. ε-Aminocaproic acid (EACA) produced no significant change of the inhibitor content. C'1 esterase inhibitor disappeared on incubation of defibrinated plasma from the patient at 37°C for 40 min, and C'1 esterase was generated. The generation time of C'1 esterase increased with increasing the concentration of EDTA in the test solution. The C'1 esterase inhibitor content of defibrinated plasma from the patient, varied with the C'1 esterase generation time, the coefficient of correlation being higher in plasma sampled before treatment with EACA. Plasminogen and α2-macroglobulin were within the normal ranges, also during attacks. EACA markedly depressed the plasminogen level, which rapidly returned to normal on withdrawal of the drug. The studies on histamine metabolism revealed no significant changes with the exception of the urinary excretion of histamine, which was moderately increased towards the end of the period studied. On the days the patient received EACA the urine never contained 1-methylimidazole-5-acetic acid which was present in all the other specimens of urine examined. The basal gastric acid secretion was increased. PMID:5701955

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus.

PubMed

Perera, Erick; Moyano, F J; Díaz, M; Perdomo-Morales, R; Montero-Alejo, V; Alonso, E; Carrillo, O; Galich, G S

2008-07-01

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

Efficient kinetic resolution of secondary alcohols using an organic solvent-tolerant esterase in non-aqueous medium.

PubMed

Gao, Wenyuan; Fan, Haiyang; Chen, Lifeng; Wang, Hualei; Wei, Dongzhi

2016-07-01

To identify an esterase-mediated kinetic resolution of secondary alcohols in non-aqueous medium. An esterase, EST4, from a marine mud metagenomic library, showed high activity and enantioselectivity for the kinetic resolution of secondary alcohols in non-aqueous medium. Using 1-phenylethanol as the model alcohol, the effects of organic solvents, acyl donors, molar ratio, temperatures and biocatalyst loading on the kinetic resolution catalyzed by the EST4 whole-cell biocatalyst were investigated and optimized. The optimized methodology was effective on resolving 16 various racemic secondary alcohols in neat n-hexane, providing excellent enantiomeric excess (up to 99.9 % ee). Moreover, EST4 exhibited a strong tolerance for high substrate concentration (up to 1 M), and the optical purity of the desired secondary alcohols was kept above 99 % ee. The esterase EST4 is a promising biocatalyst for the enantioselective synthesis of various alcohols and esters with interesting practical applications.

Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

PubMed

Fatahinia, M; Halvaeezadeh, M; Rezaei-Matehkolaei, A

2017-06-01

Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-β, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-β=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and β among all studied isolates were almost similar. In the present study, the prevalence of VVC among investigated women was higher than the previous report from Khuzestan but C. albicans has yet remained the predominant agent of VVC in this area. Given to the EAI, the virulence of C. albicans in VVC can be mediated by phospholipase, esterase and proteinases. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A functionalizable reverse thermal gel based on a polyurethane/PEG block copolymer

PubMed Central

Park, Daewon; Wu, Wei; Wang, Yadong

2010-01-01

Injectable reverse thermal gels have great potentials as biomaterials for tissue engineering and drug delivery. However, most existing gels lack functional groups that can be modified with biomolecules that can guide cell/material interactions. We created an amine-functionalized ABA block copolymer, poly(ethylene glycol)-poly(serinol hexamethylene urethane), or ESHU. This reverse thermal gel consists of a hydrophobic block (B): poly(serinol hexamethylene urethane) and a hydrophilic block (A): poly(ethylene glycol). The polymer was characterized by GPC, FTIR and 1H FTNMR. Rheological study demonstrated that ESHU solution in phosphate-buffered saline initiated phase transition at 32°C and reached maximum elastic modulus at 37°C. The in vitro degradation tests performed in PBS and cholesterol esterase solutions revealed that the polymer was hydrolyzable and the presence of cholesterol esterase greatly accelerated the hydrolysis. The in vitro cytotoxicity tests carried out using baboon smooth muscle cells demonstrated that ESHU had good cytocompatibility with cell viability indistinguishable from tissue culture treated polystyrene. Subcutaneous implantation in rats revealed well tolerated accurate inflammatory response with moderate ED-1 positive macrophages in the early stages, which largely resolved 4 weeks post-implantation. We functionalized ESHU with a hexapeptide, Ile-Lys-Val-Ala-Val-Ser (IKVAVS), which gelled rapidly at body temperature. We expect this new platform of functionalizable reverse thermal gels to provide versatile biomaterials in tissue engineering and regenerative medicine. PMID:20937526

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

PubMed Central

Hoefel, Daniel; Monis, Paul T.; Grooby, Warwick L.; Andrews, Stuart; Saint, Christopher P.

2005-01-01

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 105 bacteria ml−1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 103 active AOB ml−1 were detected in the membrane-intact fraction, and 1.40 × 104 active AOB ml−1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml−1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event. PMID:16269672

Microarray analysis of gene regulations and potential association with acephate-resistance and fitness cost in Lygus lineolaris.

PubMed

Zhu, Yu Cheng; Guo, Zibiao; He, Yueping; Luttrell, Randall

2012-01-01

The tarnished plant bug has become increasingly resistant to organophosphates in recent years. To better understand acephate resistance mechanisms, biological, biochemical, and molecular experiments were systematically conducted with susceptible (LLS) and acephate-selected (LLR) strains. Selection of a field population with acephate significantly increased resistance ratio to 5.9-fold, coupled with a significant increase of esterase activities by 2-fold. Microarray analysis of 6,688 genes revealed 329 up- and 333 down-regulated (≥2-fold) genes in LLR. Six esterase, three P450, and one glutathione S-transferase genes were significantly up-regulated, and no such genes were down-regulated in LLR. All vitellogenin and eggshell protein genes were significantly down-regulated in LLR. Thirteen protease genes were significantly down-regulated and only 3 were up-regulated in LLR. More than twice the number of catalysis genes and more than 3.6-fold of metabolic genes were up-regulated, respectively, as compared to those down-regulated with the same molecular and biological functions. The large portion of metabolic or catalysis genes with significant up-regulations indicated a substantial increase of metabolic detoxification in LLR. Significant increase of acephate resistance, increases of esterase activities and gene expressions, and variable esterase sequences between LLS and LLR consistently demonstrated a major esterase-mediated resistance in LLR, which was functionally provable by abolishing the resistance with esterase inhibitors. In addition, significant elevation of P450 gene expression and reduced susceptibility to imidacloprid in LLR indicated a concurrent resistance risk that may impact other classes of insecticides. This study demonstrated the first association of down-regulation of reproductive- and digestive-related genes with resistance to conventional insecticides, suggesting potential fitness costs associated with resistance development. This study shed new light on the understanding of the molecular basis of insecticide resistance, and the information is highly valuable for development of chemical control guidelines and tactics to minimize resistance and cross-resistance risks.

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

PubMed Central

Su, Lingqia; Woodard, Ronald W.; Chen, Jian

2013-01-01

Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinaseNS) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinaseNS showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinaseNSS130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinaseNSS130A, the cells expressing cutinaseNS showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of “cell leakage induced by the limited phospholipid hydrolysis of cutinaseNS” was proposed to explain the underlying mechanism for the extracellular release of cutinaseNS. PMID:23603671

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase.

PubMed

Roberts, I M; Montgomery, R K; Carey, M C

1984-10-01

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385, from Rhodopseudomonas palustris PSB-S.

PubMed

Luo, Xiangwen; Zhang, Deyong; Zhou, Xuguo; Du, Jiao; Zhang, Songbai; Liu, Yong

2018-05-09

Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30-46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l -1 and 0.918 ± 0.025 U·µg -1 , respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.

Metagenomic mining for thermostable esterolytic enzymes uncovers a new family of bacterial esterases.

PubMed

Zarafeta, Dimitra; Moschidi, Danai; Ladoukakis, Efthymios; Gavrilov, Sergey; Chrysina, Evangelia D; Chatziioannou, Aristotelis; Kublanov, Ilya; Skretas, Georgios; Kolisis, Fragiskos N

2016-12-19

Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Here, we have identified a new esterolytic enzyme by screening a metagenomic sample collected from a hot spring in Kamchatka, Russia. Biochemical characterization of the new esterase, termed EstDZ2, revealed that it is highly active against medium chain fatty acid esters at temperatures between 25 and 60 °C and at pH values 7-8. The new enzyme is moderately thermostable with a half-life of more than six hours at 60 °C, but exhibits exquisite stability against high concentrations of organic solvents. Phylogenetic analysis indicated that EstDZ2 is likely an Acetothermia enzyme that belongs to a new family of bacterial esterases, for which we propose the index XV. One distinctive feature of this new family, is the presence of a conserved GHSAG catalytic motif. Multiple sequence alignment, coupled with computational modelling of the three-dimensional structure of EstDZ2, revealed that the enzyme lacks the largest part of the "cap" domain, whose extended structure is characteristic for the closely related Family IV esterases. Thus, EstDZ2 appears to be distinct from known related esterolytic enzymes, both in terms of sequence characteristics, as well as in terms of three-dimensional structure.

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

PubMed

Sayer, Christopher; Finnigan, William; Isupov, Michail N; Levisson, Mark; Kengen, Servé W M; van der Oost, John; Harmer, Nicholas J; Littlechild, Jennifer A

2016-05-10

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

PubMed Central

Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

2016-01-01

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

Characterization of the Polyurethanolytic Activity of Two Alicycliphilus sp. Strains Able To Degrade Polyurethane and N-Methylpyrrolidone▿

PubMed Central

Oceguera-Cervantes, Alejandro; Carrillo-García, Agustín; López, Néstor; Bolaños-Nuñez, Sandra; Cruz-Gómez, M. Javier; Wacher, Carmen; Loza-Tavera, Herminia

2007-01-01

Two bacterial strains (BQ1 and BQ8) were isolated from decomposed soft foam. These were selected for their capacity to grow in a minimal medium (MM) supplemented with a commercial surface-coating polyurethane (PU) (Hydroform) as the carbon source (MM-PUh). Both bacterial strains were identified as Alicycliphilus sp. by comparative 16S rRNA gene sequence analysis. Growth in MM-PUh showed hyperbolic behavior, with BQ1 producing higher maximum growth (17.8 ± 0.6 mg·ml−1) than BQ8 (14.0 ± 0.6 mg·ml−1) after 100 h of culture. Nuclear magnetic resonance, Fourier transform infrared (IR) spectroscopy, and gas chromatography-mass spectrometry analyses of Hydroform showed that it was a polyester PU type which also contained N-methylpyrrolidone (NMP) as an additive. Alicycliphilus sp. utilizes NMP during the first stage of growth and was able to use it as the sole carbon and nitrogen source, with calculated Ks values of about 8 mg·ml−1. Enzymatic activities related to PU degradation (esterase, protease, and urease activities) were tested by using differential media and activity assays in cell-free supernatants of bacterial cultures in MM-PUh. Induction of esterase activity in inoculated MM-PUh, but not that of protease or urease activities, was observed at 12 h of culture. Esterase activity reached its maximum at 18 h and was maintained at 50% of its maximal activity until the end of the analysis (120 h). The capacity of Alicycliphilus sp. to degrade PU was demonstrated by changes in the PU IR spectrum and by the numerous holes produced in solid PU observed by scanning electron microscopy after bacterial culture. Changes in the PU IR spectra indicate that an esterase activity is involved in PU degradation. PMID:17693569

Influences of acephate and mixtures with other commonly used pesticides on honey bee (Apis mellifera) survival and detoxification enzyme activities.

PubMed

Yao, Jianxiu; Zhu, Yu Cheng; Adamczyk, John; Luttrell, Randall

2018-07-01

Acephate (organophosphate) is frequently used to control piercing/sucking insects in field crops in southern United States, which may pose a risk to honey bees. In this study, toxicity of acephate (formulation Bracket ® 97) was examined in honey bees through feeding treatments with sublethal (pollen residue level: 0.168 mg/L) and median-lethal (LC 50 : 6.97 mg/L) concentrations. Results indicated that adult bees treated with acephate at residue concentration did not show significant increase in mortality, but esterase activity was significantly suppressed. Similarly, bees treated with binary mixtures of acephate with six formulated pesticides (all at residue dose) consistently showed lower esterase activity and body weight. Clothianidin, λ-cyhalothrin, oxamyl, tetraconazole, and chlorpyrifos may interact with acephate significantly to reduce body weight in treated bees. The dose response data (LC50: 6.97 mg/L) revealed a relatively higher tolerance to acephate in Stoneville bee population (USA) than populations elsewhere, although in general the population is still very sensitive to the organophosphate. In addition to killing 50% of the treated bees acephate (6.97 mg/L) inhibited 79.9%, 20.4%, and 29.4% of esterase, Glutathione S-transferase (GST), and acetylcholinesterase (AChE) activities, respectively, in survivors after feeding treatment for 48 h. However, P450 activity was elevated 20% in bees exposed to acephate for 48 h. Even though feeding on sublethal acephate did not kill honey bees directly, chronic toxicity to honey bee was noticeable in body weight loss and esterase suppression, and its potential risk of synergistic interactions with other formulated pesticides should not be ignored. Published by Elsevier Inc.

Visualisation of plastid outgrowths in potato (Solanum tuberosum L.) tubers by carboxyfluorescein diacetate staining.

PubMed

Borucki, Wojciech; Bederska, Magdalena; Sujkowska-Rybkowska, Marzena

2015-05-01

We describe two types of plastid outgrowths visualised in potato tubers after carboxyfluorescein diacetate staining. Probable esterase activity of the outgrowths has been demonstrated for the first time ever. Plastid outgrowths were observed in the phelloderm and storage parenchyma cells of red potato (S. tuberosum L. cv. Rosalinde) tubers after administration of carboxyfluorescein diacetate stain. Endogenous esterases cleaved off acetic groups to release membrane-unpermeable green fluorescing carboxyfluorescein which accumulated differentially in particular cell compartments. The intensive green fluorescence of carboxyfluorescein exhibited highly branched stromules (stroma-filled plastid tubular projections of the plastid envelope) and allowed distinguishing them within cytoplasmic strands of the phelloderm cells. Stromules (1) were directed towards the nucleus or (2) penetrated the whole cells through the cytoplasmic bands of highly vacuolated phelloderm cells. Those directed towards the nucleus were flattened and adhered to the nuclear envelope. Stromule-like interconnections between two parts of the same plastids (isthmuses) were also observed. We also documented the formation of another type of the stroma-filled plastid outgrowths, referred to here as protrusions, which differed from previously defined stromules in both morphology and esterase activity. Unlike stromules, the protrusions were found to be associated with developmental processes leading to starch accumulation in the storage parenchyma cells. These results strongly suggest that stromules and protrusions exhibit esterase activity. This has been demonstrated for the first time. Morphological and biochemical features as well as possible functions of stromules and protrusions are discussed below.

Multiple functionalization of fluorescent nanoparticles for specific biolabeling and drug delivery of dopamine

NASA Astrophysics Data System (ADS)

Malvindi, Maria Ada; di Corato, Riccardo; Curcio, Annalisa; Melisi, Daniela; Rimoli, Maria Grazia; Tortiglione, Claudia; Tino, Angela; George, Chandramohan; Brunetti, Virgilio; Cingolani, Roberto; Pellegrino, Teresa; Ragusa, Andrea

2011-12-01

The development of fluorescent biolabels for specific targeting and controlled drug release is of paramount importance in biological applications due to their potential in the generation of novel tools for simultaneous diagnosis and treatment of diseases. Dopamine is a neurotransmitter involved in several neurological diseases, such as Parkinson's disease and attention deficit hyperactivity disorder (ADHD), and the controlled delivery of its agonists already proved to have beneficial effects both in vitro and in vivo. Here, we report the synthesis and multiple functionalization of highly fluorescent CdSe/CdS quantum rods for specific biolabeling and controlled drug release. After being transferred into aqueous media, the nanocrystals were made highly biocompatible through PEG conjugation and covered by a carbohydrate shell, which allowed specific GLUT-1 recognition. Controlled attachment of dopamine through an ester bond also allowed hydrolysis by esterases, yielding a smart nanotool for specific biolabeling and controlled drug release.The development of fluorescent biolabels for specific targeting and controlled drug release is of paramount importance in biological applications due to their potential in the generation of novel tools for simultaneous diagnosis and treatment of diseases. Dopamine is a neurotransmitter involved in several neurological diseases, such as Parkinson's disease and attention deficit hyperactivity disorder (ADHD), and the controlled delivery of its agonists already proved to have beneficial effects both in vitro and in vivo. Here, we report the synthesis and multiple functionalization of highly fluorescent CdSe/CdS quantum rods for specific biolabeling and controlled drug release. After being transferred into aqueous media, the nanocrystals were made highly biocompatible through PEG conjugation and covered by a carbohydrate shell, which allowed specific GLUT-1 recognition. Controlled attachment of dopamine through an ester bond also allowed hydrolysis by esterases, yielding a smart nanotool for specific biolabeling and controlled drug release. Electronic supplementary information (ESI) available: TEM images, absorption and emission spectra, ζ-potential and DLS graphics, gel electrophoresis images, cyclic voltammograms, western blot and RT-PCR data. See DOI: 10.1039/c1nr10797f

Determination of organophosphorus pesticide residues in vegetables by an enzyme inhibition method using α-naphthyl acetate esterase extracted from wheat flour*

PubMed Central

Wang, Jun-liang; Xia, Qing; Zhang, An-ping; Hu, Xiao-yan; Lin, Chun-mian

2012-01-01

The widespread use of organophosphorus pesticides (OPs) poses a great threat to human health and has made the detection of OP residues in food an important task, especially in view of the fact that easy and rapid detection methods are needed. Because OPs have inhibitory effects on the activity of α-naphthyl acetate esterase (ANAE) in plants, in this work we evaluated the possibility of detecting OPs in vegetables with ANAE extracted from commercial flour. The limits of detection (LODs) obtained for methamidophos, dichlorvos, phoxim, dimethoate, and malathion in lettuce samples with crude ANAE were 0.17, 0.11, 0.11, 0.96, and 1.70 mg/kg, respectively. Based on the maximum residue limits (MRLs) for OPs in food stipulated by Chinese laws which are 0.05, 0.20, 0.05, 1.00, and 8.00 mg/kg for methamidophos, dichlorvos, phoxim, dimethoate, and malathion, respectively, the esterase inhibition method with crude ANAE had sufficient sensitivity to detect the residues of dichlorvos, dimethoate, and malathion in lettuce, but it could not be used to guarantee the safety of the same samples if methamidophos or phoxim residue was present. The sensitivity of the method was improved by the use of esterase purified by ammonium sulfate salting-out. The LODs obtained for methamidophos and phoxim with purified esterase were lower than the MRLs for these OPs in food. This is a very promising method for the detection of OP residues in vegetables using crude or purified esterase because of its cheapness, sensitivity, and convenience. PMID:22467368

Enhancement of oral bioavailability of rivastigmine with quercetin nanoparticles by inhibiting CYP3A4 and esterases.

PubMed

Palle, Suresh; Neerati, Prasad

2017-04-01

Quercetin is a well-known flavonoid, has pharmacokinetic interaction with ester drugs due to its capability of esterase inhibition in the gut and liver. However, the interaction between quercetin nanoparticles (NQC) and rivastigmine has not been reported. Hence, the present study was performed to evaluate the effect of quercetin alone and its nanoparticles on the pharmacokinetics of rivastigmine in rats. NQC prepared by antisolvent precipitation method. The influence of quercetin on the pharmacokinetics of rivastigmine was evaluated by following methods i.e. in vitro inhibitory effect on esterase enzyme in rat liver microsomes and in vitro assessment of CYP3A activity using erythromycin-N-demethylase (EMD) assay. To confirm these findings, an in vivo pharmacokinetic study of orally administered rivastigmine in rats with quercetin and NQC pretreatments was performed. The size of NQC was observed below 300nm. Quercetin significantly (p<0.05) inhibited the esterase-mediated metabolism of rivastigmine. In in vitro assessment of CYP3A activity model the erythromycin-N-demethylation (EMD) levels in quercetin treated group were significantly reduced (p<0.05). C max , AUC 0-t and AUC 0- ∞ of rivastigmine were found to be increased in quercetin and NQC pretreated groups. Further, the CL/F and Vd/F of rivastigmine were significantly decreased. The results revealed that enhanced bioavailability of rivastigmine might be caused by the combination of their effects due to CYP3A and esterase inhibition, Therefore, concomitant administration of NQC influences the bioavailability of rivastigmine and also has synergetic effect in the treatment of Alzheimer's disease. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Crystal structure of a triacylglycerol lipase from Penicillium expansum at 1.3 A determined by sulfur SAD

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bian, Chuanbing; Yuan, Cai; Chen, Liqing

2010-04-05

Triacylglycerol lipases (EC 3.1.1.3) are present in many different organisms including animals, plants, and microbes. Lipases catalyze the hydrolysis of long-chain triglycerides into fatty acids and glycerol at the interface between the water insoluble substrate and the aqueous phase. Lipases can also catalyze the reverse esterification reaction to form glycerides under certain conditions. Lipases of microbial origin are of considerable commercial interest for wide variety of biotechnological applications in industries, including detergent, food, cosmetic, pharmaceutical, fine chemicals, and biodiesel. Nowadays, microbial lipases have become one of the most important industrial enzymes. PEL (Penicillium expansum lipase) is a fungal lipase frommore » Penicillium expansum strain PF898 isolated from Chinese soil that has been subjected to several generations of mutagenesis to increase its enzymatic activity. PEL belongs to the triacylglycerol lipases family, and its catalytic characteristics have been studied. The enzyme has been used in Chinese laundry detergent industry for several years (http://www.leveking.com). However, the poor thermal stability of the enzyme limits its application. To further study and improve this enzyme, PEL was cloned and sequenced. Furthermore, it was overexpressed in Pichia pastoris. PEL contains GHSLG sequence, which is the lipase consensus sequence Gly-X1-Ser-X2-Gly, but has a low amino acid sequence identities to other lipases. The most similar lipases are Rhizomucor miehei (PML) and Rhizopus niveus (PNL) with a 21% and 20% sequence identities to PEL, respectively. Interestingly, the similarity of PEL with the known esterases is somewhat higher with 24% sequence identity to feruloyl esterase A. Here, we report the 1.3 {angstrom} resolution crystal structure of PEL determined by sulfur SAD phasing. This structure not only presents a new lipase structure at high resolution, but also provides a structural platform to analyze the published mutagenesis results. The structure may also open up new avenues for future protein engineering study on PEL.« less

Structural Characterization and Function Determination of a Nonspecific Carboxylate Esterase from the Amidohydrolase Superfamily with a Promiscuous Ability To Hydrolyze Methylphosphonate Esters

PubMed Central

2015-01-01

The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (kcat/Km = 1.0 × 105 M–1 s–1), 2-naphthyl propionate (kcat/Km = 1.5 × 105 M–1 s–1), 1-naphthyl acetate (kcat/Km = 7.5 × 103 M–1 s–1), 4-methylumbelliferyl acetate (kcat/Km = 2.7 × 103 M–1 s–1), 4-nitrophenyl acetate (kcat/Km = 2.3 × 105 M–1 s–1), and 4-nitrophenyl butyrate (kcat/Km = 8.8 × 105 M–1 s–1). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (kcat/Km = 3.8 × 105 M–1 s–1) and isobutyl 4-nitrophenyl methylphosphonate (kcat/Km = 1.1 × 104 M–1 s–1). The (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (RP)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations. PMID:24832101

Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity.

PubMed

Robles-Mendoza, Cecilia; Zúñiga-Lagunes, Sebastian R; Ponce de León-Hill, Claudia A; Hernández-Soto, Jesús; Vanegas-Pérez, Cecilia

2011-10-01

The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (≈ 50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the use of organophosphorus pesticides in the natural habitat of the axolotl. Copyright © 2011 Elsevier B.V. All rights reserved.

Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters

PubMed Central

Martínez-Martínez, Mónica; Lores, Iván; Peña-García, Carlina; Bargiela, Rafael; Reyes-Duarte, Dolores; Guazzaroni, María-Eugenia; Peláez, Ana Isabel; Sánchez, Jesús; Ferrer, Manuel

2014-01-01

Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β-hydrolase family. The protein shares low-to-medium identity (≤ 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g−1 protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0–55 000 units g−1 protein), including (±)-menthyl-acetate, (±)-neomenthyl acetate, (±)-pantolactone, (±)-methyl-mandelate, (±)-methyl-lactate and (±)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available. PMID:24418210

Mass Spectrometry to Identify New Biomarkers of Nerve Agent Exposure

DTIC Science & Technology

2008-04-01

of its structural chemistry using p-nitrophenyl esters as substrates. Pharm Res 21:285-292. Salvi A, Carrupt PA, Mayer JM and Testa B (1997) Esterase...Otagiri, M. (2004) Esterase-like activity of serum albumin: characterization of its structural chemistry using p-nitrophenyl esters as substrates. Pharm...The diethylphosphate group was found on Tyr 411 and Tyr 138. Annual report Oksana Lockridge W81XWH-07-2-0034 13 RESULTS The structures of the

Rapid toxicity assessment using an in vivo enzyme test for Brachionus plicatilis (Rotifera).

PubMed

Moffat, B D; Snell, T W

1995-02-01

A 1-hr in vivo enzyme inhibition assay based on esterase activity has good potential for marine toxicity assessment. A test was developed for the rotifer Brachionus plicatilis based on the nonfluorescent substrate fluorescein diacetate (FDA), which is metabolized by esterases to a fluorescent product. Enzyme inhibition, as determined by reduced fluorescence, can be scored visually or quantified using a fluorometer. Quantification of fluorescence permits the calculation of NOEC, LOEC, chronic value, and IC20. The 1-hr esterase inhibition test has sensitivity comparable to that of 24-hr rotifer acute tests for several compounds. The toxicity of six compounds was examined using the quantified assay. The resulting IC20s were within a factor of 3 of the 24-hour LC50s. IC20 values ranged from 0.017 mg/l for tributyltin to 3.1 mg/l for zinc, with an average coefficient of variation of 17.8%. Electrophoretic analysis of rotifer homogenates suggested that a single C esterase (acetylesterase) was responsible for FDA metabolism in B. plicatilis. Several other aquatic species are capable of metabolizing FDA, including Brachionus calyciflorus, Mysidopsis bahia, Menidia beryllina, Pimephales promelas, Ceriodaphnia dubia, Daphnia pulex, Artemia salina, and Ophryotrocha sp. The esterase inhibition test is an attractive tool for assessing aquatic toxicity because of its speed, simplicity, sensitivity, and applicability to a broad range of aquatic species.

Carboxylesterases: General detoxifying enzymes

PubMed Central

Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

2016-01-01

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum.

PubMed

Wu, Mingbo; Peng, Xiaohong; Wen, Hua; Wang, Qin; Chen, Qianming; McKinstry, William J; Ren, Bin

2013-04-01

Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase from Lactobacillus plantarum was cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space group P1, with unit-cell parameters a = 46.5, b = 62.8, c = 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution.

Tritium labeling of a powerful methylphosphonate inhibitor of cholinesterase: synthesis and biological applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balan, A.; Barness, I.; Simon, G.

1988-02-15

7-(Methylethoxy phosphinyloxy)-1-methyl-quinolinium iodide (MEPQ), a powerful anti-cholinesterase methylphosphonate ester, was labeled with tritium (9 Ci/mmol) at the methylphosphonyl moiety (TCH2P(O)(OR)X) by an iodine-tritium replacement reaction. Kinetic measurements of the rate of inhibition of acetylcholinesterase (AChE) by (/sup 3/H)MEPQ and its rate of hydrolysis in alkaline solution confirmed the identity of (/sup 3/H)MEPQ with authentic MEPQ, which was prepared by the same reaction sequences. Gel-filtration experiments verified the radiospecificity of (/sup 3/H)MEPQ. In vitro radiolabeling of both AChE and butyrylcholinesterase along with the whole-body autoradiography of (/sup 3/H)MEPQ-treated mice suggests that (/sup 3/H)MEPQ is a convenient marker for studying biological systemsmore » containing these esterases.« less

Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution.

PubMed

Jiang, Huang; Zhang, Shaowei; Gao, Haofeng; Hu, Nan

2016-01-22

In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein. EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein.

Paraoxonase-1 activities in children and adolescents with type 1 diabetes mellitus.

PubMed

Craciun, Elena C; Leucuta, Daniel C; Rusu, Razvan L; David, Bianca A; Cret, Victoria; Dronca, Eleonora

2016-01-01

Paraoxonase-1 is an HDL-associated esterase that acts as an anti-atherogenic agent by protecting LDL from oxidation. This study investigates paraoxonase-1 activities in children and adolescents with type 1 diabetes mellitus and possible associations with other biochemical markers. The study enrolled 82 children and adolescents with type 1 diabetes mellitus and 41 controls with similar age and gender distribution. Serum paraoxonase-1 arylesterase and salt-stimulated paraoxonase activities were assessed by measuring the rates of phenyl acetate and paraoxon hydrolysis, respectively; paraoxonase-1 lactonase activity and oxidized LDL were assessed by a pH-sensitive colorimetric assay and ELISA, respectively. Glycated haemoglobin HbA1c and lipid profile were assayed with an immunoturbidimetric method and commercially available kits, respectively. We found lower paraoxonase-1 activities in diabetics when compared to controls. The decrease was statistically significant only for the lactonase activity, the difference being higher when referring to the subgroup with poor glycaemic control. The lactonase activity/HDL ratio was also lower in diabetics vs. controls, but without statistical significance. Both lactonase and arylesterase activities were negatively correlated with HbA1c in diabetics, but only the latter was statistically significant (ρ = -0.21, P = 0.055; ρ = -0.24, P = 0.03, respectively). A correlation coefficient of ρ = 0.196 (P = 0.078) was found between oxidized LDL and HbA1c. All paraoxonase-1 activities were lower in diabetic children and adolescents, but only the decrease in the lactonase activity was statistically significant. Although lipid profile and glycaemic control were altered in diabetics, no differences were observed between groups regarding oxidized LDL level.

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

PubMed

Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

2018-05-01

The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

Diagnostic and therapeutic problems associated with hereditary deficiency of the C1 esterase inhibitor.

PubMed

Molina, C; Brun, J; Coulet, M; Betail, G; Wahl, D; Hartmann, L

1977-03-01

Six patients in a family with a history of hereditary angioedema reported swelling of the extremities and recurrent abdominal pain occurring spontaneously or after trauma. Attacks of oedema involving the airways, the greatest danger with this disorder, were present only in one case. This autosomal dominant disease is due to deficient activity of the inhibitor of the first component of complement. Low levels of C4, and absence of C1 esterase inhibitor confirm the diagnosis. Two asymptomatic cases with the appropriate biochemical abnormality are reported in this study. For short term prophylaxis of attacks (before surgery expecially), fresh frozen plasma is used, or better still, C1 esterase inhibitor. For long term prophylaxis of attacks antifibrinolytic and hormonal drugs are used: in two cases, the authors obtained good results with methyltestosterone after failure of tranexamic acid.

The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

PubMed

Vincent, Florence; Yates, David; Garman, Elspeth; Davies, Gideon J; Brannigan, James A

2004-01-23

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism.

Effect of lambda cyhalothrin and temephos on detoxification enzyme systems in Culex quinquefasciatus (Diptera: Culicidae).

PubMed

Muthusamy, R; Shivakumar, M S

2015-01-01

Mosquitoes serve as vector for transmitting diseases. Among mosquitoes, Culex quinquefasciatus transmits lymphatic filariasis, yellow fever Japanese encephalitis etc. Application of chemical insecticides is still the best option for vector control programmes. Continuous use of these chemicals on mosquito reduces its effects. The present study determined the baseline susceptibility of Cx. quinquefasciatus in response to λ-cyhalothrin and temephos treatments. In addition, the biochemical mechanisms and zymogram analysis involved in insecticide detoxification among larval mosquitoes were studied. The larval bioassay indicated high LC50 value for λ-cyhalothrin (0.1484ppm) as compared to temephos (0.01092ppm). While AChE assay showed increased activity in temephos treatments, glutathione reductase (GR) and esterase levels were increased at both the treatments. Esterase quantitative analysis revealed the expression of three bands at 43kDa, 67kDa and 245kDa. The findings suggest that insensitivity of AChE, esterase and high GR activity may play an important role in developing resistance to synthetic pyrethroid and organophosphate insecticides in Cx. quinquefasciatus population.

Geranyl acetate esterase controls and regulates the level of geraniol in lemongrass (Cymbopogon flexuosus Nees ex Steud.) mutant cv. GRL-1 leaves.

PubMed

Ganjewala, Deepak; Luthra, Rajesh

2009-01-01

Essential oil isolated from lemongrass (Cymbopogon flexuosus) mutant cv. GRL-1 leaves is mainly composed of geraniol (G) and geranyl acetate (GA). The proportion of G and GA markedly fluctuates during leaf development. The proportions of GA and G in the essential oil recorded at day 10 after leaf emergence were approximately 59% and approximately 33% respectively. However, the level of GA went down from approximately 59 to approximately 3% whereas the level of G rose from approximately 33 to approximately 91% during the leaf growth period from day 10 to day 50. However, the decline in the level of GA was most pronounced in the early (day 10 to day 30) stage of leaf growth. The trend of changes in the proportion of GA and G has clearly indicated the role of an esterase that must be involved in the conversion of GA to G during leaf development. We isolated an esterase from leaves of different ages that converts GA into G and has been given the name geranyl acetate esterase (GAE). The GAE activity markedly varied during the leaf development cycle; it was closely correlated with the monoterpene (GA and G) composition throughout leaf development. GAE appeared as several isoenzymes but only three (GAE-I, GAE-II, and GAE-III) of them had significant GA cleaving activity. The GAE isoenzymes pattern was greatly influenced by the leaf developmental stages and so their GA cleaving activities. Like the GAE activity, GAE isoenzyme patterns were also found to be consistent with the monoterpene (GA and G) composition. GAE had an optimum pH at 8.5 and temperature at 30 degrees C. Besides GAE, a compound with phosphatase activity capable of hydrolyzing geranyl diphosphate (GPP) to produce geraniol has also been isolated.

Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney.

PubMed

Lamego, Joana; Ferreira, Pedro; Alves, Márcia; Matias, Ana; Simplício, Ana Luisa

2015-08-01

Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time. For evaluation of both methods when distinguishing activity of different carboxylesterases, HEK-293T transfected with the human carboxylestarase-2 (hCES2) were also tested. Application to Caco-2 or HT-29 cells demonstrated higher activity detected in cell lysates than in cell monolayers. The difference is most striking when comparing the methods at different stages of Caco-2 and HT-29 cell maturation, highlighting substrate accessibility as a limiting step in the in vivo hydrolysis rates (possibly limited by plasma and Endoplasmic Reticulum membrane permeability) with increasing relevance as the cells differentiate. Application to Hep G2 or to hCES2 transfected and non-transfected HEK-293T cells, demonstrated a tendency for higher sensitivity in living cell suspensions than that obtained with the cell lysates which indicates the importance of cell environment in the maintenance of enzyme activity. However, quantification of hCES2 activity relative to total esterase, or to total carboxylesterase activity, was not significantly different in any case. The results herein presented help to clarify which method is best suited for evaluation of carboxylesterase activity in vitro depending on the final goal of the study. Copyright © 2015 Elsevier Ltd. All rights reserved.

Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

PubMed

Winiarczyk, Krystyna; Gębura, Joanna

2016-05-01

The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1

PubMed Central

Song, Jinlong; Shi, Yanhua; Li, Kang; Zhao, Bin; Yan, Yanchun

2013-01-01

A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L−1 and 56.33 nmol min−1, respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments. PMID:24155944

Regulation of the Feruloyl Esterase (faeA) Gene from Aspergillus niger

PubMed Central

de Vries, Ronald P.; Visser, Jaap

1999-01-01

Feruloyl esterases can remove aromatic residues (e.g., ferulic acid) from plant cell wall polysaccharides (xylan, pectin) and are essential for complete degradation of these polysaccharides. Expression of the feruloyl esterase-encoding gene (faeA) from Aspergillus niger depends on d-xylose (expression is mediated by XlnR, the xylanolytic transcriptional activator) and on a second system that responds to aromatic compounds with a defined ring structure, such as ferulic acid and vanillic acid. Several compounds were tested, and all of the inducing compounds contained a benzene ring which had a methoxy group at C-3 and a hydroxy group at C-4 but was not substituted at C-5. Various aliphatic groups occurred at C-1. faeA expression in the presence of xylose or ferulic acid was repressed by glucose. faeA expression in the presence of ferulic acid and xylose was greater than faeA expression in the presence of either compound alone. The various inducing systems allow A. niger to produce feruloyl esterase not only during growth on xylan but also during growth on other ferulic acid-containing cell wall polysaccharides, such as pectin. PMID:10584009

Mechanisms of insecticide resistance in field populations of Aedes aegypti (L.) from Quintana Roo, Southern Mexico.

PubMed

Flores, Adriana E; Grajales, Jaime Salomon; Salas, Ildefonso Fernandez; Garcia, Gustavo Ponce; Becerra, Ma Haydee Loaiza; Lozano, Saul; Brogdon, William G; Black, William C; Beaty, Barry

2006-12-01

Potential insecticide-resistance mechanisms were studied with the use of biochemical assays in Aedes aegypti (L.) collected from 5 municipalities representing the north part of Quintana Roo: Benito Juarez, Cozumel, Isla Mujeres, Lazaro Cardenas, and Solidaridad. The activities of alpha and beta esterases, mixed-function oxidases (MFO), glutathione-S-transferase (GST), acethylcholinesterase (AChE), and insensitive acethylcholinesterase (iAChE) were assayed in microplates. Three replicates were performed for each enzyme and 60 males and 60 females were analyzed in each population. The New Orleans (NO) susceptible strain of Ae. aegypti was used as a susceptible reference and the threshold criteria for each enzyme were the highest NO absorbance values. In none of the 6 tests were absorbance values correlated in males and females. alpha esterases were elevated in Benito Juarez, Cozumel females and in Lazaro Cardenas males and females. beta esterases were elevated in Benito Juarez, Cozumel females and in Cozumel and Lazaro Cardenas males. Elevated esterases suggest potential insecticide-resistance mechanisms against organophosphate, carbamate, and some pyrethroid insecticides. Slightly elevated levels of MFOs appeared in Lazaro Cardenas females and in Cozumel, Isla Mujeres, and Solidaridad males. Mechanisms involving iAChE or GST were not apparent.

Effects of lateral fluid percussion injury on cholinergic markers in the newborn piglet brain.

PubMed

Donat, Cornelius K; Walter, Bernd; Kayser, Tanja; Deuther-Conrad, Winnie; Schliebs, Reinhard; Nieber, Karen; Bauer, Reinhard; Härtig, Wolfgang; Brust, Peter

2010-02-01

Traumatic brain injury is a leading cause of death and disability in children. Studies using adult animal models showed alterations of the central cholinergic neurotransmission as a result of trauma. However, there is a lack of knowledge about consequences of brain trauma on cholinergic function in the immature brain. It is hypothesized that trauma affects the relative acetylcholine esterase activity and causes a loss of cholinergic neurons in the immature brain. Severe fluid percussion trauma (FP-TBI, 3.8+/-0.3atm) was induced in 15 female newborn piglets, monitored for 6h and compared with 12 control animals. The hemispheres ipsilateral to FP-TBI obtained from seven piglets were used for acetylcholine esterase histochemistry on frozen sagittal slices, while regional cerebral blood flow and oxygen availability was determined in the remaining eight FP-TBI animals. Post-fixed slices were immunohistochemically labelled for choline acetyltransferase as well as for low-affinity neurotrophin receptor in order to characterize cholinergic neurons in the basal forebrain. Regional cerebral blood flow and brain oxygen availability were reduced during the first 2h after FP-TBI (P<0.05). In addition, acetylcholine esterase activity was significantly increased in the neocortex, basal forebrain, hypothalamus and medulla after trauma (P<0.05), whereas the number of choline acetyltransferase and low-affinity neurotrophin receptor positive cells in the basal forebrain were unaffected by the injury. Thus, traumatic brain injury evoked an increased relative activity of the acetylcholine esterase in the immature brain early after injury, without loss of cholinergic neurons in the basal forebrain. These changes may contribute to developmental impairments after immature traumatic brain injury. Copyright 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Functional characterization of salt-tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)-3-hydroxybutyrate.

PubMed

Wang, Yilong; Xu, Yongkai; Zhang, Yun; Sun, Aijun; Hu, Yunfeng

2018-06-01

The two enantiomers of ethyl 3-hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)-3-hydroxybutyrate. Herein, we also functionally characterized one novel salt-tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)-3-hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio-selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates. © 2018 Wiley Periodicals, Inc.

Optimization of hydrolysis conditions for bovine plasma protein using response surface methodology.

PubMed

Seo, Hyun-Woo; Jung, Eun-Young; Go, Gwang-Woong; Kim, Gap-Don; Joo, Seon-Tea; Yang, Han-Sul

2015-10-15

The purpose of this study was to establish optimal conditions for the hydrolysis of bovine plasma protein. Response surface methodology was used to model and optimize responses [degree of hydrolysis (DH), 2,2-diphenyl-1-picrydrazyl (DPPH) radical-scavenging activity and Fe(2+)-chelating activity]. Hydrolysis conditions, such as hydrolysis temperature (46.6-63.4 °C), hydrolysis time (98-502 min), and hydrolysis pH (6.32-9.68) were selected as the main processing conditions in the hydrolysis of bovine plasma protein. Optimal conditions for maximum DH (%), DPPH radical-scavenging activity (%) and Fe(2+)-chelating activity (%) of the hydrolyzed bovine plasma protein, were respectively established. We discovered the following three conditions for optimal hydrolysis of bovine plasma: pH of 7.82-8.32, temperature of 54.1 °C, and time of 338.4-398.4 min. We consequently succeeded in hydrolyzing bovine plasma protein under these conditions and confirmed the various desirable properties of optimal hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Variability of esterase patterns in adult flies of the saltans species group of Drosophila (subgenus Sophophora).

PubMed

Bernardo, Alessandra Augusta; Bicudo, Hermione Elly Melara de Campos

2009-09-01

Esterases are known for their involvement in several physiological processes and high degree of polymorphism, in many organisms. Such polymorphism has been used to characterize species and species groups and to study genetic changes occurred in their evolutionary history. In the present study, the esterase patterns of 19 strains from 10 species representative of the five subgroups of the saltans species group were analyzed using polyacrylamide gel electrophoresis and alpha- and beta- naphthyl acetates as substrates. Fifty-one esterase bands were detected and classified as 31 alpha-esterases, 18 beta-esterases and two alpha/beta-esterases. On the basis of the inhibition patterns using Malathion and eserine sulfate, 34 bands were classified as carboxylesterases, 14 as acethylesterases and three as cholinesterases. Ten gene loci were tentatively established on the basis of data on band position in the gel, substrate preference and inhibition pattern. Twenty bands were species-specific, the remaining being shared by species from the same or different subgroups. Bands detected exclusively in males and bands with a different frequency or degree of expression between sexes were also detected. In the gels prepared for analysis of gene expression in the body parts (head, thorax and abdomen), the degree of expression of the beta-esterases was higher in the thorax, while the alpha-esterases were expressed predominantly in the abdomen and thorax. A global view of the data available at present on the esterases of the species from the saltans group and their degree of polymorphism are presented, as well as the possibility of using some beta-esterases, because of their characteristics in the gels, as markers for species identification.

Increased excitability and metabolism in pilocarpine induced epileptic rats: effect of Bacopa monnieri.

PubMed

Mathew, Jobin; Paul, Jes; Nandhu, M S; Paulose, C S

2010-09-01

We have evaluated the acetylcholine esterase and malate dehydrogenase activity in the muscle, epinephrine, norepinephrine, insulin and T3 content in the serum of epileptic rats. Acetylcholine esterase and malate dehydrogenase activity increased in the muscle and decreased in the heart of the epileptic rats compared to control. Insulin and T3 content were increased significantly in the serum of the epileptic rats. Our results suggest that repetitive seizures resulted in increased metabolism and excitability in epileptic rats. Bacopa monnieri and Bacoside-A treatment prevents the occurrence of seizures there by reducing the impairment on peripheral nervous system. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Paraoxonase lactonase activity (PON-HTLase), asymmetric dimethylarginine (ADMA) and platelet activating factor-acetylhydrolase (PAF-AH) activity in non-obese women with PCOS.

PubMed

Bayrak, Tulin; Dursun, Polat; Bayrak, Ahmet; Gültekin, Murat; Kolusarı, Ali; Cakır, Erdinç; Ozyurt, Merve; Zeyneloğlu, Hulusi B

2012-11-01

Paraoxonase1 (PON1), exhibits both esterase activity (PON1-AREase) and homocysteine thiolactonase activity (PON1-HTLase) which respectively prevent LDL oxidation and detoxify homocysteine thiolactone (HTL). Platelet-activating factor-acetylhydrolase (PAF-AH) is an antioxidant enzyme preventing LDL oxidation by hydrolysis of oxidized phospholipids. Both of these enzymes exhibit a proatherogenic role. ADMA is an endogenous inhibitor of nitric oxide (NO) synthesis causing endothelial dysfunction. The aim was to compare non-obese PCOS patients with a BMI matched control group using the following characteristics: serum PON1-HTLase, ADMA, PAF-AH, and lipid and hormonal parameters. 77 women with PCOS and 25 healthy subject were recruited for this study, The controls were non-obese BMI and age matched with the patients. There were no significant differences with respect to age, BMI, FSH, free testosterone, DHEA, androstenadion, total cholesterol, triglycerides, HDL, LDL, VLDL, fasting glucose/insulin ratio and HOMA-IR among the groups (p > 0.05). However, total testosterone and fasting glucose levels were significantly higher in the PCOS group (p < 0.05). On the other hand, PON1-HTLase levels (39.6 ± 5.77 vs. 33.8 ± 8.2, p = 0.02) were significantly lower in the PCOS group while ADMA levels (1.14 ± 0.6 vs. 3.37 ± 6.4, p = 0.004) were significantly higher in the PCOS group. However, there was no significant difference in PAF-AH activity among the groups. Decreased PON1-HTLase and increased ADMA levels might be a relevant marker for the development of future atherosclerotic heart disease (AHD) in non-obese PCOS patients. Further studies are needed to confirm our results.

The activity of hydrolases of larval stages of Anisakis simplex (Nematoda).

PubMed

Lopieńska-Biernat, Elzbieta; Zółtowska, Krystyna; Rokicki, Jerzy

2004-01-01

Activity of hydrolases during the third and fourth larval stage of Anisakis simplex was identified by applying the API ZYM test method. In A. simplex larvae the activity of phosphatases was high, particularly that of acid phosphatase (40 nmol/mg(-1)). Among esterases lack of activity of lipase (C14) is worth noticing while the activity of esterases (C4) and (C8) was high. The activity of those later two enzymes was higher in L3 larvae than in L4 larvae. The highest activity in the subclass of glucosidases was recorded for beta-fucosidase and N-acetyl-beta-glucosaminidase. A higher activity in L3 larvae than in L4 larvae was recorded for: beta-glucuronidase and N-acetyl-beta-glucosaminidase (2-fold) and beta-fucosidase (3-fold). Differently the activity of beta-galactosidase and beta-glucosidase was higher in L4 larvae than in L3 larvae. The tests did not show activity of alpha-galactosidase, beta-glucosidase and alpha-mannosidase on both larval forms.

Degradation of Phthalate Esters by Fusarium sp. DMT-5-3 and Trichosporon sp. DMI-5-1 Isolated from Mangrove Sediments.

PubMed

Luo, Zhu-Hua; Pang, Ka-Lai; Wu, Yi-Rui; Gu, Ji-Dong; Chow, Raymond K K; Vrijmoed, L L P

2012-01-01

Phthalate esters (PAEs) are important industrial compounds mainly used as plasticizers to increase flexibility and softness of plastic products. PAEs are of major concern because of their widespread use, ubiquity in the environment, and endocrine-disrupting toxicity. In this study, two fungal strains, Fusarium sp. DMT-5-3 and Trichosporon sp. DMI-5-1 which had the capability to degrade dimethyl phthalate esters (DMPEs), were isolated from mangrove sediments in the Futian Nature Reserve of Shenzhen, China, by enrichment culture technique. These fungi were identified on the basis of spore morphology and molecular typing using 18S rDNA sequence. Comparative investigations on the biodegradation of three isomers of DMPEs, namely dimethyl phthalate (DMP), dimethyl isophthalate (DMI), and dimethyl terephthalate (DMT), were carried out with these two fungi. It was found that both fungi could not completely mineralize DMPEs but transform them to the respective monomethyl phthalate or phthalate acid. Biochemical degradation pathways for different DMPE isomers by both fungi were different. Both fungi could transform DMT to monomethyl terephthalate (MMT) and further to terephthalic acid (TA) by stepwise hydrolysis of two ester bonds. However, they could only carry out one-step ester hydrolysis to transform DMI to monomethyl isophthalate (MMI). Further metabolism of MMI did not proceed. Only Trichosporon sp. was able to transform DMP to monomethyl phthalate (MMP) but not Fusarium sp. The optimal pH for DMI and DMT degradation by Fusarium sp. was 6.0 and 4.5, respectively, whereas for Trichosporon sp., the optimal pH for the degradation of all the three DMPE isomers was at 6.0. These results suggest that the fungal esterases responsible for hydrolysis of the two ester bonds of PAEs are highly substrate specific.

Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

PubMed

De Santi, Concetta; Willassen, Nils Peder; Williamson, Adele

2016-01-01

The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

Aroma Precursors in Grapes and Wine: Flavor Release during Wine Production and Consumption.

PubMed

Parker, Mango; Capone, Dimitra L; Francis, I Leigh; Herderich, Markus J

2018-03-14

Pioneering investigations into precursors of fruity and floral flavors established the importance of terpenoid and C 13 -norisoprenoid glycosides to the flavor of aromatic wines. Nowadays flavor precursors in grapes and wine are known to be structurally diverse, encompassing glycosides, amino acid conjugates, odorless volatiles, hydroxycinnamic acids, and many others. Flavor precursors mainly originate in the grape berry but also from oak or other materials involved in winemaking. Flavors are released from precursors during crushing and subsequent production steps by enzymatic and nonenzymatic transformations, via microbial glycosidases, esterases, C-S lyases, and decarboxylases, and through acid-catalyzed hydrolysis and chemical rearrangements. Flavors can also be liberated from glycosides and amino acid conjugates by oral microbiota. Hence, it is increasingly likely that flavor precursors contribute to retronasal aroma formation through in-mouth release during consumption, prompting a shift in focus from identifying aroma precursors in grapes to understanding aroma precursors present in bottled wine.

Short peptides self-assemble to produce catalytic amyloids

NASA Astrophysics Data System (ADS)

Rufo, Caroline M.; Moroz, Yurii S.; Moroz, Olesia V.; Stöhr, Jan; Smith, Tyler A.; Hu, Xiaozhen; Degrado, William F.; Korendovych, Ivan V.

2014-04-01

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement to adopt a stable, folded conformation is likely to contribute to their relatively large size (>10,000 Da). However, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. To test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of seven-residue peptides that act as Zn2+-dependent esterases. Zn2+ helps stabilize the fibril formation, while also acting as a cofactor to catalyse acyl ester hydrolysis. These results indicate that prion-like fibrils are able to not only catalyse their own formation, but they can also catalyse chemical reactions. Thus, they might have served as intermediates in the evolution of modern-day enzymes. These results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and non-biological metal ions.

Mediating chemical reactions using polysaccharides

NASA Astrophysics Data System (ADS)

Tyler, Lauren E.

We have studied the NaBH4-mediated hydrogenation of select alkenes catalyzed by polysaccharide-stabilized nanoparticles. We compared the catalytic properties of Ni-based nanoparticles or Au/Co-based nanoparticles on the hydrogenation of cinnamic acid, cinnamide, cinnamyl alcohol, and ethyl cinnamate. We evaluated the possibility that the type of stabilizing polysaccharide surrounding the nanoparticle may affect the selectivity towards the alkene compounds that undergo the hydrogenation reaction. We found that the hydrogenation of cinnamide or ethyl cinnamate proceeded readily to 100% completion independent of the type of polysaccharide stabilizing the nanoparticle. However, the extent of the hydrogenation of cinnamyl alcohol and cinnamic acid varied greatly depending on the type of polysaccharide stabilizing the nanoparticle. In the course of these studies, we observed that some polysaccharides by themselves promoted the hydrolysis of ethyl cinnamate. Thus, we have raised the hypothesis that some polysaccharides may act as "esterases" and explored the interaction between select polysaccharides and a variety of ester compounds.

Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum

PubMed Central

Wu, Mingbo; Peng, Xiaohong; Wen, Hua; Wang, Qin; Chen, Qianming; McKinstry, William J.; Ren, Bin

2013-01-01

Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase from Lactobacillus plantarum was cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space group P1, with unit-cell paramters a = 46.5, b = 62.8, c = 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution. PMID:23545659

The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution.

PubMed

Montenegro, M F; Moral-Naranjo, M T; Páez de la Cadena, M; Campoy, F J; Muñoz-Delgado, E; Vidal, C J

2008-09-25

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.

Structure and function of the digestive system of solen grandis dunker

NASA Astrophysics Data System (ADS)

Sheng, Xiuzhen; Zhan, Wenbin; Ren, Sulian

2003-10-01

Structure and function of the digestive system of a bivalve mollusc, Solen grandis, were studied using light microscopy and histochemical methods. The wall of digestive tube consists of four layers: the mucosal epithelium, connective tissue, muscular and fibrosa or serosa (only in the portion of rectum) from the inner to the outer. The ciliated columnar epithelial cells, dispersed by cup-shaped mucous cells, rest on a thin base membrane. There are abundant blood spaces in connective tissue layer. The digestive diverticula are composed of multi-branched duct and digestive tubules. The digestive tubules are lined with digestive and basophilic secretory cells, and surrounded by a layer of smooth muscle fibers and connective tissues. Activities of acid and alkaline phosphatases, esterase and lipase are detected in the digestive cells, and the epithelia of stomach and intestine, suggesting that these cells are capable of intracellular digesting of food materials and absorbing. Besides, acid phosphatase and esterase activities are present in the posterior portion of esophagus. Phagocytes are abundant in blood spaces and the lumens of stomach and intestine, containing brown granules derived from the engulfed food materials. The present work indicates that phagocytes play important roles in ingestion and digestion of food materials, which is supported as well by the activities of acid phosphatase, esterase and lipase detected in blood spaces.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

PubMed

Varriale, Simona; Cerullo, Gabriella; Antonopoulou, Io; Christakopoulos, Paul; Rova, Ulrika; Tron, Thierry; Fauré, Régis; Jütten, Peter; Piechot, Alexander; Brás, Joana L A; Fontes, Carlos M G A; Faraco, Vincenza

2018-06-01

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Control of Tetranychus urticae Koch by extracts of three essential oils of chamomile, marjoram and Eucalyptus.

PubMed

Abd El-Moneim, M R Afify; Fatma, S Ali; Turky, A F

2012-01-01

To evaluate the acaricidal activity of extracts of three essential oils of chamomile, marjoram and Eucalyptus against Tetranychus urticae (T. urticae) Koch. Extracts of three essential oils of chamomile, marjoram and Eucalyptus with different concentrations (0.5%, 1.0%, 2.0%, 3.0% and 4.0%) were used to control T. urticae Koch. The results showed that chamomile (Chamomilla recutita) represented the most potent efficient acaricidal agent against Tetranychus followed by marjoram (Marjorana hortensis) and Eucalyptus. The LC50 values of chamomile, marjoram and Eucalyptus for adults were 0.65, 1.84 and 2.18, respectively and for eggs 1.17, 6.26 and 7.33, respectively. Activities of enzymes including glutathione-S-transferase, esterase (α-esterase and β-esterase) and alkaline phosphatase in susceptible mites were determined and activities of enzymes involved in the resistance of acaricides were proved. Protease enzyme was significantly decreased at LC50 of both chamomile and marjoram compared with positive control. Gas chromatography-mass spectrometer (GC-MS) proved that the major compositions of Chamomilla recutita are α-bisabolol oxide A (35.251%), and trans-β-farersene (7.758%), while the main components of Marjorana hortensis are terpinene-4-ol (23.860%), p-cymene (23.404%) and sabinene (10.904%). It can be concluded that extracts of three essential oils of chamomile, marjoram and Eucalyptus possess acaricidal activity against T. urticae.

Isolation and Characterization of EstC, a New Cold-Active Esterase from Streptomyces coelicolor A3(2)

PubMed Central

Brault, Guillaume; Shareck, François; Hurtubise, Yves; Lépine, François; Doucet, Nicolas

2012-01-01

The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5–9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6–11. The enzyme was active toward short-chain p-nitrophenyl esters (C2–C12), displaying optimal activity with the valerate (C5) ester (k cat/K m = 737±77 s−1 mM−1). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors. PMID:22396747

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

DOE PAGES

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; ...

2017-03-08

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Organophosphate acetylcholine esterase inhibitor poisoning from a home-made shampoo.

PubMed

Sadaka, Yair; Broides, Arnon; Tzion, Raffi Lev; Lifshitz, Matitiahu

2011-07-01

Organophosphate acetylcholine esterase inhibitor poisoning is a major health problem in children. We report an unusual cause of organophosphate acetylcholine esterase inhibitor poisoning. Two children were admitted to the pediatric intensive care unit due to organophosphate acetylcholine esterase inhibitor poisoning after exposure from a home-made shampoo that was used for the treatment of head lice. Owing to no obvious source of poisoning, the diagnosis of organophosphate acetylcholine esterase inhibitor poisoning in one of these patients was delayed. Both patients had an uneventful recovery. Organophosphate acetylcholine esterase inhibitor poisoning from home-made shampoo is possible. In cases where the mode of poisoning is unclear, direct questioning about the use of home-made shampoo is warranted, in these cases the skin and particularly the scalp should be rinsed thoroughly as soon as possible.

Limitations in Bonding to Dentin and Experimental Strategies to Prevent Bond Degradation

PubMed Central

Liu, Y.; Tjäderhane, L.; Breschi, L.; Mazzoni, A.; Li, N.; Mao, J.; Pashley, D.H.; Tay, F.R.

2011-01-01

The limited durability of resin-dentin bonds severely compromises the lifetime of tooth-colored restorations. Bond degradation occurs via hydrolysis of suboptimally polymerized hydrophilic resin components and degradation of water-rich, resin-sparse collagen matrices by matrix metalloproteinases (MMPs) and cysteine cathepsins. This review examined data generated over the past three years on five experimental strategies developed by different research groups for extending the longevity of resin-dentin bonds. They include: (1) increasing the degree of conversion and esterase resistance of hydrophilic adhesives; (2) the use of broad-spectrum inhibitors of collagenolytic enzymes, including novel inhibitor functional groups grafted to methacrylate resins monomers to produce anti-MMP adhesives; (3) the use of cross-linking agents for silencing the activities of MMP and cathepsins that irreversibly alter the 3-D structures of their catalytic/allosteric domains; (4) ethanol wet-bonding with hydrophobic resins to completely replace water from the extrafibrillar and intrafibrillar collagen compartments and immobilize the collagenolytic enzymes; and (5) biomimetic remineralization of the water-filled collagen matrix using analogs of matrix proteins to progressively replace water with intrafibrillar and extrafibrillar apatites to exclude exogenous collagenolytic enzymes and fossilize endogenous collagenolytic enzymes. A combination of several of these strategies should result in overcoming the critical barriers to progress currently encountered in dentin bonding. PMID:21220360

A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake

PubMed Central

Wang, Guozeng; Wang, Qiaohuang; Lin, Xianju; Bun Ng, Tzi; Yan, Renxiang; Lin, Juan; Ye, Xiuyun

2016-01-01

A novel esterase gene (estSL3) was cloned from the Alkalibacterium sp. SL3, which was isolated from the sediment of soda lake Dabusu. The 636-bp full-length gene encodes a polypeptide of 211 amino acid residues that is closely related with putative GDSL family lipases from Alkalibacterium and Enterococcus. The gene was successfully expressed in E. coli, and the recombinant protein (rEstSL3) was purified to electrophoretic homogeneity and characterized. rEstSL3 exhibited the highest activity towards pNP-acetate and had no activity towards pNP-esters with acyl chains longer than C8. The enzyme was highly cold-adapted, showing an apparent temperature optimum of 30 °C and remaining approximately 70% of the activity at 0 °C. It was active and stable over the pH range from 7 to 10, and highly salt-tolerant up to 5 M NaCl. Moreover, rEstSL3 was strongly resistant to most tested metal ions, chemical reagents, detergents and organic solvents. Amino acid composition analysis indicated that EstSL3 had fewer proline residues, hydrogen bonds and salt bridges than mesophilic and thermophilic counterparts, but more acidic amino acids and less hydrophobic amino acids when compared with other salt-tolerant esterases. The cold active, salt-tolerant and chemical-resistant properties make it a promising enzyme for basic research and industrial applications. PMID:26915906

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

PubMed Central

Gottschalk, Leda Maria Fortes; de Sousa Paredes, Raquel; Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant’Ana; da Silva Bon, Elba Pinto

2013-01-01

The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture. PMID:24294256

Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties.

PubMed

Orfila, Caroline; Dal Degan, Florence; Jørgensen, Bodil; Scheller, Henrik Vibe; Ray, Peter M; Ulvskov, Peter

2012-07-01

A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.

Rhanterium epapposum Oliv. essential oil: Chemical composition and antimicrobial,insect-repellent and anticholinesterase activities

USDA-ARS?s Scientific Manuscript database

Essential oils from Rhanterium epapposum Oliv. (Asteraceae) was investigated for its repellent, antimicrobial and acetyl- and butyrylcholine esterase inhibitory activities. The oil showed good repellent activity while oils demonstrated weak in antimicrobial and cholinesterase inhibitions. Terpenoids...

Use of enzyme inhibitors to evaluate the conversion pathways of ester and amide prodrugs: a case study example with the prodrug ceftobiprole medocaril.

PubMed

Eichenbaum, Gary; Skibbe, Jennifer; Parkinson, Andrew; Johnson, Mark D; Baumgardner, Dawn; Ogilvie, Brian; Usuki, Etsuko; Tonelli, Fred; Holsapple, Jeff; Schmitt-Hoffmann, Anne

2012-03-01

An approach was developed that uses enzyme inhibitors to support the assessment of the pathways that are responsible for the conversion of intravenously administered ester and amide prodrugs in different biological matrices. The methodology was applied to ceftobiprole medocaril (BAL5788), the prodrug of the cephalosporin antibiotic, ceftobiprole. The prodrug was incubated in plasma, postmitochondrial supernatant fractions from human liver (impaired and nonimpaired), kidney, and intestine as well as erythrocytes, in the presence and absence of different enzyme inhibitors (acetylcholinesterase, pseudocholinesterase, retinyl palmitoyl hydrolase, serine esterases, amidases, and cholinesterase). Hydrolysis was rapid, extensive, and not dependent on the presence of β-nicotinamide-adenine dinucleotide phosphate (reduced form) in all matrices tested, suggesting the involvement of carboxylesterases but not P450 enzymes. Hydrolysis in healthy human plasma was rapid and complete and only partially inhibited in the presence of paraoxonase inhibitors or in liver from hepatic impaired patients, suggesting involvement of nonparaoxonase pathways. The results demonstrate the utility of this approach in confirming the presence of multiple conversion pathways of intravenously administered prodrugs and in the case of BAL5788 demonstrated that this prodrug is unlikely to be affected by genetic polymorphisms, drug interactions, or other environmental factors that might inhibit or induce the enzymes involved in its conversion. Copyright © 2011 Wiley Periodicals, Inc.

Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75.

PubMed

Shah, Ziaullah; Krumholz, Lee; Aktas, Deniz Fulya; Hasan, Fariha; Khattak, Mutiullah; Shah, Aamer Ali

2013-11-01

A polyurethane (PU) degrading bacterial strain MZA-75 was isolated from soil through enrichment technique. The bacterium was identified through 16S rRNA gene sequencing, the phylogenetic analysis indicated the strain MZA-75 belonged to genus Bacillus having maximum similarity with Bacillus subtilis strain JBE0016. The degradation of PU films by strain MZA-75 in mineral salt medium (MSM) was analyzed by scanning electron microscopy (SEM), fourier transform infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM revealed the appearance of widespread cracks on the surface. FTIR spectrum showed decrease in ester functional group. Increase in polydispersity index was observed in GPC, which indicates chain scission as a result of microbial treatment. CO2 evolution and cell growth increased when PU was used as carbon source in MSM in Sturm test. Increase in both cell associated and extracellular esterases was observed in the presence of PU indicated by p-Nitrophenyl acetate (pNPA) hydrolysis assay. Analysis of cell free supernatant by gas chromatography-mass spectrometry (GC-MS) revealed that 1,4-butanediol and adipic acid monomers were produced. Bacillus subtilis strain MZA-75 can degrade the soft segment of polyester polyurethane, unfortunately no information about the fate of hard segment could be obtained. Growth of strain MZA-75 in the presence of these metabolites indicated mineralization of ester hydrolysis products into CO2 and H2O.

Impact of ester promoieties on transdermal delivery of ketorolac.

PubMed

Liu, Kuo-Sheng; Hsieh, Pei-Wen; Aljuffali, Ibrahim A; Lin, Yin-Ku; Chang, Shu-Hao; Wang, Jhi-Joung; Fang, Jia-You

2014-03-01

Different types of ketorolac ester prodrugs incorporating tert-butyl (KT), benzyl (KB), heptyl (KH), and diketorolac heptyl (DKH) promoieties were synthesized for the comparison of percutaneous penetration. The prodrugs were characterized according to their melting point, capacity factor, lipophilicity, solubility in 30% ethanol/buffer, enzymatic hydrolysis, in vitro skin permeation, hair follicle accumulation, and in vivo skin tolerance. Interactions between the prodrugs and esterases were predicted by molecular docking. Both equimolar suspensions and saturated solutions in 30% ethanol/pH 7.4 buffer were employed as the applied dose. All of the prodrugs exhibited a lower melting point than ketorolac. The lipophilicity increased in the following order: ketorolac < KT < KB < KH < DKH. The prodrugs were rapidly hydrolyzed to the parent drug in esterase medium, skin homogenate, and plasma, with KT and KB exhibiting higher degradation rates. KT exhibited the highest skin permeation, followed by KB. The flux of KT and KB exceeded that of ketorolac by 2.5-fold and twofold, respectively. KH and DKH did not improve ketorolac permeation but exhibited a sustained release behavior. KT and KH revealed selective absorption into follicles and a threefold greater follicular uptake compared with ketorolac. KB, KH, and DKH slightly but significantly increased transepidermal water loss (TEWL) after consecutive administration for 7 days, whereas ketorolac and KT exhibited no influence on TEWL. According to the experimental results, it can be concluded that an optimal balance between lipophilicity and aqueous solubility is important in the design of a successful prodrug. The acceptable skin tolerance for safe application is also an important consideration. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Effect of temperature and high pressure on the activity and mode of action of fungal pectin methyl esterase.

PubMed

Duvetter, Thomas; Fraeye, Ilse; Sila, Daniel N; Verlent, Isabel; Smout, Chantal; Clynen, Elke; Schoofs, Liliane; Schols, Henk; Hendrickx, Marc; Van Loey, Ann

2006-01-01

Pectin was de-esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal-isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure were found to stimulate PME activity. The highest rate of PME-catalyzed pectin de-esterification was obtained when combining pressures in the range 200-300 MPa with temperatures in the range 50-55 degrees C. The mode of pectin de-esterification was investigated by characterizing the pectin reaction products by enzymatic fingerprinting. No significant effect of increasing pressure (300 MPa) and/or temperature (50 degrees C) on the mode of pectin conversion was detected.

Organophosphate acetylcholine esterase inhibitor poisoning from a home-made shampoo

PubMed Central

Sadaka, Yair; Broides, Arnon; Tzion, Raffi Lev; Lifshitz, Matitiahu

2011-01-01

Organophosphate acetylcholine esterase inhibitor poisoning is a major health problem in children. We report an unusual cause of organophosphate acetylcholine esterase inhibitor poisoning. Two children were admitted to the pediatric intensive care unit due to organophosphate acetylcholine esterase inhibitor poisoning after exposure from a home-made shampoo that was used for the treatment of head lice. Owing to no obvious source of poisoning, the diagnosis of organophosphate acetylcholine esterase inhibitor poisoning in one of these patients was delayed. Both patients had an uneventful recovery. Organophosphate acetylcholine esterase inhibitor poisoning from home-made shampoo is possible. In cases where the mode of poisoning is unclear, direct questioning about the use of home-made shampoo is warranted, in these cases the skin and particularly the scalp should be rinsed thoroughly as soon as possible. PMID:21887044

Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica).

PubMed

Kanneganti, Vydehi; Gupta, Aditya Kumar

2009-04-01

Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza sativaPectin Methyl Esterase 1). It contained the structural arrangement GXYXE and GXXDFIF, found in the active groups of all PMEs. OsPME1 gene product shared varying identities, ranging from 52 % to 33 % with PMEs from other plant species belonging to Brassicaceae, Fabaceae, Amaranthaceae and Funariaceae. Southern blot analysis indicated that PME1 exists as a single copy in the rice genome. Expression pattern analysis revealed that OsPME1 is expressed only in pollen grains, during the later stages of their development and was also regulated by various abiotic stress treatments and phytohormones. Functional characterization of this pollen specific PME from rice would enable us to understand its role in pollen development.

Endophytic fungi producing of esterases: Evaluation in vitro of the enzymatic activity using pH indicator

PubMed Central

Lisboa, Helen Cristina Fávero; Biasetto, Carolina Rabal; de Medeiros, João Batista; Araújo, Ângela Regina; Silva, Dulce Helena Siqueira; Teles, Helder Lopes; Trevisan, Henrique Celso

2013-01-01

A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS). The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project “Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest”. The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1 - carboxyl-esterases) for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue), changing the color of the reaction medium (from blue to yellow), that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea as a potential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms. PMID:24516461

Effect of citrus flavonoids on HL-60 cell differentiation.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

1999-01-01

Twenty-seven Citrus flavonoids were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase, and phagocytic activities. 10 flavonoids were judged to be active (percentage of NBT reducing cells was more than 40% at a concentration of 40 microM), and the rank order of potency was natsudaidain, luteolin, tangeretin, quercetin, apigenin, 3, 3, '4, '5, 6, 7, 8-heptamethoxyflavone, nobiletin, acacetin, eriodictyol, and taxifolin. These flavonoids exerted their activity in a dose-dependent manner. HL-60 cells treated with these flavonoids differentiated into mature monocyte/macrophage. The structure-activity relationship established from comparison between flavones and flavanones revealed that ortho-catechol moiety in ring B and C2-C3 double bond had an important role for induction of differentiation of HL-60. In polymethoxylated flavones, hydroxyl group at C3 and methoxyl group at C8 enhanced the differentiation-inducing activity.

Requirement of catalytic-triad and related amino acids for the acyltransferase activity of Tanacetum cinerariifolium GDSL lipase/esterase TcGLIP for ester-bond formation in pyrethrin biosynthesis.

PubMed

Kikuta, Yukio; Yamada, Gen; Mitsumori, Tomonori; Takeuchi, Takayuki; Nakayama, Koji; Katsuda, Yoshio; Hatanaka, Akikazu; Matsuda, Kazuhiko

2013-01-01

We have recently discovered that a GDSL lipase/esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltose-binding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.

Lipophilicity plays a major role in modulating the inhibition of monoamine oxidase B by 7-substituted coumarins.

PubMed

Carotti, Angelo; Altomare, Cosimo; Catto, Marco; Gnerre, Carmela; Summo, Luciana; De Marco, Agostino; Rose, Sarah; Jenner, Peter; Testa, Bernard

2006-02-01

A series of coumarin derivatives (1-22), bearing at the 7-position ether, ketone, ester, carbamate, or amide functions of varying size and lipophilicity, were synthesized and investigated for their in vitro monoamine oxidase-A and -B (MAO-A and -B) inhibitory activities. Most of the compounds acted preferentially as MAO-B inhibitors, with IC(50) values in the micromolar to low-nanomolar range. A structure-activity-relationship (SAR) study highlighted lipophilicity as an important property modulating the MAO-B inhibition potency of 7-substituted coumarins, as shown by a linear correlation (n=20, r(2)=0.72) between pIC(50) and calculated log P values. The stability of ester-containing coumarin derivatives in rat plasma provided information on factors that either favor (lipophilicity) or decrease (steric hindrance) esterase-catalyzed hydrolysis. Two compounds (14 and 22) were selected to investigate how lipophilicity and enzymatic stability may affect in vivo MAO activities, as assayed ex vivo in rat. The most-potent and -selective MAO-B inhibitor 22 (=7-[(3,4-difluorobenzyl)oxy]-3,4-dimethyl-1-benzopyran-2(2H)-one) within the examined series significantly inhibited (>60%) ex vivo rat-liver and striatal MAO-B activities 1 h after intraperitoneal administration of high doses (100 and 300 mumol kg(-1)), revealing its ability to cross the blood-brain barrier. At the same doses, liver and striatum MAO-A was less inhibited in vivo, somehow reflecting MAO-B selectivity, as assessed in vitro. In contrast, the metabolically less stable derivative 14, bearing an isopropyl ester in the lateral chain, had a weak effect on hepatic MAO-B activity in vivo, and none on striatal MAO-B, but, surprisingly, displayed inhibitory effects on MAO-A in both peripheral and brain tissues.

Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris.

PubMed

Sriyapai, Pichapak; Kawai, Fusako; Siripoke, Somjai; Chansiri, Kosum; Sriyapai, Thayat

2015-06-12

A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%-64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0-8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2-C6), displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM(-1) · S(-1)). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

Kinetics of the inhibitory interaction of organophosphorus neuropathy inducers and non-inducers in soluble esterases in the avian nervous system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangas, Iris; Vilanova, Eugenio; Estevez, Jorge, E-mail: jorge.estevez@umh.es

2011-11-15

Some published studies suggest that low level exposure to organophosphorus esters (OPs) may cause neurological and neurobehavioral effects at long term exposure. These effects cannot be explained by action on known targets. In this work, the interactions (inhibition, spontaneous reactivation and 'ongoing inhibition') of two model OPs (paraoxon, non neuropathy-inducer, and mipafox, neuropathy-inducer) with the chicken brain soluble esterases were evaluated. The best-fitting kinetic model with both inhibitors was compatible with three enzymatic components. The amplitudes (proportions) of the components detected with mipafox were similar to those obtained with paraoxon. These observations confirm the consistency of the results and themore » model applied and may be considered an external validation. The most sensitive component (E{alpha}) for paraoxon (11-23% of activity, I{sub 50} (30 min) = 9-11 nM) is also the most sensitive for mipafox (I{sub 50} (30 min) = 4 nM). This component is spontaneously reactivated after inhibition with paraoxon. The second sensitive component to paraoxon (E{beta}, 71-84% of activity; I{sub 50} (30 min) = 1216 nM) is practically resistant to mipafox. The third component (E{gamma}, 5-8% of activity) is paraoxon resistant and has I{sub 50} (30 min) of 3.4 {mu}M with mipafox, similar to NTE (neuropathy target esterase). The role of these esterases remains unknown. Their high sensitivity suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. They will have to be considered in further metabolic and toxicological studies. -- Research Highlights: Black-Right-Pointing-Pointer Paraoxon and mipafox interactions have been evaluated with chicken soluble brain esterases. Black-Right-Pointing-Pointer The paraoxon inhibition was analyzed considering the simultaneous spontaneous reactivation. Black-Right-Pointing-Pointer The best-fitting kinetic models were compatible with a three enzymatic components. Black-Right-Pointing-Pointer The amplitudes of the components were similar in paraoxon and mipafox experiments. Black-Right-Pointing-Pointer It is suggested they may play a role in toxicity in low-level long-term exposure of these compounds.« less

[Bioaerosol concentrations and the identification of aerosolized bacteria by 16S rDNA analysis in work environments].

PubMed

Ishimatsu, Sumiyo; Abe, Hiroki; Fukuda, Kazumasa; Ishidao, Toru; Taniguchi, Hatsumi; Hori, Hajime

2007-03-01

Bioaerosols cause sick building syndrome (SBS) and allergy. Many kinds of bioaerosol impactors are used for measurement of airborne microorganism concentrations in Japan. However, because the impactors are set on agar plates, some microorganisms cannot make colonies on the plates because of their lower viability or demands of nutrition. On the other hand, by double staining using ethidium bromide (EtBr) and carboxyfluorescein diacetate (CFDA), both total cells and cells with esterase activities can be detected without incubation. In this study, we calculated total cell concentrations and percentages of cells with esterase activities by the combination of filter sampling and double staining (EtBr and CFDA) from air of a laboratory, a conference room and outdoors. Temperature and humidity in the laboratory were constantly kept by an air conditioner, but in the conference room, an air conditioner was only operated sometimes because of its low frequency of use. There were no significant differences between total cell concentrations and humidity in both rooms, but increase of the percentages of cells with esterase activities depended on rainfall before the samplings (n=15, p<0.05 by Mann-Whitney test). The increase of active microorganisms by rainfall should be considered when we evaluate the risk of bioaerosols in the workplace. There were few differences in classifications of aerosolized bacteria by 16S rDNA sequence-based homology between the laboratory and the conference room. In both rooms, few pathogenic bacteria were observed.

Control of Tetranychus urticae Koch by extracts of three essential oils of chamomile, marjoram and Eucalyptus

PubMed Central

Abd El-Moneim, MR Afify; Fatma, S Ali; Turky, AF

2012-01-01

Objective To evaluate the acaricidal activity of extracts of three essential oils of chamomile, marjoram and Eucalyptus against Tetranychus urticae (T. urticae) Koch. Methods Extracts of three essential oils of chamomile, marjoram and Eucalyptus with different concentrations (0.5%, 1.0%, 2.0%, 3.0% and 4.0%) were used to control T. urticae Koch. Results The results showed that chamomile (Chamomilla recutita) represented the most potent efficient acaricidal agent against Tetranychus followed by marjoram (Marjorana hortensis) and Eucalyptus. The LC50 values of chamomile, marjoram and Eucalyptus for adults were 0.65, 1.84 and 2.18, respectively and for eggs 1.17, 6.26 and 7.33, respectively. Activities of enzymes including glutathione-S-transferase, esterase (α-esterase and β-esterase) and alkaline phosphatase in susceptible mites were determined and activities of enzymes involved in the resistance of acaricides were proved. Protease enzyme was significantly decreased at LC50 of both chamomile and marjoram compared with positive control. Gas chromatography-mass spectrometer (GC-MS) proved that the major compositions of Chamomilla recutita are α-bisabolol oxide A (35.251%), and trans-β-farersene (7.758%), while the main components of Marjorana hortensis are terpinene-4-ol (23.860%), p-cymene (23.404%) and sabinene (10.904%). Conclusions It can be concluded that extracts of three essential oils of chamomile, marjoram and Eucalyptus possess acaricidal activity against T. urticae. PMID:23569829

Selection of Lactobacillus plantarum strains to use as starters in fermented table olives: Oleuropeinase activity and phage sensitivity.

PubMed

Zago, Miriam; Lanza, Barbara; Rossetti, Lia; Muzzalupo, Innocenzo; Carminati, Domenico; Giraffa, Giorgio

2013-05-01

Fermented table olives (Olea europaea L.) are largely diffused in the Mediterranean area. Olives are picked at different stages of maturity and after harvesting, processed to eliminate the characteristic bitterness caused by the presence of the oleuropein glucoside and to become suitable for human consumption. The spontaneous fermentation of table olives mainly depends on lactic acid bacteria (LAB), and in particular on Lactobacillus plantarum which plays an important role in the degradation of oleuropein. The hydrolysis of oleuropein is attributed to the β-glucosidase and esterase activities of the indigenous LAB microflora. This study investigated the potential of L. plantarum strains isolated from dairy products and olives to be used as starters for fermented table olives. Forty-nine strains were typed by RAPD-PCR and investigated for the presence of the β-glucosidase (bglH) gene. The full sequence of the bglH gene was carried out. All the 49 L. plantarum strains were also tested for phage resistance. A total of six strains were selected on the basis of genotypic polymorphism, bglH gene sequence analysis, and phage resistance profile. These strains were further characterized to assess the acidifying capability, the growth at different temperatures, the tolerance to different NaCl concentrations, and the oleuropeinolytic activity. Although further characterizations are required, especially concerning the influence on sensory properties, L. plantarum proved to have the potential to be used as a debittering and fermentative agent in starter culture for fermented table olives. Copyright © 2012 Elsevier Ltd. All rights reserved.

Gel filtration applied to the study of lipases and other esterases

PubMed Central

Downey, W. K.; Andrews, P.

1965-01-01

1. Sephadex G-100 and G-200 gel-filtration columns were calibrated for molecular-weight estimation with proteins of known molecular weights, and used to study the composition of several lipase or esterase preparations. 2. Enzymes from cow's milk, rat adipose tissue and pig pancreas were detected in the column effluents by their ability to liberate free acid from emulsified tributyrin at pH 8·5. 3. Four tributyrinases were detected in preparations from individual cow's milks. Molecular weights 62000, 75000 and 112000 were estimated for three of them, but although the fourth may be of unusually low molecular weight an estimate was not possible. 4. Extracts of rat adipose tissue apparently contained six tributyrinases (molecular weights 39000, 47000, 55000, 68000, 75000 and 200000) but the relative amounts of these enzymes varied widely from rat to rat. 5. Tributyrinase activity in juice expressed from pig pancreatic tissue was due mainly to one enzyme (molecular weight 42000). On the other hand, activity in extracts of acetone-dried pancreas was confined to material of molecular weight > 106, which may be an aggregated form of the lower-molecular-weight enzyme. 6. Activity in fractionated wheat-germ extracts was assayed with emulsified triacetin substrate, and was evidently due to one enzyme (molecular weight 51000). 7. Some problems arising in the application of gel filtration to the study of lipase–esterase systems were indicated. PMID:14340054

Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

PubMed

Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

2017-03-15

Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h.

Chromatographic study of highly methoxylated lime pectins deesterified by different pectin methyl-esterases.

PubMed

Ralet, M C; Bonnin, E; Thibault, J F

2001-03-25

The inter-molecular distribution of free carboxyl groups of two highly methoxylated pectins enzymatically deesterified by plant and fungus pectin methyl-esterases were investigated by size-exclusion (SEC) and ion-exchange chromatography (IEC). "Homogeneous" populations with respect to molar mass or charge density were thereby obtained and their chemical composition and physico-chemical properties (transport parameter for monovalent cations and calcium, calcium activity coefficient) were studied. Chemical analysis showed that the composition varies from one SEC fraction to another, the highest molar mass fraction being richer in rhamnose and galactose and exhibiting a slightly higher degree of methylation. Separation of pectins by IEC revealed a quite homogeneous charge density distribution for F58 contrary to P60 which exhibited a large distribution of methoxyl groups. The free carboxyl groups distributions and calcium binding behaviours of SEC and IEC fractions were shown to differ widely for highly methoxylated pectins deesterified by plant and fungus pectin methyl-esterases.

Characterization of hemicellulase and cellulase from the extremely thermophilic bacterium Caldicellulosiruptor owensensis and their potential application for bioconversion of lignocellulosic biomass without pretreatment.

PubMed

Peng, Xiaowei; Qiao, Weibo; Mi, Shuofu; Jia, Xiaojing; Su, Hong; Han, Yejun

2015-01-01

Pretreatment is currently the common approach for improving the efficiency of enzymatic hydrolysis on lignocellulose. However, the pretreatment process is expensive and will produce inhibitors such as furan derivatives and phenol derivatives. If the lignocellulosic biomass can efficiently be saccharified by enzymolysis without pretreatment, the bioconversion process would be simplified. The genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass. It gives potential opportunities for improving the efficiency of converting native lignocellulosic biomass to fermentable sugars. Both of the extracellular (extra-) and intracellular (intra-) enzymes of C. owensensis cultivated on corncob xylan or xylose had cellulase (including endoglucanase, cellobiohydrolase and β-glucosidase) and hemicellulase (including xylanase, xylosidase, arabinofuranosidase and acetyl xylan esterase) activities. The enzymes of C. owensensis had high ability for degrading hemicellulose of native corn stover and corncob with the conversion rates of xylan 16.7 % and araban 60.0 %. Moreover, they had remarkable synergetic function with the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). When the native corn stover and corncob were respectively, sequentially hydrolyzed by the extra-enzymes of C. owensensis and CTec2, the glucan conversion rates were 31.2 and 37.9 %,which were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 alone), whereas the glucan conversion rates of the steam-exploded corn stover and corncob hydrolyzed by CTec2 alone on the same loading rate were 38.2 and 39.6 %, respectively. These results show that hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment on degradation of native lignocellulosic biomass. A new process for saccharification of lignocellulosic biomass by sequential hydrolysis is demonstrated in the present research, namely hyperthermal enzymolysis (70-80 °C) by enzymes of C. owensensis followed with mesothermal enzymolysis (50-55 °C) by commercial cellulase. This process has the advantages of no sugar loss, few inhibitors generation and consolidated with sterilization. The enzymes of C. owensensis demonstrated an enhanced ability to degrade the hemicellulose of native lignocellulose. The pretreatment and detoxification steps may be removed from the bioconversion process of the lignocellulosic biomass by using the enzymes from C. owensensis.

A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

PubMed Central

Rao, Lang; Xue, Yanfen; Zheng, Yingying; Lu, Jian R.; Ma, Yanhe

2013-01-01

Background Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities. Methodology/Principal Findings Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220. Conclusion EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues. PMID:23577139

Evidence of metabolic mechanisms playing a role in multiple insecticides resistance in Anopheles stephensi populations from Afghanistan.

PubMed

Safi, Noor Halim Zahid; Ahmadi, Abdul Ali; Nahzat, Sami; Ziapour, Seyyed Payman; Nikookar, Seyed Hassan; Fazeli-Dinan, Mahmoud; Enayati, Ahmadali; Hemingway, Janet

2017-03-03

Malaria is endemic in most parts of Afghanistan and insecticide-based vector control measures are central in controlling the disease. Insecticide resistance in the main malaria vector Anopheles stephensi from Afghanistan is increasing and attempts should be made to determine the underlying resistance mechanisms for its adequate management. The contents of cytochrome P450s, esterases, glutathione S-transferases (GSTs) and acetylcholine esterase (AChE) activities were measured in the Kunar and Nangarhar populations of An. stephensi from Afghanistan and the results were compared with those of the susceptible Beech strain using the World Health Organization approved biochemical assay methods for adult mosquitoes. The cytochrome P450s enzyme ratios were 2.23- and 2.54-fold in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The enzyme ratios for esterases with alpha-naphthyl acetate were 1.45 and 2.11 and with beta-naphthyl acetate were 1.62 and 1.85 in the Kunar and Nangarhar populations respectively compared with the susceptible Beech strain. Esterase ratios with para-nitrophenyl acetate (pNPA) were 1.61 and 1.75 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The GSTs enzyme ratios were 1.33 and 1.8 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The inhibition of AChE was 70.9 in the susceptible Beech strain, and 56.7 and 51.5 in the Kunar and Nangarhar populations. The differences between all values of the enzymes activities/contents and AChE inhibition rates in the Kunar and Nangarhar populations were statistically significant when compared with those of the susceptible Beech strain. Based on the results, the reported resistance to pyrethroid and organophosphate insecticides, and tolerance to bendiocarb in the Kunar and Nangarhar populations of An. stephensi from Afghanistan are likely to be caused by a range of metabolic mechanisms, including esterases, P450s and GSTs combined with target site insensitivity in AChE.

Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

PubMed Central

Abass, Kasim Sakran

2014-01-01

Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (k cat/K m), and the K m and k cat values were 176 μM and 16,765 s−1, respectively, for S-phenyl thioacetate ester, while detected in dilution 1 : 15 was the lowest specificity constant (k cat/K m), and the K m and k cat values were 943 μM and 1154 s−1, respectively, for acetylthiocholine iodide ester. PMID:24864243

Properties of Two Novel Esterases Identified from Culture Supernatant of Penicillium purpurogenum Grown on Sugar Beet Pulp.

PubMed

Oleas, Gabriela; Callegari, Eduardo; Sepulveda, Romina; Eyzaguirre, Jaime

2016-01-01

The filamentous fungus Penicillium purpurogenum grows on a variety of natural carbon sources, such as sugar beet pulp, and secretes to the medium a large number of enzymes that degrade the carbohydrate components of lignocellulose. Sugar beet pulp is rich in pectin, and the purpose of this work is to identify novel esterases produced by the fungus, which may participate in pectin degradation. Partially purified culture supernatants of the fungus grown on sugar beet pulp were subjected to mass spectrometry analysis. Peptides thus identified, which may be part of potential esterases were probed against the proteins deduced from the fungal genome sequence. The cDNAs of two putative esterases identified were expressed in Pichia pastoris and their properties studied. One of these enzymes, named FAET, is a feruloyl esterase, while the other, PE, is classified as a pectin methyl esterase. These findings add to our knowledge of the enzymology of pectin degradation by Penicillium purpurogenum, and define properties of two novel esterases acting on de-esterification of pectin. Their availability may be useful as tools for the study of pectin structure and degradation.

Properties of Two Novel Esterases Identified from Culture Supernatant of Penicillium purpurogenum Grown on Sugar Beet Pulp

PubMed Central

Oleas, Gabriela; Callegari, Eduardo; Sepulveda, Romina; Eyzaguirre, Jaime

2017-01-01

Background The filamentous fungus Penicillium purpurogenum grows on a variety of natural carbon sources, such as sugar beet pulp, and secretes to the medium a large number of enzymes that degrade the carbohydrate components of lignocellulose. Sugar beet pulp is rich in pectin, and the purpose of this work is to identify novel esterases produced by the fungus, which may participate in pectin degradation. Methods and findings Partially purified culture supernatants of the fungus grown on sugar beet pulp were subjected to mass spectrometry analysis. Peptides thus identified, which may be part of potential esterases were probed against the proteins deduced from the fungal genome sequence. The cDNAs of two putative esterases identified were expressed in Pichia pastoris and their properties studied. One of these enzymes, named FAET, is a feruloyl esterase, while the other, PE, is classified as a pectin methyl esterase. Conclusions These findings add to our knowledge of the enzymology of pectin degradation by Penicillium purpurogenum, and define properties of two novel esterases acting on de-esterification of pectin. Their availability may be useful as tools for the study of pectin structure and degradation. PMID:28828411

Utilization of deep-sea microbial esterase PHE21 to generate chiral sec-butyl acetate through kinetic resolutions.

PubMed

Wang, Yilong; Xu, Yongkai; Zhang, Yun; Sun, Aijun; Hu, Yunfeng

2018-06-08

We previously identified and characterized 1 novel deep-sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)-3-hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)-sec-butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)-sec-butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)-sec-butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)-sec-butyl acetate reached 64% and 43%, respectively, after process optimization. Deep-sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols. © 2018 Wiley Periodicals, Inc.

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

PubMed Central

2014-01-01

Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase.

PubMed

Matsuo, Takemasa; Kobayashi, Takashi; Kimura, Yukitaka; Tsuchiyama, Moriyasu; Oh, Tadanobu; Sakamoto, Tatsuji; Adachi, Shuji

2008-12-01

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL "Amano" from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50 degrees C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25 degrees C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.

Release of Esterase Following Germination of Lettuce Seed (Lactuca sativa L.)

PubMed Central

Chandra, G. Ram; Toole, Vivian K.

1977-01-01

Light-insensitive lettuce seeds, Lactuca sativa L. cv. Great Lakes, release esterases for a period following radicle protrusion. Very little or no enzymes are released prior to 24 hours or after 48 hours of germination. As compared to intact seeds, half-seeds readily release esterases and the release is not affected by far red irradiation. Bulk of the released esterases are derived from the endosperm tissue and presumably exists in the intact seed as a component of the extraembryonic fluid. PMID:16659992

Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery

USDA-ARS?s Scientific Manuscript database

We present here a whole-cell and permeabilized E. coli cell 1' active/inactive microplate screen for ß-D-xylosidase, xylanase, endocellulase, and ferulic acid esterase enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic...

Yeasts from sub-Antarctic region: biodiversity, enzymatic activities and their potential as oleaginous microorganisms.

PubMed

Martinez, A; Cavello, I; Garmendia, G; Rufo, C; Cavalitto, S; Vero, S

2016-09-01

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).

aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species.

PubMed

Lescat, Mathilde; Hoede, Claire; Clermont, Olivier; Garry, Louis; Darlu, Pierre; Tuffery, Pierre; Denamur, Erick; Picard, Bertrand

2009-12-29

Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. We identified the gene encoding esterase B as the acetyl-esterase gene (aes) using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR) strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.

[Investigation of some virulence factors in Trichosporon spp. strains].

PubMed

Demir, Feyza; Kuştimur, Semra

2014-10-01

The frequency of fungal infections have increased recently in parallel to prolonged survival of patients with chronical infections, common use of the broad-spectrum antibiotics and cytotoxic drugs and surgical interventions. Fungi such as Trichosporon, Fusarium and Geotrichum that were previously evaluated as contaminant/colonization, become important causes of morbidity and mortality especially in neutropenic patients. The aim of this study was to investigate the presence of virulence factors such as acid proteinase, phospholipase, esterase, coagulase and hemolytic activity among Trichosporon species. A total of 40 Trichosporon strains, of them 24 (60%) were T.asahii, 6 (15%) were T.inkin and 10 (25%) were the other species (one of each of T.aquatile, T.asteroides, T.coremiiforme, T.cutaneum, T.dermatis, T.faecale, T.japonicum, T.montevideense, T.mucoides, T.ovoides) were included in the study. Identification of the isolates was performed according to microscopic morphology (blastospores, arthrospores, pseudohyphae and true hyphae) on corn meal agar media, and carbohydrate assimilation patterns (API ID32C; bioMérieux, France). Secretory acid proteinase, phospholipase and esterase activities of the strains were evaluated by 1% bovine serum albumin containing agar, by egg yolk containing solid medium, and by Tween 80 containing solid medium, respectively. Hemolytic activity of the isolates were evaluated by 5-10% sheep blood Sabouraud dextrose agar. Coagulase enzyme activity was determined by using human and rabbit plasma. In our study, all of the 40 Trichosporon spp. strains were found negative in terms of acid proteinase and phospholipase enzyme activity, however all were positive for esterase enzyme activity. Hemolytic enzyme activity were identified in a total of 15 (37.5%) strains, being "+++" in three strains (2 T.asahii, 1 T.japonicum), and "++" in 12 isolates (9 T.asahii, 1 T.inkin, 1 T.asteroides, 1 T.mentevideense). Although 11 of those 15 positive strains were T.asahii, there was no statistical difference between the species in terms of hemolytic enzyme activity (p> 0.05). Coagulase enzyme activity was detected in 5% (2/40; 1 T.asahii, 1 T.inkin) of the strains with human plasma and in 27.5% (11/40; 9 T.asahii, 1 T.inkin, 1 T.montevideense) with rabbit plasma. In conclusion, our data indicated that esterase, coagulase and hemolytic activities detected in Trichosporon spp. might play role in the pathogenesis of Trichosporon infections, however, further large-scaled clinical and mycological studies are needed to prove this relation.

Antioxidative activities of hydrolysates from edible birds nest using enzymatic hydrolysis

NASA Astrophysics Data System (ADS)

Muhammad, Nurul Nadia; Babji, Abdul Salam; Ayub, Mohd Khan

2015-09-01

Edible bird's nest protein hydrolysates (EBN) were prepared via enzymatic hydrolysis to investigate its antioxidant activity. Two types of enzyme (alcalase and papain) were used in this study and EBN had been hydrolysed with different hydrolysis time (30, 60, 90 and 120 min). Antioxidant activities in EBN protein hydrolysate were measured using DPPH, ABTS+ and Reducing Power Assay. From this study, increased hydrolysis time from 30 min to 120 min contributed to higher DH, as shown by alcalase (40.59%) and papain (24.94%). For antioxidant assay, EBN hydrolysed with papain showed higher scavenging activity and reducing power ability compared to alcalase. The highest antioxidant activity for papain was at 120 min hydrolysis time with ABTS (54.245%), DPPH (49.78%) and Reducing Power (0.0680). Meanwhile for alcalase, the highest antioxidant activity was at 30 min hydrolysis time. Even though scavenging activity for EBN protein hydrolysates were high, the reducing power ability was quite low as compared to BHT and ascorbic Acid. This study showed that EBN protein hydrolysate with alcalase and papain treatments potentially exhibit high antioxidant activity which have not been reported before.

Changes in pectin methyl esterase activity with different packaging materials and stages of fruit harvesting during cold storage of pear cv. Punjab beauty.

PubMed

Kaur, Kirandeep; Dhillon, W S; Mahajan, B V C

2014-10-01

Pear cv. Punjab Beauty has become quite popular in Punjab. Excessive softening during cold storage leading to low shelf life is the major factor limiting its wider adoption. Studies were, therefore, conducted to determine the firmness and pectin methyl esterase (PME) activity at 4 harvest dates (2nd, 3rd and 4th week of July, and 1st week of August). Various packaging materials i.e. corrugated fiber board boxes and crates with high and low density polyethylene liners, corrugated fiber board boxes, crates and wooden boxes were also evaluated for their role in extending the shelf life of fruits. The enzyme activity and fruit firmness was evaluated periodically after 30, 45, 60 and 75 days of storage at 0-1 °C and 90-95 % RH. The firmness of the fruits decreased with the increase in storage intervals but the enzyme activity increased with the storage period up to 60 days and declined thereafter. Ripening-related changes in all the harvests were characterized mainly by an increase in the solubilization of pectin with a concomitant decrease in the degree of firmness. There was a continuous increase in enzyme activity with the advancement in harvesting dates and then fell sharply in the advanced ripening stages. Highest pectin methyl esterase activity was in fruits packed in crates followed by wooden boxes and corrugated fiber board boxes while the lowest was recorded in fruits packed in corrugated fiber board boxes with high density polyethylene liners. Therefore, high density polyethylene lined CFB boxes proved to be most effective in preventing the loss in firmness.

Characterization of Cinnamoyl Esterases from Different Lactobacilli and Bifidobacteria.

PubMed

Fritsch, Caroline; Jänsch, André; Ehrmann, Matthias A; Toelstede, Simone; Vogel, Rudi F

2017-02-01

A high variety of plants that are used for food production contain esterified hydroxycinnamic acids. As their free forms display several benefits, like an enhanced absorption in human intestinal tract, anti-oxidative and anti-carcinogenic effects, an improved protein solubility and reduced discoloration, the microbial ability to cleave the ester bond is highly desired. In order to examine potential fermentation strains for this purpose, six different lactic acid bacteria and one bifidobacterial strain were screened for their ability to degrade esterified hydroxycinnamic acids because these strains are commonly used for fermentation of plant-based foods. Moreover, their cinnamoyl esterase activity was examined by molecular biological analyses. The enzymes were heterologously expressed in Escherichia coli, purified and biochemically characterized. The purified esterases with a molecular mass around 27-29 kDa had their optimum predominantly between pH 7 and 8 at 20-30 °C. Bifidobacterium animalis subsp. lactis, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus fermentum displayed activities against a broad substrate range (methyl caffeate, methyl trans-p-coumarate, chlorogenic acid as well as partially ethyl ferulate). Concerning substrate affinity, reaction velocity, thermal and pH stability, Lactobacillus gasseri showed the overall best performance. The herein studied lactic acid- and bifidobacteria are promising for the production of fermented plant-based foods with an increased quality and nutritional value.

Three-dimensional structure of homodimeric cholesterol esterase-ligand complex at 1.4 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletnev, V.; Addlagatta, A.; Wawrzak, Z.

2010-03-08

The three-dimensional structure of a Candida cylindracea cholesterol esterase (ChE) homodimer (534 x 2 amino acids) in complex with a ligand of proposed formula C{sub 23}H{sub 48}O{sub 2} has been determined at 1.4 {angstrom} resolution in space group P1 using synchrotron low-temperature data. The structure refined to R = 0.136 and R{sub free} = 0.169 and has revealed new stereochemical details in addition to those detected for the apo- and holo-forms at 1.9 and 2.0 {angstrom} resolution, respectively [Ghosh et al. (1995), Structure, 3, 279-288]. The cholesterol esterase structure is a dimer with four spatially separated interfacial contact areas andmore » two symmetry-related pairs of openings to an internal intradimer cavity. Hydrophobic active-site gorges in each subunit face each other across a central interfacial cavity. The ChE subunits have carbohydrate chains attached to their Asn314 and Asn351 residues, with two ordered N-acetyl-D-glucosoamine moieties visible at each site. The side chains of 14 residues have two alternative conformations with occupancy values of 0.5 {+-} 0.2. For each subunit the electron density in the enzyme active-site gorge is well modeled by a C{sub 23}-chain fatty acid.« less

Synergistic toxicity and physiological impact of imidacloprid alone and binary mixtures with seven representative pesticides on honey bee (Apis mellifera)

PubMed Central

Zhu, Yu Cheng; Yao, Jianxiu; Adamczyk, John; Luttrell, Randall

2017-01-01

Imidacloprid is the most widely used insecticide in the world. In this study, we used spraying methods to simulate field exposures of bees to formulated imidacloprid (Advise® 2FL) alone and binary mixtures with seven pesticides from different classes. Synergistic toxicity was detected from mixtures of Advise (58.6 mg a.i./L imidacloprid)+Domark (512.5 mg a.i. /L tetraconazole), Advise+Transform (58.5 mg a.i./L sulfoxaflor), and Advise+Vydate (68 mg a.i./L oxamyl), and mortality was significantly increased by 20%, 15%, and 26% respectively. The mixtures of Advise+Bracket (88.3 mg a.i./L acephate) and Advise+Karate (62.2 mg a.i./L L-cyhalothrin) showed additive interaction, while Advise+Belay (9.4 mg a.i./L clothianidin) and Advise+Roundup (1217.5 mg a.i./L glyphosate) had no additive/synergistic interaction. Spraying bees with the mixture of all eight pesticides increased mortality to 100%, significantly higher than all other treatments. Except Bracket which significantly suppressed esterase and acetylcholinesterase (AChE) activities, other treatments of Advise-only and mixtures with other pesticides did not suppress enzyme activities significantly, including invertase, glutathione S-transferase (GST), and esterase and AChE. Immunity-related phenoloxidase (PO) activities in survivors tended to be more variable among treatments, but mostly still statistically similar to the control. By using specific enzyme inhibitors, we demonstrated that honey bees mainly rely on cytochrome P450 monooxygenases (P450s) for detoxifying Advise, while esterases and GSTs play substantially less roles in the detoxification. This study provided valuable information for guiding pesticide selection in premixing and tank mixing in order to alleviate toxicity risk to honey bees. Our findings indicated mixtures of Advise with detoxification-enzyme-inducing pesticides may help bees to detoxify Advise, while toxicity synergists may pose further risk to bees, such as the Bracket which not only suppressed esterase and AChE activities, but also increased toxicity to bees. PMID:28467462

Synergistic toxicity and physiological impact of imidacloprid alone and binary mixtures with seven representative pesticides on honey bee (Apis mellifera).

PubMed

Zhu, Yu Cheng; Yao, Jianxiu; Adamczyk, John; Luttrell, Randall

2017-01-01

Imidacloprid is the most widely used insecticide in the world. In this study, we used spraying methods to simulate field exposures of bees to formulated imidacloprid (Advise® 2FL) alone and binary mixtures with seven pesticides from different classes. Synergistic toxicity was detected from mixtures of Advise (58.6 mg a.i./L imidacloprid)+Domark (512.5 mg a.i. /L tetraconazole), Advise+Transform (58.5 mg a.i./L sulfoxaflor), and Advise+Vydate (68 mg a.i./L oxamyl), and mortality was significantly increased by 20%, 15%, and 26% respectively. The mixtures of Advise+Bracket (88.3 mg a.i./L acephate) and Advise+Karate (62.2 mg a.i./L L-cyhalothrin) showed additive interaction, while Advise+Belay (9.4 mg a.i./L clothianidin) and Advise+Roundup (1217.5 mg a.i./L glyphosate) had no additive/synergistic interaction. Spraying bees with the mixture of all eight pesticides increased mortality to 100%, significantly higher than all other treatments. Except Bracket which significantly suppressed esterase and acetylcholinesterase (AChE) activities, other treatments of Advise-only and mixtures with other pesticides did not suppress enzyme activities significantly, including invertase, glutathione S-transferase (GST), and esterase and AChE. Immunity-related phenoloxidase (PO) activities in survivors tended to be more variable among treatments, but mostly still statistically similar to the control. By using specific enzyme inhibitors, we demonstrated that honey bees mainly rely on cytochrome P450 monooxygenases (P450s) for detoxifying Advise, while esterases and GSTs play substantially less roles in the detoxification. This study provided valuable information for guiding pesticide selection in premixing and tank mixing in order to alleviate toxicity risk to honey bees. Our findings indicated mixtures of Advise with detoxification-enzyme-inducing pesticides may help bees to detoxify Advise, while toxicity synergists may pose further risk to bees, such as the Bracket which not only suppressed esterase and AChE activities, but also increased toxicity to bees.

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2013 CFR

2013-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2014 CFR

2014-04-01

... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme... animals. (c) The enzyme is produced by a process which completely removes the organism Mucor miehei var...

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2012 CFR

2012-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2011 CFR

2011-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a process...

Serotyping and esterase typing for analysis of Listeria monocytogenes populations recovered from foodstuffs and from human patients with listeriosis in Belgium.

PubMed Central

Gilot, P; Genicot, A; André, P

1996-01-01

Listeria monocytogenes strains isolated in Belgium from different foodstuffs and in sporadic cases of human listeriosis were analyzed. The distribution of serovars differed in each of these populations. The bacteria isolated from cheeses and from human patients with listeriosis were further studied by esterase typing. The twenty esterase patterns defined were not equally distributed in these two populations. The secretion of the virulence determinant phosphatidylinositol-specific phospholipase C and the pathogenicity level of strains in immunocompromised mice could not explain the unequal distribution of esterase types. The discrimination index of esterase typing (DI = 0.868) was compared with that of serotyping (DI = 0.666) and with that of the two combined methods (DI = 0.899). PMID:8815071

Discovering novel enzymes by functional screening of plurigenomic libraries from alga-associated Flavobacteriia and Gammaproteobacteria.

PubMed

Martin, Marjolaine; Vandermies, Marie; Joyeux, Coline; Martin, Renée; Barbeyron, Tristan; Michel, Gurvan; Vandenbol, Micheline

2016-01-01

Alga-associated microorganisms, in the context of their numerous interactions with the host and the complexity of the marine environment, are known to produce diverse hydrolytic enzymes with original biochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the surface of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriia and the Gammaproteobacteria. In the present study, we constructed two "plurigenomic" (with multiple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny and their enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diverse hydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolytic isolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), an esterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role of the used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screening yields obtained for both libraries and get deeper inside the "great screen anomaly". Interestingly, the discovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases, while the two iota-carrageenases represent new members of the poorly known GH82 family (containing only 19 proteins since its description in 2000). These original results demonstrate the efficiency of our uncommon "plurigenomic" library approach and the underexplored potential of alga-associated cultivable microbiota for the identification of novel and algal-specific enzymes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Acetylation Suppresses the Proapoptotic Activity of GD3 Ganglioside

PubMed Central

Malisan, Florence; Franchi, Luigi; Tomassini, Barbara; Ventura, Natascia; Condò, Ivano; Rippo, Maria Rita; Rufini, Alessandra; Liberati, Laura; Nachtigall, Claudia; Kniep, Bernhard; Testi, Roberto

2002-01-01

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3. PMID:12486096

Studies on sterol-ester hydrolase from Fusarium oxysporum. I. Partial purification and properties.

PubMed

Okawa, Y; Yamaguchi, T

1977-05-01

1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium. 2. The cholesterol esterase from Fusarium oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography. 3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60 degrees C for 30 min, at pH 7.0). 4. The optimum pH of the enzyme was found to be around 7.0. 5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable substrate. 6. Cholesterol esters in serum were also hydrolyzed by this enzyme.

Crystal structure of human esterase D: a potential genetic marker of retinoblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dong; Li, Yang; Song, Gaojie

2009-07-10

Retinoblastoma (RB), a carcinoma of the retina, is caused by mutations in the long arm of chromosome 13, band 13q14. The esterase D (ESD) gene maps at a similar location as the RB gene locus and therefore serves as a potential marker for the prognosis of retinoblastoma. Because very little is known about the structure and function of ESD, we determined the 3-dimensional structure of the enzyme at 1.5 {angstrom} resolution using X-ray crystallography. ESD shows a single domain with an {alpha}/{beta}-hydrolase fold. A number of insertions are observed in the canonical {alpha}/{beta}-hydrolase fold. The active site is located inmore » a positively charged, shallow cleft on the surface lined by a number of aromatic residues. Superimposition studies helped identify the typical catalytic triad residues -- Ser-153, His264, and Asp230 -- involved in catalysis. Mutagenesis of any of the catalytic triad residues to alanine abolished the enzyme activity. Backbone amides of Leu54 and Met150 are involved in the formation of the oxyanion hole. Interestingly, a M150A mutation increased the enzyme activity by 62%. The structure of human ESD determined in this study will aid the elucidation of the physiological role of the enzyme in the human body and will assist in the early diagnosis of retinoblastoma. Wu, D., Li, Y., Song, G., Zhang, D., Shaw, N., Liu, Z. J. Crystal structure of human esterase D: a potential genetic marker of retinoblastoma.« less

Combination of Xylanase and Debranching Enzymes Specific to Wheat Arabinoxylan Improve the Growth Performance and Gut Health of Broilers.

PubMed

Lei, Zhao; Shao, Yuxin; Yin, Xiaonan; Yin, Dafei; Guo, Yuming; Yuan, Jianmin

2016-06-22

Arabinoxylan (AX) is the major antinutritional factor of wheat. This study evaluated the synergistic effects of xylanase and debranching enzymes (arabinofuranosidase [ABF] and feruloyl esterase [FAE]) on AX. During in vitro tests, the addition of ABF or FAE accelerated the hydrolysis of water-soluble AX (WE-AX) and water-insoluble AX (WU-AX) and produced more xylan oligosaccharides (XOS) than xylanase alone. XOS obtained from WE-AX stimulated greater proliferation of Lactobacillus brevis and Bacillus subtilis than did fructo-oligosaccharides (FOS) and glucose. During in vivo trials, xylanase increased the average daily growth (ADG), decreased the feed-conversion ratio (FCR), and reduced the digesta viscosity of jejunum and intestinal lesions of broilers fed a wheat-based diet on day 36. ABF or FAE additions further improved these effects. Broilers fed a combination of xylanase, ABF, and FAE exhibited the best growth. In conclusion, the synergistic effects among xylanase, ABF, and FAE increased AX degradation, which improve the growth performance and gut health of broilers.

Simultaneous use of in silico design and a correlated mutation network as a tool to efficiently guide enzyme engineering.

PubMed

Nobili, Alberto; Tao, Yifeng; Pavlidis, Ioannis V; van den Bergh, Tom; Joosten, Henk-Jan; Tan, Tianwei; Bornscheuer, Uwe T

2015-03-23

In order to improve the efficiency of directed evolution experiments, in silico multiple-substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3-phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta-analysis of the docking conformations in wild-type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15-fold improved enantioselectivity was found. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Composition and biodegradation of a synthetic oil spilled on the perennial ice cover of Lake Fryxell, Antarctica.

PubMed

Jaraula, Caroline M B; Kenig, Fabien; Doran, Peter T; Priscu, John C; Welch, Kathleen A

2009-04-15

A helicopter crashed in January 2003 on the 5 m-thick perennial ice cover of Lake Fryxell, spilling synthetic turbine oil Aeroshell 500. Molecular compositions of the oils were analyzed by gas chromatography-mass spectrometry and compared to the composition of contaminants in ice, meltwater, and sediments collected a year after the accident. Aeroshell 500 is based on C20-C33 Pentaerythritol triesters (PET) with C5-C10 fatty acids susbstituents and contain a number of antioxidant additives, such as tricresyl phosphates. Biodegradation of this oil in the ice cover occurs when sediments are present PETs with short fatty acids substituents are preferentially degraded, whereas long chain fatty acids seem to hinder esters from hydrolysis by esterase derived from the microbial assemblage. It remains to be seen if the microbial ecosystem can degrade tricresyl phosphates. These more recalcitrant PET species and tricresyl phosphates are likely to persist and comprise the contaminants that may eventually cross the ice cover to reach the pristine lake water.

Characterization of a novel deep-sea microbial esterase EstC10 and its use in the generation of ( R)-methyl2-chloropropionate

NASA Astrophysics Data System (ADS)

Gong, Yanhui; Ma, Sanmei; Wang, Yongfei; Xu, Yongkai; Sun, Aijun; Zhang, Yun; Hu, Yunfeng

2018-03-01

A novel esterase EstC10 from Bacillus sp. CX01 isolated from the deep sea of the Western Pacific Ocean and the functionalities of EstC10 was characterized. At present, the reports about the kinetic resolution of racemic methyl 2-chloropropionate were quite rare. So we developed deep-sea microbial esterase EstC10 as a novel biocatalyst in the kinetic resolution of racemic methyl 2-chloropropionate and generate ( R)-methyl 2-chloropropionate with high enantiomeric excess (>99%) after the optimization of process parameters such as pH, temperature, organic co-solvents, surfactants, substrate concentration and reaction time. Notably, the optimal substrate concentration (80 mmol/L) of esterase EstC10 was higher than the kinetic resolution of another esterase, Est12-7 (50 mmol/L). The novel microbial esterase EstC10 identified from the deep sea was a promising green biocatalyst in the generation of ( R)-methyl 2-chloropropionate as well of many other valuable chiral chemicals in industry.

Simultaneous determination of triamcinolone hexacetonide and triamcinolone acetonide in rabbit plasma using a highly sensitive and selective UPLC-MS/MS method.

PubMed

Sun, Wei; Ho, Stacy; Fang, Xiaojun Rick; O'Shea, Thomas; Liu, Hanlan

2018-05-10

An ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was successfully developed and qualified for the simultaneous determination of triamcinolone hexacetonide (TAH) and triamcinolone acetonide (TAA, the active metabolite of TAH) in rabbit plasma. To prevent the hydrolysis of TAH to TAA ex vivo during sample collection and processing, we evaluated the effectiveness of several esterase inhibitors to stabilize TAH in plasma. Phenylmethanesulfonyl fluoride (PMSF) at 2.0 mM was chosen to stabilize TAH in rabbit plasma. The developed method is highly sensitive with a lower limit of quantitation of 10.0 pg/mL for both TAA and TAH using a 300 μL plasma aliquot. The method demonstrated good linearity, accuracy, precision, sensitivity, selectivity, recovery, matrix effects, dilution integrity, carryover, and stability. Linearity was obtained over the range of 10-2500 pg/mL. Both intra- and inter-run coefficients of variation were less than 9.1% and accuracies across the assay range were all within 100 ± 8.4%. The run time is under 5 minutes. The method was successfully implemented to support a rabbit pharmacokinetic study of TAH and TAA following a single intra-articular administration of TAH (Aristospan ® ). Copyright © 2018 Elsevier B.V. All rights reserved.

Enzymes mediating resistance to lambda-cyhalothrin in Eriopis connexa (Coleoptera: Coccinellidae).

PubMed

Rodrigues, Agna R S; Siqueira, Herbert A A; Torres, Jorge B

2014-03-01

Resistance to widely used insecticide, lambda-cyhalothrin, was recently reported in the predatory lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae). However, to understand whether metabolic mechanisms underlie such resistance, synergism bioassays and in vitro studies were carried out by using inhibitors and model substrates for enzymatic assays, respectively. The LD50s estimated for susceptible and resistant populations ηg of lambda-cyhalothrin/insect, and thus, a 22-fold difference in resistance ratio. Synergism ratios for the susceptible population with piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP), and S,S,S-tributylphosphorotrithioate (DEF) were respectively 33.8-, 0.24-, 0.35-, and 4.25-fold, while for the resistant population, they were 1463.0-, 0.79-, 0.85-, and 282.6-fold, respectively. The synergized resistance ratios were 0.50-, 2.00-, 6.75-, and 8.77-fold with PBO, DEF, DEM, and TPP, respectively, while resistance was virtually suppressed with DEF. The esterase exhibited 4.16-, 4.03-, and 5.38-fold greater activity towards formation of α-naphthol, β-naphthol, and 4-nitrophenol in the resistant population of E. connexa than in the susceptible population. The activity of esterase depended on concentrations of DEF applied, either using α-naphthol or β-naphthol, which completely inhibited the activity at 636 ηM. The PBO inhibited the β-naphthol formation in approximately 50%, suggesting it as inhibitor of esterases. The activities of glutathione-S-transferase were similar and corresponded to 0.36-0.47 ηmol(-1) min(-1)μg of protein, for S and R populations, respectively. Similarly, the activities of cytochrome P450-dependent microsomal monooxygenases were 0.04 and 0.05 ηmol(-1) min(-1)μg of protein. The native gel indicated that the formation of β-naphthol was completely inhibited by methyl-paraoxon, but only partially inhibited by eserine, TPP, and PBO. Although other studies with DEF and PBO have demonstrated strong inhibition of type B carboxylesterase associated with insecticide resistance, the results reported here do not rule out metabolism by cytochrome P450-dependent microsomal monooxygenases as a factor conferring E. connexa resistance to lambda-cyhalothrin and confirmed that PBO may also act by inhibiting esterases of insects. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure of the catalytic domain of glucuronoyl esterase Cip2 from Hypocrea jecorina

USDA-ARS?s Scientific Manuscript database

The structure of the catalytic domain of glucuronoyl esterase Cip2 from the fungus Hypocrea jecorina was determined at a resolution of 1.9 Angstroms. This is the first structure of the newly established carbohydrate esterase family 15. The structure has revealed the residues Ser278–His411–Glu301 pre...

Changes of oxidase and hydrolase activities in pecan leaves elicited by black pecan aphid (Hemiptera: Aphididae) feeding.

PubMed

Chen, Yigen; Ni, Xinzhi; Cottrell, Ted E; Wood, Bruce W; Buntin, G David

2009-06-01

The black pecan aphid, Melanocallis caryaefoliae (Davis) (Hemiptera: Aphididae), is a foliar feeder of pecan, Carya illinoinensis (Wangenh.) K. Koch (Juglandaceae). The pest causes chlorosis of leaflet lamina, physiological damage to foliage and trees, and commonly limits the profitability of commercial pecan orchard enterprises. However, key aspects of this host-pest interaction are poorly understood. We report here the effects of M. caryaefoliae feeding on the foliar activity of oxidative (i.e., catalase, lipoxygenase [LOX]-1 and 3, and peroxidase) and hydrolytic (i.e., esterase) enzymes in relation to the degree of aphid resistance among pecan varieties. The 2-yr study showed that M. caryaefoliae-infested foliage exhibited elevated peroxidase activity only in susceptible ('Desirable', 'Sumner', and 'Schley'), but not in resistant ('Cape Fear', 'Gloria Grande', and 'Money Maker') genotypes. Susceptible genotypes also exhibited more severe leaf chlorosis in response to M. caryaefoliae feeding than the resistant genotypes; however, the aphid feeding did not influence catalase or esterase activity in all varieties, except the increase of esterase activity in Desirable and Gloria Grande. Melanocallis caryaefoliae feeding also influences activity of two lipoxygenase isozymes, with LOX3 being more frequently induced than LOX1. Foliar LOX3 activity was more frequently induced by M. caryaefoliae feeding in the moderately resistant 'Oconee' and highly resistant Money Maker and Cape Fear than in the susceptible genotypes. Therefore, the elevation of peroxidase is likely to be associated with aphid susceptibility and contributed to the severe leaf chlorosis, whereas the increase of LOX3 activity might be associated with aphid resistance in pecan. These findings contribute to our understanding of the etiology of M. caryaefoliae-elicited leaf chlorosis on pecan foliage. Such information may also be used to develop enzyme markers for identifying black pecan aphid resistance and/or susceptibility in pecan germplasm.

Absorption enhancement of adefovir dipivoxil by incorporating MCT and ethyl oleate complex oil phase in emulsion

PubMed Central

Li, Ping; Yu, Hong-zhen; Zhang, Xin-xin; Gan, Li; Zhu, Chun-liu; Gan, Yong

2010-01-01

Aim: To improve the oral absorption of adefovir dipivoxil (ADV) by employing MCT and the esterase inhibitor ethyl oleate (EO) as a complex oil phase in emulsion. Methods: EO was used as the esterase inhibitor, and its inhibitory effect on esterase activity was assessed in rat intestinal homogenates. ADV emulsions with or without EO were prepared. The emulsions' protective effect against intestinal metabolism was evaluated in rat luminal contents, ex vivo, as well as in vivo. Results: The IC50 of EO in intestinal mucosal homogenates was 2.2 mg/mL. The emulsions exhibited significant protective effects in rat luminal contents compared to a simple suspension (98.7%, 96.3%, 95.7% vs 74.7%, P<0.01). The permeability calculated from the emulsion containing EO was significantly different (11.4×10−6 vs 7.4/8.0×10−6, P<0.05) from the simple suspension or the emulsion without EO in an ex vivo assay. A bioavailability study in vivo revealed that emulsions containing both EO and MCT as a complex oil phase demonstrated 1.6- and 1.5-fold enhancements in area under the curve (AUC0–12) values (5358 vs 3386/3618, P<0.05), respectively, when compared with emulsions containing EO or MCT as a single oil phase. Conclusion: Heterotic lipid formulations (emulsions) with an esterase inhibitor (ie, EO) may be useful in protecting ester prodrugs from intestinal metabolism and increasing their oral bioavailability. PMID:20562905

Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

PubMed

Pereira, Mariana Rangel; Mercaldi, Gustavo Fernando; Maester, Thaís Carvalho; Balan, Andrea; Lemos, Eliana Gertrudes de Macedo

2015-01-01

Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

Enzymatic production of ferulic acid from defatted rice bran by using a combination of bacterial enzymes.

PubMed

Uraji, Misugi; Kimura, Masayo; Inoue, Yosikazu; Kawakami, Kayoko; Kumagai, Yuya; Harazono, Koichi; Hatanaka, Tadashi

2013-11-01

Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an α-L-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.

Methodological constraints in interpreting serum paraoxonase-1 activity measurements: an example from a study in HIV-infected patients.

PubMed

Parra, Sandra; Marsillach, Judit; Aragonès, Gerard; Rull, Anna; Beltrán-Debón, Raúl; Alonso-Villaverde, Carlos; Joven, Jorge; Camps, Jordi

2010-03-25

Paraoxonase-1 (PON1) is an antioxidant enzyme that attenuates the production of the monocyte chemoattractant protein-1 (MCP-1) in vitro. Although oxidation and inflammation are closely related processes, the association between PON1 and MCP-1 has not been completely characterised due, probably, to that the current use of synthetic substrates for PON1 measurement limits the interpretation of the data. In the present study, we explored the relationships between the circulating levels of PON1 and MCP-1 in human immunodeficiency virus-infected patients in relation to the multifunctional capabilities of PON1. We measured selected variables in 227 patients and in a control group of 409 participants. Serum PON1 esterase and lactonase activities were measured as the rates of hydrolysis of paraoxon and of 5-(thiobutyl)-butyrolactone, respectively. Oxidised LDL and MCP-1 concentrations were determined by enzyme-linked immunosorbent assay. High-density lipoproteins cholesterol, apolipoprotein A-I, and C-reactive protein concentrations were measured by standard automated methods. There were significant relationships between PON1 activity and several indices of oxidation and inflammation in control subjects and in infected patients. However, these relationships varied not only with disease status but also on the type of substrate used for PON1 measurement. The present study is a cautionary tale highlighting that results of clinical studies on PON1 may vary depending on the methods used as well as the disease studied. Until more specific methods using physiologically-akin substrates are developed for PON1 measurement, we suggest the simultaneous employment of at least two different substrates in order to improve the reliability of the results obtained.

Accelerated hydrolysis of substituted cellulose for potential biofuel production: kinetic study and modeling.

PubMed

Mu, Bingnan; Xu, Helan; Yang, Yiqi

2015-11-01

In this work, kinetics of substitution accelerated cellulose hydrolysis with multiple reaction stages was investigated to lay foundation for mechanism study and molecular design of substituting compounds. High-efficiency hydrolysis of cellulose is critical for cellulose-based bioethanol production. It is known that, substitution could substantially decrease activation energy and increase reaction rate of acidic hydrolysis of glycosidic bonds in cellulose. However, reaction kinetics and mechanism of the accelerated hydrolysis were not fully revealed. In this research, it was proved that substitution therefore accelerated hydrolysis only occurred in amorphous regions of cellulose fibers, and was a process with multiple reaction stages. With molar ratio of substitution less than 1%, the overall hydrolysis rate could be increased for around 10 times. We also quantified the relationship between the hydrolysis rate of individual reaction stage and its major influences, including molar ratio of substitution, activation energy of acidic hydrolysis, pH and temperature. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Colletotrichum gloeosporioides-induced esterase gene of nonclimacteric pepper (Capsicum annuum) fruit during ripening plays a role in resistance against fungal infection.

PubMed

Ko, Moon Kyung; Jeon, Woong Bae; Kim, Kwang Sang; Lee, Hyun Hwa; Seo, Hyo Hyoun; Kim, Young Soon; Oh, Boung-Jun

2005-07-01

Ripe fruits of pepper (Capsicum annuum) are resistant to the anthracnose fungus, Colletotrichum gloeosporioides, whereas unripe-mature fruits are susceptible. A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between the ripe fruit of pepper and C. gloeosporioides was previously cloned. Deduced amino acid sequence of PepEST cDNA showed homology to both esterases and lipases, and contained -HGGGF- and -GXSXG- motifs and a catalytic triad. Inhibition of PepEST activity by a specific inhibitor of serine hydrolase demonstrated that a serine residue is critical for the enzyme activity. Expression of PepEST gene was fruit-specific in response to C. gloeosporioides inoculation, and up-regulated by wounding or jasmonic acid treatment during ripening. PepEST mRNA and protein was differentially accumulated in ripe vs. unripe fruit from 24 h after inoculation when C. gloeosporioides is invading into fruits. Immunochemical examination revealed that PepEST accumulation was localized in epidermal and cortical cell layers in infected ripe fruit, but rarely even in epidermal cells in infected unripe one. Over-expression of PepEST in transgenic Arabidopsis plants caused restriction of Alternaria brassicicola colonization by inhibition of spore production, resulting in enhanced resistance against A.brassicicola. These results suggest that PepEST is involved in the resistance of ripe fruit against C.gloeosporioides infection.

Characterization of a Novel Subgroup of Extracellular Medium-Chain-Length Polyhydroxyalkanoate Depolymerases from Actinobacteria

PubMed Central

Gangoiti, Joana; Santos, Marta; Prieto, María Auxiliadora; de la Mata, Isabel; Llama, María J.

2012-01-01

Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases. PMID:22865072

Assessment of erythrocyte acetylcholine esterase activities in painters.

PubMed

Khan, Mohd Imran; Mahdi, Abbas Ali; Islam, Najmul; Rastogi, Subodh Kumar; Negi, M P S

2009-04-01

Thirty-five male painters in the age group of 20-50 years occupationally engaged in domestic and commercial painting for 5-12 years having blood lead levels (BLL)

THE INHIBITION OF PLASMIN, PLASMA KALLIKREIN, PLASMA PERMEABILITY FACTOR, AND THE C'1r SUBCOMPONENT OF THE FIRST COMPONENT OF COMPLEMENT BY SERUM C'1 ESTERASE INHIBITOR

PubMed Central

Ratnoff, Oscar D.; Pensky, Jack; Ogston, Derek; Naff, George B.

1969-01-01

The fraction of human serum designated as C'1 esterase inhibitor is known to inhibit the action of C'1 esterase, a plasma kallikrein, and PF/Dil, an enzyme in plasma enhancing cutaneous vascular permeability. In the present study, C'1 esterase inhibitor has been found to block the actions of plasmin and the C'1r subcomponent of the first component of complement, and to retard the generation of PF/Dil. No inhibition of blood clotting or of the generation of plasmin was demonstrable. PMID:4178758

The biological activity of alpha-mangostin, a larvicidal botanic mosquito sterol carrier protein-2 inhibitor.

PubMed

Larson, Ryan T; Lorch, Jeffrey M; Pridgeon, Julia W; Becnel, James J; Clark, Gary G; Lan, Que

2010-03-01

alpha-Mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening, alpha-Mangostin was tested for its larvicidal activity against third instar larvae of six mosquito species, and the median lethal concentration values range from 0.84 to 2.90 ppm. The residual larvicidal activity of alpha-mangostin was examined under semifield conditions. The results indicated that alpha-mangostin was photolytic with a half-life of 53 min in water under full sunlight exposure. The effect of alpha-mangostin on activities of major detoxification enzymes such as P450, glutathione S-transferase, and esterase was investigated. The results showed that alpha-mangostin significantly elevated activities of P450 and glutathione S-transferase in larvae, whereas it suppressed esterase activity. Toxicity of alpha-mangostin against young rats was studied, and there was no detectable adverse effect at dosages as high as 80 mg/kg. This is the first multifaceted study of the biological activity of alpha-mangostin in mosquitoes. The results suggest that alpha-mangostin may be a lead compound for the development of a new organically based mosquito larvicide.

Interactive toxicity of chlorpyrifos and parathion in neonatal rats: Role of esterases in exposure sequence-dependent toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kacham, R.; Karanth, S.; Baireddy, P.

2006-01-15

We previously reported that sequence of exposure to chlorpyrifos and parathion in adult rats can markedly influence toxic outcome. In the present study, we evaluated the interactive toxicity of chlorpyrifos (8 mg/kg, po) and parathion (0.5 mg/kg, po) in neonatal (7 days old) rats. Rats were exposed to the insecticides either concurrently or sequentially (separated by 4 h) and sacrificed at 4, 8, and 24 h after the first exposure for biochemical measurements (cholinesterase activity in brain, plasma, and diaphragm and carboxylesterase activity in plasma and liver). The concurrently-exposed group showed more cumulative lethality (15/24) than either of the sequentialmore » dosing groups. With sequential dosing, rats treated initially with chlorpyrifos prior to parathion (C/P) exhibited higher lethality (7/23) compared to those treated with parathion before chlorpyrifos (P/C; 1/24). At 8 h after initial dosing, brain cholinesterase inhibition was significantly greater in the C/P group (59%) compared to the P/C group (28%). Diaphragm and plasma cholinesterase activity also followed a relatively similar pattern of inhibition. Carboxylesterase inhibition in plasma and liver was relatively similar among the treatment groups across time-points. Similar sequence-dependent differences in brain cholinesterase inhibition were also noted with lower binary exposures to chlorpyrifos (2 mg/kg) and parathion (0.35 mg/kg). In vitro and ex vivo studies compared relative oxon detoxification of carboxylesterases (calcium-insensitive) and A-esterases (calcium-sensitive) in liver homogenates from untreated and insecticide pretreated rats. Using tissues from untreated rats, carboxylesterases detoxified both chlorpyrifos oxon and paraoxon, while A-esterases only detoxified chlorpyrifos oxon. With parathion pretreatment, A-esterases still detoxified chlorpyrifos oxon while liver from chlorpyrifos pretreated rats had little apparent effect on paraoxon. We conclude that while neonatal rats are less capable than adults at detoxifying many organophosphorus insecticides including chlorpyrifos and parathion, toxicant-selective differences in detoxification play a role in sequence-dependent toxicity in both neonatal and adult rats with these two insecticides.« less

Plasma esterases in the tegu lizard Tupinambis merianae (Reptilia, Teiidae): impact of developmental stage, sex, and organophosphorus in vitro exposure.

PubMed

Basso, Agustín; Attademo, Andrés M; Lajmanovich, Rafael C; Peltzer, Paola M; Junges, Celina; Cabagna, Mariana C; Fiorenza, Gabriela S; Sanchez-Hernandez, Juan Carlos

2012-01-01

In this study, we determined normal serum butyrylcholinesterase (BChE) and carboxylesterase (CbE) activities in Tupinambis merianae in order to obtain reference values for organophosphorus pesticide monitoring. Forty-two T. merianae individuals were grouped by sex and size to identify potential differences in their enzyme levels to allow for proper representation of normal values for females, males, juveniles, and hatchlings. Mean CbE was determined using two model substrates: alpha-naphtylacetate (α-NA) and p-nitrophenyl valerate (4-NPV). BChE and CbE sensitivity to malaoxon (Mx) was also evaluated as well as the possibility of BChE reactivation with pyridine-2-aldoxime methochloride (2-PAM). Mean adult females' BChE was significantly higher than adult males, juveniles, and hatchlings. No significant differences were found between groups regarding CbE. CbE (4-NPV) activity showed slightly negative correlation with lizard snout-vent length, while BChE and CbE (α-NA) showed no correlation with body size. Apparent IC(50) values for BChE and CbE (α-NA) suggested different sensitivities among groups. CbE (4-NPV) could not be inhibited. All Mx-inhibited groups treated with 2-PAM in a final concentration of 2.8 mM showed clear signs of reactivation. In conclusion, the results demonstrate that (1) plasma esterase activity did not vary with age and sex, except for BChE activity, and (2) because biological and environmental variables could be confounding factors in the response of plasma cholinesterases, complementary biomarkers like CbE inhibition and oxime-induced reactivation of esterases are strongly recommended.

Understanding how the complex molecular architecture of mannan-degrading hydrolases contributes to plant cell wall degradation.

PubMed

Zhang, Xiaoyang; Rogowski, Artur; Zhao, Lei; Hahn, Michael G; Avci, Utku; Knox, J Paul; Gilbert, Harry J

2014-01-24

Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.

Species differences in the hydrolysis of 2-cyanoethylene oxide, the epoxide metabolite of acrylonitrile.

PubMed

Kedderis, G L; Batra, R

1993-04-01

The carcinogenic effects of acrylonitrile in rats are believed to be mediated by its DNA-reactive epoxide metabolite, 2-cyanoethylene oxide (CEO). Previous studies have shown that conjugation with glutathione is the major detoxication pathway for both acrylonitrile and CEO. This study investigated the role of epoxide hydrolase in the hydrolysis of CEO by HPLC analysis of the products from [2,3-14C]CEO. CEO is a relatively stable epoxide with a half-life of 99 min at 37 degrees C in sodium phosphate buffer (0.1 M), pH 7.3. Incubation with hepatic microsomes or cytosols from male F-344 rats or B6C3F1 mice did not enhance the rate of hydrolysis of CEO (0.69 nmol/min). Human hepatic microsomes significantly increased the rate of hydrolysis of CEO, whereas human hepatic cytosols did not. Human hepatic microsomal hydrolysis activity was heat-sensitive and potently inhibited by 1,1,1-trichloropropene oxide (IC50 of 23 microM), indicating that epoxide hydrolase was the catalyst. The hydrolysis of CEO catalyzed by hepatic microsomes from six individuals exhibited normal saturation kinetics with KM ranging from 0.6 to 3.2 mM and Vmax from 8.3 to 18.8 nmol hydrolysis products/min/mg protein. Pretreatment of rodents with phenobarbital or acetone induced hepatic microsomal hydrolysis activity toward CEO, whereas treatment with beta-naphthoflavone, dexamethasone or acrylonitrile itself was without effect. These data show that humans possess an additional detoxication pathway for CEO that is not active in rodents (but is inducible). The presence of an active epoxide hydrolase hydrolysis activity toward CEO in humans should be considered in assessments of cancer risk from acrylonitrile exposure.

Simultaneous measurement of two enzyme activities using infrared spectroscopy: A comparative evaluation of PARAFAC, TUCKER and N-PLS modeling.

PubMed

Baum, Andreas; Hansen, Per Waaben; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

2013-08-06

Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which are active on specific substrates generating a multitude of products. In this paper we use the plant polymer, pectin, to present a method to quantify enzyme activity of two pectolytic enzymes by monitoring their superimposed spectral evolutions simultaneously. The data is analyzed by three chemometric multiway methods, namely PARAFAC, TUCKER3 and N-PLS, to establish simultaneous enzyme activity assays for pectin lyase and pectin methyl esterase. Correlation coefficients Rpred(2) for prediction test sets are 0.48, 0.96 and 0.96 for pectin lyase and 0.70, 0.89 and 0.89 for pectin methyl esterase, respectively. The retrieved models are compared and prediction test sets show that especially TUCKER3 performs well, even in comparison to the supervised regression method N-PLS. Copyright © 2013 Elsevier B.V. All rights reserved.

Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture.

PubMed

Ross, Heather A; Wright, Kathryn M; McDougall, Gordon J; Roberts, Alison G; Chapman, Sean N; Morris, Wayne L; Hancock, Robert D; Stewart, Derek; Tucker, Gregory A; James, Euan K; Taylor, Mark A

2011-01-01

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties.

Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture

PubMed Central

Ross, Heather A.; Wright, Kathryn M.; McDougall, Gordon J.; Roberts, Alison G.; Chapman, Sean N.; Morris, Wayne L.; Hancock, Robert D.; Stewart, Derek; Tucker, Gregory A.; James, Euan K.; Taylor, Mark A.

2011-01-01

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties. PMID:20855456

Standard Gibbs energy of metabolic reactions: II. Glucose-6-phosphatase reaction and ATP hydrolysis.

PubMed

Meurer, Florian; Do, Hoang Tam; Sadowski, Gabriele; Held, Christoph

2017-04-01

ATP (adenosine triphosphate) is a key reaction for metabolism. Tools from systems biology require standard reaction data in order to predict metabolic pathways accurately. However, literature values for standard Gibbs energy of ATP hydrolysis are highly uncertain and differ strongly from each other. Further, such data usually neglect the activity coefficients of reacting agents, and published data like this is apparent (condition-dependent) data instead of activity-based standard data. In this work a consistent value for the standard Gibbs energy of ATP hydrolysis was determined. The activity coefficients of reacting agents were modeled with electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT). The Gibbs energy of ATP hydrolysis was calculated by combining the standard Gibbs energies of hexokinase reaction and of glucose-6-phosphate hydrolysis. While the standard Gibbs energy of hexokinase reaction was taken from previous work, standard Gibbs energy of glucose-6-phosphate hydrolysis reaction was determined in this work. For this purpose, reaction equilibrium molalities of reacting agents were measured at pH7 and pH8 at 298.15K at varying initial reacting agent molalities. The corresponding activity coefficients at experimental equilibrium molalities were predicted with ePC-SAFT yielding the Gibbs energy of glucose-6-phosphate hydrolysis of -13.72±0.75kJ·mol -1 . Combined with the value for hexokinase, the standard Gibbs energy of ATP hydrolysis was finally found to be -31.55±1.27kJ·mol -1 . For both, ATP hydrolysis and glucose-6-phosphate hydrolysis, a good agreement with own and literature values were obtained when influences of pH, temperature, and activity coefficients were explicitly taken into account in order to calculate standard Gibbs energy at pH7, 298.15K and standard state. Copyright © 2017 Elsevier B.V. All rights reserved.

Intracellular Signaling by Hydrolysis of Phospholipids and Activation of Protein Kinase C

NASA Astrophysics Data System (ADS)

Nishizuka, Yasutomi

1992-10-01

Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels. This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids, particularly choline phospholipids, by phospholipase D and phospholipase A_2 may also take part in cell signaling. The products of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.

The Hydrolysis of Carbonyl Sulfide at Low Temperature: A Review

PubMed Central

Zhao, Shunzheng; Yi, Honghong; Tang, Xiaolong; Jiang, Shanxue; Gao, Fengyu; Zhang, Bowen; Zuo, Yanran; Wang, Zhixiang

2013-01-01

Catalytic hydrolysis technology of carbonyl sulfide (COS) at low temperature was reviewed, including the development of catalysts, reaction kinetics, and reaction mechanism of COS hydrolysis. It was indicated that the catalysts are mainly involved metal oxide and activated carbon. The active ingredients which can load on COS hydrolysis catalyst include alkali metal, alkaline earth metal, transition metal oxides, rare earth metal oxides, mixed metal oxides, and nanometal oxides. The catalytic hydrolysis of COS is a first-order reaction with respect to carbonyl sulfide, while the reaction order of water changes as the reaction conditions change. The controlling steps are also different because the reaction conditions such as concentration of carbonyl sulfide, reaction temperature, water-air ratio, and reaction atmosphere are different. The hydrolysis of carbonyl sulfide is base-catalyzed reaction, and the force of the base site has an important effect on the hydrolysis of carbonyl sulfide. PMID:23956697

Requirement for Pectin Methyl Esterase and Preference for Fragmented over Native Pectins for Wall-associated Kinase-activated, EDS1/PAD4-dependent Stress Response in Arabidopsis*

PubMed Central

Kohorn, Bruce D.; Kohorn, Susan L.; Saba, Nicholas J.; Martinez, Victoriano Meco

2014-01-01

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. PMID:24855660

Enhancement of the Rate of Pyrophosphate Hydrolysis by Nonenzymatic Catalysts and by Inorganic Pyrophosphatase*

PubMed Central

Stockbridge, Randy B.; Wolfenden, Richard

2011-01-01

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, 31P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PPi) in the presence and absence of Mg2+ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPPi2− proceeds with a rate constant of 2.8 × 10−10 s−1, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s−1 under the same conditions. The resulting rate enhancement (2 × 1012-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH‡ = −16.6 kcal/mol). The presence of Mg2+ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼106-fold. Transfer to dimethyl sulfoxide accelerated PPi hydrolysis by reducing the enthalpy of activation. Mg2+ increased the rate of PPi hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation. PMID:21460215

The diagnostic accuracy of the rapid dipstick test to predict asymptomatic urinary tract infection of pregnancy.

PubMed

Eigbefoh, J O; Isabu, P; Okpere, E; Abebe, J

2008-07-01

Untreated urinary tract infection can have devastating maternal and neonatal effects. Thus, routine screening for bacteriuria is advocated. This study was designed to evaluate the diagnostic accuracy of the rapid dipstick test to predict urinary tract infection in pregnancy with the gold standard of urine microscopy, culture and sensitivity acting as the control. The urine dipstick test uses the leucocyte esterase, nitrite and test for protein singly and in combination. The result of the dipstick was compared with the gold standard, urine microscopy, culture and sensitivity using confidence interval for proportions. The reliability and validity of the urine dipstick was also evaluated. Overall, the urine dipstick test has a poor correlation with urine culture (p = 0.125, CI 95%). The same holds true for individual components of the dipstick test. The overall sensitivity of the urine dipstick test was poor at 2.3%. Individual sensitivity of the various components varied between 9.1% for leucocyte esterase and the nitrite test to 56.8% for leucocyte esterase alone. The other components of the dipstick test, the test of nitrite, test for protein and combination of the test (leucocyte esterase, nitrite and proteinuria) appear to decrease the sensitivity of the leucocyte esterase test alone. The ability of the urine dipstick test to correctly rule out urinary tract infection (specificity) was high. The positive predictive value for the dipstick test was high, with the leucocyte esterase test having the highest positive predictive value compared with the other components of the dipstick test. The negative predictive value (NPV) was expectedly highest for the leucocyte esterase test alone with values higher than the other components of the urine dipstick test singly and in various combinations. Compared with the other parameters of the urine dipstick test, singly and in combination, leucocyte esterase appears to be the most accurate (90.25%). The dipstick test has a limited use in screening for asymptomatic bacteriuria. The leucocyte esterase test component of the dipstick test appears to have the highest reliability and validity. The other parameters of the dipstick test decreases the reliability and validity of the leucocyte esterase test. A positive test merits empirical antibiotics, while a negative test is an indication for urine culture. The urine dipstick test if positive will also be useful in follow-up of patient after treatment of urinary tract infection. This is useful in poor resource setting especially in the third world where there is a dearth of trained personnel and equipment for urine culture.

Heterologous production and characterization of a chlorogenic acid esterase from Ustilago maydis with a potential use in baking.

PubMed

Nieter, Annabel; Kelle, Sebastian; Takenberg, Meike; Linke, Diana; Bunzel, Mirko; Popper, Lutz; Berger, Ralf G

2016-10-15

Ustilago maydis, an edible mushroom growing on maize (Zea mays), is consumed as the food delicacy huitlacoche in Mexico. A chlorogenic acid esterase from this basidiomycete was expressed in good yields cultivating the heterologous host Pichia pastoris on the 5L bioreactor scale (reUmChlE; 45.9UL(-1)). In contrast to previously described chlorogenic acid esterases, the reUmChlE was also active towards feruloylated saccharides. The enzyme preferred substrates with the ferulic acid esterified to the O-5 position of arabinose residues, typical of graminaceous monocots, over the O-2 position of arabinose or the O-6 position of galactose residues. Determination of kcat/Km showed that the reUmChlE hydrolyzed chlorogenic acid 18-fold more efficiently than methyl ferulate, p-coumarate or caffeate. Phenolic acids were released by reUmChlE from natural substrates, such as destarched wheat bran, sugar beet pectin and coffee pulp. Treatment of wheat dough using reUmChlE resulted in a noticeable softening indicating a potential application of the enzyme in bakery and confectionery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

PubMed

Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

2016-06-28

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

Isozvme specificity during germination and early growth of knobcone pine

Treesearch

M. Thompson Conkle

1971-01-01

Five enzyme classes from 11 developmental stages of germinating embryos were separated by starch gel electrophoresis. Alcohol dehydrogenase isozymes found in embryos of dry seed were most active at the time of radicle emergence; activity decreased thereafter, fading below the level of detection when seed coats were shed. Peroxidase isozymes were absent and esterase...

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase.

PubMed

Zwane, Eunice N; van Zyl, Petrus J; Duodu, Kwaku G; Rose, Shaunita H; Rumbold, Karl; van Zyl, Willem H; Viljoen-Bloom, Marinda

2017-03-01

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487 U/mg, K m  = 0.43 mM, K cat  = 0.48/min on methyl ferulate). The 36-kDa At FAEA protein showed maximum ferulic acid esterase activity at 50 °C and pH 6, suggesting potential application in industrial processes. A crude At FAEA preparation extracted 26.56 and 8.86 mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p -coumaric and caffeic acid from triticale bran. The cost-effective production of At FAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p -coumaric acid) from plant substrates. The potential for larger-scale production of At FAEA was demonstrated with the A. niger D15[ AtfaeA ] strain yielding a higher enzyme activity (185.14 vs. 83.48 U/ml) and volumetric productivity (3.86 vs. 1.74 U/ml/h) in fed-batch than batch fermentation.

Structure-activity correlations for organophosphorus ester anticholinesterases. Part 2: CNDO/2 calculations applied to ester hydrolysis rates

NASA Technical Reports Server (NTRS)

Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

1984-01-01

Quantitative structure-activity relationships are presented for the hydrolysis of organophosphorus esters, RR'P(O)X, where R and R' are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. CNDO/2 calculations provide values for molecular parameters that correlate with alkaline hydrolysis rates. For each subset of esters with the same leaving group, X, the CNDO-derived net atomic charge at the central phosphorus atom correlates well with the alkaline hydrolysis rate constants. For the whole set of esters with different leaving groups, equations are derived that relate charge, orbital energy and bond order to the hydrolysis rate constants.

Study of the kinetic parameters for synthesis and hydrolysis of pharmacologically active salicin isomer catalyzed by baker's yeast maltase

NASA Astrophysics Data System (ADS)

Veličković, D. V.; Dimitrijević, A. S.; Bihelović, F. J.; Jankov, R. M.; Milosavić, N.

2011-12-01

One of the key elements for understanding enzyme reactions is determination of its kinetic parameters. Since transglucosylation is kinetically controlled reaction, besides the reaction of synthesis, very important is the reaction of enzymatic hydrolysis of created product. Therefore, in this study, kinetic parameters for synthesis and secondary hydrolysis of pharmacologically active α isosalicin by baker's yeast maltase were calculated, and it was shown that specifity of maltase for hydrolysis is approximately 150 times higher then for synthesis.

NeuA sialic acid O-acetylesterase activity modulates O-acetylation of capsular polysaccharide in group B Streptococcus.

PubMed

Lewis, Amanda L; Cao, Hongzhi; Patel, Silpa K; Diaz, Sandra; Ryan, Wesley; Carlin, Aaron F; Thon, Vireak; Lewis, Warren G; Varki, Ajit; Chen, Xi; Nizet, Victor

2007-09-21

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) was enhanced by CTP and Mg(2+), the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac(2) followed by CMP activation of Neu5Ac or activation of Neu5,9Ac(2) followed by de-O-acetylation of CMP-Neu5,9Ac(2). Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.

Antioxidant, antihypertensive and antimicrobial properties of ovine milk caseinate hydrolyzed with a microbial protease.

PubMed

Corrêa, Ana Paula F; Daroit, Daniel J; Coelho, Julise; Meira, Stela M M; Lopes, Fernanda C; Segalin, Jéferson; Risso, Patrícia H; Brandelli, Adriano

2011-09-01

Bioactive peptides might be released from precursor proteins through enzymatic hydrolysis. These molecules could be potentially employed in health and food products. In this investigation, ovine milk caseinate hydrolysates obtained with a novel microbial protease derived from Bacillus sp. P7 were evaluated for antioxidant, antimicrobial, and angiotensin I-converting enzyme (ACE)-inhibitory activities. Antioxidant activity measured by the 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid method increased with hydrolysis time up to 2 h, remaining stable for up to 4 h. Hydrolysates showed low 2,2-diphenyl-1-picrylhydrazyl radical-scavenging abilities, with higher activity (31%) reached after 1 h of hydrolysis. Fe(2+) -chelating ability was maximum for 0.5 h hydrolysates (83.3%), decreasing thereafter; and the higher reducing power was observed after 1 h of hydrolysis. ACE-inhibitory activity was observed to increase up to 2 h of hydrolysis (94% of inhibition), declining afterwards. 3 h hydrolysates were shown to inhibit the growth of Bacillus cereus, Corynebacterium fimi, Aspergillus fumigatus, and Penicillium expansum. Ovine caseinate hydrolyzed with Bacillus sp. P7 protease presented antioxidant, antihypertensive, and antimicrobial activities. Hydrolysis time was observed to affect the evaluated bioactivities. Such hydrolysates might have potential applications in the food industry. Copyright © 2011 Society of Chemical Industry.

Ubiquitinated Proteins Activate the Proteasomal ATPases by Binding to Usp14 or Uch37 Homologs*

PubMed Central

Peth, Andreas; Kukushkin, Nikolay; Bossé, Marc; Goldberg, Alfred L.

2013-01-01

Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction. PMID:23341450

The role of adhesive materials and oral biofilm in the failure of adhesive resin restorations.

PubMed

Pinna, Roberto; Usai, Paolo; Filigheddu, Enrica; Garcia-Godoy, Franklin; Milia, Egle

2017-10-01

To critically discuss adhesive materials and oral cariogenic biofilm in terms of their potential relevance to the failures of adhesive restorations in the oral environment. The literature regarding adhesive restoration failures was reviewed with particular emphasis on the chemistry of adhesive resins, weakness in dentin bonding, water fluids, cariogenic oral biofilm and the relations that influence failures. Particular attention was paid to evidence derived from clinical studies. There was much evidence that polymerization shrinkage is one of the main drawbacks of composite formulations. Stress results in debonding and marginal leakage into gaps with deleterious effects in bond strength, mechanical properties and the whole stability of restorations. Changes in resins permit passage of fluids and salivary proteins with a biological breakdown of the restorations. Esterases enzymes in human saliva catalyze exposed ester groups in composite producing monomer by-products, which can favor biofilm accumulation and secondary caries. Adhesive systems may not produce a dense hybrid layer in dentin. Very often this is related to the high viscous solubility and low wettability in dentin of the hydrophobic BisGMA monomer. Thus, dentin hybrid layer may suffer from hydrolysis using both the Etch&Rinse and Self-Etching adhesive systems. In addition, exposed and non-resin enveloped collagen fibers may be degraded by activation of the host-derived matrix metalloproteinase. Plaque accumulation is significantly influenced by the surface properties of the restorations. Biofilm at the contraction gap has demonstrated increased growth of Streptococcus mutans motivated by the chemical hydrolysis of the adhesive monomers at the margins. Streptococcus mutans is able to utilize some polysaccharides from the biofilm to increase the amount of acid in dental plaque with an increase in virulence and destruction of restorations. Stability of resin restorations in the oral environment is highly dependent on the structure of the monomers used in composite and adhesive systems. Still, the issues related to microleakage of fluids into the gap and bacteria leaching from the surface of composites represent the main causes of failure of adhesive restorations. Modifications of adhesive materials are necessary to address their instability in the oral environment.

Effects of a simulated martian UV flux on the cyanobacterium, Chroococcidiopsis sp. 029.

PubMed

Cockell, Charles S; Schuerger, Andrew C; Billi, Daniela; Friedmann, E Imre; Panitz, Corinna

2005-04-01

Dried monolayers of Chroococcidiopsis sp. 029, a desiccation-tolerant, endolithic cyanobacterium, were exposed to a simulated martian-surface UV and visible light flux, which may also approximate to the worst-case scenario for the Archean Earth. After 5 min, there was a 99% loss of cell viability, and there were no survivors after 30 min. However, this survival was approximately 10 times higher than that previously reported for Bacillus subtilis. We show that under 1 mm of rock, Chroococcidiopsis sp. could survive (and potentially grow) under the high martian UV flux if water and nutrient requirements for growth were met. In isolated cells, phycobilisomes and esterases remained intact hours after viability was lost. Esterase activity was reduced by 99% after a 1-h exposure, while 99% loss of autofluorescence required a 4-h exposure. However, cell morphology was not changed, and DNA was still detectable by 4',6-diamidino-2-phenylindole staining after an 8-h exposure (equivalent to approximately 1 day on Mars at the equator). Under 1 mm of simulant martian soil or gneiss, the effect of UV radiation could not be detected on esterase activity or autofluorescence after 4 h. These results show that under the intense martian UV flux the morphological signatures of life can persist even after viability, enzymatic activity, and pigmentation have been destroyed. Finally, the global dispersal of viable, isolated cells of even this desiccation-tolerant, ionizing-radiation-resistant microorganism on Mars is unlikely as they are killed quickly by unattenuated UV radiation when in a desiccated state. These findings have implications for the survival of diverse microbial contaminants dispersed during the course of human exploratory class missions on the surface of Mars.

Insecticide resistance to permethrin and malathion and associated mechanisms in Aedes aegypti mosquitoes from St. Andrew Jamaica

PubMed Central

Francis, Sheena; Saavedra-Rodriguez, Karla; Perera, Rushika; Paine, Mark; Black, William C.

2017-01-01

The emergence of novel diseases spread by the Aedes aegypti mosquito in Jamaica and the Caribbean, has prompted studies on insecticide resistance towards effective management of the vector. Though Jamaica has been using the organophosphate insecticide malathion in its vector control program for more than 30 years, resistance to the pesticide has not been tested in over a decade. We analyzed resistance to malathion and the pyrethroid insecticide, permethrin on mosquitoes collected across St. Andrew, Jamaica, and analyzed the molecular basis of resistance. The Center for Disease Control (CDC) bioassay revealed that Ae. aegypti mosquitoes from St. Andrew, Jamaica were resistant to permethrin (15 μg/bottle) with mortalities at 0–8% at 30 minute exposure time, while contact with malathion (50 μg/bottle) revealed ≤ 50% mortality at 15 minutes, which increased to 100% at 45 minutes. The standard susceptible New Orleans (NO) strain exhibited 100% mortality within15 minutes. The activities of multifunction oxidases and p-nitro phenyl-acetate esterases were significantly greater in most Jamaican populations in comparison to the NO strain, while activities of glutathione-S-transferase, acetylcholinesterase, α-esterase and ß-esterase activity were relatively equal, or lower than that of the control strain. The frequency of knockdown resistance mutations in the voltage dependent sodium channel gene were measured. All collections were fixed for Cys1,534 while 56% of mosquitoes were Ile1,016/Val1,016 heterozygotes, and 33% were Ile1,016 homozygotes. Aedes aegypti from St. Andrew Jamaica are resistant to permethrin with variations in the mode of mechanism, and possibly developing resistance to malathion. Continued monitoring of resistance is critically important to manage the spread of the vector in the country. PMID:28650966

Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past.

PubMed

Ribera, Judit; Estupiñán, Mónica; Fuentes, Alba; Fillat, Amanda; Martínez, Josefina; Diaz, Pilar

2017-01-01

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.

Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past

PubMed Central

Ribera, Judit; Estupiñán, Mónica; Fuentes, Alba; Fillat, Amanda; Martínez, Josefina

2017-01-01

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions. PMID:28742841

Metabolic pathways and pharmacokinetics of natural medicines with low permeability.

PubMed

Zeng, Mei; Yang, Lan; He, Dan; Li, Yao; Shi, Mingxin; Zhang, Jingqing

2017-11-01

Drug metabolism plays an important role in the drug disposal process. Differences in pharmacokinetics among individuals are the basis for personalized medicine. Natural medicines, formed by long-term evolution of nature, prioritize the action of a target protein with a drug. Natural medicines are valued for structural diversity, low toxicity, low cost, and definite biological activities. Metabolic pathway and pharmacokinetic research of natural medicines is highly beneficial for clinical dose adjustment and the development of personalized medicine. This review was performed using a systematic search of all available literature. It provides an overview and discussion of metabolic pathways and the pharmacokinetics of natural medicines with low permeability. The related enzymes and factors affecting them are analyzed. The series of metabolic reactions, including phase I reactions(oxidation hydrolysis, and reduction reactions) and phase II reactions (binding reactions), catalyzed by intracellular metabolic enzymes (such as CYP450, esterase, SULT, and UGT enzymes) in tissues (such as liver and gastro-intestinal tract) or in the body fluid environment were examined. The administration route, drug dose, and delivery system had a large influence on absorption, metabolism, and pharmacokinetics. Natural medicines with low permeability had distinctive metabolisms and pharmacokinetics. The metabolic and in vivo kinetic properties were favorably modified by choosing suitable drug delivery systems, administration routes and drug doses, among other variables. This study provides valuable information for clinicians and pharmacists to guide patients safe, effective, and rational drug use. The research of metabolism and pharmacokinetics is significant in guiding personalized clinical medicine.

Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining.

PubMed

Fang, Yaowei; Wang, Shujun; Liu, Shu; Jiao, Yuliang

2015-09-01

An esterase gene, encoding a 325-amino-acid protein (SAestA), was mined form obligate marine actinomycete strain Salinispora arenicola CNP193 genome sequence. Phylogenetic analysis of the deduced amino acid sequence showed that the enzyme belonged to the family IV of lipolytic enzymes. The gene was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The molecular weight of His-tagged SAestA is ∼38 kDa. SAestA-His6 was active in a temperature (5-40 °C) and pH range (7.0-11.0), and maximal activity was determined at pH 9.0 and 30 °C. The activity was severely inhibited by Hg(2+), Cu(2+), and Zn(2+). In particular, this enzyme showed remarkable stability in presence of organic solvents (25%, v/v) with log P>2.0 even after incubation for 7 days. All these characteristics suggested that SAestA may be a potential candidate for application in industrial processes in aqueous/organic media. Copyright © 2015. Published by Elsevier B.V.

Esterases of laboratory-reared and field-collected cotton boll weevils, Anthonomus grandis Boh.: polymorphism of adult esterases and formal genetics of esterase II.

PubMed

Biggers, C J; Bancroft, H R

1977-04-01

The esterases of the cotton boll weevil were separated by polyacrylamide gel electrophoresis into four major regions. These were named Est I-IV in order of migration from anode to origin. Polymorphism was observed in all regions. The Est II region was shown to consist of no more than two bands (fast and slow). The inheritance of the fast and slow bands of Est II was demonstrated to be controlled by codominant autosomal alleles. Analysis of the gene frequency of the Est II region showed that one field population was consistent with the Hardy-Weinberg law (P = 0.995), while a second field population was not at equilibrium (P less than 0.001).

Overproduced esterases in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Vietnam.

PubMed

Pasteur, N; Marquine, M; Hoang, T H; Nam, V S; Failloux, A B

2001-09-01

The electrophoretic polymorphism of loci encoding for 10 enzymes was studied in Culex p. quinquefasciatus Say from six localities of Vietnam. The analysis of 11 "neutral genes" showed that differentiation among samples was low, but significant (Fst = 0.06), and significantly related to geographic distance between sample sites. These results are similar to those observed in other countries (Europe and west Africa). A single type of overproduced esterases (A2-B2) was observed, and its frequency was high (60-100%) in all samples. This situation is in sharp contrast with that observed in other countries of South East Asia (China, South Korea and Japan), where two or more types of overproduced esterases have been reported. A map summarizing the geographic distribution of Asian Cr. p. quinquefasciatus with overproduced esterases is provided.

Descriptive parameters for revealing substitution patterns of sugar beet pectins using pectolytic enzymes.

PubMed

Remoroza, C; Buchholt, H C; Gruppen, H; Schols, H A

2014-01-30

Enzymatic fingerprinting was applied to sugar beet pectins (SBPs) modified by either plant or fungal pectin methyl esterases and alkali catalyzed de-esterification to reveal the ester distributions over the pectin backbone. A simultaneous pectin lyase (PL) treatment to the commonly used endo-polygalacturonase (endo-PG) degradation showed to be effective in degrading both high and low methylesterified and/or acetylated homogalaturonan regions of SBP simultaneously. Using LC-HILIC-MS/ELSD, we studied in detail all the diagnostic oligomers present, enabling us to discriminate between differently prepared sugar beet pectins having various levels of methylesterification and acetylation. Furthermore, distinction between commercially extracted and de-esterified sugar beet pectin having different patterns of substitution was achieved by using novel descriptive pectin parameters. In addition to DBabs approach for nonmethylesterified sequences degradable by endo-PG, the "degree of hydrolysis" (DHPG) representing all partially saturated methylesterified and/or acetylated galacturonic acid (GalA) moieties was introduced as a new parameter. Consequently, the description DHPL has been introduced to quantify all esterified unsaturated GalA oligomers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability

NASA Astrophysics Data System (ADS)

Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

2015-09-01

Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82_V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.

Heterologous Expression of Two Ferulic Acid Esterases from Penicillium Funiculosum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knoshaug, E. P.; Selig, M. J.; Baker, J. O.

2008-01-01

Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for theirmore » potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.« less

Heterologous Expression of Two Ferulic Acid Esterases from Penicillium funiculosum

NASA Astrophysics Data System (ADS)

Knoshaug, Eric P.; Selig, Michael J.; Baker, John O.; Decker, Stephen R.; Himmel, Michael E.; Adney, William S.

Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

Geomicrobiology of cores from Suruí Mangrove--Guanabara Bay--Brazil.

PubMed

Fontana, Luiz Francisco; Mendonça Filho, João Graciano; Netto, Annibal Duarte Pereira; Sabadini-Santos, Elisamara; de Figueiredo, Alberto Garcia; Crapez, Mirian Araújo Carlos

2010-10-01

The aim of this work was to quantify the biopolymers associated to esterase enzymes and identify bacterial respiratory activity in four cores collected in Suruí Mangrove, Guanabara Bay - RJ. Biopolymer concentration was 1000 times lower than previously reported in the literature, indicating the need for creating and establishing eutrophication indicative rates and records compatible with tropical coastal systems. The biochemical representative relationships in the cores were equivalent to those from studies on coastal marine environments made in the Northern Hemisphere. The esterase enzymes in the sediment proved efficient in the mineralization of biopolymers, even with preferentially anaerobic metabolic physiology. Despite the lack of incipient geomicrobiological studies, the results highlighted the possible application of microbiology to a better understanding of geological processes. Copyright © 2010 Elsevier Ltd. All rights reserved.

DOSE-RESPONSE MODELING FOR THE ASSESSMENT OF CUMULATIVE RISK DUE TO EXPOSURE TO N-METHYL CARBAMATE PESTICIDES

EPA Science Inventory

The US EPAs N-Methyl Carbamate Cumulative Risk Assessment (NMCRA) assesses the effect on acetylcholine esterase (AChE) activity of exposure to 10 N-methyl carbamate (NMC) pesticides through dietary, drinking water, and residential exposures.

A whole genome screening and RNA interference identify a juvenile hormone esterase-like gene of the diamondback moth, Plutella xylostella.

PubMed

Gu, Xiaojun; Kumar, Sunil; Kim, Eunjin; Kim, Yonggyun

2015-09-01

Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis. A whole genome of the diamondback moth (DBM), Plutella xylostella, has been annotated and proposed 11 JHE candidates. Sequence analysis using conserved motifs commonly found in other JHEs proposed a putative JHE (Px004817). Px004817 (64.61 kDa, pI=5.28) exhibited a characteristic JHE expression pattern by showing high peak at the early last instar, at which JHE enzyme activity was also at a maximal level. RNA interference of Px004817 reduced JHE activity and interrupted pupal development with a significant increase of larval period. This study identifies Px004817 as a JHE-like gene of P. xylostella. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evidence of sibling species in the brown planthopper complex (Nilaparvata lugens) detected from short and long primer random amplified polymorphic DNA fingerprints.

PubMed

Latif, M A; Soon Guan, Tan; Mohd Yusoh, Omar; Siraj, Siti Shapor

2008-08-01

The inheritance of 31 amplicons from short and long primer RAPD was tested for segregating ratios in two families of the brown planthopper, Nilaparvata lugens, and they were found to be inherited in a simple Mendelian fashion. These markers could now be used in population genetics studies of N. lugens. Ten populations of N. lugens were collected from five locations in Malaysia. Each location had two sympatric populations. Cluster and principal coordinate analyses based on genetic distance along with AMOVA revealed that the rice-infesting populations (with high esterase activity) at five localities clustered together as a group, and Leersia-infesting populations (with low esterase activity) at the same localities formed another distinct cluster. Two amplicons from primers OPD03 (0.65 kb) and peh#6 (1.0 kb) could be considered diagnostic bands, which were fixed in the Leersia-infesting populations. These results represent evidence of a sibling species in the N. lugens complex.

Phenolic acid esterases, coding sequences and methods

DOEpatents

Blum, David L.; Kataeva, Irina; Li, Xin-Liang; Ljungdahl, Lars G.

2002-01-01

Described herein are four phenolic acid esterases, three of which correspond to domains of previously unknown function within bacterial xylanases, from XynY and XynZ of Clostridium thermocellum and from a xylanase of Ruminococcus. The fourth specifically exemplified xylanase is a protein encoded within the genome of Orpinomyces PC-2. The amino acids of these polypeptides and nucleotide sequences encoding them are provided. Recombinant host cells, expression vectors and methods for the recombinant production of phenolic acid esterases are also provided.

Phosphoester hydrolysis: the incoming substrate turns the bridging hydroxido nucleophile into a terminal one.

PubMed

Gouré, Eric; Carboni, Michaël; Troussier, Angélique; Lebrun, Colette; Pécaut, Jacques; Jacquot, Jean-François; Dubourdeaux, Patrick; Clémancey, Martin; Blondin, Geneviève; Latour, Jean-Marc

2015-05-26

Identifying the active nucleophile in hydrolysis reactions catalyzed by binuclear hydrolases is a recurrent problem and a matter of intense debate. We report on the phosphate ester hydrolysis by a Fe(III)Fe(II) complex of a binucleating ligand. This complex presents activities in the range of those observed for similar biomimetic compounds in the literature. The specific electronic properties of the Fe(III)Fe(II) complex allowed us to use (1)H NMR and Mössbauer spectroscopies to investigate the nature of the various species present in the solution in the pH range of 5-10. Both techniques showed that the hydrolysis activity is associated to a μ-hydroxido Fe(III)Fe(II) species. Further (1)H NMR experiments show that binding of anions or the substrate changes this bonding mode suggesting that a terminal hydroxide is the likely nucleophile in these hydrolysis reactions. This view is further supported by the structure determination of the hydrolysis product. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biological and chemical analysis of the toxic potency of pesticides in rainwater.

PubMed

Hamers, T; Smit, M G; Murk, A J; Koeman, J H

2001-11-01

A newly developed method for measuring the integrated esterase inhibiting potency of rainwater samples was applied in practice, and the results are compared to the toxic potency calculated from concentrations of 31 organophosphate (OP) and carbamate pesticides, out of a total of 66 chemically analyzed pesticides. In addition, the general toxic potency of the rainwater samples was evaluated in a microtiter luminescence assay with Vibrio fischeri bacteria. Rainwater samples were collected over four consecutive 14-day periods in both open and wet-only samplers. The esterase inhibiting potency of the open rainwater samples (expressed as ng dichlorvos-equivalents/l) corresponded well with the chemical analyses of the rainwater samples collected by both types of samplers (r = 0.83-0.86). By far, the highest esterase inhibiting potency was found in a sample collected in an area with intense horticultural activities in June, and was attributed to high concentrations of dichlorvos, mevinphos, pirimiphos-methyl and methiocarb. The esterase inhibiting potency of this sample was equivalent to a dichlorvos concentration of 1380 ng/l in the rainwater, which is almost 2000 times higher than the maximum permissible concentration (MPC) of dichlorvos set for surface water in Netherlands. Maximum individual concentrations of dichlorvos and pirimiphos-methyl even exceeded the EC50 for Daphnia, suggesting that pesticides in rainwater pose a risk for aquatic organisms. Not all responses of the luminescence-assay for general toxicity could be explained by the analyzed pesticide concentrations. The bio-assays enable a direct assessment the toxic potency of all individual compounds present in the complex mixture of rainwater pollutants, even if they are unknown or present at concentrations below the detection limit. Therefore, they are valuable tools for prescreening and hazard characterization purposes.

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

DOE PAGES

Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem; ...

2017-10-25

Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of thesemore » pruned enzymes with a series of substrates. A substantial rate enhancement of ~1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7–10 8-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.« less

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem

Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of thesemore » pruned enzymes with a series of substrates. A substantial rate enhancement of ~1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7–10 8-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.« less

Cholinesterases: structure of the active site and mechanism of the effect of cholinergic receptor blockers on the rate of interaction with ligands

NASA Astrophysics Data System (ADS)

Antokhin, A. M.; Gainullina, E. T.; Taranchenko, V. F.; Ryzhikov, S. B.; Yavaeva, D. K.

2010-10-01

Modern views on the structure of cholinesterase active sites and the mechanism of their interaction with organophosphorus inhibitors are considered. The attention is focused on the mechanism of the effect of cholinergic receptor blockers, acetylcholine antagonists, on the rate of interaction of acetylcholine esterase with organophosphorus inhibitors.

Differentiation-promoting activity of pomegranate (Punica granatum) fruit extracts in HL-60 human promyelocytic leukemia cells.

PubMed

Kawaii, Satoru; Lansky, Ephraim P

2004-01-01

Differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies. Flavonoids are a group of differentiation-inducing chemicals with a potentially lower toxicology profile than retinoids. Flavonoid-rich polyphenol fractions from the pomegranate (Punica granatum) fruit exert anti-proliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in vitro and in vivo. Here we tested flavonoid-rich fractions from fresh (J) and fermented (W) pomegranate juice and from an aqueous extraction of pomegranate pericarps (P) as potential differentiation-promoting agents of human HL-60 promyelocytic leukemia cells. Four assays were used to assess differentiation: nitro blue tetrazolium reducing activity, nonspecific esterase activity, specific esterase activity, and phagocytic activity. In addition, the effect of these extracts on HL-60 proliferation was evaluated. Extracts W and P were strong promoters of differentiation in all settings, with extract J showing only a relatively mild differentiation-promoting effect. The extracts had proportional inhibitory effects on HL-60 cell proliferation. The results highlight an important, previously unknown, mechanism of the cancer preventive and suppressive potential of pomegranate fermented juice and pericarp extracts.

Acetylcholine-hydrolyzing activities in soluble brain fraction: Characterization with reversible and irreversible inhibitors.

PubMed

Estévez, Jorge; Selva, Verónica; Benabent, Mónica; Mangas, Iris; Sogorb, Miguel Ángel; Vilanova, Eugenio

2016-11-25

Some effects of organophosphorus compounds (OPs) esters cannot be explained through actions on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In soluble chicken brain fraction, three components (Eα, Eβ and Eγ) of pheny lvalerate esterase activity (PVase) were kinetically discriminated and their relationship with acetylcholine-hydrolyzing activity (cholinesterase activity) were studied in previous works. In this work, four enzymatic components (CS1, CS2, CS3 and CS4) of cholinesterase activity have been discriminated in soluble fraction, according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA and to reversible inhibitors ethopropazine and BW284C51. Cholinesterase component CS1 can be related to the Eα component of PVase activity and identified as butyrylcholinesterase (BuChE). No association and similarities can be stablished among the other PVase component (Eβ and Eγ) with the other cholinesterase components (CS2, CS3, CS4). The kinetic analysis has allowed us to stablish a method for discriminating the enzymatic component based on a simple test with two inhibitors. It can be used as biomarker in toxicological studies and for monitoring these cholinesterase components during isolation and molecular identification processes, which will allow OP toxicity to be understood by a multi-target approach. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Survival and persistence of nonspore-forming biothreat agents in water.

PubMed

Gilbert, S E; Rose, L J

2012-09-01

To determine whether nonspore-forming biothreat agents can survive and persist in potable water that does not contain a disinfectant.  Autoclaved, de-chlorinated Atlanta municipal water was inoculated with eight isolates of bacterial biothreat agents (10⁶ CFU ml⁻¹). The inoculated water samples were incubated at 5, 8 (Francisella tularensis only) or 25°C and assayed for viability by culture and by the presence of metabolic activity as measured by esterase activity (ScanRDI, AES Chemunex). Viability as determined by culture varied from 1 to 30 days, depending upon the organism and the temperature of the water. All organisms were determined viable as measured by esterase activity for the entire 30 days, regardless of the incubation temperature.  Francisella tularensis was culturable for at least 21 days if held at 8°C. The remaining nonspore-forming bacterial biothreat agents were found to be metabolically active for at least 30 days in water held at 5 or 25°C.  The data can assist public health officials to determine the safety of drinking water after contamination with a biothreat agent. No claim to US Government works. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics

PubMed Central

Shirke, Abhijit N.; Basore, Danielle; Holton, Samantha; Su, An; Baugh, Evan; Butterfoss, Glenn L.; Makhatadze, George

2016-01-01

Cutinases are esterases of industrial importance for applications in recycling and surface modification of polyesters. The cutinase from Thielavia terrestris (TtC) is distinct in terms of its ability to retain its stability and activity in acidic pH. Stability and activity in acidic pHs are desirable for esterases as the pH of the reaction tends to go down with the generation of acid. The pH stability and activity are governed by the charged state of the residues involved in catalysis or in substrate binding. In this study, we performed the detailed structural and biochemical characterization of TtC coupled with surface charge analysis to understand its acidic tolerance. The stability of TtC in acidic pH was rationalized by evaluating the contribution of charge interactions to the Gibbs free energy of unfolding at varying pHs. The activity of TtC was found to be limited by substrate binding affinity, which is a function of the surface charge. Additionally, the presence of glycosylation affects the biochemical characteristics of TtC owing to steric interactions with residues involved in substrate binding. PMID:26758295

Design, characterisation and application of alginate-based encapsulated pig liver esterase.

PubMed

Pauly, Jan; Gröger, Harald; Patel, Anant V

2018-06-05

Encapsulation of hydrolases in biopolymer-based hydrogels often suffers from low activities and encapsulation efficiencies along with high leaching and unsatisfactory recycling properties. Exemplified for the encapsulation of pig liver esterase the coating of alginate and chitosan beads have been studied by creating various biopolymer hydrogel beads. Enzyme activity and encapsulation efficiency were notably enhanced by chitosan coating of alginate beads while leaching remained nearly unchanged. This was caused by the enzymatic reaction acidifying the matrix, which increased enzyme retention through enhanced electrostatic enzyme-alginate interaction but decreased activity through enzyme deactivation. A practical and ready-to-use method for visualising pH in beads during reaction by co-encapsulation of a conventional pH indicator was also found. Our method proves that pH control inside the beads can only be realised by buffering. The resulting beads provided a specific activity of 0.267 μmol ∙ min -1 ∙ mg -1 , effectiveness factor 0.88, encapsulation efficiency of 88%, 5% leaching and good recycling properties. This work will contribute towards better understanding and application of encapsulated hydrolases for enzymatic syntheses. Copyright © 2018 Elsevier B.V. All rights reserved.

Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs with the Active Sites of Acetylcholine-Receptor and -Esterase*

PubMed Central

Mautner, Henry G.; Bartels, Eva

1970-01-01

p-Nitrobenzene diazonium fluoroborate (NDF) is a potent inhibitor of the carbamylcholine-induced depolarization of the electroplax and of acetylcholinesterase. It probably forms covalent bonds with the acetylcholine-receptor and -esterase at the active site of the proteins. Its inhibitory strength is at least the same as that of trimethylammonium diazonium fluoroborate (TDF). The p-acetoxy analog, with its weaker electron-withdrawing group, is about ten times weaker as an inhibitor than the trimethylammonium or p-nitro analogs, both of which have strong electron-withdrawing groups. After treatment of the electroplax preparation with dithiothreitol, NDF remains an irreversible receptor-inhibitor, while TDF becomes a potent reversible receptor-activator. TDF is self-inhibitory: applied before reduction, it no longer depolarizes. Although the first observations on TDF suggested that the compound labels both proteins by virtue of the steric complementary of its trimethylammonium group to a negative subsite in the proteins, the present study indicates that it is the positively charged diazonium group that reacts with the active sites of the proteins to form a covalent bond with an appropriate amino-acid residue. PMID:5272331

Symmetry broken and rebroken during the ATP hydrolysis cycle of the mitochondrial Hsp90 TRAP1

PubMed Central

Elnatan, Daniel; Betegon, Miguel; Liu, Yanxin; Ramelot, Theresa; Kennedy, Michael A; Agard, David A

2017-01-01

Hsp90 is a homodimeric ATP-dependent molecular chaperone that remodels its substrate ‘client’ proteins, facilitating their folding and activating them for biological function. Despite decades of research, the mechanism connecting ATP hydrolysis and chaperone function remains elusive. Particularly puzzling has been the apparent lack of cooperativity in hydrolysis of the ATP in each protomer. A crystal structure of the mitochondrial Hsp90, TRAP1, revealed that the catalytically active state is closed in a highly strained asymmetric conformation. This asymmetry, unobserved in other Hsp90 homologs, is due to buckling of one of the protomers and is most pronounced at the broadly conserved client-binding region. Here, we show that rather than being cooperative or independent, ATP hydrolysis on the two protomers is sequential and deterministic. Moreover, dimer asymmetry sets up differential hydrolysis rates for each protomer, such that the buckled conformation favors ATP hydrolysis. Remarkably, after the first hydrolysis, the dimer undergoes a flip in the asymmetry while remaining in a closed state for the second hydrolysis. From these results, we propose a model where direct coupling of ATP hydrolysis and conformational flipping rearranges client-binding sites, providing a paradigm of how energy from ATP hydrolysis can be used for client remodeling. DOI: http://dx.doi.org/10.7554/eLife.25235.001 PMID:28742020

Internal Hydrolysis Indicator for Sample Specific Monitoring of β-Glucuronidase Activity.

PubMed

Taylor, Lacy L; Flint, Noah A; Ma, Vinh; Hill, Brandy M; Clark, Chantry J; Strathmann, Frederick G

2017-06-01

Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing β-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the β-Glucuronidase enzyme. In this study resorufin β-D-glucuronide, a substrate of the β-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin β-D-glucuronide. The β-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin β-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The β-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

PubMed

Kohorn, Bruce D; Kohorn, Susan L; Saba, Nicholas J; Martinez, Victoriano Meco

2014-07-04

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Understanding the molecular mechanism of aryl acylamidase activity of acetylcholinesterase - An in silico study.

PubMed

Chinnadurai, Raj Kumar; Saravanaraman, Ponne; Boopathy, Rathanam

2015-08-15

Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and aryl acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates (o-nitroacetanilide, and o-nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900kJ/mol/nm). Among the two substrates, o-nitrotrifluoroacetanilide required lesser force (380kJ/mol/nm) than o-nitroacetanilide the (550kJ/mol/nm) for its binding, thus validating o-nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetically-determined polymorphism of nonspecific esterases and phosphoglucomutase in eight introduced breeds of the silkworm, Bombyx mori, raised in Bulgaria.

PubMed

Staykova, Teodora

2008-01-01

Isoenzymes are very suitable markers for the study of the inter-breed diversity of the silkworm Bombyx mon L. (Lepidoptera: Bombycidae). More than 250 breeds are raised in Bulgaria, which are not very well studied with regard to their isoenzymic polymorphism. Polymorphism of nonspecific esterases from pupal haemolymph was analyzed, as well as of phosphoglucomutase from different organs of larvae, pupae and imago, from eight introduced breeds. Electrophoresis in polyacrylamide gels was used. A polylocus control of nonspecific esterases, and possible monolocus control of phosphoglucomutase was ascertained. Biallele and triallele polymorphism of phosphoglucomutase locus and in three of the esterase loci was determined. The allelic frequencies of the polymorphic loci in each breed were analyzed. Inter-breed differences were found in different allelic frequencies, different heterozygosity and polymorphism.

Immobilization of Enzymes in Polymer Supports.

ERIC Educational Resources Information Center

Conlon, Hugh D.; Walt, David R.

1986-01-01

Two experiments in which an enzyme is immobilized onto a polymeric support are described. The experiments (which also demonstrate two different polymer preparations) involve: (1) entrapping an enzyme in an acrylamide polymer; and (2) reacting the amino groups on the enzyme's (esterase) lysine residues with an activated polymer. (JN)

Preparation of Egg White Liquid Hydrolysate (ELH) and Its Radical-Scavenging Activity

PubMed Central

Noh, Dong Ouk; Suh, Hyung Joo

2015-01-01

In the present study, an optimum protease was selected to hydrolyze the egg white liquid protein for the antioxidant peptides. Alcalase treatment yielded the highest amount of α-amino groups (15.27 mg/mL), while the control (no enzymatic hydrolysis) showed the lowest amount of α-amino groups (1.53 mg/mL). Alcalase also gave the highest degree of hydrolysis (DH) value (43.2%) and was more efficient for egg white liquid hydrolysis than the other enzymes. The Alcalase hydrolysate had the highest radical-scavenging activity (82.5%) at a concentration of 5.0 mg/mL. The conditions for enzymatic hydrolysis of egg white liquid with Alcalase were selected as substrate : water ratio of 2:1. Five percent Alacalse treatment did not show significant (P>0.05) increases of DH and α-amino nitrogen content after 24 h-hydrolysis. Thirty two hour-hydrolysis with 5% Alcalase is sufficient to make antioxidative egg white liquid hydrolysate from egg white liquid. DPPH and ABTS radical-scavenging activities were significantly (P<0.05) higher after enzymatic digestion. These results suggest that active peptides released from egg-white protein are effective radical-scavengers. Thus, this approach may be useful for the preparation of potent antioxidant products. PMID:26451355

Discovery of G protein signaling.

PubMed

Selinger, Zvi

2008-01-01

The mechanism of transmembrane signaling by the receptor-activated adenylyl cyclase was an enigma. It was suggested that hydrolysis of GTP is a turn-off mechanism that resets the active adenylyl cyclase to the inactive state. To test this hypothesis, we developed a specific GTPase assay and found that the catecholamine adrenergic agonists stimulated the hydrolysis of GTP. To resolve the question of how the hormone concurrently stimulates GTP hydrolysis and activates the adenylyl cyclase, we suggested the regulatory GTPase cycle. Thus, because the hormone facilitates the binding of GTP, which is subsequently hydrolyzed, the regulatory cycle results in a hormone-stimulated GTPase activity. This model also predicts that two mechanisms could account for stimulation of adenylyl cyclase activity-either by the familiar hormone stimulation of the activation reaction or by an inhibition of the turn-off reaction. Indeed, we showed that cholera toxin enhances adenylyl cyclase activity by inhibition of GTP hydrolysis. Finally, we also showed that the hormone-activated receptor stimulates adenylyl cyclase activity by facilitating the exchange of bound GDP for free GTP. Thus, we presented, for the first time, an explicit mechanism for receptor action.

Catalytic hydrolysis of ammonia borane via cobalt palladium nanoparticles.

PubMed

Sun, Daohua; Mazumder, Vismadeb; Metin, Önder; Sun, Shouheng

2011-08-23

Monodisperse 8 nm CoPd nanoparticles (NPs) with controlled compositions were synthesized by the reduction of cobalt acetylacetonate and palladium bromide in the presence of oleylamine and trioctylphosphine. These NPs were active catalysts for hydrogen generation from the hydrolysis of ammonia borane (AB), and their activities were composition dependent. Among the 8 nm CoPd catalysts tested for the hydrolysis of AB, the Co(35)Pd(65) NPs exhibited the highest catalytic activity and durability. Their hydrolysis completion time and activation energy were 5.5 min and 27.5 kJ mol(-1), respectively, which were comparable to the best Pt-based catalyst reported. The catalytic performance of the CoPd/C could be further enhanced by a preannealing treatment at 300 °C under air for 15 h with the hydrolysis completion time reduced to 3.5 min. This high catalytic performance of Co(35)Pd(65) NP catalyst makes it an exciting alternative in pursuit of practical implementation of AB as a hydrogen storage material for fuel cell applications. © 2011 American Chemical Society

Using urinary leucocyte esterase tests as an indicator of infection with gonorrhoea or chlamydia in asymptomatic males in a primary health care setting.

PubMed

Rahman, Md Saifur; Beever, Warwick; Skov, Steven; Boffa, John

2014-02-01

To evaluate a leucocyte esterase test as a predictor of gonorrhoea or chlamydia in asymptomatic Aboriginal males at the Central Australian Aboriginal Congress Male Clinic (Ingkintja), first-void urine samples and clinical information were collected from consecutive asymptomatic males presenting to the Ingkintja in Alice Springs between March 2008 and December 2009. Urine was tested immediately with a leucocyte esterase test dipstick and then by polymerase chain reaction for gonorrhoea and chlamydia. Among the 292 specimens from asymptomatic males, 15.4% were positive for gonorrhoea or chlamydia. In this group, compared with polymerase chain reaction result for gonorrhoea or chlamydia, leucocyte esterase test alone and in combination with age ≤35 years showed sensitivities of 66.7% and 60%, specificities of 90.7% and 94.7%, positive predictive values of 56.6% and 67.5%, negative predictive values of 93.7% and 92.8% and the area under receiver operating characteristics curve values of 0.79 and 0.85, respectively. Leucocyte esterase tests can reasonably be used as a basis for immediate empirical treatment for gonorrhoea or chlamydia in asymptomatic central Australian Aboriginal men under 35 years of age.

Identification and functional characterization of esterases in Euschistus heros (Hemiptera, Pentatomidae) and their relationship with thiamethoxam and lambda-cyhalothrin.

PubMed

Hegeto, L A; Ronqui, L; Lapenta, A S; Albuquerque, F A

2015-09-22

The brown stink bug Euschistus heros is the most abundant species of the soybean-sucking bugs, and causes large economic losses. Applying different chemical groups of organosynthetic insecticides for its control increases the potential for resistance. Esterases are a group of enzymes that play a variety of roles in insects, and some of them are related to the metabolism of xenobiotics. The aim of this study was to analyze the esterase isoenzyme system of this species and investigate its response to Engeo™ Pleno (thiamethoxam and lambda-cyhalothrin), which is the most widely used pesticide in soybean crops. Two strains were analyzed: the EB strain, which had been free of insecticides for several generations; and the MA strain, which was collected in a location exposed to agrochemicals. By analyzing the polyacrylamide gel electrophoresis profile, seven different esterases in adults and nymphs of both strains were found. Eight gene loci were responsible for the synthesis of these enzymes. The differences in esterases between the two strains and enzyme changes in insects exposed to Engeo™ Pleno suggest that EST-2 and EST-4 are related to the metabolism of the agrochemical used and are mechanisms of resistance.

Hydrogen-bonded intermediates and transition states during spontaneous and acid-catalyzed hydrolysis of the carcinogen (+)-anti-BPDE.

PubMed

Palenik, Mark C; Rodriguez, Jorge H

2014-07-07

Understanding mechanisms of (+)-anti-BPDE detoxification is crucial for combating its mutagenic and potent carcinogenic action. However, energetic-structural correlations of reaction intermediates and transition states during detoxification via hydrolysis are poorly understood. To gain mechanistic insight we have computationally characterized intermediate and transition species associated with spontaneous and general-acid catalyzed hydrolysis of (+)-anti-BPDE. We studied the role of cacodylic acid as a proton donor in the rate limiting step. The computed activation energy (ΔG‡) is in agreement with the experimental value for hydrolysis in a sodium cacodylate buffer. Both types of, spontaneous and acid catalyzed, BPDE hydrolysis can proceed through low-entropy hydrogen bonded intermediates prior to formation of transition states whose energies determine reaction activation barriers and rates.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

PubMed

Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

2011-01-01

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance. Copyright © 2010 Elsevier Ltd. All rights reserved.

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

PubMed

Tyagi, Sadhna; Pleiss, Juergen

2006-06-25

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

Insecticidal and acetylcholine esterase inhibition activity of Apiaceae plant essential oils and their constituents against adults of German cockroach (Blattella germanica).

PubMed

Yeom, Hwa-Jeong; Kang, Jae Soon; Kim, Gil-Hah; Park, Il-Kwon

2012-07-25

We evaluated the insecticidal and acetylcholine esterase (AChE) inhibition activity of 11 Apiaceae plant essential oils and their constituents in adult male and female Blattella germanica. Of the 11 Apiaceae plant essential oils tested, dill (Anethum graveolens), carvi (Carum carvi), and cumin (Cuminum cyminum) demonstrated >90% fumigant toxicity against adult male German cockroaches at a concentration of 5 mg/filter paper. In a contact toxicity test, dill (Anethum graveolens), carvi (Carum carvi), cumin (Cuminum cyminum), and ajowan (Trachyspermum ammi) produced strong insecticidal activity against adult male and female German cockroaches. Among the test compounds, (S)-(+)-carvone, 1,8-cineole, trans-dihydrocarvone, cuminaldehyde, trans-anethole, p-cymene, and γ-terpinene demonstrated strong fumigant toxicity against adult male and female B. germanica. In a contact toxicity test, carveol, cuminaldehyde, (S)-(+)-carvone, trans-anethole, thymol, and p-cymene showed strong contact toxicity against adult male and female B. germanica. IC(50) values of α-pinene, carvacrol, and dihydrocarvone against female AChE were 0.28, 0.17, and 0.78 mg/mL, respectively. The toxicity of the blends of constituents identified in 4 active oils indicated that carvone, cuminaldehyde, and thymol were major contributors to the fumigant activity or contact toxicity of the artificial blend.

Effects of plant lectin from cobra lily, Arisaema curvatum Kunth on development of melon fruit fly, Bactrocera cucurbitae (Coq.).

PubMed

Singh, Kuljinder; Kaur, Manpreet; Rup, Pushpinder J; Singh, Jatinder

2008-11-01

The lectin from tubers of cobra lily, Arisaema curvatum Kunth was purified by affinity chromatography using asialofetuin-linked amino activated porous silica beads. The concentration dependent effect of lectin was studied on second instar larvae (64-72 hr) of Bactrocera cucurbitae (Coq.). The treatment not only resulted in a significant reduction in the percentage pupation and emergence of the adults from treated larvae but it also prolonged the remaining larval development period. A very low LC50 value, 39 mgl(-1) of lectin was obtained on the basis of adult emergence using probit analysis. The activity of three hydrolase enzymes (esterases, acid and alkaline phosphatases), one oxidoreductase (catalase) and one group transfer enzyme (GSTs: Glutathione S-transferases) was assayed in second instar larvae under the influence of the LC50 of lectin at increasing exposure intervals (0, 24, 48 and 72 hr). The Arisaema curvatum lectin significantly decreased the activity of all the enzymes except for esterases, where the activity increased as compared to control at all exposure intervals. The decrease in pupation and emergence as well as significant suppression in the activities of two hydrolases, one oxidoreductase and one GST enzyme in treated larvae of B. cucurbitae indicated that this lectin has anti-metabolic effect on the melon fruit fly larvae.

Pectinase from Trichoderma reesei QM 9414

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haltmeier, T.; Leisola, M.; Ulmer, D.

1983-06-01

The present study was undertaken to determine whether T. reesei produces pectinolytic enzymes and, if so, to determine their action pattern and specificity. The aim was also to find out the practical importance of pectinolytic activity in the hydrolysis of cellulosic materials. It was found that the presence of pectinase does not appear to facilitate the hydrolysis of cellulose in plant material through any cell-separating mechanism. It is concluded that cellulase and xylanase activities are the important activities in the practical hydrolysis of cellulosic materials and that lignin is the real limiting factor. (Refs. 12).

Thrombotic events associated with C1 esterase inhibitor products in patients with hereditary angioedema: investigation from the United States Food and Drug Administration adverse event reporting system database.

PubMed

Gandhi, Pranav K; Gentry, William M; Bottorff, Michael B

2012-10-01

To investigate reports of thrombotic events associated with the use of C1 esterase inhibitor products in patients with hereditary angioedema in the United States. Retrospective data mining analysis. The United States Food and Drug Administration (FDA) adverse event reporting system (AERS) database. Case reports of C1 esterase inhibitor products, thrombotic events, and C1 esterase inhibitor product-associated thrombotic events (i.e., combination cases) were extracted from the AERS database, using the time frames of each respective product's FDA approval date through the second quarter of 2011. Bayesian statistical methodology within the neural network architecture was implemented to identify potential signals of a drug-associated adverse event. A potential signal is generated when the lower limit of the 95% 2-sided confidence interval of the information component, denoted by IC₀₂₅ , is greater than zero. This suggests that the particular drug-associated adverse event was reported to the database more often than statistically expected from reports available in the database. Ten combination cases of thrombotic events associated with the use of one C1 esterase inhibitor product (Cinryze) were identified in patients with hereditary angioedema. A potential signal demonstrated by an IC₀₂₅ value greater than zero (IC₀₂₅ = 2.91) was generated for these combination cases. The extracted cases from the AERS indicate continuing reports of thrombotic events associated with the use of one C1 esterase inhibitor product among patients with hereditary angioedema. The AERS is incapable of establishing a causal link and detecting the true frequency of an adverse event associated with a drug; however, potential signals of C1 esterase inhibitor product-associated thrombotic events among patients with hereditary angioedema were identified in the extracted combination cases. © 2012 Pharmacotherapy Publications, Inc.

Enhanced catalytic activity of the nanostructured Co-W-B film catalysts for hydrogen evolution from the hydrolysis of ammonia borane.

PubMed

Li, Chao; Wang, Dan; Wang, Yan; Li, Guode; Hu, Guijuan; Wu, Shiwei; Cao, Zhongqiu; Zhang, Ke

2018-08-15

In this work, nanostructured Co-W-B films are successfully synthesized on the foam sponge by electroless plating method and employed as the catalysts with enhanced catalytic activity towards hydrogen evolution from the hydrolysis of ammonia borane (NH 3 BH 3 , AB) at room temperature. The particle size of the as-prepared Co-W-B film catalysts is varied by adjusting the depositional pH value to identify the most suitable particle size for hydrogen evolution of AB hydrolysis. The Co-W-B film catalyst with the particle size of about 67.3 nm shows the highest catalytic activity and can reach a hydrogen generation rate of 3327.7 mL min -1 g cat -1 at 298 K. The activation energy of the hydrolysis reaction of AB is determined to be 32.2 kJ mol -1 . Remarkably, the as-obtained Co-W-B film is also a reusable catalyst preserving 78.4% of their initial catalytic activity even after 5 cycles in hydrolysis of AB at room temperature. Thus, the enhanced catalytic activity illustrates that the Co-W-B film is a promising catalyst for AB hydrolytic dehydrogenation in fuel cells and the related fields. Copyright © 2018 Elsevier Inc. All rights reserved.

The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

PubMed

Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

2013-08-01

The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Regulation of synaptic acetylcholine concentrations by acetylcholine transport in rat striatal cholinergic transmission.

PubMed

Muramatsu, Ikunobu; Uwada, Junsuke; Masuoka, Takayoshi; Yoshiki, Hatsumi; Sada, Kiyonao; Lee, Kung-Shing; Nishio, Matomo; Ishibashi, Takaharu; Taniguchi, Takanobu

2017-10-01

In addition to hydrolysis by acetylcholine esterase (AChE), acetylcholine (ACh) is also directly taken up into brain tissues. In this study, we examined whether the uptake of ACh is involved in the regulation of synaptic ACh concentrations. Superfusion experiments with rat striatal segments pre-incubated with [ 3 H]choline were performed using an ultra-mini superfusion vessel, which was developed to minimize superfusate retention within the vessel. Hemicholinium-3 (HC-3) at concentrations less than 1 μM, selectively inhibited the uptake of [ 3 H]choline by the high affinity-choline transporter 1 and had no effect on basal and electrically evoked [ 3 H]efflux in superfusion experiments. In contrast, HC-3 at higher concentrations, as well as tetraethylammonium (>10 μM), which inhibited the uptake of both [ 3 H]choline and [ 3 H]ACh, increased basal [ 3 H]overflow and potentiated electrically evoked [ 3 H]efflux. These effects of HC-3 and tetraethylammonium were also observed under conditions where tissue AChE was irreversibly inactivated by diisopropylfluorophosphate. Specifically, the potentiation of evoked [ 3 H]efflux was significantly higher in AChE-inactivated preparations and was attenuated by atropine. On the other hand, striatal segments pre-incubated with [ 3 H]ACh failed to increase [ 3 H]overflow in response to electrical stimulation. These results show that synaptic ACh concentrations are significantly regulated by the postsynaptic uptake of ACh, as well as by AChE hydrolysis and modulation of ACh release mediated through presynaptic muscarinic ACh receptors. In addition, these data suggest that the recycling of ACh-derived choline may be minor in cholinergic terminals. This study reveals a new mechanism of cholinergic transmission in the central nervous system. © 2017 International Society for Neurochemistry.

β-Glucuronidase-coupled assays of glucuronoyl esterases.

PubMed

Fraňová, Lucia; Puchart, Vladimír; Biely, Peter

2016-10-01

Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries. Copyright © 2016 Elsevier Inc. All rights reserved.

Angio-oedema in dentistry: management of two cases using C1 esterase inhibitor.

PubMed

Socker, Michal; Boyle, Carole; Burke, Mary

2005-01-01

Angio-oedema is a rare condition; it may be a hereditary or acquired form. It results from biochemical defects which cause excessive activation of the complement cascade and result in deep swellings in the skin and alimentary tract, called angio-oedema. These swellings are painful rather than itchy and not associated with urticaria, which helps to differentiate angio-oedema from allergic reactions. Even mild trauma can give rise to swelling, which may be life-threatening in the oral region. Management of two cases, one hereditary and the other acquired angio-oedema, are reported to demonstrate the use of C1 esterase inhibitor prophylaxis. It is important that patients giving a history of angio-oedema are thoroughly investigated and, in discussion with the patient's medical team, appropriate prophylactic measures are taken to prevent swelling.

Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

PubMed Central

Diaz-Meco, M T; Dominguez, I; Sanz, L; Municio, M M; Berra, E; Cornet, M E; Garcia de Herreros, A; Johansen, T; Moscat, J

1992-01-01

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1. Images PMID:1309592

Effect of NaHCO3 treatments on the activity of cell wall-degrading enzymes produced by Penicillium digitatum during the pathogenesis process on grapefruit.

PubMed

Venditti, Tullio; D'hallewin, Guy; Ladu, Gianfranca; Petretto, Giacomo L; Pintore, Giorgio; Labavitch, John M

2018-03-25

The present study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with NaHCO 3 , at two different pH (5 and 8.3), on cell wall-degrading enzymes activity, over a period of 72 h. The treatment with NaHCO 3 , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, if compared to the control or the same treatment at pH 5. On the contrary, the pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5 treated plugs up to 72 h. The rapid increase in pectin methyl esterase activity, under alkaline conditions, is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. This article is protected by copyright. All rights reserved.

Evaluation of active and passive transport processes in corneas extracted from preserved rabbit eyes.

PubMed

Majumdar, Soumyajit; Hingorani, Tushar; Srirangam, Ramesh

2010-04-01

In vitro transcorneal permeability studies are an important screening tool in drug development. The objective of this research is to examine the feasibility of using corneas isolated from preserved rabbit eyes as a model for permeability evaluation. Eyes from male New Zealand White rabbits were used immediately or were stored overnight in phosphate-buffered saline (PBS) or Hanks balanced salt solution (HBSS) over wet ice. Integrity of isolated corneas was evaluated by measuring the TEER and by determining the permeability of paracellular and transcellular markers. Active transport was assessed by measuring transcorneal permeability of selected amino acids. Esterase activity was estimated using p-nitrophenyl assay. In all cases, corneas from freshly enucleated eyes were compared to those isolated from the day-old preserved eyes. Transcellular and paracellular passive diffusion was not affected by the storage medium and observed to be similar in the fresh and preserved eye models. However, amino acid transporters demonstrated lower functional activity in corneas excised from eyes preserved in PBS. Moreover, preserved eyes displayed almost 1.5-fold lower esterase activity in the corneal tissue. Thus, corneas isolated from day-old eyes, preserved in HBSS, closely mimics freshly excised rabbit corneas in terms of both active and passive transport characteristics but possesses slightly reduced enzymatic activity. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Effect of alkali lignins with different molecular weights from alkali pretreated rice straw hydrolyzate on enzymatic hydrolysis.

PubMed

Li, Yun; Qi, Benkun; Luo, Jianquan; Wan, Yinhua

2016-01-01

This study investigated the effect of alkali lignins with different molecular weights on enzymatic hydrolysis of lignocellulose. Different alkali lignins fractions, which were obtained from cascade ultrafiltration, were added into the dilute acid pretreated (DAP) and alkali pretreated (AP) rice straws respectively during enzymatic hydrolysis. The results showed that the addition of alkali lignins enhanced the hydrolysis and the enhancement for hydrolysis increased with increasing molecular weights of alkali lignins, with maximum enhancement being 28.69% for DAP and 20.05% for AP, respectively. The enhancement was partly attributed to the improved cellulase activity, and filter paper activity increased by 18.03% when adding lignin with highest molecular weight. It was found that the enhancement of enzymatic hydrolysis was correlated with the adsorption affinity of cellulase on alkali lignins, and the difference in surface charge and hydrophobicity of alkali lignins were responsible for the difference in affinity between cellulase and lignins. Copyright © 2015 Elsevier Ltd. All rights reserved.

The reliability and validity of using the urine dipstick test by patient self-assessment for urinary tract infection screening in spinal cord injury patients.

PubMed

Duanngai, Krit; Sirasaporn, Patpiya; Ngaosinchai, Siriwan Surapaitoon

2017-01-01

The aim of this is to evaluate the reliability of the urine dipstick test by patients' self-assessment for urinary tract infection (UTI) screening and to determine the validity of urine dipstick test. Rehabilitation Department, Srinagarind Hospital, Thailand. A diagnostic study. This study compared the urine dipstick test (index test) with the National Institute on Disability and Rehabilitation Research (NIDRR) criteria (gold standard test) in spinal cord injury (SCI) patients. The urine dipstick test informed positive and negative results. Besides the NIDRR criteria classified as UTI and no UTI. The interrater reliability was measured in the sense of Kappa whereas the validity of urine dipstick test was reported in terms of sensitivity, specificity, positive likelihood ratio (LR) (+LR), negative LR (-LR), positive predictive value (PPV), and negative predictive value (NPV). Out of the 56 participants, the kappa of urine dipstick test for leukocyte esterase, nitrite, and combined leukocyte esterase and nitrite were 0.09, 0.21, and 0.52, respectively. The nitrite urine dipstick test showed the highest sensitivity (90%). The combined leukocyte esterase and nitrite urine dipstick test gave the highest specificity (87%), PPV (60%), NPV (93%), and +LR (5.63). The interrater reliability of combined leukocyte esterase and nitrite urine dipstick test was moderate agreement. The combined leukocyte esterase and nitrite urine dipstick test showed high level of both sensitivity and specificity. The combined leukocyte esterase and nitrite urine dipstick test should be promoted for patients' self-assessment for UTI screening in SCI patients.

Molecular population genetics of X-linked genes in Drosophila pseudoobscura.

PubMed Central

Kovacevic, M; Schaeffer, S W

2000-01-01

This article presents a nucleotide sequence analysis of 500 bp determined in each of five X-linked genes, runt, sisterlessA, period, esterase 5, and Heat-shock protein 83, in 40 Drosophila pseudoobscura strains collected from two populations. Estimates of the neutral migration parameter for the five loci show that gene flow among D. pseudoobscura populations is sufficient to homogenize inversion frequencies across the range of the species. Nucleotide diversity at each locus fails to reject a neutral model of molecular evolution. The sample of 40 chromosomes included six Sex-ratio inversions, a series of three nonoverlapping inversions that are associated with a strong meiotic drive phenotype. The selection driven by the Sex-ratio meiotic drive element has not fixed variation across the X chromosome of D. pseudoobscura because, while significant linkage disequilibrium was observed within the sisterlessA, period, and esterase 5 genes, we did not find evidence for nonrandom association among loci. The Sex-ratio chromosome was estimated to be 25,000 years old based on the decomposition of linkage disequilibrium between esterase 5 and Heat-shock protein 83 or 1 million years old based on the net divergence of esterase 5 between Standard and Sex-ratio chromosomes. Genetic diversity was depressed within esterase 5 within Sex-ratio chromosomes, while the four other genes failed to show a reduction in heterozygosity in the Sex-ratio background. The reduced heterogeneity in esterase 5 is due either to its location near one of the Sex-ratio inversion breakpoints or that it is closely linked to a gene or genes responsible for the Sex-ratio meiotic drive system. PMID:10978282

Comparison of the free and bound phenolic profiles and cellular antioxidant activities of litchi pulp extracts from different solvents

PubMed Central

2014-01-01

Background The phenolic contents and antioxidant activities of fruits could be underestimated if the bound phenolic compounds are not considered. In the present study, the extraction efficiencies of various solvents were investigated in terms of the total content of the free and bound phenolic compounds, as well as the phenolic profiles and antioxidant activities of the extracts. Methods Five different solvent mixtures were used to extract the free phenolic compounds from litchi pulp. Alkaline and acidic hydrolysis methods were compared for the hydrolysis of bound phenolic compounds from litchi pulp residue. The phenolic compositions of the free and bound fractions from the litchi pulp were identified using HPLC-DAD. The antioxidant activities of the litchi pulp extracts were determined by oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Results Of the solvents tested, aqueous acetone extracted the largest amount of total free phenolic compounds (210.7 mg GAE/100 g FW) from litchi pulp, followed sequentially by aqueous mixtures of methanol, ethanol and ethyl acetate, and water itself. The acid hydrolysis method released twice as many bound phenolic compounds as the alkaline hydrolysis method. Nine phenolic compounds were detected in the aqueous acetone extract. In contrast, not all of these compounds were found in the other four extracts. The classification and content of the bound phenolic compounds released by the acid hydrolysis method were higher than those achieved by the alkaline hydrolysis. The aqueous acetone extract showing the highest ORAC value (3406.9 μmol TE/100 g FW) for the free phenolic extracts. For the CAA method, however, the aqueous acetone and methanol extracts (56.7 and 55.1 μmol QE/100 g FW) showed the highest levels of activity of the five extracts tested. The ORAC and CAA values of the bound phenolic compounds obtained by acid hydrolysis were 2.6- and 1.9-fold higher than those obtained using the alkaline hydrolysis method. Conclusions The free and bound phenolic contents and profiles and antioxidant activities of the extracts were found to be dependent on the extraction solvent used. Litchi exhibited good cellular antioxidant activity and could be a potentially useful natural source of antioxidants. PMID:24405977

Interfacial binding of cutinase rather than its catalytic activity determines the steady state interfacial tension during oil drop lipid hydrolysis.

PubMed

Flipsen, J A; van Schaick, M A; Dijkman, R; van der Hijden, H T; Verheij, H M; Egmond, M R

1999-02-01

Hydrolysis of triglycerides by cutinase from Fusarium solani pisi causes in oil drop tensiometer experiments a decrease of the interfacial tension. A series of cutinase variants with amino acid substitutions at its molecular surface yielded different values of the steady state interfacial tension. This tension value poorly correlated with the specific activity as such nor with the total activity (defined as the specific activity multiplied by the amount of enzyme bound) of the cutinase variants. Moreover, it appeared that at activity levels above 15% of that of wild type cutinase the contribution of hydrolysis to the decrease of the tension is saturating. A clear positive correlation was found between the interfacial tension plateau value and the interfacial binding of cutinase, as determined with attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR). These results indicate that the interfacial steady state level is not determined by the rate of hydrolysis, but mainly by the interfacial binding of cutinase.

Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

PubMed

Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

2016-03-04

Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.