Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

PubMed Central

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-01-01

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

PubMed

Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

2013-05-07

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

The PPST and NTE as Predictors of Student Teacher Performance.

ERIC Educational Resources Information Center

Salzman, Stephanie A.

The Pre-Professional Skills Test (PPST) and the National Teacher Examinations (NTE) were examined as predictors of student classroom performance as measured by three instruments from the "Teacher Performance Assessment Instruments" (TPAIs) of W. Capie et al. (1979). Subjects were 305 teacher education students taking the student teaching…

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.

In vitro study of the neuropathic potential of the organophosphorus compounds trichlorfon and acephate.

PubMed

Fernandes, Laís S; Emerick, Guilherme L; dos Santos, Neife Aparecida G; de Paula, Eloísa Silva; Barbosa, Fernando; dos Santos, Antonio Cardozo

2015-04-01

Organophosphorus-induced delayed neuropathy (OPIDN) is a central and peripheral distal axonopathy characterized by ataxia and paralysis. Trichlorfon and acephate are two organophosphorus compounds (OPs) used worldwide as insecticide and which cause serious effects to non-target species. Despite that, the neuropathic potential of these OPs remains unclear. The present study addressed the neurotoxic effects and the neuropathic potential of trichlorfon and acephate in SH-SY5Y human neuroblastoma cells, by evaluating inhibition and aging of neuropathy target esterase (NTE), inhibition of acetylcholinesterase (AChE), neurite outgrowth, cytotoxicity and intracellular calcium. Additionally, the effects observed were compared to those of two well-studied OPs: mipafox (known as neuropathic) and paraoxon (known as non-neuropathic). Trichlorfon and mipafox presented the lowest percentage of reactivation of inhibited NTE and the lowest ratio IC50 NTE/IC50 AChE. Moreover, they caused inhibition and aging of at least 70% of the activity of NTE at sub-lethal concentrations. All these effects have been associated with induction of OPIDN. When assayed at these concentrations, trichlorfon and mipafox reduced neurite outgrowth and increased intracellular calcium, events implicated in the development of OPIDN. Acephate caused effects similar to those caused by paraoxon (non-neuropathic OP) and was only able to inhibit 70% of NTE activity at lethal concentrations. These findings suggest that trichlorfon is potentially neuropathic, whereas acephate is not. Copyright © 2015 Elsevier Ltd. All rights reserved.

46 CFR 151.50-76 - Hydrochloric acid, spent (NTE 15%).

Code of Federal Regulations, 2011 CFR

2011-10-01

... must provide for the free passage of air and gases to the vent or vents. (f) No lights may be used... 46 Shipping 5 2011-10-01 2011-10-01 false Hydrochloric acid, spent (NTE 15%). 151.50-76 Section 151.50-76 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS...

46 CFR 151.50-76 - Hydrochloric acid, spent (NTE 15%).

Code of Federal Regulations, 2012 CFR

2012-10-01

... must provide for the free passage of air and gases to the vent or vents. (f) No lights may be used... 46 Shipping 5 2012-10-01 2012-10-01 false Hydrochloric acid, spent (NTE 15%). 151.50-76 Section 151.50-76 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS...

46 CFR 151.50-76 - Hydrochloric acid, spent (NTE 15%).

Code of Federal Regulations, 2014 CFR

2014-10-01

... must provide for the free passage of air and gases to the vent or vents. (f) No lights may be used... 46 Shipping 5 2014-10-01 2014-10-01 false Hydrochloric acid, spent (NTE 15%). 151.50-76 Section 151.50-76 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS...

46 CFR 151.50-76 - Hydrochloric acid, spent (NTE 15%).

Code of Federal Regulations, 2013 CFR

2013-10-01

... must provide for the free passage of air and gases to the vent or vents. (f) No lights may be used... 46 Shipping 5 2013-10-01 2013-10-01 false Hydrochloric acid, spent (NTE 15%). 151.50-76 Section 151.50-76 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS...

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.

[Human ecology and nutritional anthropometry of adult Xavánte Indians in Mato Grosso, Brazil].

PubMed

Gugelmin, S A; Santos, R V

2001-01-01

This study compares anthropometric and ecological profiles of two Xavánte indigenous communities in Mato Grosso, Central Brazil. The research describes time allocation patterns and involves an anthropometric survey (including body mass, stature, and BMI) in adults over 20 years of age. Data from Etéñitépa (also known as Pimentel Barbosa) were collected in 1994. Field work at São José was conducted in 1998 and 1999. Compared with the São José group, Xavánte in Etéñitépa do more subsistence activities like farming, fishing, hunting, and gathering. The São José Xavánte do more paid work and generally engage in less physical activity. Average stature in the two communities is similar, but there are major differences in mean body mass and BMI. The São José group has average BMI values well over those of the Etéñitépa group in practically all age brackets. Obesity prevalence rates were high in both men (24.6%) and women (41.3%) in São José, while in Etéñitépa the rates were only 2.5% and 4.8%, respectively. The authors conclude that the different nutritional profiles in the two communities result from specific patterns of social, political, and economic interactions with Brazilian society.

The NTE As a Measure of General Academic Ability: Implications for Teacher Education.

ERIC Educational Resources Information Center

Loadman, William E.; Deville, Craig W.

In 1987 the Ohio State Board of Education adopted new requirements for teacher education and teacher certification. This included the implementation of competency testing in Ohio, using the National Teacher Examination (NTE) as the certification examination. Correlation and regression analyses were performed on data collected between 1982 and 1989…

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 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

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

The NTeQ ISD Model: A Tech-Driven Model for Digital Natives (DNs)

ERIC Educational Resources Information Center

Williams, C.; Anekwe, J. U.

2017-01-01

Integrating Technology for enquiry (NTeQ) instructional development model (ISD), is believed to be a technology-driven model. The authors x-rayed the ten-step model to reaffirm the ICT knowledge demand of the learner and the educator; hence computer-based activities at various stages of the model are core elements. The model also is conscious of…

40 CFR Appendix I to Subpart T - Sample Graphical Summary of NTE Emission Results

Code of Federal Regulations, 2010 CFR

2010-07-01

... Emission Results I Appendix I to Subpart T Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... (CONTINUED) Manufacturer-Run In-Use Testing Program for Heavy-Duty Diesel Engines Pt. 86, Subpt. T, App. I Appendix I to Subpart T—Sample Graphical Summary of NTE Emission Results The following figure shows an...

40 CFR Appendix I to Subpart T - Sample Graphical Summary of NTE Emission Results

Code of Federal Regulations, 2011 CFR

2011-07-01

... Emission Results I Appendix I to Subpart T Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... (CONTINUED) Manufacturer-Run In-Use Testing Program for Heavy-Duty Diesel Engines Pt. 86, Subpt. T, App. I Appendix I to Subpart T—Sample Graphical Summary of NTE Emission Results The following figure shows an...

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

PubMed

Mangas, I; Vilanova, E; Benabent, M; Estévez, J

2014-02-10

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

HZE Radiation Non-Targeted Effects on the Microenvironment That Mediate Mammary Carcinogenesis.

PubMed

Barcellos-Hoff, Mary Helen; Mao, Jian-Hua

2016-01-01

Clear mechanistic understanding of the biological processes elicited by radiation that increase cancer risk can be used to inform prediction of health consequences of medical uses, such as radiotherapy, or occupational exposures, such as those of astronauts during deep space travel. Here, we review the current concepts of carcinogenesis as a multicellular process during which transformed cells escape normal tissue controls, including the immune system, and establish a tumor microenvironment. We discuss the contribution of two broad classes of radiation effects that may increase cancer: radiation targeted effects that occur as a result of direct energy deposition, e.g., DNA damage, and non-targeted effects (NTE) that result from changes in cell signaling, e.g., genomic instability. It is unknown whether the potentially greater carcinogenic effect of high Z and energy (HZE) particle radiation is a function of the relative contribution or extent of NTE or due to unique NTE. We addressed this problem using a radiation/genetic mammary chimera mouse model of breast cancer. Our experiments suggest that NTE promote more aggressive cancers, as evidenced by increased growth rate, transcriptomic signatures, and metastasis, and that HZE particle NTE are more effective than reference γ-radiation. Emerging evidence suggest that HZE irradiation dampens antitumor immunity. These studies raise concern that HZE radiation exposure not only increases the likelihood of developing cancer but also could promote progression to more aggressive cancer with a greater risk of mortality.

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

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.

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

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, F.H.; Moore, J.C.

1999-05-25

A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, Frances H.; Moore, Jeffrey C.

1998-01-01

A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, Frances H.; Moore, Jeffrey C.

1999-01-01

A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, F.H.; Moore, J.C.

1998-04-21

A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases. These enzymes exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

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

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.

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.

Isolation and characterization of juvenile hormone esterase from gypsy moth (Lymantria dispar)

Treesearch

Algimantas P. Valaitis; Joan Jolliff

1991-01-01

Insect metamorphosis is under precise hormonal control. During the last larval stadium, the degradation of juvenile hormone by juvenile hormone esterase (JHE) is essential for the initiation of pupation. Therefore, we have targeted this system for disruption with a strategy to produce a recombinant gypsy moth virus which expresses JHE. In order to clone and insert the...

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.

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.

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

[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.

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

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.

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

Non-Targeted Effects and the Dose Response for Heavy Ion Tumorigenesis

NASA Technical Reports Server (NTRS)

Chappelli, Lori J.; Cucinotta, Francis A.

2010-01-01

BACKGROUND: There is no human epidemiology data available to estimate the heavy ion cancer risks experienced by astronauts in space. Studies of tumor induction in mice are a necessary step to estimate risks to astronauts. Previous experimental data can be better utilized to model dose response for heavy ion tumorigenesis and plan future low dose studies. DOSE RESPONSE MODELS: The Harderian Gland data of Alpen et al.[1-3] was re-analyzed [4] using non-linear least square regression. The data set measured the induction of Harderian gland tumors in mice by high-energy protons, helium, neon, iron, niobium and lanthanum with LET s ranging from 0.4 to 950 keV/micron. We were able to strengthen the individual ion models by combining data for all ions into a model that relates both radiation dose and LET for the ion to tumor prevalence. We compared models based on Targeted Effects (TE) to one motivated by Non-targeted Effects (NTE) that included a bystander term that increased tumor induction at low doses non-linearly. When comparing fitted models to the experimental data, we considered the adjusted R2, the Akaike Information Criteria (AIC), and the Bayesian Information Criteria (BIC) to test for Goodness of fit.In the adjusted R2test, the model with the highest R2values provides a better fit to the available data. In the AIC and BIC tests, the model with the smaller values of the summary value provides the better fit. The non-linear NTE models fit the combined data better than the TE models that are linear at low doses. We evaluated the differences in the relative biological effectiveness (RBE) and found the NTE model provides a higher RBE at low dose compared to the TE model. POWER ANALYSIS: The final NTE model estimates were used to simulate example data to consider the design of new experiments to detect NTE at low dose for validation. Power and sample sizes were calculated for a variety of radiation qualities including some not considered in the Harderian Gland data

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.

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

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.

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.

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.

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.

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

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

Lipoamidase activity in normal and mutagenized pancreatic cholesterol esterase (bile salt-stimulated lipase).

PubMed Central

Hui, D Y; Hayakawa, K; Oizumi, J

1993-01-01

Purified human milk lipoamidase was digested with endoproteinase Lys-C and the digested peptides were subjected to gasphase microsequence analysis. The sequencing of three isolated peptides of human milk lipoamidase revealed the identity of this protein with human milk bile salt-stimulated lipase (pancreatic cholesterol esterase). The identity of the cholesterol esterase with lipoamidase was confirmed by expressing a recombinant form of rat pancreatic cholesterol esterase and testing for lipoamidase activity of the recombinant protein. The results showed that the recombinant cholesterol esterase displayed both lipolytic and lipoamidase activities and was capable of hydrolysing triacetin and lipoyl-4-aminobenzoate (LPAB). The mechanisms of the esterase and amidase activities of the enzyme were further tested by determining enzyme activity in a mutagenized cholesterol esterase with a His435-->Gln435 substitution. This mutation has been shown previously to abolish enzyme activity against esterase substrates [DiPersio, Fontaine and Hui (1991) J. Biol. Chem. 266, 4033-4036]. We showed that the mutagenized protein was effective in hydrolysing the amidase substrate LPAB and displayed similar enzyme kinetics to those of the native enzyme. These data indicate that the mechanism for the cholesterol esterase hydrolysis of lipoamides is different from that of the hydrolysis of substrates with an ester linkage. The presence of an enzyme in the gastrointestinal tract capable of both ester and amide hydrolysis suggests an important role for this protein in the digestion and absorption processes. PMID:8471055

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...

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

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

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, 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...

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

Non-Targeted Effects Models Predict Significantly Higher Mars Mission Cancer Risk than Targeted Effects Models

DOE PAGES

Cucinotta, Francis A.; Cacao, Eliedonna

2017-05-12

Cancer risk is an important concern for galactic cosmic ray (GCR) exposures, which consist of a wide-energy range of protons, heavy ions and secondary radiation produced in shielding and tissues. Relative biological effectiveness (RBE) factors for surrogate cancer endpoints in cell culture models and tumor induction in mice vary considerable, including significant variations for different tissues and mouse strains. Many studies suggest non-targeted effects (NTE) occur for low doses of high linear energy transfer (LET) radiation, leading to deviation from the linear dose response model used in radiation protection. Using the mouse Harderian gland tumor experiment, the only extensive data-setmore » for dose response modelling with a variety of particle types (>4), for the first-time a particle track structure model of tumor prevalence is used to investigate the effects of NTEs in predictions of chronic GCR exposure risk. The NTE model led to a predicted risk 2-fold higher compared to a targeted effects model. The scarcity of data with animal models for tissues that dominate human radiation cancer risk, including lung, colon, breast, liver, and stomach, suggest that studies of NTEs in other tissues are urgently needed prior to long-term space missions outside the protection of the Earth’s geomagnetic sphere.« less

Non-Targeted Effects Models Predict Significantly Higher Mars Mission Cancer Risk than Targeted Effects Models

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

Cucinotta, Francis A.; Cacao, Eliedonna

Cancer risk is an important concern for galactic cosmic ray (GCR) exposures, which consist of a wide-energy range of protons, heavy ions and secondary radiation produced in shielding and tissues. Relative biological effectiveness (RBE) factors for surrogate cancer endpoints in cell culture models and tumor induction in mice vary considerable, including significant variations for different tissues and mouse strains. Many studies suggest non-targeted effects (NTE) occur for low doses of high linear energy transfer (LET) radiation, leading to deviation from the linear dose response model used in radiation protection. Using the mouse Harderian gland tumor experiment, the only extensive data-setmore » for dose response modelling with a variety of particle types (>4), for the first-time a particle track structure model of tumor prevalence is used to investigate the effects of NTEs in predictions of chronic GCR exposure risk. The NTE model led to a predicted risk 2-fold higher compared to a targeted effects model. The scarcity of data with animal models for tissues that dominate human radiation cancer risk, including lung, colon, breast, liver, and stomach, suggest that studies of NTEs in other tissues are urgently needed prior to long-term space missions outside the protection of the Earth’s geomagnetic sphere.« less

Regiospecific Ester Hydrolysis by Orange Peel Esterase - An Undergraduate Experiment.

NASA Astrophysics Data System (ADS)

Bugg, Timothy D. H.; Lewin, Andrew M.; Catlin, Eric R.

1997-01-01

A simple but effective experiment has been developed to demonstrate the regiospecificity of enzyme catalysis using an esterase activity easily isolated from orange peel. The experiment involves the preparation of diester derivatives of para-, meta- and ortho-hydroxybenzoic acid (e.g. methyl 4-acetoxy-benzoic acid). The derivatives are incubated with orange peel esterase, as a crude extract, and with commercially available pig liver esterase and porcine pancreatic lipase. The enzymatic hydrolysis reactions are monitored by thin layer chromatography, revealing which of the two ester groups is hydrolysed, and the rate of the enzyme-catalysed reaction. The results of a group experiment revealed that in all cases hydrolysis was observed with at least one enzyme, and in most cases the enzymatic hydrolysis was specific for production of either the hydroxy-ester or acyl-acid product. Specificity towards the ortho-substituted series was markedly different to that of the para-substituted series, which could be rationalised in the case of pig liver esterase by a published active site model.

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.

Structural Mechanism for the Temperature-Dependent Activation of the Hyperthermophilic Pf2001 Esterase.

PubMed

Varejão, Nathalia; De-Andrade, Rafael A; Almeida, Rodrigo V; Anobom, Cristiane D; Foguel, Debora; Reverter, David

2018-02-06

Lipases and esterases constitute a group of enzymes that catalyze the hydrolysis or synthesis of ester bonds. A major biotechnological interest corresponds to thermophilic esterases, due to their intrinsic stability at high temperatures. The Pf2001 esterase from Pyrococcus furiosus reaches its optimal activity between 70°C and 80°C. The crystal structure of the Pf2001 esterase shows two different conformations: monomer and dimer. The structures reveal important rearrangements in the "cap" subdomain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, the dimer interface is essential for the formation of the hydrophobic channel for substrate selectivity, as confirmed by mutagenesis and kinetic analysis. We also provide evidence for dimer formation at high temperatures, a process that correlates with its enzymatic activation. Thus, we propose a temperature-dependent activation mechanism of the Pf2001 esterase via dimerization that is necessary for the substrate channel formation in the active-site cleft. Copyright © 2017 Elsevier Ltd. All rights reserved.

The non-targeted effects of radiation are perpetuated by exosomes.

PubMed

Al-Mayah, Ammar; Bright, Scott; Chapman, Kim; Irons, Sarah; Luo, Ping; Carter, David; Goodwin, Edwin; Kadhim, Munira

2015-02-01

Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma. Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood. The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein-RNA inactivation. These were added to separate populations of unirradiated cells. The BE was partially inhibited when either exosome protein or exosome RNA were inactivated separately, whilst combined RNA-protein inhibition significantly reduced or eliminated the BE. These results demonstrate that exosomes are associated with long-lived signalling of the NTE of IR. Both RNA and protein molecules of exosomes work in a synergistic manner to initiate NTE

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.

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.

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.

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.

Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness

PubMed Central

Kmoch, S.; Majewski, J.; Ramamurthy, V.; Cao, S.; Fahiminiya, S.; Ren, H.; MacDonald, I.M.; Lopez, I.; Sun, V.; Keser, V.; Khan, A.; Stránecký, V.; Hartmannová, H.; Přistoupilová, A.; Hodaňová, K.; Piherová, L.; Kuchař, L.; Baxová, A.; Chen, R.; Barsottini, O.G.P.; Pyle, A.; Griffin, H.; Splitt, M.; Sallum, J.; Tolmie, J.L.; Sampson, J.R.; Chinnery, P.; Canada, Care4Rare; Banin, E.; Sharon, D.; Dutta, S.; Grebler, R.; Helfrich-Foerster, C.; Pedroso, J.L.; Kretzschmar, D.; Cayouette, M.; Koenekoop, R.K.

2015-01-01

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photo-receptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness. PMID:25574898

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.

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.

Mycobacteriocins produced by rapidly growing mycobacteria are Tween-hydrolyzing esterases.

PubMed Central

Saito, H; Tomioka, H; Watanabe, T; Yoneyama, T

1983-01-01

Smegmatocin, a protein produced by Mycobacterium smegmatis ATCC 14468, was found to have an esterase activity, hydrolyzing Tween 80, polyoxyethylene sorbitan monooleate, added to the assay medium for various "bacteriocins" from mycobacteria. Because M. diernhoferi ATCC 19340 (indicator strain for smegmatocin) is highly susceptible to oleic acid and smegmatocin requires Tween 80 for manifestation of its anti-M. diernhoferi activity, it is likely that smegmatocin-mediated antimicrobial action is caused by oleic acid generated by hydrolysis of Tween 80 by the inherent esterase action of smegmatocin. Other mycobacteriocins from rapidly growing mycobacteria also have inherent esterase activity against Tween 80 and require Tween 80 for expression of antimycobacterial action. Smegmatocin was found to hydrolyze various polyoxyethylene (sorbitan) fatty acyl esters but not sorbitan monooleate and glyceryl esters. Images PMID:6826523

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.

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

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

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.

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 ...

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.

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.

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.

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.

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.

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

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.

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

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.

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...

Phospholipase B activity and organophosphorus compound toxicity in cultured neural cells

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

Read, David J.; Langford, Lynda; Barbour, Helen R.

2007-03-15

Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [{sup 14}C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [{sup 14}C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTEmore » inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [{sup 14}C]phosphatidylcholine to [{sup 14}C]GroPCho over 24 h was relatively small (2.3-14%). Consequently, even with > 80% inhibition of [{sup 14}C]GroPCho production, increased [{sup 14}C]phosphatidylcholine was not detected. At concentrations of 1-10 {mu}M, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2-3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia.« less

3 Benzyl-6-chloropyrone: a suicide inhibitor of cholesterol esterase

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

Saint, C.; Gallo, I.; Kantorow, M.

Cholesterol, absorbed from the intestine, appears in lymph as the ester. Cholesterol esterase is essential for this process, since depletion of the enzyme blocks and repletion restores, absorption. Selective inhibitors of cholesterol esterase may thus prove useful in reducing cholesterol uptake. A series of potential suicide substrates were synthesized which, following cleavage by the enzyme, would attack the putative nucleophile in the active site. One of these, 3-benzyl-6-chloropyrone (3BCP), inhibited both synthesis and hydrolysis of /sup 14/C-cholesteryl oleate with an I/sub 50/ of approximately 150 ..mu..M. The inactivation was time-dependent and characteristic of a suicide mechanism. The ..cap alpha.. pyronemore » structure (lactone analog) is cleaved by a serine-hydroxyl in the active site. This generates an enoyl chloride which inactivates the imidazole believed to play a part in the catalytic function of the enzyme. Inhibition by 3BCP is selective for cholesterol esterase. The activity of pancreatic lipase as not affected by concentrations up to 1 mM.« less

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.

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.

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.

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

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.

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.

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.

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

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

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.

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

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.

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).

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

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.

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

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.

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.

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.

Identifying Safer Anti-Wear Triaryl Phosphate Additives for Jet Engine Lubricants

PubMed Central

Baker, Paul E.; Cole, Toby B.; Cartwright, Megan; Suzuki, Stephanie M.; Thummel, Kenneth E.; Lin, Yvonne S.; Co, Aila L.; Rettie, Allan E.; Kim, Jerry H.; Furlong, Clement E.

2013-01-01

Individuals aboard jet aircraft may be exposed to potentially toxic triaryl organophosphate anti-wear lubricant additives (TAPs) that are converted by cytochromes P450 into toxic metabolites. Consequences of exposure could be reduced by using less toxic TAPs. Our goal was to determine whether an in vitro assay for inhibition of butyrylcholinesterase (BChE) by bioactivated TAPs would be predictive of inhibition of serine active-site enzymes in vivo. The in vitro assay involved TAP bioactivation with liver microsomes and NADPH, followed by incubation with human BChE and measurement of BChE activity. Of 19 TAPs tested, tert-butylated isomers produced the least BChE inhibition. To determine the relevance of these results in vivo, mice were exposed to Durad 125 (D125; a commercial mixture of TAP esters) or to TAPs demonstrating low or no BChE inhibition when assayed in vitro. Inhibition of BChE by bioactivated TAPs in vitro correlated well with inhibition of other serine active-site enzymes in vivo, with the exception of brain acetylcholinesterase and neuropathy target esterase (NTE), which were not inhibited by any TAP tested following single exposures. A recombinant catalytic domain of NTE (rNEST) exhibited classical kinetic properties of NTE. The metabolite of tri-(o-cresyl) phosphate (ToCP), 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP), inhibited rNEST in vitro, but with an IC50 value almost 6-times higher than for inhibition of BChE. Physiologically-relevant concentrations of the flavonoid, naringenin, dramatically reduced D125 bioconversion in vitro. The in vitro assay should provide a valuable tool for prescreening candidate TAP anti-wear additives, identifying safer additives and reducing the number of animals required for in vivo toxicity testing. PMID:23085349

Identifying safer anti-wear triaryl phosphate additives for jet engine lubricants.

PubMed

Baker, Paul E; Cole, Toby B; Cartwright, Megan; Suzuki, Stephanie M; Thummel, Kenneth E; Lin, Yvonne S; Co, Aila L; Rettie, Allan E; Kim, Jerry H; Furlong, Clement E

2013-03-25

Individuals aboard jet aircraft may be exposed to potentially toxic triaryl organophosphate anti-wear lubricant additives (TAPs) that are converted by cytochromes P450 into toxic metabolites. Consequences of exposure could be reduced by using less toxic TAPs. Our goal was to determine whether an in vitro assay for inhibition of butyrylcholinesterase (BChE) by bioactivated TAPs would be predictive of inhibition of serine active-site enzymes in vivo. The in vitro assay involved TAP bioactivation with liver microsomes and NADPH, followed by incubation with human BChE and measurement of BChE activity. Of 19 TAPs tested, tert-butylated isomers produced the least BChE inhibition. To determine the relevance of these results in vivo, mice were exposed to Durad 125 (D125; a commercial mixture of TAP esters) or to TAPs demonstrating low or no BChE inhibition when assayed in vitro. Inhibition of BChE by bioactivated TAPs in vitro correlated well with inhibition of other serine active-site enzymes in vivo, with the exception of brain acetylcholinesterase and neuropathy target esterase (NTE), which were not inhibited by any TAP tested following single exposures. A recombinant catalytic domain of NTE (rNEST) exhibited classical kinetic properties of NTE. The metabolite of tri-(o-cresyl) phosphate (ToCP), 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP), inhibited rNEST in vitro, but with an IC(50) value almost 6-times higher than for inhibition of BChE. Physiologically-relevant concentrations of the flavonoid naringenin dramatically reduced D125 bioconversion in vitro. The in vitro assay should provide a valuable tool for prescreening candidate TAP anti-wear additives, identifying safer additives and reducing the number of animals required for in vivo toxicity testing. Copyright © 2012 Elsevier Ireland Ltd. 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

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

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.

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

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.

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

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.

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.

Esterase Isoenzyme Profiles in Acute and Chronic Leukemias.

PubMed

Drexler, H G; Gignac, S M; Hoffbrand, A V; Minowada, J

1991-01-01

Using isoelectric focusing (IEF) a number of carboxylic esterase isoenzymes (EC 3.1.1.1) with isoelectric points between pH 4.5-8.0 can be separated. One particular isoenzyme with an isoelectric point at about pH 6.0, the Mono-band, can be selectively and completely inhibited by sodium fluoride; this isoenzyme comprises a number of closely related subcomponents and may appear in more than one band on the gel. We analyzed the expression of typical esterase isoenzyme patterns in cells from a large panel of leukemias which were tested under identical conditions by IEF on horizontal thin-layer polyacrylamide gels with an ampholyte of pH 2-11. The 442 cases of acute and chronic myeloid and lymphoid leukemia (AML/AMMoL, CML/CMML, ALL, CLL) were classified according to clinical, morpho-cytochemical and immunophenotyping criteria. While bands between pH 4.5-5.5 appeared not to be specific for lineage or stage of differentiation, isoenzymes between pH 6.6-7.7 provided information on the type of leukemia involved. Seven typical isoenzyme patterns termed Mono1/Mono2 (fo monocyte-associated), My1/My2 (myeloid), Lym1/Lym2 (lymphoid) and Und (undifferentiated) could be discerned. Lym and Und patterns are characterized by fewer bands with a weaker staining intensity than Mono and My patterns. Nearly all cases of lymphoid leukemias (acute and chronic) expressed only Lym or Und esterase isoenzyme patterns, but no Mono or My patterns. Cases of acute or chronic myeloid and (myelo)monocytic leukemia showed strong isoenzyme staining displaying predominantly Mono or My isoenzyme patterns. The isoenzyme patterns found in CML in lymphoid or myeloid blast crisis corresponded to those seen in the respective acute leukemias, ALL or AML. The Mono-band was found in most cases of leukemias with monocytic elements (AMMoL 80%, CML 44%, CMML 100%), in the occasional case of CML-myeloid blast crisis or AML, but in none of the cases of ALL or CLL. This isoenzyme is a distinctive, specific marker for

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.

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.

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

Cultural diversification promotes rapid phenotypic evolution in Xavánte Indians

PubMed Central

Hünemeier, Tábita; Gómez-Valdés, Jorge; Ballesteros-Romero, Mónica; de Azevedo, Soledad; Martínez-Abadías, Neus; Esparza, Mireia; Sjøvold, Torstein; Bonatto, Sandro L.; Salzano, Francisco Mauro; Bortolini, Maria Cátira; González-José, Rolando

2012-01-01

Shifts in social structure and cultural practices can potentially promote unusual combinations of allele frequencies that drive the evolution of genetic and phenotypic novelties during human evolution. These cultural practices act in combination with geographical and linguistic barriers and can promote faster evolutionary changes shaped by gene–culture interactions. However, specific cases indicative of this interaction are scarce. Here we show that quantitative genetic parameters obtained from cephalometric data taken on 1,203 individuals analyzed in combination with genetic, climatic, social, and life-history data belonging to six South Amerindian populations are compatible with a scenario of rapid genetic and phenotypic evolution, probably mediated by cultural shifts. We found that the Xavánte experienced a remarkable pace of evolution: the rate of morphological change is far greater than expected for its time of split from their sister group, the Kayapó, which occurred around 1,500 y ago. We also suggest that this rapid differentiation was possible because of strong social-organization differences. Our results demonstrate how human groups deriving from a recent common ancestor can experience variable paces of phenotypic divergence, probably as a response to different cultural or social determinants. We suggest that assembling composite databases involving cultural and biological data will be of key importance to unravel cases of evolution modulated by the cultural environment. PMID:22184238

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

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

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore

PubMed Central

Marquardt, Joseph R.; Perkins, Jennifer L.; Beuoy, Kyle J.; Fisk, Harold A.

2016-01-01

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus. PMID:27339139

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore.

PubMed

Marquardt, Joseph R; Perkins, Jennifer L; Beuoy, Kyle J; Fisk, Harold A

2016-07-12

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus.

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.

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.

Overexpression of esterase D in kidney from trisomy 13 fetuses.

PubMed Central

Loughna, S; Bennett, P; Gau, G; Nicolaides, K; Blunt, S; Moore, G

1993-01-01

Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D was found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses. Images Figure 1 Figure 2 Figure 3 PMID:8213811

Overexpression of esterase D in kidney from trisomy 13 fetuses

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

Loughna, S.; Moore, G.; Gau, G.

1993-10-01

Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D wasmore » found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses. 34 refs., 3 figs., 2 tabs.« less

Activation of Neuregulin 1/ErbB Signaling Is Involved in the Development of TOCP-Induced Delayed Neuropathy.

PubMed

Xu, Hai-Yang; Wang, Pan; Sun, Ying-Jian; Xu, Ming-Yuan; Zhu, Li; Wu, Yi-Jun

2018-01-01

Organophosphate-induced delayed neuropathy (OPIDN) is characterized by progressive axonal degeneration and demyelination of the spinal cord and sciatic nerves. The neuregulin 1/epidermal growth factor receptor (ErbB) signaling pathway is crucial for axonal myelination. In this study, we investigated whether the neuregulin 1/ErbB signaling pathway mediated the progression of OPIDN. Adult hens were given tri- o -cresyl phosphate (TOCP), a typical neuropathic organophosphorus compound, to induce OPIDN. The ErbB inhibitor lapatinib was administered to hens 4 h prior to and 4 days after TOCP exposure. The neuregulin 1/ErbB signaling pathway was examined for their role in maintaining spinal cord and sciatic nerve fiber integrity. Schwann cell line sNF96.2 was used as the in vitro cell model. The in vivo results showed that TOCP (750 mg/kg body weight, p.o .) induced prominent ataxia and significant axon degeneration in the spinal cord and sciatic nerves. Lapatinib (25 mg/kg body weight, p.o .) treatment attenuated OPIDN clinically and histopathlogically and partially prevented the TOCP-induced activation of neuregulin 1/ErbB signaling pathway. Lapatinib also prevented the TOCP-induced inhibition of neuropathy target esterase (NTE), a key enzyme during the development of OPIDN, and the disturbed metabolism of phosphatidylcholine in sciatic nerves. In addition, lapatinib was shown, in vitro , to protect sNF96.2 cells from TOCP-induced dedifferentiation through neuregulin 1/ErbB signaling. Our results suggest that neuregulin 1/ErbB, through regulation of NTE activity in the peripheral nervous system, mediates the progression of OPIDN. Thus, this signal may serve as a potential target for the treatment of OPIDN.

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

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).

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.

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...

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.

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.

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

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.

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.

Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase

PubMed Central

Swartz, Kenneth; Zhang, Yiguan; Valeriote, Frederick; Chen, Ben; Shaw, Jiajiu

2013-01-01

UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25 °C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/Vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis–Menten kinetics showed that the Km values of UTL-5g were 2.07 mM with PLE and 0.37 mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA. PMID:24126042

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.

Usefulness of Leukocyte Esterase Test Versus Rapid Strep Test for Diagnosis of Acute Strep Pharyngitis.

PubMed

Nibhanipudi, Kumara V

2015-01-01

A study to compare the usage of throat swab testing for leukocyte esterase on a test strip(urine dip stick-multi stick) to rapid strep test for rapid diagnosis of Group A Beta hemolytic streptococci in cases of acute pharyngitis in children. The testing of throat swab for leukocyte esterase on test strip currently used for urine testing may be used to detect throat infection and might be as useful as rapid strep. All patients who come with a complaint of sore throat and fever were examined clinically for erythema of pharynx, tonsils and also for any exudates. Informed consent was obtained from the parents and assent from the subjects. 3 swabs were taken from pharyngo-tonsillar region, testing for culture, rapid strep & Leukocyte Esterase. Total number is 100. Cultures 9(+); for rapid strep== 84(-) and16 (+); For LE== 80(-) and 20(+) From data configuration Rapid Strep versus LE test don't seem to be a random (independent) assignment but extremely aligned. The Statistical results show rapid and LE show very agreeable results. Calculated Value of Chi Squared Exceeds Tabulated under 1 Degree Of Freedom (P<.0.0001) reject Null HYPOTHESIS and Conclude Alternative Conclusions: Leukocyte esterase on throat swab is as useful as rapid strep test for rapid diagnosis of strep pharyngitis on test strip currently used for urine dip stick causing acute pharyngitis in children.

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.

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.

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.

Esterase detoxification of acetylcholinesterase inhibitors using human liver samples in vitro

EPA Science Inventory

Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are consider...

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.

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.

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

A new esterase for the cleavage of pivalic acid-containing prodrug esters of cephalosporins.

PubMed

Sauber, K; Aretz, W; Meiwes, J; Wollmann, T

1996-07-01

An extracellular esterase from the actinomycetes Amycolatopsis orientalis was found by screening. It is capable of splitting the isomeric mixture (K/J) of (I, Scheme 1) into 7-amino-3-methoxymethyl-3-cephem-4-carboxylic acid, pivalic acid, and acetaldehyde with a high yield. The purified enzyme of 55.4 Kd by SDS-PAGE shows an N-terminal sequence of VRTCADLVRTYDLPGAVTH. The isoelectric point is 8.9 +/- 0.1. It can be immobilized with good yield to VA-Epoxy Biosynth. Besides the above-mentioned reaction, the esterase cleaves many other esters such as methyl-2-chloropropionic acid.

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.

Usefulness of Leukocyte Esterase Test Versus Rapid Strep Test for Diagnosis of Acute Strep Pharyngitis

PubMed Central

2015-01-01

Objective: A study to compare the usage of throat swab testing for leukocyte esterase on a test strip(urine dip stick-multi stick) to rapid strep test for rapid diagnosis of Group A Beta hemolytic streptococci in cases of acute pharyngitis in children. Hypothesis: The testing of throat swab for leukocyte esterase on test strip currently used for urine testing may be used to detect throat infection and might be as useful as rapid strep. Methods: All patients who come with a complaint of sore throat and fever were examined clinically for erythema of pharynx, tonsils and also for any exudates. Informed consent was obtained from the parents and assent from the subjects. 3 swabs were taken from pharyngo-tonsillar region, testing for culture, rapid strep & Leukocyte Esterase. Results: Total number is 100. Cultures 9(+); for rapid strep== 84(-) and16 (+); For LE== 80(-) and 20(+) Statistics: From data configuration Rapid Strep versus LE test don’t seem to be a random (independent) assignment but extremely aligned. The Statistical results show rapid and LE show very agreeable results. Calculated Value of Chi Squared Exceeds Tabulated under 1 Degree Of Freedom (P<.0.0001) reject Null Hypothesis and Conclude Alternative Conclusions: Leukocyte esterase on throat swab is as useful as rapid strep test for rapid diagnosis of strep pharyngitis on test strip currently used for urine dip stick causing acute pharyngitis in children. PMID:27335975

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

Influence of ammonium salts on the lipase/esterase activity assay using p-nitrophenyl esters as substrates.

PubMed

De Yan, Hong; Zhang, Yin Jun; Liu, Hong Cai; Zheng, Jian Yong; Wang, Zhao

2013-01-01

p-Nitrophenyl esters with a short-chain carboxylic group, such as p-nitrophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB), could be effectively hydrolyzed by ammonium salts. p-Nitrophenyl esters were usually used as substrates to assay the lipase/esterase activity. Ammonium sulfate precipitation was often used to purify proteins, and some ammonium salts were usually used as nitrogen sources or inorganic salts for the lipase/esterase production. To study the effect of ammonium salts on the assay of the lipase/esterase activity, the contributing factors of hydrolysis of p-NPA/p-NPB catalyzed by ammonium salts were investigated. The lipase activities were compared in the presence and absence of ammonium sulfate. The hydrolysis reaction could be catalyzed under neutral and alkaline circumstances. The hydrolysis rate increased with the increase in the reaction temperature or the concentration of ammonium ion. When p-NPA was employed as the substrate for the analysis of the lipase/esterase activity, the effect of ammonium sulfate on the analysis could be neutralized by setting a control when the concentration of ammonium sulfate was less than 40% saturation. However, when the concentration of ammonium sulfate increased from 40% to 100% saturation, the enzyme activities decreased about 13-40%, which could not be ignored for accurate analysis of the enzyme activity. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase

USDA-ARS?s Scientific Manuscript database

The objective was to phylogenetically classify diverse strains of A. pullulans and determine their production of feruloyl esterase. Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were as...

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.

Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase.

PubMed

Swartz, Kenneth; Zhang, Yiguan; Valeriote, Frederick; Chen, Ben; Shaw, Jiajiu

2013-12-01

UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25°C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis-Menten kinetics showed that the Km values of UTL-5g were 2.07mM with PLE and 0.37mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA. Copyright © 2013 Elsevier B.V. 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

Glucuronoyl esterases: diversity, properties and biotechnological potential. A review.

PubMed

Monrad, Rune Nygaard; Eklöf, Jens; Krogh, Kristian B R M; Biely, Peter

2018-05-08

Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 (CE15) are involved in microbial degradation of lignocellulosic plant materials. GEs are capable of degrading complex polymers of lignin and hemicellulose cleaving ester bonds between glucuronic acid residues in xylan and lignin alcohols. GEs promote separation of lignin, hemicellulose and cellulose which is crucial for efficient utilization of biomass as an energy source and feedstock for further processing into products or chemicals. Genes encoding GEs are found in both fungi and bacteria, but, so far, bacterial GEs are essentially unexplored, and despite being discovered >10 years ago, only a limited number of GEs have been characterized. The first laboratory scale example of improved xylose and glucuronic acid release by the synergistic action of GE with cellulolytic enzymes was only reported recently (improved C5 sugar and glucuronic acid yields) and, until now, not much is known about their biotechnology potential. In this review, we discuss the diversity, structure and properties of microbial GEs and consider the status of their action on natural substrates and in biological systems in relation to their future industrial use.

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.

Esterase detoxification of acetylcholinesterase inhibitors by human or rat liver in vitro

EPA Science Inventory

Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON) are considered...

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.

Acute promyelocytic leukemia, hypogranular variant, with uncharacteristic staining with chloroacetate esterase.

PubMed

Dunphy, C H; Polski, J M; Johns, G; Evans, H L; Gardner, L J

2001-06-01

A diagnosis of the hypogranular variant of acute promyelocytic leukemia (APLv) may be difficult to establish based on cytomorphology alone. However, the great majority of cases have a classical immunophenotype by flow cytometric immunophenotyping (FCI) (CD13+, CD33+, dim CD64+, HLA-DR-, and CD34-) and a classical enzyme cytochemical (EC) staining pattern. [intensely staining with myeloperoxidase, Sudan Black B, and chloroacetate esterase (CAE) and negative with alpha'-naphthyl acetate and butyrate esterases]. Although the immunophenotype of APLv by FCI has varied in the literature (HLA-DR +/- and CD34 +/-), the EC staining pattern has remained constant. We report a case of APLv with characteristic cytomorphology, compatible FCI data (CD13+, CD33+, dim CD64+, HLA-DR +/-, and CD34-), chromosomal detection of t(15; 17), and molecular detection of the PML/RAR alpha fusion gene; however, staining of the leukemic cells with CAE was quite uncharacteristic. We describe our findings.

Electrophysiologic changes following treatment with organophosphorus-induced delayed neuropathy-producing agents in the adult hen.

PubMed

Robertson, D G; Schwab, B W; Sills, R D; Richardson, R J; Anderson, R J

1987-03-15

Although clinical, pathological, and biochemical effects of organophosphorus-induced delayed neuropathy (OPIDN) have been intensively investigated in the adult hen, detailed electrophysiological studies are lacking. Adult white leghorn hens were treated with a single oral dose of either 30 mg/kg tri-2-cresyl phosphate (TOCP), 750 mg/kg TOCP, 4 mg/kg di-n-butyl-2,2-dichlorovinyl phosphate (DBCV), or 30 mg/kg di-n-butyl-2,2-dichlorovinyl phosphinate (DBCV-P). The 750 mg/kg TOCP and DBCV, but not the 30 mg/kg TOCP and DBCV-P, treatments resulted in clinical signs of OPIDN and mild to marked damage of the tibial nerve 21 days after dose. Twenty-four hr lymphocyte neurotoxic esterase (NTE) inhibition was used as an index of brain NTE inhibition for the various organophosphorus compound (OP) treatment. Twenty-four hr lymphocyte NTE inhibition for 30 mg/kg TOCP, 750 mg/kg TOCP, DBCV, and DBCV-P was 54.1, 87.1, 84.8, and 68.3%, respectively. Twenty-one days after dose, the TOCP-treated hens exhibited some abnormalities in conduction velocity and action potential duration in the tibial or sciatic nerves. No abnormalities were observed in action potential parameters of either the DBCV or DBCV-P treatments. Neurotoxic OP (TOCP and DBCV) treatment resulted in decreased refractoriness in the tibial nerve, increased refractoriness in the sciatic nerve, and elevated strength duration threshold for both nerves. These changes were not present in nerves from DBCV-P (a non-neurotoxic NTE inhibitor)-treated hens. These results suggest that refractory period and strength duration abnormalities in peripheral nerve correlate well with the production of OPIDN and are evident without coincident clinical signs or histopathology.

Susceptibility of various areas of the nervous system of hens to TOCP-induced delayed neuropathy.

PubMed

Classen, W; Gretener, P; Rauch, M; Weber, E; Krinke, G J

1996-01-01

Sensitivity of in-life parameters, biochemical endpoints, and susceptibility of various areas of the chicken nervous system to delayed neuropathy induced by tri-orthocresyl phosphate (TOCP) was assessed. Groups of hens were exposed to a single oral dose of TOCP of 0, 50, 200 or 500 mg/kg and the animals observed for 21 days. Perfusion fixed, paraffin embedded tissue sections were stained with Bodian's silver and Luxol blue and semi-thin epoxy sections with toluidine blue. Sciatic and tibial nerves, lumbosacral, midthoracic, and upper cervical spinal cord, medulla oblongata and cerebellum were examined using a semiquantitative scoring system. In pair-dosed hens inhibition of brain and spinal cord neurotoxic esterase (NTE) and cholinesterase and of plasma and erythrocyte cholinesterases was determined 24 hr and 48 hr after administration. At all dose levels NTE in brain and spinal cord and plasma cholinesterase was inhibited markedly. Quantitative inhibition of NTE was seen also in absence of neuropathy. Ataxia and body weight loss occurred in high-dose animals only, while dose-related neuropathy was seen in the distal tibial nerve, medulla oblongata and cerebellum. Ataxia was correlated best with neuropathy in peripheral nerves while degeneration of nerve fibers in the cerebellum, seen best in mid-longitudinal sections, was the most sensitive histological indicator of TOCP-induced delayed neuropathy. The particular susceptibility of spinocerebellar neurons was recognized long ago, but often has been neglected in delayed neurotoxicity studies and respective guidelines. Optimal sensitivity of toxicity tests is a prerequisite for risk assessment, can be cost efficient, and nowadays should be a main interest of animal welfare in order to reduce animals' suffering. Based on these data, determination of NTE inhibition together with histopathological examination of longitudinal sections of distal tibial nerves, mid-longitudinal sections of rostral cerebellum and cross

[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.

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.

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.

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.

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.

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

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.

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.

[Electrophoretic forms of glucose-6-phosphate dehydrogenase, acid phosphatase and esterase in Amoeba species amoebas].

PubMed

Sopina, V A

2000-01-01

Glucose-6-phosphate dehydrogenase (G6PD), acid phosphatase and esterases in free-living amoebae of 7 Amoeba species were investigated with the use of disc-electrophoresis in polyacrylamide gel. The evidence provided is suggestive that the electrophoretic isoenzyme patterns of acid phosphatase and esterases (and G6PD in some cases), in addition to a few morphological characters, can serve as a taxonomic criterion for species identification within this genus, as well as for revealing erroneously classified species and strains. It is suggested that A. indica is an independent species whose preliminary diagnosis has been given in this paper. It is concluded that A. discoides and A. lescherae are strains of A. proteus, rather than two independent species. A and As-102 amoebian strains, kept in the collection of protozoan strains and species of the Institute of Cytology RAS and referred to as strains of A. proteus, belong in reality to another Amoeba species and even to another genus within the family Amoebidae. This conclusion has been documented by results of our analysis of electrophoretic patterns of acid phosphatase and esterases in these strains.

Enzymatic acylation of flavonoid glycosides by a carbohydrate esterase of family 16.

PubMed

Biely, Peter; Cziszárová, Mária; Wong, Ken K Y; Fernyhough, Alan

2014-11-01

The acetyl esterase of Trichoderma reesei belonging to carbohydrate esterase (CE) family 16 catalyzes transacylations to carbohydrate moieties of flavonoid glycosides, esculin and rutin. The enzyme recognizes as acyl donors vinyl esters of short carboxylic acids. Esculin was acylated at position 3 of the glucosyl residue in aqueous solutions saturated with vinyl acetate and vinyl propionate. The yields of esculin monoacetate and monopropionate of esculin in aqueous medium (esculin 40 mM, enzyme 40 µg/ml, 40 °C, 3 days) were 67 and 55 %, respectively. Replacement of water by 2-propanol was required for a similar acylation of rutin at 4 mM concentration. The yields of rutin monoacetate and propionate were 60 and 30 %, respectively. The results indicate that the enzyme could be used for an easy modification of solubility and hydrophobicity of glycosylated compounds, including drugs and functional food additives.

Biocatalytic Synthesis of Poly(δ-Valerolactone) Using a Thermophilic Esterase from Archaeoglobus fulgidus as Catalyst

PubMed Central

Cao, Hong; Han, Haobo; Li, Guangquan; Yang, Jiebing; Zhang, Lingfei; Yang, Yan; Fang, Xuedong; Li, Quanshun

2012-01-01

The ring-opening polymerization of δ-valerolactone catalyzed by a thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically evaluated. Through the optimization of reaction conditions, poly(δ-valerolactone) was produced in 97% monomer conversion, with a number-average molecular weight of 2225 g/mol, in toluene at 70 °C for 72 h. This paper has produced a new biocatalyst for the synthesis of poly(δ-valerolactone), and also deeper insight has been gained into the mechanism of thermophilic esterase-catalyzed ring-opening polymerization. PMID:23202895

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...

Comparison of neurotoxic effects and potential risks from oral administration or ingestion of tricresyl phosphate and jet engine oil containing tricresyl phosphate.

PubMed

Mackerer, C R; Barth, M L; Krueger, A J; Chawla, B; Roy, T A

1999-07-09

Neurotoxicity of tricresyl phosphates (TCPs) and jet engine oil (JEO) containing TCPs were evaluated in studies conducted in both rat and hen. Results for currently produced samples ("conventional" and "low-toxicity") were compared with published findings on older samples to identify compositional changes and relate those changes to neurotoxic potential. Finally, a human risk assessment for exposure by oral ingestion of currently produced TCPs in JEO at 3% (JEO + 3%) was conducted. TCPs and certain other triaryl phosphates administered as single doses inhibited brain neuropathy target esterase (B-NTE; neurotoxic esterase) in the rat and the hen (hen 3.25 times as sensitive), and both species were deemed acceptable for initial screening purposes. Neither rat nor hen was sensitive enough to detect statistically significant inhibition of B-NTE after single doses of IEO + 3% "conventional" TCP. Subacute administration of 2 g/kg/d of JEO + 3% "conventional" TCP to the hen produced B-NTE inhibition (32%), which did not result in organophosphorus-induced delayed neurotoxicity (OPIDN). Subchronic administration of JEO + 3% TCP but not JEO + 1% TCP at 2 g/kg/d produced OPIDN. Thus, the threshold for OPIDN was between 20 and 60 mg "conventional" TCP/kg/d in JEO for 10 wk. The current "conventional" TCPs used in JEO and new "low-toxicity" TCPs now used in some JEO are synthesized from phenolic mixtures having reduced levels of ortho-cresol and ortho-xylenols resulting in TCPs of very high content of meta- and para-substituted phenyl moieties; this change in composition results in lower toxicity. The "conventional" TCPs still retain enough inhibitory activity to produce OPIDN, largely because of the presence of ortho-xylyl moieties; the "low-toxicity" TCPs are largely devoid of ortho substituents and have extremely low potential to cause OPIDN. The TCPs produced in the 1940s and 1950s were more than 400 times as toxic as the "low-toxicity" TCPs produced today. Analysis of the

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.

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.

Chasing Ghosts in Space Radiobiology Research: The Lost Focus on Non-Targeted Effects

NASA Astrophysics Data System (ADS)

Cucinotta, Francis; Saganti, Premkumar; Cacao, Eliedonna

2016-07-01

The doses and dose-rates of astronaut exposures to galactic cosmic rays (GCR) are accurately known, and lead to particle hits per cell nucleus from high charge and energy (HZE) particles of much less than one hit per cell per week. A large number of experiments have shown that additivity of biological effects is a valid assumption for space radiation exposures, while experiments at higher doses and dose-rates than occur in space continue to be a focus of the majority of space radiobiology research. Furthermore HZE particle exposures with mono-energetic particles manifest themselves as a mixed-radiation field due to the contributions of delta-rays and the random impact parameter of a particles track core to DNA and non-DNA targets in cells and tissues. The mixed-field manifestation of mono-energetic HZE particle exposures is well known from theoretical studies of microdosimetry and track structure. Additional mixed-field effects occur for single species experiments due to nuclear fragmentation in particle accelerator beam-lines and biological samples along with energy straggling. In contrast to these well known aspects of space radiobiology there are many open questions on the contribution of non-targeted effects to low dose and dose-rate exposures. Non-targeted effects (NTEs) include bystander effects and genomic instability, and have been shown to be the most important outstanding question for reducing uncertainties in space radiation cancer risk assessment. The dose-rate and radiation quality dependence of NTE's has not been established, while there is an over-arching need to develop 21st century experimental models of human cancer risk. We review possible mechanisms of NTE's and how new experiments to address these issues could be designed.

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.

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.

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.

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

Contributions of a unique β-clamp to substrate recognition illuminates the molecular basis of exolysis in ferulic acid esterases.

PubMed

Gruninger, Robert J; Cote, Chris; McAllister, Tim A; Abbott, D Wade

2016-04-01

Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging to Anaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 Å (1 Å=0.1 nm)] and absence (1.60 Å) of ferulic acid. AmCE1 adopts an α/β-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the β-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the β-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition. © 2016 Authors; published by

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.

Esterase-sensitive prodrugs with tunable release rates and direct generation of hydrogen sulfidea

PubMed Central

Zheng, Yueqin; Yu, Bingchen; Ji, Kaili; Pan, Zhixiang; Chittavong, Vayou

2016-01-01

Prodrugs that release hydrogen sulfide upon esterase-mediated cleavage of an ester group followed by lactonization are described herein. By modifying the ester group and thus its susceptibility to esterase, and structural features critical to the lactonization rate, H2S release rates can be tuned. Such prodrugs directly release hydrogen sulfide without the involvement of perthiol species, which are commonly encountered with existing H2S donors. Additionally, such prodrugs can easily be conjugated to another non-steroidal anti-inflammatory agent, leading to easy synthesis of hybrid prodrugs. As a biological validation of the H2S prodrugs, the anti-inflammatory effects of one such prodrug were examined by studying its ability to inhibit LPS-induced TNF-α production in RAW 264.7 cells. This type of H2S prodrugs shows great potential as both research tools and therapeutic agents. PMID:26822005

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

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

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

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.

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.

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.

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.

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

Preliminary X-ray analysis of twinned crystals of the Q88Y25_Lacpl esterase from Lactobacillus plantarum WCFS1

PubMed Central

Álvarez, Yanaisis; Esteban-Torres, María; Acebrón, Iván; de las Rivas, Blanca; Muñoz, Rosario; Martínez-Ripoll, Martín; Mancheño, José M.

2011-01-01

Q88Y25_Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxyl­esterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-­terminally His6-tagged Q88Y25_Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His6-tagged Q88Y25_Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 Å resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 Å. PMID:22102251

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

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

Functional Characterization of a Novel Marine Microbial Esterase and its Utilization in the Enantioselective Preparation of (R)-Methyl 2-Chloropropionate.

PubMed

Cao, Yingying; Deng, Dun; Sun, Aijun; Zhang, Yun; Hu, Yunfeng

2016-09-01

Chiral 2-chloropropanoic acids and their ester derivatives are crucial intermediates in the synthesis of many chemicals, especially herbicides. The enzymatic synthesis of chiral 2-chloropropanoic acids and their ester derivatives by esterases was not easily achieved, because the structural difference between the two enantiomers was too small to be recognized by esterases. Herein, we report the expression and functional characterization of one novel low temperature-resistant esterase EST12-7 identified from the genome of Pseudonocardia antitumoralis SCSIO 01299 isolated from the sediments of the South China Sea. Biocatalyst EST12-7 could hydrolyze racemic methyl 2-chloropropinate and generate optically pure (R)-methyl 2-chloropropinate with high enantiomeric excess (>99 %) and conversion (>49 %) after process optimization. Notably, the addition of different surfactants and using surfactants of different concentrations in the kinetic resolution catalyzed by EST12-7 could greatly affect the enantiomeric excess and conversion rate of product (R)-methyl 2-chloropropinate.

Isolation of an N-acetyl-DL-phenylalanine beta-naphthyl esterase from rabbit peritoneal polymorphonuclear leukocytes.

PubMed

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

1975-09-22

An N-acetyl-DL-phenylalanine beta-naphthyl esterase has been purified 26-fold from rabbit peritoneal polymorphonuclear leukocytes. The purified enzyme was inhibited by 10(-7) M p-nitrophenylethyl-5-chloropentylphosphonate. The apparent Km for hydrolysis of N-acetyl-DL-phenylalanine beta-naphthyl ester is 71 muM. Optimal reaction rates were observed at pH 6-8. No divalent cation requirement for the activation of the enzyme activity was observed. The esterase activity was neither inhibited nor stimulated by bacterial factor, complement component C5a, guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) which are attractants or repellents for polymorphonuclear leukocytes. High chemotactic activity was observed in the partially purified fraction of the enzyme. The chemotactic activity, like the enzyme activity, was completely inhibited by 10(-7) M phosphonate.

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.

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.

Pseudomonas aeruginosa biofilm growth inhibition on medical plastic materials by immobilized esterases and acylase.

PubMed

Kisch, Johannes Martin; Utpatel, Christian; Hilterhaus, Lutz; Streit, Wolfgang R; Liese, Andreas

2014-09-05

Biofilms are matrix-encapsulated cell aggregates that cause problems in technical and health-related areas; for example, 65 % of all human infections are biofilm associated. This is mainly due to their ameliorated resistance against antimicrobials and immune systems. Pseudomonas aeruginosa, a biofilm-forming organism, is commonly responsible for nosocomial infections. Biofilm development is partly mediated by signal molecules, such as acyl-homoserine lactones (AHLs) in Gram-negative bacteria. We applied horse liver esterase, porcine kidney acylase, and porcine liver esterase; these can hydrolyze AHLs, thereby inhibiting biofilm formation. As biofilm infections are often related to foreign material introduced into the human body, we immobilized the enzymes on medical plastic materials. Biofilm formation was quantified by Crystal Violet staining and confocal laser scanning microscopy, revealing up to 97 % (on silicone), 54 % (on polyvinyl chloride), and 77 % (on polyurethane) reduced biomass after 68 h growth. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

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

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

Effects of high hydrostatic pressure and temperature increase on Escherichia coli spp. and pectin methyl esterase inactivation in orange juice.

PubMed

Torres, E F; González-M, G; Klotz, B; Rodrigo, D

2016-03-01

The aim of this study was to evaluate the effect of high hydrostatic pressure treatment combined with moderate processing temperatures (25 ℃-50 ℃) on the inactivation of Escherichia coli O157: H7 (ATCC 700728), E. coli K12 (ATCC 23716), and pectin methyl esterase in orange juice, using pressures of 250 to 500 MPa with times ranging between 1 and 30 min. Loss of viability of E. coli O157:H7 increased significantly as pressure and treatment time increased, achieving a 6.5 log cycle reduction at 400 MPa for 3 min at 25 ℃ of treatment. With regard to the inactivation of pectin methyl esterase, the greatest reduction obtained was 90.05 ± 0.01% at 50 ℃ and 500 MPa of pressure for 15 min; therefore, the pectin methyl esterase enzyme was highly resistant to the treatments by high hydrostatic pressure. The results obtained in this study showed a synergistic effect between the high pressure and moderate temperatures in inactivating E. coli cells. © The Author(s) 2016.

Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass

USDA-ARS?s Scientific Manuscript database

The Aspergillus niger ferulic acid esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interactio...

Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes.

PubMed Central

Trapani, J A; Klein, J L; White, P C; Dupont, B

1988-01-01

A cDNA clone encoding a human serine esterase gene was isolated from a library constructed from poly(A)+ RNA of allogeneically stimulated, interleukin 2-expanded peripheral blood mononuclear cells. The clone, designated HSE26.1, represents a full-length copy of a 0.9-kilobase mRNA present in human cytotoxic cells but absent from a wide variety of noncytotoxic cell lines. Clone HSE26.1 contains an 892-base-pair sequence, including a single 741-base-pair open reading frame encoding a putative 247-residue polypeptide. The first 20 amino acids of the polypeptide form a leader sequence. The mature protein is predicted to have an unglycosylated Mr of approximately equal to 26,000 and contains a single potential site for N-linked glycosylation. The nucleotide and predicted amino acid sequences of clone HSE26.1 are homologous with all murine and human serine esterases cloned thus far but are most similar to mouse granzyme B (70% nucleotide and 68% amino acid identity). HSE26.1 protein is expressed weakly in unstimulated peripheral blood mononuclear cells but is strongly induced within 6-hr incubation in medium containing phytohemagglutinin. The data suggest that the protein encoded by HSE26.1 plays a role in cell-mediated cytotoxicity. Images PMID:3261871

Crystallization and preliminary X-ray analysis of a novel halotolerant feruloyl esterase identified from a soil metagenomic library

PubMed Central

Chen, Shang-ke; Wang, Kui; Liu, Yuhuan; Hu, Xiaopeng

2012-01-01

Feruloyl esterase cleaves the ester linkage formed between ferulic acid and polysaccharides in plant cell walls and thus has wide potential industrial applications. A novel feruloyl esterase (EstF27) identified from a soil metagenomic library was crystallized and a complete data set was collected from a single cooled crystal using an in-house X-ray source. The crystal diffracted to 2.9 Å resolution and belonged to space group P212121, with unit-cell parameters a = 94.35, b = 106.19, c = 188.51 Å, α = β = γ = 90.00°. A Matthews coefficient of 2.55 Å3 Da−1, with a corresponding solvent content of 51.84%, suggested the presence of ten protein subunits in the asymmetric unit. PMID:22750860

Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications.

PubMed

Mathew, Sindhu; Abraham, T Emilia

2004-01-01

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.

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

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.

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

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.

Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis

PubMed Central

2012-01-01

Background GDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP (Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes. Results In this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the

Crystallization and preliminary X-ray crystallographic analysis of a putative feruloyl esterase from Talaromyces cellulolyticus

PubMed Central

Watanabe, Masahiro; Ishikawa, Kazuhiko

2014-01-01

Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate-binding module connected through a serine/threonine-rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium acetate, 14% PEG 8000 as the precipitant. The crystal diffracted to 2.6 Å resolution and the crystal system is primitive orthorhombic, with unit-cell parameters a = 90.9, b = 123.4, c = 135.4 Å. Four molecules are assumed to be present per asymmetric unit, corresponding to a Matthews coefficient of 2.50 Å3 Da−1 and a solvent content of 50.88%(v/v). PMID:25484222

Crystallization and preliminary X-ray crystallographic analysis of a putative feruloyl esterase from Talaromyces cellulolyticus.

PubMed

Watanabe, Masahiro; Ishikawa, Kazuhiko

2014-12-01

Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate-binding module connected through a serine/threonine-rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium acetate, 14% PEG 8000 as the precipitant. The crystal diffracted to 2.6 Å resolution and the crystal system is primitive orthorhombic, with unit-cell parameters a = 90.9, b = 123.4, c = 135.4 Å. Four molecules are assumed to be present per asymmetric unit, corresponding to a Matthews coefficient of 2.50 Å(3) Da(-1) and a solvent content of 50.88%(v/v).

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

ISOLATION OF JUVENILE HORMONES ESTERASE AND ITS PARTIAL CDNA CLONE FROM THE BEETLE, TENEBRIO MOLITOR. (R825433)

EPA Science Inventory

Juvenile hormone esterase (JHE) plays an essential role in insect development. It is partially responsible for the clearance of juvenile hormone (JH) which regulates various aspects of insect development and reproduction. Because of its role in regulating JH titer, this enzyme...

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.

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.

The Alpha-Defensin Immunoassay and Leukocyte Esterase Colorimetric Strip Test for the Diagnosis of Periprosthetic Infection

PubMed Central

Wyatt, M.C.; Beswick, A.D.; Kunutsor, S.K.; Wilson, M.J.; Whitehouse, M.R.; Blom, A.W.

2016-01-01

Background: Synovial biomarkers have recently been adopted as diagnostic tools for periprosthetic joint infection (PJI), but their utility is uncertain. The purpose of this systematic review and meta-analysis was to synthesize the evidence on the accuracy of the alpha-defensin immunoassay and leukocyte esterase colorimetric strip test for the diagnosis of PJI compared with the Musculoskeletal Infection Society diagnostic criteria. Methods: We performed a systematic review to identify diagnostic technique studies evaluating the accuracy of alpha-defensin or leukocyte esterase in the diagnosis of PJI. MEDLINE and Embase on Ovid, ACM, ADS, arXiv, CERN DS (Conseil Européen pour la Recherche Nucléaire Document Server), CrossRef DOI (Digital Object Identifier), DBLP (Digital Bibliography & Library Project), Espacenet, Google Scholar, Gutenberg, HighWire, IEEE Xplore (Institute of Electrical and Electronics Engineers digital library), INSPIRE, JSTOR (Journal Storage), OAlster (Open Archives Initiative Protocol for Metadata Harvesting), Open Content, Pubget, PubMed, and Web of Science were searched for appropriate studies indexed from inception until May 30, 2015, along with unpublished or gray literature. The classification of studies and data extraction were performed independently by 2 reviewers. Data extraction permitted meta-analysis of sensitivity and specificity with construction of receiver operating characteristic curves for each test. Results: We included 11 eligible studies. The pooled diagnostic sensitivity and specificity of alpha-defensin (6 studies) for PJI were 1.00 (95% confidence interval [CI], 0.82 to 1.00) and 0.96 (95% CI, 0.89 to 0.99), respectively. The area under the curve (AUC) for alpha-defensin and PJI was 0.99 (95% CI, 0.98 to 1.00). The pooled diagnostic sensitivity and specificity of leukocyte esterase (5 studies) for PJI were 0.81 (95% CI, 0.49 to 0.95) and 0.97 (95% CI, 0.82 to 0.99), respectively. The AUC for leukocyte esterase and PJI

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

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.

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

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

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.

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

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.

Esterase- and pH-responsive poly(β-amino ester)-capped mesoporous silica nanoparticles for drug delivery.

PubMed

Fernando, Isurika R; Ferris, Daniel P; Frasconi, Marco; Malin, Dmitry; Strekalova, Elena; Yilmaz, M Deniz; Ambrogio, Michael W; Algaradah, Mohammed M; Hong, Michael P; Chen, Xinqi; Nassar, Majed S; Botros, Youssry Y; Cryns, Vincent L; Stoddart, J Fraser

2015-04-28

Gating of mesoporous silica nanoparticles (MSNs) with the stimuli-responsive poly(β-amino ester) has been achieved. This hybrid nanocarrier releases doxorubicin (DOX) under acidic conditions or in the presence of porcine liver esterase. The DOX loaded poly(β-amino ester)-capped MSNs reduce cell viability when tested on MDA-MB-231 human breast cancer cells.

Glycerophosphocholine catabolism as a new route for choline formation for phosphatidylcholine synthesis by the Kennedy pathway.

PubMed

Fernández-Murray, J Pedro; McMaster, Christopher R

2005-11-18

In eukaryotes, neuropathy target esterase (Nte1p in yeast) deacylates phosphatidylcholine derived exclusively from the CDP-choline pathway to produce glycerophosphocholine (GroPCho) and release two fatty acids. The metabolic fate of GroPCho in eukaryotic cells is currently not known. Saccharomyces cerevisiae contains two open reading frames predicted to contain glycerophosphodiester phosphodiesterase domains, YPL110c and YPL206c. Pulse-chase experiments were conducted to monitor GroPCho metabolic fate under conditions known to alter CDP-choline pathway flux and consequently produce different rates of formation of GroPCho. From this analysis, it was revealed that GroPCho was metabolized to choline, with this choline serving as substrate for renewed synthesis of phosphatidylcholine. YPL110c played the major role in this metabolic pathway. To extend and confirm the metabolic studies, the ability of the ypl110cDelta and ypl206cDelta strains to utilize exogenous GroPCho or glycerophosphoinositol as the sole source of phosphate was analyzed. Consistent with our metabolic profiling, the ypl206cDelta strain grew on both substrates with a similar rate to wild type, whereas the ypl110cDelta strain grew very poorly on GroPCho and with moderately reduced growth on glycerophosphoinositol.

A cold-induced pectin methyl-esterase inhibitor gene contributes negatively to freezing tolerance but positively to salt tolerance in Arabidopsis.

PubMed

Chen, Jian; Chen, Xuehui; Zhang, Qingfeng; Zhang, Yidan; Ou, Xiangli; An, Lizhe; Feng, Huyuan; Zhao, Zhiguang

2018-03-01

Plant pectin methyl-esterase (PME) and PME inhibitor (PMEI) belong to large gene families whose members are proposed to be widely involved in growth, development, and stress responses; however, the biological functions of most PMEs and PMEIs have not been characterized. In this study, we studied the roles of CbPMEI1, a cold-induced pectin methyl-esterase inhibitor (PMEI) gene from Chorispora bungeana, under freezing and salt stress. The putative CbPMEI1 peptide shares highest similarity (83%) with AT5G62360 (PMEI13) of Arabidopsis. Overexpression of either CbPMEI1 or PMEI13 in Arabidopsis decreased tissue PME activity and enhanced the degree of methoxylation of cell wall pectins, indicating that both genes encode functional PMEIs. CbPMEI1 and PMEI13 were induced by cold but repressed by salt stress and abscisic acid, suggesting distinct roles of the genes in freezing and salt stress tolerance. Interestingly, transgenic Arabidopsis plants overexpressing CbPMEI1 or PMEI13 showed decreased freezing tolerance, as indicated by survival and electrolyte leakage assays. On the other hand, the salt tolerance of transgenic plants was increased, showing higher rates of germination, root growth, and survival under salinity conditions as compared with non-transgenic wild-type plants. Although the transgenic plants were freezing-sensitive, they showed longer roots than wild-type plants under cold conditions, suggesting a role of PMEs in balancing the trade-off between freezing tolerance and growth. Thus, our study indicates that CbPMEI1 and PMEI13 are involved in root growth regulation under cold and salt stresses, and suggests that PMEIs may be potential targets for genetic engineering aimed to improve fitness of plants under stress conditions. Copyright © 2018 Elsevier GmbH. All rights reserved.

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.

Response of digestive enzymes and esterases of ecotoxicological concern in earthworms exposed to chlorpyrifos-treated soils.

PubMed

Sanchez-Hernandez, Juan C; Ríos, Juan Manuel; Attademo, Andrés M

2018-03-01

Assessment of organophosphorus (OP) pesticide exposure in non-target organisms rarely involves non-neural molecular targets. Here we performed a 30-d microcosm experiment with Lumbricus terrestris to determine whether the activity of digestive enzymes (phosphatase, β-glucosidase, carboxylesterase and lipase) was sensitive to chlorpyrifos (5 mg kg -1 wet soil). Likewise, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the wall muscle and gastrointestinal tissues as indicators of OP exposure. Chlorpyrifos inhibited the acid phosphatase (34% of controls), carboxylesterase (25.6%) and lipase activities (31%) in the gastrointestinal content. However, in the gastrointestinal tissue, only the carboxylesterase and lipase activities were significantly depressed (42-67% carboxylesterase inhibition in the foregut and crop/gizzard, and 15% lipase inhibition in the foregut). Chlorpyrifos inhibited the activity of both cholinesterases in the gastrointestinal tissues, whereas the AChE activity was affected in the wall muscle. These results suggested chlorpyrifos was widely distributed throughout the earthworm body after 30 d of incubation. Interestingly, we found muscle carboxylesterase activity strongly inhibited (92% of control) compared with that detected in the gastrointestinal tissues of the same OP-exposed individuals. This finding was explained by the occurrence of pesticide-resistant esterases in the gastrointestinal tissues, which were evidenced by zymography. Our results suggest that digestive processes of L. terrestris may be altered by chlorpyrifos, as a consequence of the inhibitory action of the insecticide on some digestive enzymes.

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) esterase (AChE) levels both in plasma and red blood cell (RBC) lysate. BLL were determined using a graphite furnace atomic absorption spectrometer. The results showed that BLL were 7.7 times higher in the painters as compared with that of the control group. Significant decreases in RBC and plasma AChE were observed in the exposed group in comparison with controls. RBC and plasma AChE showed a decrease of 18.4% and 18%, respectively, in the exposed group. The findings also indicated a significant negative correlation of both RBC and plasma AChE activities with BLL. The marked reduction observed in both RBC and plasma AChE activity may account for disruption of cholinergic function and result in neurotoxicity among the painters.

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

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.

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 ...

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

Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar.

PubMed

Saleme, Marina de Lyra Soriano; Cesarino, Igor; Vargas, Lívia; Kim, Hoon; Vanholme, Ruben; Goeminne, Geert; Van Acker, Rebecca; Fonseca, Fernando Campos de Assis; Pallidis, Andreas; Voorend, Wannes; Junior, José Nicomedes; Padmakshan, Dharshana; Van Doorsselaere, Jan; Ralph, John; Boerjan, Wout

2017-11-01

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis ( Arabidopsis thaliana ) and later in Medicago truncatula However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar ( Populus tremula × Populus alba ) resulted in up to 25% reduced lignin deposition, increased levels of p -hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries. © 2017 American Society of Plant Biologists. All Rights Reserved.

Depletion of juvenile hormone esterase extends larval growth in Bombyx mori.

PubMed

Zhang, Zhongjie; Liu, Xiaojing; Shiotsuki, Takahiro; Wang, Zhisheng; Xu, Xia; Huang, Yongping; Li, Muwang; Li, Kai; Tan, Anjiang

2017-02-01

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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...

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

Assessment of neurotoxic effects of tri-cresyl phosphates (TCPs) and cresyl saligenin phosphate (CBDP) using a combination of in vitro techniques.

PubMed

Hausherr, Vanessa; Schöbel, Nicole; Liebing, Julia; van Thriel, Christoph

2017-03-01

Environmental exposures to tri-cresyl phosphates (TCPs) and the possible formation of toxic metabolites (e.g. cresyl saligenin phosphate; CBDP) may cause a variety of neurotoxic effects in humans. As reported for other organophosphorus compounds (OPs), the inhibition of acetylcholine esterase (AChE) has also been proposed as the underlying mechanism for TCP neurotoxicity. The ortho-isomer, ToCP and its metabolite CBDP are also known to affect neuropathy target esterase (NTE) leading to organophosphate-induced delayed neuropathy (OPIDN). Recently, in vitro testing has led to the identification of other molecular targets and alternative mechanisms of ToCP toxicity. The metabolite CBDP and other isomers, as well as commercial mixtures have not been tested for such additional modes of actions. Accordingly, the present study investigates alterations of neurobiological correlates of central nervous processes using different in vitro techniques. The three symmetric TCP isomers - ToCP, TpCP, and TmCP - that contain a methyl group at the ortho-, para-, or meta-position of the aromatic ring system, respectively, together with a commercial TCP mixture, and CBDP were all tested using concentrations not exceeding their cytotoxic concentrations. Isolated cortical neurons were kept in culture for 6days followed by 24h incubation with different concentrations of the test compounds. Thus, all endpoints were assessed after 7days in vitro (DIV 7), at which time cell viability, neurite microstructure, and the function of glutamate receptors and voltage-gated calcium cannels (VGCC) were measured. While the cytotoxic potential of the TCP isomers and their mixture were comparable (IC 50 ≥80μM), CBDP was more cytotoxic (IC 50 : 15μM) to primary cortical neurons. In contrast, CBDP (up to 10μM) did not compromise the microstructure of neurites. Ten μM of ToCP significantly reduced the size and complexity of neurite networks, but neither TmCP and TpCP nor the mixture affected this

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

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.

Development of octreotide-conjugated polymeric prodrug of bufalin for targeted delivery to somatostatin receptor 2 overexpressing breast cancer in vitro and in vivo

PubMed Central

Liu, Tao; Jia, Tingting; Yuan, Xia; Liu, Cheng; Sun, Jian; Ni, Zhenhua; Xu, Jian; Wang, Xuhui; Yuan, Yi

2016-01-01

Background Development of polymeric prodrugs of small molecular anticancer drugs has become one of the most promising strategies to overcome the intrinsic shortcomings of small molecular anticancer drugs and improve their anticancer performance. Materials and methods In the current work, we fabricated a novel octreotide (Oct)-modified esterase-sensitive tumor-targeting polymeric prodrug of bufalin (BUF) and explored its anticancer performance against somatostatin receptor 2 overexpressing breast cancer. Results The obtained tumor-targeting polymeric prodrug of BUF, P(oligo[ethylene glycol] monomethyl ether methacrylate [OEGMA]-co-BUF-co-Oct), showed a nanosize dimension and controlled drug release features in the presence of esterase. It was demonstrated by in vitro experiment that P(OEGMA-co-BUF-co-Oct) showed enhanced cytotoxicity, cellular uptake, and apoptosis in comparison with those of free BUF. In vivo experiment further revealed the improved accumulation of drugs in tumor tissues and enhanced anticancer performance of P(OEGMA-co-BUF-co-Oct). Conclusion Taken together, this study indicated that polymeric prodrug of BUF holds promising potential toward the treatment of somatostatin receptor 2 overexpressing breast cancer. PMID:27284243

Hereditary Angioedema Caused By C1-Esterase Inhibitor Deficiency: A Literature-Based Analysis and Clinical Commentary on Prophylaxis Treatment Strategies

PubMed Central

2011-01-01

Hereditary angioedema (HAE) caused by C1-esterase inhibitor deficiency is an autosomal-dominant disease resulting from a mutation in the C1-inhibitor gene. HAE is characterized by recurrent attacks of intense, massive, localized subcutaneous edema involving the extremities, genitalia, face, or trunk, or submucosal edema of upper airway or bowels. These symptoms may be disabling, have a dramatic impact on quality of life, and can be life-threatening when affecting the upper airways. Because the manifestations and severity of HAE are highly variable and unpredictable, patients need individualized care to reduce the burden of HAE on daily life. Although effective therapy for the treatment of HAE attacks has been available in many countries for more than 30 years, until recently, there were no agents approved in the United States to treat HAE acutely. Therefore, prophylactic therapy is an integral part of HAE treatment in the United States and for selected patients worldwide. Routine long-term prophylaxis with either attenuated androgens or C1-esterase inhibitor has been shown to reduce the frequency and severity of HAE attacks. Therapy with attenuated androgens, a mainstay of treatment in the past, has been marked by concern about potential adverse effects. C1-esterase inhibitor works directly on the complement and contact plasma cascades to reduce bradykinin release, which is the primary pathologic mechanism in HAE. Different approaches to long-term prophylactic therapy can be used to successfully manage HAE when tailored to meet the needs of the individual patient. PMID:23283143

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.

Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens1

PubMed Central

Lanz, Wayne W.; Williams, Phletus P.

1973-01-01

An obligately anaerobic ruminal bacterial isolate was selected from 18 tributyrin-degrading isolates and identified as Butyrivibrio fibrisolvens strain 53. The culture in late exponential phase contained enzymes which could be released by sonic disruption. These enzymes degraded substrates at a rate in the order 1-naphthyl acetate (NA) > 1-naphthyl butyrate > 1-naphthyl propionate but did not degrade 1-naphthyl palmitate or 1-naphthyl phosphate. The enzymes on NA were neither stimulated nor inhibited by CoCl2, MgCl2, and MnCl (each varied from 10−6 to 10−4 M). CaCl at 10−3 M stimulated esterase activity by 16%. Aliphatic substrates were hydrolyzed at a rate in the order triacetin > tributyrin > tripropionin, and ethyl acetate > ethyl formate. Similarly, aromatic fluorescein diesters were degraded at a rate in the order acetyl > propionyl > caproyl > butyryl > capryl > lauryl. Polyacrylamide gel electrophoretic zymograms indicated that the enzyme composite contained cathodally migrating bands. By column chromatography, these enzymes were separated into six NA-degrading fractions. Fraction V contained an esterase which had an optimal temperature of 39 C, a Km of 7.6 × 10−4 on NA, and a molecular weight of about 66,000. This enzyme was inhibited by paraoxon (41%, 10−4 M), eserine (17%, 10−2 M), NaF (17%, 10−2 M), and diisopropyl fluorophosphate (62%, 10−4 M) but not by 1-naphthyl N-methyl carbamate at 8.4 × 10−4 M. PMID:4734862

LipC (Rv0220) Is an Immunogenic Cell Surface Esterase of Mycobacterium tuberculosis

PubMed Central

Shen, Guomiao; Singh, Krishna; Chandra, Dinesh; Serveau-Avesque, Carole; Maurin, Damien; Canaan, Stéphane; Singla, Rupak; Behera, Digambar

2012-01-01

We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif “GXSXG” characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV−) and HIV-positive (HIV+) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD+) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines. PMID:22038913

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.

Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar1[OPEN

PubMed Central

2017-01-01

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis (Arabidopsis thaliana) and later in Medicago truncatula. However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar (Populus tremula × Populus alba) resulted in up to 25% reduced lignin deposition, increased levels of p-hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries. PMID:28878037

Oxygen ionic conductivity of NTE materials of cubic Zr 1- xLn xW 2- yMo yO 8- x/2 (Ln = Er, Yb)

NASA Astrophysics Data System (ADS)

Li, Hai-Hua; Xia, Hai-Ting; Jing, Xi-Ping; Zhao, Xin-Hua

2008-08-01

Cubic Zr 1- xLn xW 2- yMo yO 8- x/2 (Ln = Er: x = 0.01, 0.02, 0.03; y = 0; Ln = Yb: x = 0.02, 0.03; y = 0.4) solid solutions, well-known negative thermal expansion (NTE) materials were prepared by using conventional solid state reactions. The morphology and the composition of the fracture surfaces of the ceramic pellets were determined by SEM and EDX technology. The conductance properties of the pellets, such as conductivity and conductance activation energy, were studied by AC impedance spectroscopy and the materials perform clearly oxygen ionic conduction with the conductivity of about 10 -4 S cm -1 at 673 K, a comparable value to that of ceria based solid electrolytes. The substitution of Mo for W enhanced the thermal stability of ZrW 2O 8, so that the conductivity of Zr 0.98Yb 0.02W 1.6Mo 0.4O 7.99 ceramic can be measured up to 873 K, which is about 5.9 × 10 -4 S cm -1.

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

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

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.

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.

Switching catalysis from hydrolysis to perhydrolysis in P. fluorescens esterase

PubMed Central

Yin, De Lu (Tyler); Bernhardt, Peter; Morley, Krista L.; Jiang, Yun; Cheeseman, Jeremy D.; Purpero, Vincent; Schrag, Joseph D.; Kazlauskas, Romas J.

2010-01-01

Many serine hydrolases catalyze perhydrolysis – the reversible formation of per-acids from carboxylic acids and hydrogen peroxide. Recently we showed that a single amino acid substitution in the alcohol binding pocket - L29P - in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. Angew. Chem. Intl. Ed. 2005, 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two x-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active-site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of ε-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction – hydrolysis of peracetic acid to acetic acid and hydrogen peroxide – occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed two fold higher kcat, but Km also increased so the specificity constant, kcat/Km, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate), but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of ε-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for

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.

Characterization of the aes gene of Escherichia coli encoding an enzyme with esterase activity.

PubMed Central

Peist, R; Koch, A; Bolek, P; Sewitz, S; Kolbus, T; Boos, W

1997-01-01

malQ mutants of Escherichia coli lacking amylomaltase cannot grow on maltose. They express the maltose system constitutively and are sensitive to maltose when grown on another carbon source. In an attempt to isolate a multicopy suppressor that would result in growth on maltose, we transformed a malQ mutant with a gene bank of E. coli DNA which had been digested with Sau3a and cloned in pBR322. We screened the transformants on MacConkey maltose plates. A colony was isolated that appeared to be resistant to maltose and was pink on these plates, but it was still unable to grow on minimal medium with maltose as the carbon source. The plasmid was isolated, and the gene causing this phenotype was characterized. The deduced amino acid sequence of the encoded protein shows homology to that of lipases and esterases. We termed the gene aes, for acetyl esterase. Extracts of cells harboring plasmid-encoded aes under its own promoter exhibit a fivefold higher capacity to hydrolyze p-nitrophenyl acetate than do extracts of cells of plasmid-free strains. Similarly, strains harboring plasmid-encoded aes are able to grow on triacetyl glycerol (triacetin) whereas the plasmid-free strains are not. The expression of plasmid-encoded aes resulted in strong repression of the maltose transport genes in malT+ strains (10-fold reduction), but not in a malT(Con) strain which is independent of the inducer. Also, overproduction of MalT counteracted the Aes-dependent repression, indicating a direct interaction between MalT and Aes. PMID:9401025

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

PubMed

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Purification and substrate specificity of polymorphic forms of esterase D from human erythrocytes.

PubMed Central

Scott, E M; Wright, R C

1978-01-01

Esterase D (EsD), purified from human erythrocytes and tested with a variety of substrates, hydrolyzed only triacetin, tributyrin, and certain soluble aryl esters of aliphatic acids. Esters of 4-methylumbelliferone were easily the best substrates. When the three genetically different isozymes were compared, the less common forms, EsD 2 and EsD 2-1, were less stable than EsD 1. With some substrates, the Michaelis constant of the EsD 2 form differed from that of the EsD 1 form. The EsD 2-1 hybrid form was usually, but not invariably, intermediate in properties. The physiologic significance of the genetic variability of this enzyme is unknown. PMID:623100

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).

Nontrauma emergency surgery: optimal case mix for general surgery and acute care surgery training.

PubMed

Cherry-Bukowiec, Jill R; Miller, Barbra S; Doherty, Gerard M; Brunsvold, Melissa E; Hemmila, Mark R; Park, Pauline K; Raghavendran, Krishnan; Sihler, Kristen C; Wahl, Wendy L; Wang, Stewart C; Napolitano, Lena M

2011-11-01

To examine the case mix and patient characteristics and outcomes of the nontrauma emergency (NTE) service in an academic Division of Acute Care Surgery. An NTE service (attending, chief resident, postgraduate year-3 and postgraduate year-2 residents, and two physician assistants) was created in July 2005 for all urgent and emergent inpatient and emergency department general surgery patient consults and admissions. An NTE database was created with prospective data collection of all NTE admissions initiated from November 1, 2007. Prospective data were collected by a dedicated trauma registrar and Acute Physiology and Chronic Health Evaluation-intensive care unit (ICU) coordinator daily. NTE case mix and ICU characteristics were reviewed for the 2-year time period January 1, 2008, through December 31, 2009. During the same time period, trauma operative cases and procedures were examined and compared with the NTE case mix. Thousand seven hundred eight patients were admitted to the NTE service during this time period (789 in 2008 and 910 in 2009). Surgical intervention was required in 70% of patients admitted to the NTE service. Exploratory laparotomy or laparoscopy was performed in 449 NTE patients, comprising 37% of all surgical procedures. In comparison, only 118 trauma patients (5.9% of admissions) required a major laparotomy or thoracotomy during the same time period. Acuity of illness of NTE patients was high, with a significant portion (13%) of NTE patients requiring ICU admission. NTE patients had higher admission Acute Physiology and Chronic Health Evaluation III scores [61.2 vs. 58.8 (2008); 58.2 vs. 55.8 (2009)], increased mortality [(9.71% vs. 4.89% (2008); 6.78% vs. 5.16% (2009)], and increased readmission rates (15.5% vs. 7.4%) compared with the total surgical ICU (SICU) admissions. In an era of declining operative caseload in trauma, the NTE service provides ample opportunity for complex general surgery decision making and operative procedures for

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

An Atypical α/β-Hydrolase Fold Revealed in the Crystal Structure of Pimeloyl-Acyl Carrier Protein Methyl Esterase BioG from Haemophilus influenzae.

PubMed

Shi, Jie; Cao, Xinyun; Chen, Yaozong; Cronan, John E; Guo, Zhihong

2016-12-06

Pimeloyl-acyl carrier protein (ACP) methyl esterase is an α/β-hydrolase that catalyzes the last biosynthetic step of pimeloyl-ACP, a key intermediate in biotin biosynthesis. Intriguingly, multiple nonhomologous isofunctional forms of this enzyme that lack significant sequence identity are present in diverse bacteria. One such esterase, Escherichia coli BioH, has been shown to be a typical α/β-hydrolase fold enzyme. To gain further insights into the role of this step in biotin biosynthesis, we have determined the crystal structure of another widely distributed pimeloyl-ACP methyl esterase, Haemophilus influenzae BioG, at 1.26 Å. The BioG structure is similar to the BioH structure and is composed of an α-helical lid domain and a core domain that contains a central seven-stranded β-pleated sheet. However, four of the six α-helices that flank both sides of the BioH core β-sheet are replaced with long loops in BioG, thus forming an unusual α/β-hydrolase fold. This structural variation results in a significantly decreased thermal stability of the enzyme. Nevertheless, the lid domain and the residues at the lid-core interface are well conserved between BioH and BioG, in which an analogous hydrophobic pocket for pimelate binding as well as similar ionic interactions with the ACP moiety are retained. Biochemical characterization of site-directed mutants of the residues hypothesized to interact with the ACP moiety supports a similar substrate interaction mode for the two enzymes. Consequently, these enzymes package the identical catalytic function under a considerably different protein surface.

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

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.

The Alpha-Defensin Immunoassay and Leukocyte Esterase Colorimetric Strip Test for the Diagnosis of Periprosthetic Infection: A Systematic Review and Meta-Analysis.

PubMed

Wyatt, M C; Beswick, A D; Kunutsor, S K; Wilson, M J; Whitehouse, M R; Blom, A W

2016-06-15

Synovial biomarkers have recently been adopted as diagnostic tools for periprosthetic joint infection (PJI), but their utility is uncertain. The purpose of this systematic review and meta-analysis was to synthesize the evidence on the accuracy of the alpha-defensin immunoassay and leukocyte esterase colorimetric strip test for the diagnosis of PJI compared with the Musculoskeletal Infection Society diagnostic criteria. We performed a systematic review to identify diagnostic technique studies evaluating the accuracy of alpha-defensin or leukocyte esterase in the diagnosis of PJI. MEDLINE and Embase on Ovid, ACM, ADS, arXiv, CERN DS (Conseil Européen pour la Recherche Nucléaire Document Server), CrossRef DOI (Digital Object Identifier), DBLP (Digital Bibliography & Library Project), Espacenet, Google Scholar, Gutenberg, HighWire, IEEE Xplore (Institute of Electrical and Electronics Engineers digital library), INSPIRE, JSTOR (Journal Storage), OAlster (Open Archives Initiative Protocol for Metadata Harvesting), Open Content, Pubget, PubMed, and Web of Science were searched for appropriate studies indexed from inception until May 30, 2015, along with unpublished or gray literature. The classification of studies and data extraction were performed independently by 2 reviewers. Data extraction permitted meta-analysis of sensitivity and specificity with construction of receiver operating characteristic curves for each test. We included 11 eligible studies. The pooled diagnostic sensitivity and specificity of alpha-defensin (6 studies) for PJI were 1.00 (95% confidence interval [CI], 0.82 to 1.00) and 0.96 (95% CI, 0.89 to 0.99), respectively. The area under the curve (AUC) for alpha-defensin and PJI was 0.99 (95% CI, 0.98 to 1.00). The pooled diagnostic sensitivity and specificity of leukocyte esterase (5 studies) for PJI were 0.81 (95% CI, 0.49 to 0.95) and 0.97 (95% CI, 0.82 to 0.99), respectively. The AUC for leukocyte esterase and PJI was 0.97 (95% CI, 0.95 to 0

Development of an amperometric biosensor based on acetylcholine esterase covalently bound to a new support material.

PubMed

Khayyami, M; Pérez Pita, M T; Peña Garcia, N; Johansson, G; Danielsson, B; Larsson, P O

1998-01-01

A new type of amperometric biosensor based on immobilised acetylcholine esterase was designed and constructed. The enzyme was immobilised on a flow-through working electrode, which was prepared from reticulated vitreous carbon (RVC) or from a composite material consisting of RVC and superporous agarose. The sensor was operated in FIA mode using acetylthiocholine as a substrate. The sensor responded to inhibitors such as paraoxon-10(-9) mol was detected by the sensor in a non-optimised configuration. The practical lifetime of the sensor was at least 1 month.

Anisotropic Negative Thermal Expansion Behavior of the As-Fabricated Ti-Rich and Equiatomic Ti-Ni Alloys Induced by Preferential Grain Orientation

NASA Astrophysics Data System (ADS)

Zhao, Zhong-Xun; Ma, Xiao; Cao, Shan-Shan; Ke, Chang-Bo; Zhang, Xin-Ping

2017-12-01

The present study focuses on the anisotropic negative thermal expansion (NTE) behaviors of Ti-rich (Ti54Ni46) and equiatomic Ti-Ni (Ti50Ni50) alloys fabricated by vacuum arc melting and without subsequent plastic deformation. Both alloys exhibit NTE responses in vertical and horizontal directions, and the total strains and CTEs of the NTE stage along the two mutually perpendicular measuring directions are obviously different, indicating obvious anisotropic NTE behavior of the alloys. Besides, the numerical differences between the starting temperature of NTE and austenitic transformation and between the finishing temperature of NTE and austenitic transformation are very small, which indicates that an apparent relationship exists between the NTE behavior and the phase transformation. The microstructure in the vertical cross sections shows obviously preferential orientation characteristics: Ti2Ni phases of both alloys grow along the vertical direction, and B19' martensite of Ti50Ni50 alloy has distinct preferential orientation, which results from a large temperature gradient between the top and the bottom of the button ingots during solidification. The microstructure with preferential orientation induces the anisotropic NTE behavior of the samples.

Anisotropic Negative Thermal Expansion Behavior of the As-Fabricated Ti-Rich and Equiatomic Ti-Ni Alloys Induced by Preferential Grain Orientation

NASA Astrophysics Data System (ADS)

Zhao, Zhong-Xun; Ma, Xiao; Cao, Shan-Shan; Ke, Chang-Bo; Zhang, Xin-Ping

2018-03-01

The present study focuses on the anisotropic negative thermal expansion (NTE) behaviors of Ti-rich (Ti54Ni46) and equiatomic Ti-Ni (Ti50Ni50) alloys fabricated by vacuum arc melting and without subsequent plastic deformation. Both alloys exhibit NTE responses in vertical and horizontal directions, and the total strains and CTEs of the NTE stage along the two mutually perpendicular measuring directions are obviously different, indicating obvious anisotropic NTE behavior of the alloys. Besides, the numerical differences between the starting temperature of NTE and austenitic transformation and between the finishing temperature of NTE and austenitic transformation are very small, which indicates that an apparent relationship exists between the NTE behavior and the phase transformation. The microstructure in the vertical cross sections shows obviously preferential orientation characteristics: Ti2Ni phases of both alloys grow along the vertical direction, and B19' martensite of Ti50Ni50 alloy has distinct preferential orientation, which results from a large temperature gradient between the top and the bottom of the button ingots during solidification. The microstructure with preferential orientation induces the anisotropic NTE behavior of the samples.

Distribution and probable physiological role of esterases in reproductive, digestive, and fat-body tissues of the adult cotton boll weevil, Anthonomus grandis Boh.

PubMed

Jones, B R; Bancroft, H R

1986-06-01

Polyacrylamide gel electrophoresis was used to examine gut, Malpighian tube, fat-body, testes, and ovarioles tissues of the adult cotton boll weevil, Anthonomus grandis Boh. Esterases for which the inheritance has been reported previously by Terranova using whole-body homogenates were detected in dissected tissues and the probable physiological function of each allozyme is suggested. EST-1 occurs most frequently in ovarioles and female fat bodies. EST-2 is most often found in fat bodies and may be important in lipid turnover. No sex difference was observed. EST-3S is found in fat bodies and reproductive tissue, while EST-3F is always located in gut tissues, indicating that EST-3 is not controlled by a single autosomal locus with two codominant alleles as previously reported. EST-4, the most abundant esterase, can be detected in gut tissue at any age and is probably involved in digestion. EST-5 contains four allozymes which appear most frequently in testes and may be important during reproduction.

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

PubMed Central

Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W.; Almeida-Souza, Hebréia O.; Tu, Aye; Rao, Basuthkar J.; Feldstein, Paul A.; Bruening, George; Goulart, Luiz R.; Dandekar, Abhaya M.

2016-01-01

Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms. PMID:26753904

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines.

PubMed

Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W; Almeida-Souza, Hebréia O; Tu, Aye; Rao, Basuthkar J; Feldstein, Paul A; Bruening, George; Goulart, Luiz R; Dandekar, Abhaya M

2016-01-12

Pierce's disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review.

PubMed

Ramírez-Velasco, Lorena; Armendáriz-Ruiz, Mariana; Rodríguez-González, Jorge Alberto; Müller-Santos, Marcelo; Asaff-Torres, Ali; Mateos-Díaz, Juan Carlos

2016-01-01

Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

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.

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.

Feruloyl esterases from Schizophyllum commune to treat food industry side-streams.

PubMed

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

2016-11-01

Agro-industrial side-streams are abundant and renewable resources of hydroxycinnamic acids with potential applications as antioxidants and preservatives in the food, health, cosmetic, and pharmaceutical industries. Feruloyl esterases (FAEs) from Schizophyllum commune were functionally expressed in Pichia pastoris with extracellular activities of 6000UL(-1). The recombinant enzymes, ScFaeD1 and ScFaeD2, released ferulic acid from destarched wheat bran and sugar beet pectin. Overnight incubation of coffee pulp released caffeic (>60%), ferulic (>80%) and p-coumaric acid (100%) indicating applicability for the valorization of food processing wastes and enhanced biomass degradation. Based on substrate specificity profiling and the release of diferulates from destarched wheat bran, the recombinant FAEs were characterized as type D FAEs. ScFaeD1 and ScFaeD2 preferably hydrolyzed feruloylated saccharides with ferulic acid esterified to the O-5 position of arabinose residues and showed an unprecedented ability to hydrolyze benzoic acid esters. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of Morphological Character and Esterase Isozyme Pattern in Second-Generation Black Rice Plant Irradiated to Gamma Rays

NASA Astrophysics Data System (ADS)

Hartanti, R. S.; Putri, T. A. N.; Zulfa, F.; Sutarno; Suranto

2017-04-01

Black rice is one of the functional foods due to its high anthocyanin content. Black rice grain was irradiated by gamma rays with a dose of 200 Gy and 300 Gy. The main purpose of this irradiation is to induce mutation to the black rice plant in order to achieve the improved organism. This study was undertaken to elucidate the morphological character and esterase isozyme pattern of black rice plant after irradiated by gamma rays. There were morphological differences on leaves, stems and grains between irradiated and non irradiated black rice plant. Gamma radiation dose of 200 Gy showed the significant influence of the length of the stem, number of internodes, and length of leaves. The radiation dose of 300 Gy showed the significant influence of the decrease value of diameter of 3rd internodes, number of branches and width of leaves. Flowering time is getting faster as increasing radiation dose. At the age of 74 days after planting there are 9.15% plants of 200 Gy radiation dose that have flowered faster than normal plants. This value increased into 11.45% at the dose of radiation 300 Gy. There were differences in the esterase banding pattern between radiation dose of 200 Gy and 300 Gy than the control plants, indicated that randomly mutation has occurred.

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.

Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae

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

Aparna, Gudlur; Chatterjee, Avradip; Jha, Gopaljee

2007-08-01

The crystallization and preliminary crystallographic studies of LipA, a lipase/esterase secreted by X. oryzae pv. oryzae during its infection of rice plants, are reported. Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant–microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positivemore » tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 Å and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 Å, β = 90.8°. Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 Å, β = 92.6° and diffract to 1.86 Å. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.« less

Enzymatic degradation of lignin-carbohydrate complexes (LCCs): model studies using a fungal glucuronoyl esterase from Cerrena unicolor.

PubMed

d'Errico, Clotilde; Jørgensen, Jonas O; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

2015-05-01

Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and ɣ-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding ɣ-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass. © 2014 Wiley Periodicals, Inc.

Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris.

PubMed

Cedillo, Víctor Barba; Plou, Francisco J; Martínez, María Jesús

2012-06-07

The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture. Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris, and the hydrolytic activity of the recombinant protein (OPE*) studied. After the initial screening of different clones expressing the enzyme, only one was selected for showing the highest production rate. Different culture conditions were tested to improve the expression of the recombinant enzyme. Complex media were better than minimal media for production, but in any case the levels of enzymatic activity were higher (7-fold in the best case) than those obtained from O. piceae. The purified enzyme had a molecular mass of 76 kDa, higher than that reported for the native enzyme under SDS-PAGE (60 kDa). Steady-state kinetic characterization of the recombinant protein showed improved catalytic efficiency for this enzyme as compared to the native one, for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). Different causes for this were studied, as the increased glycosylation degree of the recombinant enzyme, their secondary structures or the oxidation of methionine residues. However, none of these could explain the improvements found in the recombinant protein. N-terminal sequencing of OPE* showed that two populations of this enzyme were expressed, having either 6 or 8 amino acid residues more than the native one. This fact affected the aggregation behaviour of the recombinant protein, as was corroborated by analytical ultracentrifugation, thus improving the catalytic efficiency of this enzyme. P. pastoris resulted to be an optimum biofactory for the heterologous production of recombinant

Negative thermal expansion in TiF3 from the first-principles prediction

NASA Astrophysics Data System (ADS)

Wang, Lei; Yuan, Peng-Fei; Wang, Fei; Sun, Qiang; Liang, Er-Jun; Jia, Yu; Guo, Zheng-Xiao

2014-08-01

In negative thermal expansion (NTE) materials, rhombohedral TiF3 as a new member is predicted from first-principles calculation. The NTE behavior of rhombohedral TiF3 occurs at low temperatures. In our work, the NTE mechanism is elaborated in accordance with vibrational modes. It is confirmed that the rigid unit mode (RUM) of internal TiF6 octahedra in low-frequency optical range is most responsible for the NTE properties.

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.

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.

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...

High Throughput Screening of Esterases, Lipases and Phospholipases in Mutant and Metagenomic Libraries: A Review.

PubMed

Peña-García, Carlina; Martínez-Martínez, Mónica; Reyes-Duarte, Dolores; Ferrer, Manuel

2016-01-01

Nowadays, enzymes can be efficiently identified and screened from metagenomic resources or mutant libraries. A set of a few hundred new enzymes can be found using a simple substrate within few months. Hence, the establishment of collections of enzymes is no longer a big hurdle. However, a key problem is the relatively low rate of positive hits and that a timeline of several years from the identification of a gene to the development of a process is the reality rather than the exception. Major problems are related to the time-consuming and cost-intensive screening process that only very few enzymes finally pass. Accessing to the highest possible enzyme and mutant diversity by different, but complementary approaches is increasingly important. The aim of this review is to deliver state-of-art status of traditional and novel screening protocols for targeting lipases, esterases and phospholipases of industrial relevance, and that can be applied at high throughput scale (HTS) for at least 200 distinct substrates, at a speed of more than 105 - 108 clones/day. We also review fine-tuning sequence analysis pipelines and in silico tools, which can further improve enzyme selection by an unprecedent speed (up to 1030 enzymes). If the hit rate in an enzyme collection could be increased by HTS approaches, it can be expected that also the very further expensive and time-consuming enzyme optimization phase could be significantly shortened, as the processes of enzyme-candidate selection by such methods can be adapted to conditions most likely similar to the ones needed at industrial scale.

Recent applications of nuclear track emulsion technique

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

Zarubin, P. I., E-mail: zarubin@lhe.jinr.ru

A survey of recent results obtained using the nuclear track emulsion (NTE) technique in low energy applications is given. NTE irradiation with 60 MeV {sup 8}He nuclei provides identification of their decays at stopping, evaluation of the possibility of α range spectrometry, and observation of drift of thermalized {sup 8}He atoms. Correlations of α particles studied in {sup 12}C → 3α splitting induced by 14.1 MeV neutrons indicate the presence of a superposition of 0{sup +} and 2{sup +} states of the {sup 8}Be nucleus in the ground state of {sup 12}C. Angular correlations of fragments are studied in boron-enrichedmore » NTE, and the prospects of NTE application in radioactivity and nuclear fission research are discussed. It is proposed to use an automated microscope to search for collinear tripartition of heavy nuclei implanted in NTE. Surface irradiation of NTE by a {sup 252}Cf source is started. Planar events containing fragment pairs and long range α particles, as well as fragment triples, are studied. NTE samples are calibrated using Kr and Xe ions with an energy of 1.2 and 3 A MeV.« less

Targeting the GD3 acetylation pathway selectively induces apoptosis in glioblastoma

PubMed Central

Birks, Suzanne M.; Danquah, John Owusu; King, Linda; Vlasak, Reinhardt; Gorecki, Dariusz C.; Pilkington, Geoffrey J.

2011-01-01

The expression of ganglioside GD3, which plays crucial roles in normal brain development, decreases in adults but is upregulated in neoplastic cells, where it regulates tumor invasion and survival. Normally a buildup of GD3 induces apoptosis, but this does not occur in gliomas due to formation of 9-O-acetyl GD3 by the addition of an acetyl group to the terminal sialic acid of GD3; this renders GD3 unable to induce apoptosis. Using human biopsy-derived glioblastoma cell cultures, we have carried out a series of molecular manipulations targeting GD3 acetylation pathways. Using immunocytochemistry, flow cytometry, western blotting, and transwell assays, we have shown the existence of a critical ratio between GD3 and 9-O-acetyl GD3, which promotes tumor survival. Thus, we have demonstrated for the first time in primary glioblastoma that cleaving the acetyl group restores GD3, resulting in a reduction in tumor cell viability while normal astrocytes remain unaffected. Additionally, we have shown that glioblastoma viability is reduced due to the induction of mitochondrially mediated apoptosis and that this occurs after mitochondrial membrane depolarization. Three methods of cleaving the acetyl group using hemagglutinin esterase were investigated, and we have shown that the baculovirus vector transduces glioma cells as well as normal astroctyes with a relatively high efficacy. A recombinant baculovirus containing hemagglutinin esterase could be developed for the clinic as an adjuvant therapy for glioma. PMID:21807667

Evolution of insecticide resistance in non-target black flies (Diptera: Simuliidae) from Argentina.

PubMed

Montagna, Cristina Mónica; Gauna, Lidia Ester; D'Angelo, Ana Pechen de; Anguiano, Olga Liliana

2012-06-01

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.

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

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

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

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

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

Diagnostic Value of Leukocyte Esterase Test Strip Reagents for Rapid Clinical Diagnosis of Spontaneous Bacterial Peritonitis in Patients Admitted to Hospital Emergency Departments in Iran.

PubMed

Hashemian, Amir Masoud; Ahmadi, Koorosh; Zamani Moghaddam, Hamid; Zakeri, Hosein; Davoodi Navakh, Seyed Akbar; Sharifi, Mohammad Davood; Bahrami, Abdollah

2015-10-01

Spontaneous bacterial peritonitis (SBP) is a common and important clinical problem and is life-threatening in decompensated liver disease. Ascites fluid test by leukocyte esterase test strip has been recently proposed as an effective and rapid method to diagnose SBP in patients with cirrhosis. This study aimed to evaluate sensitivity and specificity of leukocyte esterase test strip in the diagnosis of SBP. The population of this research was all patients with cirrhosis and ascites admitted to the emergency room at Imam Reza (AS) hospital, Mashhad. A written consent was taken for inclusion in the study. 50 mL ascites sample was taken from all patients for use in a urine test strip (LER) (Urine Test Strips Convergys®Urine Matrix 11). The patient's ascites samples were evaluated for cell counting. Positive dipstick test for LER in this study considered as grade 3 +. The values of WBC > 500 cell/mm(3) or PMN > 250 cell/mm(3) considered as positive result of the gold standard method for the diagnosis of SBP. In this study, 100 patients with ascites due to cirrhosis, with an average age of 38.9 ± 6.54 years were evaluated. Twenty cases had positive results, of whom 17 cases were also detected based on the standard diagnostic criteria and other three cases were healthy individuals. Thus, sensitivity, specificity, positive and negative predictive values, and accuracy of the method were 95%, 96.3%, 85%, 97.5% and 95%, respectively. The use of leukocyte esterase urine dipstick test can be a quick and easy method in early diagnosis of SBP to start the treatment until preparation of SBP-cell count results.

40 CFR 86.1370-2007 - Not-To-Exceed test procedures.

Code of Federal Regulations, 2012 CFR

2012-07-01

... that include discrete regeneration events and that send a recordable electronic signal indicating the start and end of the regeneration event, determine the minimum averaging period for each NTE event that... averaging period is used to determine whether the individual NTE event is a valid NTE event. For engines...

Structural analysis of thermostabilizing mutations of cocaine esterase

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

Narasimhan, Diwahar; Nance, Mark R.; Gao, Daquan

Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstablemore » at physiological temperatures ({tau}{sub 1/2} {approx} 13 min at 37 C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of these engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans.« less

Bacterial cocaine esterase: a protein-based therapy for cocaine overdose and addiction

PubMed Central

Narasimhan, Diwahar; Woods, James H; Sunahara, Roger K

2012-01-01

Cocaine is highly addictive and there are no pharmacotherapeutic drugs available to treat acute cocaine toxicity or chronic abuse. Antagonizing an inhibitor such as cocaine using a small molecule has proven difficult. The alternative approach is to modify cocaine’s pharmacokinetic properties by sequestering or hydrolyzing it in serum and limiting access to its sites of action. We took advantage of a bacterial esterase (CocE) that has evolved to hydrolyze cocaine and have developed it as a therapeutic that rapidly and specifically clears cocaine from the subject. Native enzyme was unstable at 37°C, thus limiting CocE’s potential. Innovative computational methods based on the protein’s structure helped elucidate its mechanism of destabilization. Novel protein engineering methodologies were applied to substantially improve its stability in vitro and in vivo. These improvements rendered CocE as a powerful and efficacious therapeutic to treat cocaine intoxication and lead the way towards developing a therapy for addiction. PMID:22300094

40 CFR 86.1930 - What special provisions apply from 2005 through 2009?

Code of Federal Regulations, 2010 CFR

2010-07-01

... (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF EMISSIONS FROM NEW AND IN-USE HIGHWAY VEHICLES AND ENGINES... specifically describes the family as being designed to comply with NTE requirements. (2) If you participate in... standards in § 86.007-11, determine the applicable NTE standards as follows: (i) If any numerical NTE...

Lightweight Mechanical Metamaterials with Tunable Negative Thermal Expansion

NASA Astrophysics Data System (ADS)

Wang, Qiming; Jackson, Julie A.; Ge, Qi; Hopkins, Jonathan B.; Spadaccini, Christopher M.; Fang, Nicholas X.

2016-10-01

Ice floating on water is a great manifestation of negative thermal expansion (NTE) in nature. The limited examples of natural materials possessing NTE have stimulated research on engineered structures. Previous studies on NTE structures were mostly focused on theoretical design with limited experimental demonstration in two-dimensional planar geometries. In this work, aided with multimaterial projection microstereolithography, we experimentally fabricate lightweight multimaterial lattices that exhibit significant negative thermal expansion in three directions and over a temperature range of 170 degrees. Such NTE is induced by the structural interaction of material components with distinct thermal expansion coefficients. The NTE can be tuned over a large range by varying the thermal expansion coefficient difference between constituent beams and geometrical arrangements. Our experimental results match qualitatively with a simple scaling law and quantitatively with computational models.

N-TERMINALLY ELONGATED SpliInx2 AND SpliInx3 REDUCE BACULOVIRUS-TRIGGERED APOPTOSIS VIA HEMICHANNEL CLOSURE.

PubMed

Chen, Ya-Bin; Xiao, Wei; Li, Ming; Zhang, Yan; Yang, Yang; Hu, Jian-Sheng; Luo, Kai-Jun

2016-05-01

The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate. An insect Bac-to-Bac expression system, pFastBac(TM) HT A, allows us to construct an N-terminally elongated SpliInx2 (Nte-Inx2) and SpliInx3 (Nte-Inx3). Here, we demonstrated that recombinant baculovirus Bac-Nte-Inx2 (reBac-Net-Inx2) and Bac-Nte-Inx3 (reBac-Nte-Inx3) closed the endogenous hemichannel on the Sf9 cell surface. Importantly, primary baculovirus infections significantly caused early apoptosis, and this apoptosis was reduced by hemichannel-closed Sf9 cells at 24-h post-infection (PI). Although N-terminal-elongated residue led to the increase in the phosphorylated sites in both Nte-Inx2 and Nte-Inx3 and an additional transmembrane domain in Nte-Inx3, both the proteins localized on the cell surface, suggesting Nte-Inxs proteins could mediate hemichannel closure. Further supporting evidence showed that hemichannel closure was dependent on N-Inxs expressed by baculovirus polyhedrin promoter, which began to express at 18-24 h PI. These results identify an unconventional function of N-terminal-elongated innexins that could act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from intracellular to extracellular compartment. © 2016 Wiley Periodicals, Inc.

Size effects on negative thermal expansion in cubic ScF{sub 3}

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

Yang, C.; Guo, X. G.; Zhang, K.

2016-07-11

Scandium trifluoride (ScF{sub 3}), adopting a cubic ReO{sub 3}-type structure at ambient pressure, undergoes a pronounced negative thermal expansion (NTE) over a wide range of temperatures (10 K–1100 K). Here, we report the size effects on the NTE properties of ScF{sub 3}. The magnitude of NTE is reduced with diminishing the crystal size. As revealed by the specific heat measurement, the low-energy phonon vibrations which account for the NTE behavior are stiffened as the crystal size decreases. With decreasing the crystal size, the peaks in high-energy X-ray pair distribution function (PDF) become broad, which cannot be illuminated by local symmetry breaking. Instead,more » the broadened PDF peaks are strongly indicative of enhanced atomic displacements which are suggested to be responsible for the stiffening of NTE-related lattice vibrations. The present study suggests that the NTE properties of ReO{sub 3}-type and other open-framework materials can be effectively adjusted by controlling the crystal size.« less

The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation

PubMed Central

Posse, Viktor; Hoberg, Emily; Dierckx, Anke; Shahzad, Saba; Koolmeister, Camilla; Larsson, Nils-Göran; Wilhelmsson, L. Marcus; Hällberg, B. Martin; Gustafsson, Claes M.

2014-01-01

Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated. PMID:24445803

Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

PubMed

Rocchi, V; Janni, M; Bellincampi, D; Giardina, T; D'Ovidio, R

2012-03-01

Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

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

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

Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint.

PubMed

Combes, Guillaume; Barysz, Helena; Garand, Chantal; Gama Braga, Luciano; Alharbi, Ibrahim; Thebault, Philippe; Murakami, Luc; Bryne, Dominic P; Stankovic, Stasa; Eyers, Patrick A; Bolanos-Garcia, Victor M; Earnshaw, William C; Maciejowski, John; Jallepalli, Prasad V; Elowe, Sabine

2018-03-19

Monopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1-dependent SAC functions and propose that Mps1 activation likely occurs sequentially through dimerization of a "prone-to-autophosphorylate" Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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

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.

High-pressure processing of apple juice: kinetics of pectin methyl esterase inactivation.

PubMed

Riahi, Esmaeil; Ramaswamy, Hosahalli S

2003-01-01

High-pressure (HP) inactivation kinetics of pectin methyl esterase (PME) in apple juice were evaluated. Commercial PME was dispensed in clarified apple juice, sealed in dual peel sterilizable plastic bags, and subjected to different high-pressure processing conditions (200-400 MPa, 0-180 min). Residual enzyme activity was determined by a titration method estimating the rate of free carboxyl group released by the enzyme acting on pectin substrate at pH 7.5 (30 degrees C). The effects of pressure level and pressure holding time on enzyme inactivation were significant (p < 0.05). PME from the microbial source was found to be more resistant (p < 0.05) to pressure inactivation than PME from the orange peel. Almost a full decimal reduction in the activity of commercial PME was achieved by HP treatment at 400 MPa for 25 min. Inactivation kinetics were evaluated on the basis of a dual effect model involving a pressure pulse effect and a first-order rate model, and the pressure sensitivity of rate constants was modeled by using the z-value concept.

New insights into the negative thermal expansion: Direct experimental evidence for the “guitar-string” effect in cubic ScF 3

DOE PAGES

Hu, Lei; Chen, Jun; Sanson, Andrea; ...

2016-06-23

The understanding of the negative thermal expansion (NTE) mechanism remains challenging but critical for the development of NTE materials. This study sheds light on NTE of ScF 3, one of the most outstanding materials with NTE. The local dynamics of ScF 3 has been investigated by a combined analysis of synchrotron-based X-ray total scattering, ex-tended X-ray absorption fine structure and neutron powder diffraction. Very interestingly, we observe that i) the Sc-F nearest-neighbor distance strongly expands with increasing temperature while the Sc-Sc next-nearest-neighbor distance contracts, ii) the thermal ellipsoids of relative vibrations be-tween Sc-F nearest-neighbors are highly elongated in the directionmore » perpendicular to the Sc-F bond, indicating that the Sc-F bond is much softer to bend than to stretch, and iii) there is mainly dynamically transverse motion of fluorine atoms, rather than static shifts. Here, these results are the direct experimental evidence for the NTE mechanism, in which the rigid unit is not necessary for the occurrence of NTE, and the key role is played by the transverse thermal vibrations of fluorine atoms through the “guitar-string” effect.« less

A Thermally Stable Form of Bacterial Cocaine Esterase: A Potential Therapeutic Agent for Treatment of Cocaine Abuse

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

Brim, Remy L.; Nance, Mark R.; Youngstrom, Daniel W.

Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 C, which represents a 340-fold improvement over wt and is 15-fold greatermore » than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-{angstrom} resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.« less

Role of neutrophilic inflammation in ozone-induced epithelial alterations in the nasal airways of rats

NASA Astrophysics Data System (ADS)

Cho, Hye Youn

Ozone is a principal oxidant air pollutant in photochemical smog. Epithelial cells lining the centriacinar region of lung and the proximal aspects of nasal passage are primary target sites for ozone-induced injury in laboratory animals. Acute exposure of rats to high ambient concentrations of ozone (e.g., 0.5 ppm) results in neutrophilic inflammation, epithelial hyperplasia and mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE) lining the proximal nasal airways. The principal purpose of the present study was to investigate the role of pre-metaplastic cellular responses, especially neutrophilic inflammation, in the pathogenesis of ozone-induced MCM in rat NTE. For this purpose, three specific hypotheses-based whole-animal inhalation studies were conducted. Male F344/N rats were exposed in whole-body inhalation chambers to 0 (filtered air) or 0.5 ppm ozone for 1-3 days (8 h/day). Histochemical, immunochemical, molecular and morphometric techniques were used to investigate the ozone-induced cellular and molecular events in the NTE. Two in vitro studies were also conducted to examine the effects of ozone-inducible cytokines (i.e., tumor necrosis factor-alpha; TNF- a, and interleukin-6; IL-6) on mucin gene (rMuc-5AC) expression. Ozone induced a rapid increase of rMuc-5AC mRNA in nasal tissues within hours after the start of exposure. It preceded the appearance of MCM, and persisted with MCM. Ozone-induced neutrophilic inflammation accompanied the mucin gene upregulation, but was resolved when MCM first appeared in the NTE. Antibody-mediated depletion of circulating neutrophils attenuated ozone-induced MCM, although it did not affect the ozone-induced epithelial hyperplasia and mucin mRNA upregulation. In another study, it was found that preexisting neutrophilic rhinitis induced by endotoxin augmented the ozone-induced MCM. However, pre-existing rhinitis did not alter the severity of ozone-induced epithelial hyperplasia and mucin gene upregulation

Potential biological targets for bioassay development in drug discovery of Sturge-Weber syndrome.

PubMed

Mohammadipanah, Fatemeh; Salimi, Fatemeh

2017-04-29

Sturge-Weber Syndrome (SWS) is among the neurocutaneous diseases, which has several clinical manifestations of ocular (glaucoma), cutaneous (port-wine stain), neurological (seizures) and vascular problems. Molecular mechanisms of SWS pathogenesis are initiated by the somatic mutation in GNAQ. Therefore, no definite treatments exist for the SWS and treatment options only mitigate the intensity of its clinical manifestations. Biological assay design for drug discovery against this syndrome demands comprehensive knowledge on mechanisms which are involved in its pathogenesis. By analysis of the interrelated molecular targets of SWS, some in vitro bioassay systems can be allotted for drug screening against this syndrome. Development of such platforms of bioassay can bring along the implementation of high throughput screening of natural or synthetic compounds in drug discovery programs. Regarding the fact that study of biological targets and their integration in biological assay design can facilitate the process of effective drug discovery; some potential biological targets and their respective biological assay for SWS drug discovery are propounded in this review. For this purpose, some biological targets for SWS drug discovery such as acetylcholine esterase, alkaline phosphatase, gamma-aminobutyricacidergic, Hypoxia-Inducible Factor (HIF) -1α and 2α are suggested. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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.

Development of the methodology of exhaust emissions measurement under RDE (Real Driving Emissions) conditions for non-road mobile machinery (NRMM) vehicles

NASA Astrophysics Data System (ADS)

Merkisz, J.; Lijewski, P.; Fuc, P.; Siedlecki, M.; Ziolkowski, A.

2016-09-01

The paper analyzes the exhaust emissions from farm vehicles based on research performed under field conditions (RDE) according to the NTE procedure. This analysis has shown that it is hard to meet the NTE requirements under field conditions (engine operation in the NTE zone for at least 30 seconds). Due to a very high variability of the engine conditions, the share of a valid number of NTE windows in the field test is small throughout the entire test. For this reason, a modification of the measurement and exhaust emissions calculation methodology has been proposed for farm vehicles of the NRMM group. A test has been developed composed of the following phases: trip to the operation site (paved roads) and field operations (including u-turns and maneuvering). The range of the operation time share in individual test phases has been determined. A change in the method of calculating the real exhaust emissions has also been implemented in relation to the NTE procedure.

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.

Two Decades of Negative Thermal Expansion Research: Where Do We Stand?

PubMed Central

Lind, Cora

2012-01-01

Negative thermal expansion (NTE) materials have become a rapidly growing area of research over the past two decades. The initial discovery of materials displaying NTE over a large temperature range, combined with elucidation of the mechanism behind this unusual property, was followed by predictions that these materials will find use in various applications through controlled thermal expansion composites. While some patents have been filed and devices built, a number of obstacles have prevented the widespread implementation of NTE materials to date. This paper reviews NTE materials that contract due to transverse atomic vibrations, their potential for use in controlled thermal expansion composites, and known problems that could interfere with such applications. PMID:28817027

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

A New Strategy to Reduce Influenza Escape: Detecting Therapeutic Targets Constituted of Invariance Groups

PubMed Central

Lao, Julie; Vanet, Anne

2017-01-01

The pathogenicity of the different flu species is a real public health problem worldwide. To combat this scourge, we established a method to detect drug targets, reducing the possibility of escape. Besides being able to attach a drug candidate, these targets should have the main characteristic of being part of an essential viral function. The invariance groups that are sets of residues bearing an essential function can be detected genetically. They consist of invariant and synthetic lethal residues (interdependent residues not varying or slightly varying when together). We analyzed an alignment of more than 10,000 hemagglutinin sequences of influenza to detect six invariance groups, close in space, and on the protein surface. In parallel we identified five potential pockets on the surface of hemagglutinin. By combining these results, three potential binding sites were determined that are composed of invariance groups located respectively in the vestigial esterase domain, in the bottom of the stem and in the fusion area. The latter target is constituted of residues involved in the spring-loaded mechanism, an essential step in the fusion process. We propose a model describing how this potential target could block the reorganization of the hemagglutinin HA2 secondary structure and prevent viral entry into the host cell. PMID:28257108

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury.

PubMed

Wygrecka, Malgorzata; Kosanovic, Djuro; Wujak, Lukasz; Reppe, Katrin; Henneke, Ingrid; Frey, Helena; Didiasova, Miroslava; Kwapiszewska, Grazyna; Marsh, Leigh M; Baal, Nelli; Hackstein, Holger; Zakrzewicz, Dariusz; Müller-Redetzky, Holger C; de Maat, Steven; Maas, Coen; Nolte, Marc W; Panousis, Con; Schermuly, Ralph T; Seeger, Werner; Witzenrath, Martin; Schaefer, Liliana; Markart, Philipp

2017-07-15

Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.

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.

Reactivity of Acetylcholine Esterase in inner Ear Maculae of Fish after Development at Hypergravity

NASA Astrophysics Data System (ADS)

Feucht, I.; Hilbig, R.; Anken, R.

It has been shown earlier that the growth of inner ear otoliths of larval fish is (among other environmental factors) guided by the gravity vector. This guidance most probably is effected by the efferent vestibular system in the brainstem, because a transection of the nervus vestibularis has been shown to effect a cessation of the supply of calcium to the otoliths. The efferent innervation of fish inner ear maculae uses the synaptic transmitter acetylcholine (ACh). Therefore, we were - in order to further assess the role of the efferent system for otolith growth - prompted to determine ACh esterase-reactivity in the sensory epithelium of the utricle and the saccule (as well as in a non-gravity relevant brain region for control) in larval cichlid fish (Oreochromis mossambicus), which had been maintained at hypergravity during their development. The respective data will be communicated at the meeting. Acknowledgement: This work was financially supported by the German Aerospace Center (DLR) (FKZ: 50 WB 9997).

Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein.

PubMed

Pignatelli, Jaime; Alonso-Padilla, Julio; Rodríguez, Dolores

2013-12-23

Hemagglutinin-esterases (HE) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. HE proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. The role of the HE protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. The phylogenetic analysis of HE coding sequences from porcine torovirus (PToV) field strains revealed the existence of two distinct HE lineages. In a previous study, PToV virus strains with HE proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. In this work, we report antigenic differences between the two HE lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of PToV infection in the farms.

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.

Characterization of Feruloyl Esterases Produced by the Four Lactobacillus Species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, Isolated from Ensiled Corn Stover.

PubMed

Xu, Zhenshang; He, Huiying; Zhang, Susu; Guo, Tingting; Kong, Jian

2017-01-01

Lactic acid bacteria (LAB) play important roles in silage fermentation, which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms. However, LAB are not able to degrade cellulose and hemicellulose. Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility. In the present study, we isolated four Lactobacillus strains ( L. amylovorus CGMCC 11056, L. acidophilus CCTCC AB2010208, L. farciminis CCTCC AB2016237 and L. fermentum CCTCC AB2010204) with feruloyl esterase (FAE) activities from ensiled corn stover (CS) by a plate screening assay. The genes encoding FAEs were cloned and hetero-expressed in Escherichia coli . The optimal temperature and pH of these purified enzymes ranged from 45 to 50°C and from 7.0 to 8.0, respectively. They could hydrolyze hydroxycinnamoyl esters in a substrate-specific manner when methyl ferulate, methyl caffeate, methyl ρ-coumarate and methyl sinapinate were used as substrates. Moreover, these four FAEs were able to hydrolyze CS to release hydroxycinnamic acids. Furthermore, these strains could degrade hydroxycinnamic esters, and L. amylovorus CGMCC 11056 was the most efficient strain among these four isolates. These results provided a new target for the development of inoculants to improve silage quality and digestibility.

Characterization of Feruloyl Esterases Produced by the Four Lactobacillus Species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, Isolated from Ensiled Corn Stover

PubMed Central

Xu, Zhenshang; He, Huiying; Zhang, Susu; Guo, Tingting; Kong, Jian

2017-01-01

Lactic acid bacteria (LAB) play important roles in silage fermentation, which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms. However, LAB are not able to degrade cellulose and hemicellulose. Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility. In the present study, we isolated four Lactobacillus strains (L. amylovorus CGMCC 11056, L. acidophilus CCTCC AB2010208, L. farciminis CCTCC AB2016237 and L. fermentum CCTCC AB2010204) with feruloyl esterase (FAE) activities from ensiled corn stover (CS) by a plate screening assay. The genes encoding FAEs were cloned and hetero-expressed in Escherichia coli. The optimal temperature and pH of these purified enzymes ranged from 45 to 50°C and from 7.0 to 8.0, respectively. They could hydrolyze hydroxycinnamoyl esters in a substrate-specific manner when methyl ferulate, methyl caffeate, methyl ρ-coumarate and methyl sinapinate were used as substrates. Moreover, these four FAEs were able to hydrolyze CS to release hydroxycinnamic acids. Furthermore, these strains could degrade hydroxycinnamic esters, and L. amylovorus CGMCC 11056 was the most efficient strain among these four isolates. These results provided a new target for the development of inoculants to improve silage quality and digestibility. PMID:28626449

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.

Induction of Chromosomal Aberrations at Fluences of Less Than One HZE Particle per Cell Nucleus

NASA Technical Reports Server (NTRS)

Hada, Megumi; Chappell, Lori J.; Wang, Minli; George, Kerry A.; Cucinotta, Francis A.

2014-01-01

The assumption of a linear dose response used to describe the biological effects of high LET radiation is fundamental in radiation protection methodologies. We investigated the dose response for chromosomal aberrations for exposures corresponding to less than one particle traversal per cell nucleus by high energy and charge (HZE) nuclei. Human fibroblast and lymphocyte cells where irradiated with several low doses of <0.1 Gy, and several higher doses of up to 1 Gy with O (77 keV/ (long-s)m), Si (99 keV/ (long-s)m), Fe (175 keV/ (long-s)m), Fe (195 keV/ (long-s)m) or Fe (240 keV/ (long-s)m) particles. Chromosomal aberrations at first mitosis were scored using fluorescence in situ hybridization (FISH) with chromosome specific paints for chromosomes 1, 2 and 4 and DAPI staining of background chromosomes. Non-linear regression models were used to evaluate possible linear and non-linear dose response models based on these data. Dose responses for simple exchanges for human fibroblast irradiated under confluent culture conditions were best fit by non-linear models motivated by a non-targeted effect (NTE). Best fits for the dose response data for human lymphocytes irradiated in blood tubes were a NTE model for O and a linear response model fit best for Si and Fe particles. Additional evidence for NTE were found in low dose experiments measuring gamma-H2AX foci, a marker of double strand breaks (DSB), and split-dose experiments with human fibroblasts. Our results suggest that simple exchanges in normal human fibroblasts have an important NTE contribution at low particle fluence. The current and prior experimental studies provide important evidence against the linear dose response assumption used in radiation protection for HZE particles and other high LET radiation at the relevant range of low doses.

Effects of Cr substitution on negative thermal expansion and magnetic properties of antiperovskite Ga1-xCrxN0.83Mn3 compounds

NASA Astrophysics Data System (ADS)

Guo, Xinge; Tong, Peng; Lin, Jianchao; Yang, Cheng; Zhang, Kui; Lin, Shuai; Song, Wenhai; Sun, Yuping

2018-03-01

Negative thermal expansion (NTE) and magnetic properties were investigated for antiperovskite Ga1-xCrxN0.83Mn3 compounds. As x increases, the temperature span (ΔT) of NTE related with Γ5g antiferromagnetic (AFM) order is expanded and shifted to lower temperatures. At x = 0.1, NTE happens between 256 K and 318 K (ΔT = 62 K) with an average linear coefficient of thermal expansion, αL = -46 ppm/K. The ΔT is expanded to 81 K (151 K- 232 K) in x = 0.2 with αL = -22.6 ppm/K. Finally, NTE is no longer visible for x ≥ 0.3. Ferromagnetic order is introduced by Cr doping and continuously strengthened with increasing x, which may impede the AFM ordering and thus account for the broadening of NTE temperature window. Moreover, our specific heat measurement suggests the electronic density of states at the Fermi level is enhanced upon Cr doping, which favors the FM order rather than the AFM one.

Acetylcholine esterase inhibitors and melanin synthesis inhibitors from Salvia officinalis.

PubMed

Sallam, Amal; Mira, Amira; Ashour, Ahmed; Shimizu, Kuniyoshi

2016-09-15

Salvia officinalis is a traditionally used herb with a wide range of medicinal applications. Many phytoconstituents have been isolated from S. officinalis, mainly phenolic diterpenes, which possess many biological activities. This study aimed to evaluate the ability of the phenolic diterpenes of S. officinalis to inhibit acetylcholine esterase (AChE) as well as their ability to inhibit melanin biosynthesis in B16 melanoma cells. The phenolic diterpenes isolated from the aerial parts of S. officinalis were tested for their effect on melanin biosynthesis in B16 melanoma cell lines. They were also tested for their ability to inhibit AChE using Ellman's method. Moreover, a molecular docking experiment was used to investigate the binding affinity of the isolated phenolic diterpenes to the amino acid residues at the active sites of AChE. Seven phenolic diterpenes-sageone, 12-methylcarnosol, carnosol, 7b-methoxyrosmanol, 7a-methoxyrosmanol, isorosmanol and epirosmanol-were isolated from the methanolic extract of the aerial parts of S. officinalis. Isorosmanol showed a melanin-inhibiting activity as potent as that of arbutin. Compounds 7a-methoxyrosmanol and isorosmanol inhibited AChE activity by 50% and 65%, respectively, at a concentration of 500 µM. The results suggest that isorosmanol is a promising natural compound for further studies on development of new medications which might be useful in ageing disorders such as the declining of cognitive functions and hyperpigmentation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Conservation, fiber digestibility, and nutritive value of corn harvested at 2 cutting heights and ensiled with fibrolytic enzymes, either alone or with a ferulic acid esterase-producing inoculant.

PubMed

Lynch, J P; Baah, J; Beauchemin, K A

2015-02-01

The aim of this study was to determine the effects of the use of a fibrolytic enzyme product, applied at ensiling either alone or in combination with a ferulic acid esterase-producing bacterial additive, on the chemical composition, conservation characteristics, and in vitro degradability of corn silage harvested at either conventional or high cutting height. Triplicate samples of corn were harvested to leave stubble of either a conventional (15cm; NC) or high (45cm; HC) height above ground. Sub-samples of chopped herbage were ensiled untreated or with a fibrolytic enzyme product containing xylanases and cellulases applied either alone (ENZ) or in combination with a ferulic acid esterase-producing silage inoculant (ENZ+FAEI). The fibrolytic enzyme treatment was applied at 2mL of enzyme product/kg of herbage dry matter (DM), and the inoculant was applied at 1.3×10(5) cfu/g of fresh herbage. Samples were packed into laboratory-scale silos, stored for 7, 28, or 70 d, and analyzed for fermentation characteristics, and samples ensiled for 70 d were also analyzed for DM losses, chemical composition, and in vitro ruminal degradability. After 70 d of ensiling, the fermentation characteristics of corn silages were generally unaffected by cutting height, whereas the neutral detergent fiber, acid detergent fiber, and ash concentrations were lower and the starch concentration greater for silages made with crops harvested at HC compared with NC. After 70 d of ensiling, the acetic acid, ethanol concentrations, and the number of yeasts were greater, and the pH and neutral detergent fiber concentrations were lower, in silages produced using ENZ or ENZ+FAEI than the untreated silages, whereas ENZ+FAEI silages also incurred higher DM losses. No effect of additive treatment was observed on in vitro degradability indices after 48h ruminal incubation. The use of a fibrolytic enzyme product, either alone or in combination with a ferulic acid esterase-producing inoculant, at ensiling

The Origin of Uni-axial Negative Thermal Expansion in a Layered Perovskite

NASA Astrophysics Data System (ADS)

Ablitt, Chris; Craddock, Sarah; Senn, Mark; Mostofi, Arash; Bristowe, Nicholas

Using first-principles calculations within the quasi-harmonic approximation (QHA), we explain the origin of experimentally observed uni-axial negative thermal expansion (NTE) in a layered perovskite: the Ruddlesden-Popper (RP) oxide Ca2MnO4, which has anti-ferromagnetic ordering at low temperatures and is closely related to Ca3Mn2O7, which exhibits hybrid improper ferroelectricity and uni-axial NTE in competing phases. Dynamic tilts of MnO6 octahedra, common in many complex oxides, drive the expansion of the a axis and contraction of the c axis of the tetragonal NTE phase. We find that ferroelastic RP phases with a frozen octahedral rotation are unusually compliant to particular combinations of strains along different axes. The atomic mechanism responsible is characteristic of the perovskite/rock-salt interfaces present in the RP structure. We show that the contribution from this anisotropic elasticity must be taken into account in order to accurately predict NTE over the temperature range observed in experiment. A similar compliance to cooperative strains is found in other systems with uni-axial NTE. The development of this mechanistic understanding of NTE in complex oxides may pave the way for designing tunable multifunctional materials. The authors would like to acknowledge support from the EPSRC and the Centre for Doctoral Training in Theory and Simulation of Materials.

Effects of temperature, ultraviolet radiation and pectin methyl esterase on aerobic methane release from plant material.

PubMed

Bruhn, D; Mikkelsen, T N; Obro, J; Willats, W G T; Ambus, P

2009-11-01

This study examines the effects of different irradiance types on aerobic methane (CH(4)) efflux rates from terrestrial plant material. Furthermore, the role of the enzyme pectin methyl esterase (PME) on CH(4) efflux potential was also examined. Different types of plant tissue and purified pectin were incubated in glass vials with different combinations of irradiation and/or temperature. Purified dry pectin was incubated in solution, and with or without PME. Before and after incubation, the concentration of CH(4) was measured with a gas chromatograph. Rates of CH(4) emission were found to depend exponentially on temperature and linearly on UV-B irradiance. UV-B had a greater stimulating effect than UV-A, while visible light had no effect on emission rates. PME was found to substantially reduce the potential for aerobic CH(4) emissions upon demethylation of pectin.

Structural communication between the chromophore-binding pocket and the N-terminal extension in plant phytochrome phyB.

PubMed

Velázquez Escobar, Francisco; Buhrke, David; Fernandez Lopez, Maria; Shenkutie, Sintayehu Manaye; von Horsten, Silke; Essen, Lars-Oliver; Hughes, Jon; Hildebrandt, Peter

2017-05-01

The N-terminal extension (NTE) of plant phytochromes has been suggested to play a functional role in signaling photoinduced structural changes. Here, we use resonance Raman spectroscopy to study the effect of the NTE on the chromophore structure of B-type phytochromes from two evolutionarily distant plants. NTE deletion seems to have no effect on the chromophore in the inactive Pr state, but alters the torsion of the C-D ring methine bridge and the surrounding hydrogen bonding network in the physiologically active Pfr state. These changes are accompanied by a shift of the conformational equilibrium between two Pfr substates, which might affect the thermal isomerization rate of the C-D double bond and, thus, account for the effect of the NTE on the dark reversion kinetics. © 2017 Federation of European Biochemical Societies.

Time course of electrophysiologic effects induced by di-n-butyl-2,2-dichlorovinyl phosphate (DBCV) in the adult hen.

PubMed

Robertson, D G; Mattson, A M; Bestervelt, L L; Richardson, R J; Anderson, R J

1988-01-01

Previous work in our laboratory indicated that di-n-butyl-2,2-dichlorovinyl phosphate (DBCV) produced electrophysiologic changes in hen peripheral nerve that coincided with the development of histopathologic changes and neurologic signs of peripheral neuropathy. The purpose of the present study was to follow the time course for the development of the electrophysiologic changes and to determine whether pretreatment with the phosphinate analog of DBCV (DBCV-P), a nonageable organophosphorus compound, prevented these effects. Although significant electrophysiologic deficits occurred in the tibial and sciatic nerve 24 h after DBCV treatment, the most marked changes coincided with the onset of clinical signs of organophosphorus-induced delayed neuropathy (14-21 d). The sciatic and tibial nerves were equally susceptible to DBCV in producing deficits characterized by changes in the relative refractory period and an increased strength-duration threshold. Pretreatment with DBCV-P prevented the clinical signs and also attenuated the electrophysiologic deficits induced by DBCV treatment. These data suggest that electrophysiologic deficits occur before clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) and may be indicative of a link between neurotoxic esterase (NTE) inhibition and onset of overt clinical toxicity.

When the Wheels Touch Earth and the Flight is Through, Pilots Find One Eye is Better Than Two?

NASA Technical Reports Server (NTRS)

Valimont, Brian; Wise, John A.; Nichols, Troy; Best, Carl; Suddreth, John; Cupero, Frank

2009-01-01

This study investigated the impact of near to eye displays on both operational and visual performance by employing a human-in-the-loop simulation of straight-in ILS approaches while using a near to eye (NTE) display. The approaches were flown in simulated visual and instrument conditions while using either a biocular NTE or a monocular NTE display on either the dominant or non dominant eye. The pilot s flight performance, visual acuity, and ability to detect unsafe conditions on the runway were tested.

Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis.

PubMed

Ren, Hui; Xing, Zhen; Yang, Jiebing; Jiang, Wei; Zhang, Gang; Tang, Jun; Li, Quanshun

2016-06-18

Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol). Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone) was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

[Anaesthesic management of vaginal delivery in a parturient with C1 esterase deficiency].

PubMed

Libert, N; Schérier, S; Dubost, C; Franck, L; Rouquette, I; Tortosa, J-C; Rousseau, J-M

2009-04-01

Hereditary and acquired angioedema (HAE/AAE) are the clinical translation of a qualitative or a quantitative deficit of C1 esterase inhibitor (C1 INH). The frequency and severity of clinical manifestations vary greatly, ranging from a moderate swelling of the extremities to obstruction of upper airway. Anaesthesiologists and intensivists must be prepared to manage acute manifestations of this disease in case of life-threatening laryngeal edema. Surgery, physical trauma and labour are classical triggers of the disease. The anaesthesiologists should be aware of the drugs used as prophylaxis and treatment of acute attacks when considering labour and caesarean section. Androgens are contraindicated during pregnancy. If prophylaxis is required, tranexamic acid may be used with caution. The safest obstetric approach appears to be to administer a predelivery infusion of C1 INH concentrate. It is important to avoid manipulation of the airway as much as possible by relying on regional techniques. We report the case of a patient suffering from an HAE discovered during pregnancy. The management included administration of C1 INH during labor and early epidural analgesia for pain relief. A short review of the pathophysiology and therapeutic options follows.

In-utero exposure to metformin for type 2 diabetes or polycystic ovary syndrome: A prospective comparative observational study.

PubMed

Diav-Citrin, Orna; Steinmetz-Shoob, Salit; Shechtman, Svetlana; Ornoy, Asher

2018-05-29

To evaluate the rate of major anomalies after first trimester (T1)-metformin exposure. Comparative, observational cohort study done at the Israeli Teratology Information Service between 2000 and 2013. 170 T1-metformin-exposed pregnancies [119 for diabetes and 51 for polycystic ovary syndrome (PCOS)] were prospectively followed-up and compared with 93 pregnancies of T1-insulin treated women and 530 non-teratogenic exposed (NTE) pregnancies. The differences in the rate of major anomalies excluding genetic/cytogenetic, and spontaneously resolved cardiovascular anomalies were not significant [4.4% (2/45) - metformin-PCOS, 1.1% (1/90) - metformin-diabetes, 2.5% (2/80) - insulin, and 1.7% (9/519) - NTE; OR adj metformin / NTE 1.77; 95% CI 0.45-7.01; OR adj insulin / NTE 1.69; 95% CI 0.35-8.11]. The rate of Cesarean section was higher in both the metformin-diabetes 51/90 (56.7%) and insulin 45/79 (57.0%) groups compared with the NTE group [138/503 (27.4%)]. Metformin-T1-exposure per se is not associated with an increased risk of major anomalies. Copyright © 2018 Elsevier Inc. All rights reserved.

Understanding the Origins of Large Negative Thermal Expansion in Ferroelectric Perovskites from First Principles

NASA Astrophysics Data System (ADS)

Ritz, Ethan; Benedek, Nicole

Many of the functional properties of ABO3 perovskite oxides (for example, ferroelectricity) are strongly linked to particular phonon modes in the material. In addition, in many cases it is possible to formulate simple guidelines or `rules of thumb' that link crystal structure and chemistry to specific lattice dynamical characteristics. The thermal transport properties of perovskites are thus potentially highly tunable and dynamically controllable with external fields. We use first-principles density functional theory to reveal new details related to the origin of the large negative thermal expansion (NTE) observed for ferroelectric PbTiO3. Although the origin of NTE in this material is often ascribed to ferroelectricity (which arises from the freezing in of a soft, zone-center optical phonon), our results suggest that zone-boundary modes play a major role in driving NTE. In addition, hybridization between different electronic states has a significant effect on the lattice dynamics of PbTiO3 in general, and its NTE behavior in particular. Our work has implications for the understanding of, discovery and design of NTE in perovskites and other families of inorganic materials. This work was supported in part by a NASA Space Technology Research Fellowship.

Inverse analysis of giant macroscopic negative thermal expansion of Ca2RuO4‑ y ceramics based on elasticity and structural topology optimization

NASA Astrophysics Data System (ADS)

Takezawa, Akihiro; Takenaka, Koshi; Zhang, Xiaopeng

2018-05-01

Ca2RuO4‑ y ceramics exhibit a large volumetric negative thermal expansions (NTE), although the crystallographic volume contraction on heating is much smaller than the NTE. Therefore, we examine the differences in the mechanisms underlying the volumetric thermal expansion for ruthenate ceramics and crystals in the context of the elasticity. We identify the possible microstructure of ruthenate ceramics composed of crystal grains and cavities using structural topology optimization. We conclude that the measured large volumetric NTE of ruthenate ceramics is certainly possible via anisotropic crystallographic thermal expansion through an elastic mechanism.

Pectin methyl esterase and natural microflora of fresh mixed orange and carrot juice treated with pulsed electric fields.

PubMed

Rodrigo, D; Barbosa-Cánovas, G V; Martínez, A; Rodrigo, M

2003-12-01

The effects of pulsed electric fields (PEFs) on pectin methyl esterase (PME), molds and yeast, and total flora in fresh (nonpasteurized) mixed orange and carrot juice were studied. The PEF effect was more extensive when juices with high levels of initial PME activity were subjected to treatment and when PEF treatment (at 25 kV/cm for 340 micros) was combined with a moderate temperature (63 degrees C), with the maximum level of PME inactivation being 81.4%. These conditions produced 3.7 decimal reductions in molds and yeast and 2.4 decimal reductions in total flora. Experimental inactivation data for PME, molds and yeast, and total flora were fitted to Bigelow, Hülsheger, and Weibull inactivation models by nonlinear regression. The best fit (lowest mean square error) was obtained with the Weibull model.

Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual-Action Prodrug for the Treatment of Osteoporosis.

PubMed

Xie, Haibo; Chen, Gang; Young, Robert N

2017-08-24

A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

Two-dimensional nanoscale correlations in the strong negative thermal expansion material ScF 3

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

Handunkanda, Sahan U.; Occhialini, Connor A.; Said, Ayman H.

We present diffuse x-ray scattering data on the strong negative thermal expansion (NTE) material ScF3 and find that two-dimensional nanoscale correlations exist at momentum-space regions associated with possibly rigid rotations of the perovskite octahedra. We address the extent to which rigid octahedral motion describes the dynamical fluctuations behind NTE by generalizing a simple model supporting a single floppy mode that is often used to heuristically describe instances of NTE. We find this model has tendencies toward dynamic inhomogeneities and its application to recent and existing experimental data suggest an intricate link between the nanometer correlation length scale, the energy scalemore » for octahedral tilt fluctuations, and the coefficient of thermal expansion in ScF3. We then investigate the breakdown of the rigid limit and propose a resolution to an outstanding debate concerning the role of molecular rigidity in strong NTE materials.« less

Long-term effects of ionizing radiation after the Chernobyl accident: Possible contribution of historic dose.

PubMed

Omar-Nazir, Laila; Shi, Xiaopei; Moller, Anders; Mousseau, Timothy; Byun, Soohyun; Hancock, Samuel; Seymour, Colin; Mothersill, Carmel

2018-08-01

The impact of the Chernobyl NPP accident on the environment is documented to be greater than expected, with higher mutation rates than expected at the current, chronic low dose rate. In this paper we suggest that the historic acute exposure and resulting non-targeted effects (NTE) such as delayed mutations and genomic instability could account at least in part for currently measured mutation rates and provide an initial test of this concept. Data from Møller and Mousseau on the phenotypic mutation rates of Chernobyl birds 9-11 generations post the Chernobyl accident were used and the reconstructed dose response for mutations was compared with delayed reproductive death dose responses (as a measure of genomic instability) in cell cultures exposed to a similar range of doses. The dose to birds present during the Chernobyl NPP accident was reconstructed through the external pathway due to Cs-137 with an estimate of the uncertainty associated with such reconstruction. The percentage of Chernobyl birds several generations after the accident without mutations followed the general shape of the clonogenic survival percentage of the progeny of irradiated cells, and it plateaued at low doses. This is the expected result if NTE of radiation are involved. We suggest therefore, that NTE induced by the historic dose may play a role in generating mutations in progeny many generations following the initial disaster. Copyright © 2018 Elsevier Inc. All rights reserved.

Neurotoxic and teratogenic effects of an organophosphorus insecticide (phenyl phosphonothioic acid-O-ethyl -O-[4-nitrophenyl] ester) on mallard development

USGS Publications Warehouse

Hoffman, D.J.; Sileo, L.

1984-01-01

Phenyl phosphonothioic acid-O-ethyl-O-[4-nitrophenyl] ester (EPN) is one of the 10 most frequently used organophosphorus insecticides and causes delayed neurotoxicity in adult chickens and mallards. Small amounts of organophosphorus insecticides placed on birds' eggs are embryotoxic and teratogenic. For this reason, the effects of topical egg application on EPN were examined on mallard (Anas platyrhynchos) embryo development. Mallard eggs were treated topically at 72 hr of incubation with 25 microliter of a nontoxic oil vehicle or with EPN in the vehicle at concentrations of approximately 12, 36, or 108 micrograms/g egg, equivalent to one, three, and nine times the agricultural level of application used to spray crops. Treatment with EPN resulted in 22 to 44% mortality over this dose range by 18 days of development compared with 4 and 5% for untreated and vehicle-treated controls. EPN impaired embryonic growth and was highly teratogenic: 37-42% of the surviving embryos at 18 days were abnormal with cervical and axial scoliosis as well as severe edema. Brain weights were significantly lower in EPN-treated groups at different stages of development including hatchlings. Brain neurotoxic esterase (NTE) activity was inhibited by as much as 91% at 11 days, 81% at 18 days, and 79% in hatchlings. Examination of brain NTE activity during the course of normal development revealed an increase of nearly sixfold from Day 11 through hatching. The most rapid increase occurred between Day 20 and hatching. Brain acetylcholinesterase (AChE) activity was inhibited by as much as 41% at 11 days, 47% at 18 days, and 20% in hatchlings. Plasma cholinesterase and alkaline phosphatase activities were inhibited and plasma aspartate aminotransferase activity was increased at one or more stages of development. Hatchlings from EPN-treated eggs were weaker and slower to right themselves. Histopathological examination did not reveal demyelination and axonopathy of the spinal cord that was

Negative thermal expansion correlated with polyhedral movements and distortions in orthorhombic Y{sub 2}Mo{sub 3}O{sub 12}

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

Wang, Lei; Wang, Fei; Yuan, Peng-Fei

Graphical abstract: Our work confirms the negative thermal expansion (NTE) behavior of the orthorhombic Y{sub 2}Mo{sub 3}O{sub 12} in this range 0–1000 K. The orthorhombic Y{sub 2}Mo{sub 3}O{sub 12} has an open framework structure where MoO{sub 4} tetrahedra and YO{sub 6} octahedra are connected by oxygen atoms. The previous mechanisms for the NTE behavior of orthorhombic Y{sub 2}Mo{sub 3}O{sub 12} are that the translational mode (see (b)) of the O bridge atoms in Y-O-Mo linkages will cause the linkages to be bent, reducing the space between polyhedra and making the volumetric shrinkage. Furthermore, the internal polyhedral distortions have been reportedmore » experimentally. It is necessary to reveal the relationship between NTE and polyhedral movements, distortions. From the vibrational properties, we get that the different vibrational eigenvectors of oxygen atoms relative to Y or Mo atoms can lead internal polyhedra to distort unevenly (see (c)). Herein, an extended 3D model of the connected unit YO{sub 6}-MoO{sub 4} based on the Y-O-Mo linkage is proposed (see (a)). It presents a simultaneous dynamic process, i.e. the YO{sub 6} octahedra and MoO{sub 4} tetrahedra distort unevenly, along with both polyhedra being closer which makes the volumetric contraction. This model is helpful to improve the mechanisms of NTE and may be applied in the whole A{sub 2}M{sub 3}O{sub 12} family. - Highlights: • The NTE properties of Y{sub 2}Mo{sub 3}O{sub 12} are confirmed using a first-principles calculation. • The optical branch with the lowest frequency is most responsible for the NTE. • The relationship between NTE and polyhedral movements, distortions is elucidated. • An extended 3D model of the connected unit YO{sub 6}-MoO{sub 4} is proposed. - Abstract: The internal polyhedral distortions have been reported experimentally in orthorhombic Y{sub 2}Mo{sub 3}O{sub 12} as a negative thermal expansion (NTE) material. To reveal the relationship between NTE and

A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

PubMed Central

Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca

2015-01-01

Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. PMID:25746986

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.

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

Molecular and biochemical characterization of a sand fly population from Sri Lanka: evidence for insecticide resistance due to altered esterases and insensitive acetylcholinesterase.

PubMed

Surendran, S N; Karunaratne, S H P P; Adams, Z; Hemingway, J; Hawkes, N J

2005-08-01

With an increasing incidence of cutaneous leishmaniasis in Sri Lanka, particularly in northern provinces, insecticide-mediated vector control is under consideration. Optimizing such a strategy requires the characterization of sand fly populations in target areas with regard to species composition and extant resistance, among other parameters. Sand flies were collected by human bait and cattle-baited net traps on Delft Island, used as an illegal transit location by many refugees returning to the north of Sri Lanka from southern India where leishmaniasis is endemic. For species identification, genomic DNA was extracted and a fragment of the ribosomal 18S gene amplified. The sequence from all flies analysed matched that of Phlebotomus argentipes Annandale & Brunetti, the primary vector in India and the most likely vector in Sri Lanka. Independent morphological analysis also identified P. argentipes. To establish the current susceptibility status of vector species, data were obtained at the biochemical level, from which potential cross-resistance to alternative insecticides can be predicted. The Delft Island collection was assayed for the activities of four enzyme systems involved in insecticide resistance (acetylcholinesterase, non-specific carboxylesterases, glutathione-S-transferases and cytochrome p450 monooxygenases), establishing baselines against which subsequent collections can be evaluated. There was preliminary evidence for elevated esterases and altered acetylcholinesterase in this population, the first report of these resistance mechanisms in sand flies to our knowledge, which probably arose from the malathion-based spraying regimes of the Anti-Malarial Campaign.

Magnetically driven negative thermal expansion in antiperovskite Ga1-xMnxN0.8Mn3 (0.1 ≤ x ≤ 0.3)

NASA Astrophysics Data System (ADS)

Guo, X. G.; Lin, J. C.; Tong, P.; Wang, M.; Wu, Y.; Yang, C.; Song, B.; Lin, S.; Song, W. H.; Sun, Y. P.

2015-11-01

Negative thermal expansion (NTE) was investigated for Ga1-xMnxN0.8Mn3 (0.1 ≤ x ≤ 0.3). As x increases, the temperature range where lattice contracts upon heating becomes broad and shifts to lower temperatures. The coefficient of linear thermal expansion beyond -40 ppm/K with a temperature interval of ˜50 K was obtained around room temperature in x = 0.2 and 0.25. Local lattice distortion which was thought to be intimately related to NTE is invisible in the X-ray pair distribution function of x = 0.3. Furthermore, a zero-field-cooling exchange bias was observed as a result of competing ferromagnetic (FM) and antiferromagnetic (AFM) orders. The concomitant FM order serves as an impediment to the growth of the AFM order, and thus broadens the temperature range of NTE. Our result suggests that NTE can be achieved in antiperovskite manganese nitrides by manipulating the magnetic orders without distorting the local structure.

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.

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

76 FR 50287 - Request for Public Comments To Compile the National Trade Estimate Report on Foreign Trade...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-12

... (2011 SPS Report) and the 2011 Report on Technical Barriers to Trade (2011 TBT Report) respectively. The TPSC invites written comments from the public on issues that USTR should examine in preparing the NTE..., Technical Barriers to Trade, USTR (202-395-4498). SUPPLEMENTARY INFORMATION: The NTE sets out an inventory...

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.

A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

PubMed

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

2015-05-01

Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mode of de-esterification of alkaline and acidic pectin methyl esterases at different pH conditions.

PubMed

Duvetter, Thomas; Fraeye, Ilse; Sila, Daniel N; Verlent, Isabel; Smout, Chantal; Hendrickx, Marc; Van Loey, Ann

2006-10-04

Highly esterified citrus pectin was de-esterified at pH 4.5 and 8.0 by a fungal pectin methyl esterase (PME) that was shown to have an acidic isoelectric pH (pI) and an acidic pH optimum and by a plant PME that was characterized by an alkaline pI and an alkaline pH optimum. Interchain and intrachain de-esterification patterns were studied by digestion of the pectin products with endo-polygalacturonase and subsequent analysis using size exclusion and anion-exchange chromatography. No effect of pH was observed on the de-esterification mode of either of the two enzymes. Acidic, fungal PME converted pectin according to a multiple-chain mechanism, with a limited degree of multiple attack at the intrachain level, both at pH 4.5 and at pH 8.0. A multiple-attack mechanism, with a high degree of multiple attack, was more appropriate to describe the action mode of alkaline, plant PME, both at pH 4.5 and at pH 8.0.

Resistance Mechanisms to Chlorpyrifos and F392W Mutation Frequencies in the Acetylcholine Esterase Ace1 Allele of Field Populations of the Tobacco Whitefly, Bemisia tabaci in China

PubMed Central

Zhang, Ning-ning; Liu, Cai-feng; Yang, Fang; Dong, Shuang-lin; Han, Zhao-jun

2012-01-01

The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides. PMID:22954331

Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

PubMed

Sonnleitner, Elisabeth; Valentini, Martina; Wenner, Nicolas; Haichar, Feth el Zahar; Haas, Dieter; Lapouge, Karine

2012-01-01

The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

40 CFR 86.1912 - How do I determine whether an engine meets the vehicle-pass criteria?

Code of Federal Regulations, 2010 CFR

2010-07-01

... deficiency area or limited testing region, will not be added together to make a 30 second or longer event... section for at least 90 percent of the valid NTE sampling events, as defined in paragraph (b) of this section. For 2007 through 2009 model year engines, the average emissions from every NTE sampling event...

40 CFR 86.1912 - How do I determine whether an engine meets the vehicle-pass criteria?

Code of Federal Regulations, 2011 CFR

2011-07-01

... region, will not be added together to make a 30 second or longer event. Exclude any portion of a sampling... section for at least 90 percent of the valid NTE sampling events, as defined in paragraph (b) of this section. For 2007 through 2009 model year engines, the average emissions from every NTE sampling event...

Colossal Negative Thermal Expansion in Electron-Doped PbVO3 Perovskites.

PubMed

Yamamoto, Hajime; Imai, Takashi; Sakai, Yuki; Azuma, Masaki

2018-07-02

Colossal negative thermal expansion (NTE) with a volume contraction of about 8 %, the largest value reported so far for NTE materials, was observed in an electron-doped giant tetragonal perovskite compound Pb 1-x Bi x VO 3 (x=0.2 and 0.3). A polar tetragonal (P4mm) to non-polar cubic structural transition took place upon heating. The coefficient of thermal expansion (CTE) and the working temperature could be tuned by changing the Bi content, and La substitution decreased the transition temperature to room temperature. Pb 0.76 La 0.04 Bi 0.20 VO 3 exhibited a unit cell volume contraction of 6.7 % from 200 K to 420 K. Interestingly, further gigantic NTE of about 8.5 % was observed in a dilametric measurement of a Pb 0.76 La 0.04 Bi 0.20 VO 3 polycrystalline sample. The pronounced NTE in the sintered body should be attributed to an anisotropic lattice parameter change. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distribution of O-Acetylated Sialic Acids among Target Host Tissues for Influenza Virus

PubMed Central

Barnard, Karen N.; Ossiboff, Robert J.; Khedri, Zahra; Feng, Kurtis H.; Yu, Hai; Chen, Xi; Varki, Ajit

2017-01-01

ABSTRACT Sialic acids (Sias) are important glycans displayed on the cells and tissues of many different animals and are frequent targets for binding and modification by pathogens, including influenza viruses. Influenza virus hemagglutinins bind Sias during the infection of their normal hosts, while the encoded neuraminidases and/or esterases remove or modify the Sia to allow virion release or to prevent rebinding. Sias naturally occur in a variety of modified forms, and modified Sias can alter influenza virus host tropisms through their altered interactions with the viral glycoproteins. However, the distribution of modified Sia forms and their effects on pathogen-host interactions are still poorly understood. Here we used probes developed from viral Sia-binding proteins to detect O-acetylated (4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl) Sias displayed on the tissues of some natural or experimental hosts for influenza viruses. These modified Sias showed highly variable displays between the hosts and tissues examined. The 9-O-acetyl (and 7,9-) modified Sia forms were found on cells and tissues of many hosts, including mice, humans, ferrets, guinea pigs, pigs, horses, dogs, as well as in those of ducks and embryonated chicken egg tissues and membranes, although in variable amounts. The 4-O-acetyl Sias were found in the respiratory tissues of fewer animals, being primarily displayed in the horse and guinea pig, but were not detected in humans or pigs. The results suggest that these Sia variants may influence virus tropisms by altering and selecting their cell interactions. IMPORTANCE Sialic acids (Sias) are key glycans that control or modulate many normal cell and tissue functions while also interacting with a variety of pathogens, including many different viruses. Sias are naturally displayed in a variety of different forms, with modifications at several positions that can alter their functional interactions with pathogens. In addition, Sias are often modified or

40 CFR 1042.101 - Exhaust emission standards for Category 1 engines and Category 2 engines.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 0.19 kW ≥ 3700 2015 0.06 1.8 0.19 (b) Averaging, banking, and trading. You may generate or use emission credits under the averaging, banking, and trading (ABT) program as described in subpart H of this...) Determine the applicable NTE zone and subzones as described in § 1042.515. Determine NTE multipliers for...

Integrating Technology into the Classroom: A Teacher's Perspective.

ERIC Educational Resources Information Center

Clark, Frances T.

1998-01-01

The iNtegrating Technology for inQuiry (NTeQ) model teaches students to use computers to solve problems, as adults do in the workplace and home. This article presents a third grade teacher's experience using the NTeQ model for a thematic unit in which student entrepreneurs developed and marketed a new pizza requiring no refrigeration or cooking.…

Simulating Space Radiation-Induced Breast Tumor Incidence Using Automata.

PubMed

Heuskin, A C; Osseiran, A I; Tang, J; Costes, S V

2016-07-01

Estimating cancer risk from space radiation has been an ongoing challenge for decades primarily because most of the reported epidemiological data on radiation-induced risks are derived from studies of atomic bomb survivors who were exposed to an acute dose of gamma rays instead of chronic high-LET cosmic radiation. In this study, we introduce a formalism using cellular automata to model the long-term effects of ionizing radiation in human breast for different radiation qualities. We first validated and tuned parameters for an automata-based two-stage clonal expansion model simulating the age dependence of spontaneous breast cancer incidence in an unexposed U.S. We then tested the impact of radiation perturbation in the model by modifying parameters to reflect both targeted and nontargeted radiation effects. Targeted effects (TE) reflect the immediate impact of radiation on a cell's DNA with classic end points being gene mutations and cell death. They are well known and are directly derived from experimental data. In contrast, nontargeted effects (NTE) are persistent and affect both damaged and undamaged cells, are nonlinear with dose and are not well characterized in the literature. In this study, we introduced TE in our model and compared predictions against epidemiologic data of the atomic bomb survivor cohort. TE alone are not sufficient for inducing enough cancer. NTE independent of dose and lasting ∼100 days postirradiation need to be added to accurately predict dose dependence of breast cancer induced by gamma rays. Finally, by integrating experimental relative biological effectiveness (RBE) for TE and keeping NTE (i.e., radiation-induced genomic instability) constant with dose and LET, the model predicts that RBE for breast cancer induced by cosmic radiation would be maximum at 220 keV/μm. This approach lays the groundwork for further investigation into the impact of chronic low-dose exposure, inter-individual variation and more complex space radiation

The thrombogenicity of C1 esterase inhibitor (human): review of the evidence.

PubMed

Crowther, Mark; Bauer, Kenneth A; Kaplan, Allen P

2014-01-01

Thromboembolic events associated with human plasma-derived C1 esterase inhibitor (C1-INH) use in patients with hereditary angioedema (HAE) have been reported in the U.S. Food and Drug Administration (FDA) Adverse Event Reporting System database. The purpose of this article is to review and assess the strength of available evidence regarding the thrombogenicity of human plasma-derived C1-INH. A PubMed search was conducted of English language articles from January 1990 to December 2013 reporting the thrombogenicity of C1-INH. Original research articles were selected if the following criteria were met: (1) C1-INH was the focus of the study and (2) the authors addressed the pro- or antithrombotic potential of C1-INH. Additional articles on the clinical use of C1-INH in disease states other than HAE were obtained using reference lists of selected articles. Pivotal studies and prescribing information for C1-INH products were also reviewed. Limited animal and clinical data suggest that C1-INH, particularly at high doses of up to 500 U/kg (compared with the U.S. FDA-approved 20-U/kg dose), may be prothrombotic. In contrast, C1-INH has been used in some patients with myocardial infarction, ischemic stroke, sepsis, and capillary leak syndrome at off-label supratherapeutic doses (up to 100 U/kg) without evidence of a thrombogenic effect. Based on our review, thromboembolic events reported with C1-INH use are rare and patients with HAE who experienced such events often have underlying thromboembolic risk factors.

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.

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system.

PubMed

Ruppert, Martin; Woll, Jörn; Giritch, Anatoli; Genady, Ezzat; Ma, Xueyan; Stöckigt, Joachim

2005-11-01

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

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

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

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

HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

PubMed

Levesque-Tremblay, Gabriel; Müller, Kerstin; Mansfield, Shawn D; Haughn, George W

2015-03-01

Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy. © 2015 American Society of Plant Biologists. All Rights Reserved.

In vino silentium? Individual, situational, and alcohol-related factors in reporting violence to the police.

PubMed

Brennan, Iain R

2011-01-01

This study identifies the individual, situational, and alcohol-related factors associated with reporting violent victimization to the police. Factors positively associated with reporting included older age and incident severity (the assailant's use of a weapon, incurring injury that required attendance at an emergency department). Factors negatively associated with reporting included higher educational qualifications, assault in the nighttime economy (NTE), and drinking more than two alcoholic drinks immediately prior to victimization. It is possible that drinkers engage in "moratorium" on reporting violence in the NTE. Recognizing and reducing the acceptability of violence in the NTE may help reduce incidence of alcohol-related violence. Organizations that use police records of violence to inform practice and policy should account for uneven distributions in reporting behavior when analyzing trends in violence.

Matrix metalloproteinase inhibitor modulates esterase-catalyzed degradation of resin-dentin interfaces.

PubMed

Serkies, Kyle B; Garcha, Reena; Tam, Laura E; De Souza, Grace M; Finer, Yoav

2016-12-01

Assess the modulating effect of matrix metalloproteinase (MMP) inhibition on simulated human salivary enzyme (SHSE)-catalyzed degradation of interfacial fracture-toughness (FT) of self-etched and total-etched resin-dentin interfaces. Miniature short-rod FT specimens (N=10/group) containing a resin composite bonded to human dentin, using a self-etch (Easy Bond, EB) or a total-etch (Scotchbond, SB) adhesives, were prepared with and without application of an MMP inhibitor (galardin). Specimens were non-incubated or incubated in phosphate buffered saline (PBS) or SHSE for 7, 30, 90, or 180-days. FT data were obtained using a universal testing machine. Incubation media were analyzed by high performance liquid chromatography (HPLC) for the presence of a 2,2-bis-[4-2(2-hydroxy-3-methacryloxypropoxy)phenyl]-propane (bisGMA)-derived degradation product, bis-hydroxy-propoxy-phenyl-propane (bisHPPP). Fractographic analysis was performed by scanning electron microscopy and image processing software (ImageJ). Statistical analysis was performed by ANOVA and Tukey's (p<0.05). More bisHPPP was detected in SHSE vs. PBS for both adhesive systems (p<0.05). EB specimens yielded no difference in FT and failed preferentially in the resin after >30-days (p<0.05). SB specimens yielded lower FT values after 180-days with SHSE ±galardin vs. 0-days/no-galardin (p<0.05) and failed preferentially in the hybrid-layer after >30-days (p<0.05). Galardin mildly modulated the change in fracture mode for both systems. Esterase-catalyzed degradation of total-etch interfaces is modulated by MMP-inhibition, however, self-etch interfaces possess greater biostability under simulated intra-oral conditions, regardless of MMP inhibition. This could be related to different chemical compositions and/or mode of adhesion. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Luminescence and antibacterial studies of silver nanoparticles using the esterases-containing latex of E. Tirucalli plant via green route

NASA Astrophysics Data System (ADS)

Sudheerkumar, K. H.; Dhananjaya, N.; Reddy Yadav, L. S.

2016-04-01

Silver nanoparticles (Ag NPs) synthesized from silver nitrate solutions using the esterase-containing latex of the E. Tirucalli plant widely found in a large region in Karnataka, India. Plant-mediated synthesis of nanoparticles is a green chemistry approach that intercom-nects nanotechnology and plant biotechnology. The effect of extract concentration, contact time, and temperature on the reaction rate and the shape of the Ag nanoparticles was investigated. The nanoparticles have been characterized by powder X-ray diffraction, UV-visible spectroscopy, photoluminescence spectroscopy and morphology by scanning electron microscope, transmission electron microscopy, as a function of the ratio of silver ions to reducing agent molecules. Powder X-ray diffraction patterns show that the crystal structure obtained is face-centered cubic (fcc). The morphology of the silver nanoparticle was uniform with well-distributed elliptical particles with a range from 15 to 25nm. Ag NPs exhibit significant antibacterial activity against Bacillus cereus using the agar well diffusion method.

The origin of uniaxial negative thermal expansion in layered perovskites

NASA Astrophysics Data System (ADS)

Ablitt, Chris; Craddock, Sarah; Senn, Mark S.; Mostofi, Arash A.; Bristowe, Nicholas C.

2017-10-01

Why is it that ABO3 perovskites generally do not exhibit negative thermal expansion (NTE) over a wide temperature range, whereas layered perovskites of the same chemical family often do? It is generally accepted that there are two key ingredients that determine the extent of NTE: the presence of soft phonon modes that drive contraction (have negative Grüneisen parameters); and anisotropic elastic compliance that predisposes the material to the deformations required for NTE along a specific axis. This difference in thermal expansion properties is surprising since both ABO3 and layered perovskites often possess these ingredients in equal measure in their high-symmetry phases. Using first principles calculations and symmetry analysis, we show that in layered perovskites there is a significant enhancement of elastic anisotropy due to symmetry breaking that results from the combined effect of layering and condensed rotations of oxygen octahedra. This feature, unique to layered perovskites of certain symmetry, is what allows uniaxial NTE to persist over a large temperature range. This fundamental insight means that symmetry and the elastic tensor can be used as descriptors in high-throughput screening and to direct materials design.

Large magnetovolume effect induced by ferromagnetic-antiferromagnetic competition in a cobaltite perovskite

NASA Astrophysics Data System (ADS)

Miao, Ping; Lin, Xiaohuan; Koda, Akihiro; Lee, Sanghyun; Ishikawa, Yoshihisa; Torii, Shuki; Yonemura, Masao; Mochiku, Takashi; Sagayama, Hajime; Itoh, Shinichi; Wang, Yinxia; Kadono, Ryosuke; Kamiyama, Takashi

Materials that show negative thermal expansion (NTE) have significant industrial merit because they can be used to fabricate composites whose dimensions remain invariant upon heating. In some materials, NTE is concomitant with the spontaneous magnetization, known as the magnetovolume effect (MVE). Here we report a new class of MVE material; namely, a layered perovskite PrBaCo2O5.5+ x (0 <= x <= 0.41),in which strong NTE (β -3.3 × 10-5 K-1 at x = 0.24) is triggered by embedding ferromagnetic (F) clusters into the antiferromagnetic (AF) matrix. The strongest MVE is found near the boundary between F and AF phases in the phase diagram, indicating the essential role of competing interaction between the F-clusters and the AF-matrix. Furthermore, the MVE is not limited to the PrBaCo2O5.5+ x but is also observed in the NdBaCo2O5.5+ x . The present study provides a new approach to obtaining MVE and offers a path to the design of NTE materials. The study was financed by the S-type project (No. 2014S05) of KEK.

Large Magnetovolume Effect Induced by Embedding Ferromagnetic Clusters into Antiferromagnetic Matrix of Cobaltite Perovskite.

PubMed

Miao, Ping; Lin, Xiaohuan; Koda, Akihiro; Lee, Sanghyun; Ishikawa, Yoshihisa; Torii, Shuki; Yonemura, Masao; Mochiku, Takashi; Sagayama, Hajime; Itoh, Shinichi; Ikeda, Kazutaka; Otomo, Toshiya; Wang, Yinxia; Kadono, Ryosuke; Kamiyama, Takashi

2017-07-01

Materials that show negative thermal expansion (NTE) have significant industrial merit because they can be used to fabricate composites whose dimensions remain invariant upon heating. In some materials, NTE is concomitant with the spontaneous magnetization due to the magnetovolume effect (MVE). Here the authors report a new class of MVE material; namely, a layered perovskite PrBaCo 2 O 5.5+ x (0 ≤ x ≤ 0.41), in which strong NTE [β ≈ -3.6 × 10 -5 K -1 (90-110 K) at x = 0.24] is triggered by embedding ferromagnetic (F) clusters into the antiferromagnetic (AF) matrix. The strongest MVE is found near the boundary between F and AF phases in the phase diagram, indicating the essential role of competition between the F-clusters and the AF-matrix. Furthermore, the MVE is not limited to the PrBaCo 2 O 5.5+ x but is also observed in the NdBaCo 2 O 5.5+ x . The present study provides a new approach to obtaining MVE and offers a path to the design of NTE materials. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

PubMed

Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

2012-04-15

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more

The NTE and Teacher Certification

ERIC Educational Resources Information Center

McDaniel, Thomas R.

1977-01-01

Although in U.S. v. State of South Carolina the Court has established important guidelines legitimating the use of validated National Teacher Examination scores in the certification of teachers, some problems and questions remain. (Author/IRT)

Anthropometric evaluation of indigenous Brazilian children under 60 months of age using NCHS/1977 and WHO/2005 growth curves.

PubMed

Orellana, Jesem D Y; Santos, Ricardo V; Coimbra, Carlos E A; Leite, Maurício S

2009-01-01

To perform a comparative analysis of anthropometric data from Suruí, Xavánte and Wari' indigenous children under 60 months of age using the NCHS/1977 and the WHO/2005 growth curves. Anthropometric measurements followed standard procedures and the data obtained were converted into z scores using the Epi-Info (Version 3.4) and WHO-Anthro (Version Beta) softwares. The indices height/age (H/A), weight/age (W/A) and weight/height (W/H) were descriptors of nutritional status for all children under 60 months of age, as well as the body mass index (BMI) for children 24-59 months old. The frequencies of Suruí children < -2 z scores for H/A were 31.4 (NCHS/1977) and 38.6% (WHO/2005); Xavánte 30.9 and 42.3%; Wari' 61.7 and 68.3%. The frequencies of Suruí children < -2 z scores for W/A were 12.4 (NCHS/1977) and 8.5% (WHO/2005); Xavánte 16.5 and 11.6%; Wari' 51.7 and 45.0%. None of the Suruí children were < -2 z scores for W/H (NCHS/1977 and WHO/2005); the frequencies of Xavánte children were 1.7 and 3.3% and Wari' 1.7 and 0.0%. The frequencies of Suruí children > 2 z scores for W/H were 3.9 (NCHS/1977) and 3.9% (WHO/2005); Xavánte 0.0 and 0.8%; Wari' 0.0 and 0.0%. The frequency of Suruí children aged 24-59 months > 2 z scores for BMI was 5.4% (WHO/2005); Xavánte 9.5%; and Wari' 0.0%. Our findings revealed important differences in the results from nutritional assessment, according to the set of growth curves used; however, the use of both growth curves revealed a high prevalence of malnutrition. Therefore, future studies with indigenous populations should present their results using two sets of growth curves to allow consistent comparison.

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution

PubMed Central

Zeng, Qinghong; Langereis, Martijn A.; van Vliet, Arno L. W.; Huizinga, Eric G.; de Groot, Raoul J.

2008-01-01

The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer–monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design. PMID:18550812

Computational Design of a Thermostable Mutant of Cocaine Esterase via Molecular Dynamics Simulations

PubMed Central

Huang, Xiaoqin; Gao, Daquan; Zhan, Chang-Guo

2015-01-01

Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing the naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ~11 min at physiological temperature (37°C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the RMSD and RMSF of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modeling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37°C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability may be considered as a valuable tool for computational design of thermostable mutants of an enzyme. PMID:21373712

Computational design of a thermostable mutant of cocaine esterase via molecular dynamics simulations.

PubMed

Huang, Xiaoqin; Gao, Daquan; Zhan, Chang-Guo

2011-06-07

Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ∼11 min at physiological temperature (37 °C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the root-mean square deviation and root-mean square fluctuation of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modelling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37 °C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability relationship may be considered as a valuable tool for the computational design of thermostable mutants of an enzyme.

Measurement of myeloid maturation by flow cytochemistry in HL-60 leukemia: esterase is inducible, myeloperoxidase is not.

PubMed

Ross, D W

1986-05-01

The phenomenon of leukemic cell maturation requires a measurement of myeloid maturation to understand the process and to exploit it as a means of therapy for leukemia. The HL-60 leukemic cell line was used as a model of induced leukemic cell maturation in order to develop a method of quantitating granulocytic and monocytic maturation in response to drug therapy. An automated flow cytochemistry system (Hemalog-D) was employed to measure mean cell volume, myeloperoxidase (MPO), and nonspecific esterase (NSE). For granulocytic maturation induced by vitamin A or DMSO, MPO and cell volume decreased by 50%, maintaining a constant mean cellular MPO concentration throughout maturation from promyelocyte to neutrophil-like forms. For monocytic maturation induced by low-dose ARA-c, the mean NSE increased substantially, while cell volume remained constant. Unlike MPO concentration, NSE was truly inducible and thus a useful quantitative measure of maturation caused by low-dose ARA-c. Flow cytochemistry and cytofluorometry may be developed to allow for quantitative monitoring of therapeutic trials of induced maturation in human leukemias. However, this will require adapting these techniques to the complexity of human leukemias in vivo, and the necessity of handling heterogeneous populations encountered in bone marrow samples.

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

Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca).

PubMed

Osorio, Sonia; Castillejo, Cristina; Quesada, Miguel A; Medina-Escobar, Nieves; Brownsey, Geoff J; Suau, Rafael; Heredia, Antonio; Botella, Miguel A; Valpuesta, Victoriano

2008-04-01

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.

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.

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion.

PubMed

Willats, W G; Orfila, C; Limberg, G; Buchholt, H C; van Alebeek, G J; Voragen, A G; Marcus, S E; Christensen, T M; Mikkelsen, J D; Murray, B S; Knox, J P

2001-06-01

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.

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.

Pectin methyl esterase treatment on high-methoxy pectin for making fruit jam with reduced sugar content.

PubMed

Wang, Yuh-Tai; Lien, Ling-Lan; Chang, Ya-Chu; Wu, James Swi-Bea

2013-01-01

Pectin methyl esterase (PME) has been postulated to catalyse the transacylation reaction between pectin molecules. The present study aimed to prove the occurrence of this reaction. The feasibility of applying PME-catalysed transacylation between high-methoxy pectin molecules in making fruit jam with reduced sugar content was also investigated. PME treatment increased the turbidity and particle size in pectin solution and the molecular weight of pectin, while it decreased the number of methoxy ester linkages and the intensity of the CH₃ absorption peak in the Fourier transform infrared spectrum without changes in the number of total ester linkages in pectin molecules. These findings support the occurrence of PME-catalysed transacylation between pectin molecules. Higher values of hardness, gumminess and chewiness were found in a jam containing PME-treated citrus pectin (10 g L⁻¹) and sugar (350 g L⁻¹) as compared with either a jam containing untreated citrus pectin (10 g L⁻¹) and sugar (350 g L⁻¹) or strawberry jam containing pectin (10 g L⁻¹) from the fruit and sugar (650 g L⁻¹). The demand for sugar in jam making can be greatly reduced by the use of PME-treated high-methoxy pectin. Copyright © 2012 Society of Chemical Industry.

Potential human cholesterol esterase inhibitor design: benefits from the molecular dynamics simulations and pharmacophore modeling studies.

PubMed

John, Shalini; Thangapandian, Sundarapandian; Lee, Keun Woo

2012-01-01

Human pancreatic cholesterol esterase (hCEase) is one of the lipases found to involve in the digestion of large and broad spectrum of substrates including triglycerides, phospholipids, cholesteryl esters, etc. The presence of bile salts is found to be very important for the activation of hCEase. Molecular dynamic simulations were performed for the apoform and bile salt complexed form of hCEase using the co-ordinates of two bile salts from bovine CEase. The stability of the systems throughout the simulation time was checked and two representative structures from the highly populated regions were selected using cluster analysis. These two representative structures were used in pharmacophore model generation. The generated pharmacophore models were validated and used in database screening. The screened hits were refined for their drug-like properties based on Lipinski's rule of five and ADMET properties. The drug-like compounds were further refined by molecular docking simulation using GOLD program based on the GOLD fitness score, mode of binding, and molecular interactions with the active site amino acids. Finally, three hits of novel scaffolds were selected as potential leads to be used in novel and potent hCEase inhibitor design. The stability of binding modes and molecular interactions of these final hits were re-assured by molecular dynamics simulations.

Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus.

PubMed

Siedlecka, Anna; Wiklund, Susanne; Péronne, Marie-Amélie; Micheli, Fabienne; Lesniewska, Joanna; Sethson, Ingmar; Edlund, Ulf; Richard, Luc; Sundberg, Björn; Mellerowicz, Ewa J

2008-02-01

Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula x tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.

Pectin methyl esterases and pectins in normal and hyperhydric shoots of carnation cultured in vitro.

PubMed

Saher, Shady; Piqueras, Abel; Hellin, Eladio; Olmos, Enrique

2005-02-01

Control and hyperhydric micropropagated plantlets from three carnation cultivars have been used to study their pectin composition and the activity of pectin methyl esterases (PMEs; EC 3.1.1.11). Pectins are a highly heterogeneous group of polymers that contribute to cell adhesion, cell wall architecture, and cell wall mechanical strength. Pectins control cell wall porosity and cell wall ionic status and are implicated in intercellular space development. The degree of esterification of pectins is controlled by the activity of cell wall PMEs; their different actions can affect the properties of the cell wall, which have been considered important with respect to controlling the development of hyperhydricity. The total pectins of hyperhydric leaves of the three varieties were significantly reduced in comparison with controls. The pectate fraction was significantly increased in hyperhydric leaves of all varieties while soluble pectins and protopectins were significantly lower. The PME activity of hyperhydric leaves was higher (4-10 times) compared to controls of the three varieties. Isoelectric focusing of PME isozymes revealed the presence of three isoforms; neutral PME activity was the major isozyme in control and hyperhydric leaves of the three varieties, whilst a decrease in the activity of the acidic isoforms was observed in hyperhydric leaves. The different PME activities could regulate some of the structural changes related to hyperhydricity in micropropagated carnation plants.

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

PubMed

Petrovskaya, Lada E; Novototskaya-Vlasova, Ksenia A; Spirina, Elena V; Durdenko, Ekaterina V; Lomakina, Galina Yu; Zavialova, Maria G; Nikolaev, Evgeny N; Rivkina, Elizaveta M

2016-05-01

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Foundational errors in the Neutral and Nearly-Neutral theories of evolution in relation to the Synthetic Theory: is a new evolutionary paradigm necessary?

PubMed

Valenzuela, Carlos Y

2013-01-01

The Neutral Theory of Evolution (NTE) proposes mutation and random genetic drift as the most important evolutionary factors. The most conspicuous feature of evolution is the genomic stability during paleontological eras and lack of variation among taxa; 98% or more of nucleotide sites are monomorphic within a species. NTE explains this homology by random fixation of neutral bases and negative selection (purifying selection) that does not contribute either to evolution or polymorphisms. Purifying selection is insufficient to account for this evolutionary feature and the Nearly-Neutral Theory of Evolution (N-NTE) included negative selection with coefficients as low as mutation rate. These NTE and N-NTE propositions are thermodynamically (tendency to random distributions, second law), biotically (recurrent mutation), logically and mathematically (resilient equilibria instead of fixation by drift) untenable. Recurrent forward and backward mutation and random fluctuations of base frequencies alone in a site make life organization and fixations impossible. Drift is not a directional evolutionary factor, but a directional tendency of matter-energy processes (second law) which threatens the biotic organization. Drift cannot drive evolution. In a site, the mutation rates among bases and selection coefficients determine the resilient equilibrium frequency of bases that genetic drift cannot change. The expected neutral random interaction among nucleotides is zero; however, huge interactions and periodicities were found between bases of dinucleotides separated by 1, 2... and more than 1,000 sites. Every base is co-adapted with the whole genome. Neutralists found that neutral evolution is independent of population size (N); thus neutral evolution should be independent of drift, because drift effect is dependent upon N. Also, chromosome size and shape as well as protein size are far from random.

Common and distant structural characteristics of feruloyl esterase families from Aspergillus oryzae.

PubMed

Udatha, D B R K Gupta; Mapelli, Valeria; Panagiotou, Gianni; Olsson, Lisbeth

2012-01-01

Feruloyl esterases (FAEs) are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic enzymes. FAEs have gained increased attention in the area of biocatalytic transformations for the synthesis of value added compounds with medicinal and nutritional applications. Following the increasing attention on these enzymes, a novel descriptor based classification system has been proposed for FAEs resulting into 12 distinct families and pharmacophore models for three FAE sub-families have been developed. The feruloylome of Aspergillus oryzae contains 13 predicted FAEs belonging to six sub-families based on our recently developed descriptor-based classification system. The three-dimensional structures of the 13 FAEs were modeled for structural analysis of the feruloylome. The three genes coding for three enzymes, viz., A.O.2, A.O.8 and A.O.10 from the feruloylome of A. oryzae, representing sub-families with unknown functional features, were heterologously expressed in Pichia pastoris, characterized for substrate specificity and structural characterization through CD spectroscopy. Common feature-based pharamacophore models were developed according to substrate specificity characteristics of the three enzymes. The active site residues were identified for the three expressed FAEs by determining the titration curves of amino acid residues as a function of the pH by applying molecular simulations. Our findings on the structure-function relationships and substrate specificity of the FAEs of A. oryzae will be instrumental for further understanding of the FAE families in the novel classification system. The developed pharmacophore models could be applied for virtual screening of compound databases for short listing the putative substrates prior to docking studies or for post

New Modeling Approaches to Investigate Cell Signaling in Radiation Response

NASA Technical Reports Server (NTRS)

Plante, Ianik; Cucinotta, Francis A.; Ponomarev, Artem L.

2011-01-01

Ionizing radiation damages individual cells and tissues leading to harmful biological effects. Among many radiation-induced lesions, DNA double-strand breaks (DSB) are considered the key precursors of most early and late effects [1] leading to direct mutation or aberrant signal transduction processes. In response to damage, a flow of information is communicated to cells not directly hit by the radiation through signal transduction pathways [2]. Non-targeted effects (NTE), which includes bystander effects and genomic instability in the progeny of irradiated cells and tissues, may be particularly important for space radiation risk assessment [1], because astronauts are exposed to a low fluence of heavy ions and only a small fraction of cells are traversed by an ion. NTE may also have important consequences clinical radiotherapy [3]. In the recent years, new simulation tools and modeling approaches have become available to study the tissue response to radiation. The simulation of signal transduction pathways require many elements such as detailed track structure calculations, a tissue or cell culture model, knowledge of biochemical pathways and Brownian Dynamics (BD) propagators of the signaling molecules in their micro-environment. Recently, the Monte-Carlo simulation code of radiation track structure RITRACKS was used for micro and nano-dosimetry calculations [4]. RITRACKS will be used to calculate the fraction of cells traversed by an ion and delta-rays and the energy deposited in cells in a tissue model. RITRACKS also simulates the formation of chemical species by the radiolysis of water [5], notably the .OH radical. This molecule is implicated in DNA damage and in the activation of the transforming growth factor beta (TGF), a signaling molecule involved in NTE. BD algorithms for a particle near a membrane comprising receptors were also developed and will be used to simulate trajectories of signaling molecules in the micro-environment and characterize autocrine

Inhibition of Polyisoprenylated Methylated Protein Methyl Esterase by Synthetic Musks Induces Cell Degeneration

PubMed Central

Ayuk-Takem, Lambert; Amissah, Felix; Aguilar, Byron J.; Lamango, Nazarius S.

2013-01-01

Synthetic fragrances are persistent environmental pollutants that tend to bioaccumulate in animal tissues. They are widely used in personal care products and cleaning agents. Worldwide production of Galaxolide and Tonalide are in excess of 4500 tons annually. Because of their widespread production and use, they have been detected in surface waters and fish in the US and Europe. Consumption of contaminated water and fish from such sources leads to bioaccumulation and eventual toxicity. Since fragrances and flavors bear structural similarities to polyisoprenes, it was of interest to determine whether toxicity by Galaxolide and Tonalide may be linked with polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition. A concentration-dependent study of PMPMEase inhibition by Galaxolide and Tonalide as well as their effects on the degeneration of cultured cells were conducted. Galaxolide and Tonalide inhibited purified porcine liver PMPMEase with Ki values of 11 and 14 µM, respectively. Galaxolide and Tonalide also induced human cancer cell degeneration with EC50 values of 26 and 98 µM (neuroblastoma SH-SY5Y cells) and 58 and 14 µM (lung cancer A549 cells), respectively. The effects on cell viability correlate well with the inhibition of PMPMEase activity in the cultured cells. Molecular docking analysis revealed that the binding interactions are most likely between the fragrance molecules and hydrophobic amino acids in the active site of the enzyme. These results appear to suggest that the reported neurotoxicity of these compounds may be associated with their inhibition of PMPMEase. Exposure to fragrances may pose a significant risk to individuals predisposed to developing degenerative disorders. PMID:22489002

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.

Preterm infant thermal care: differing thermal environments produced by air versus skin servo-control incubators.

PubMed

Thomas, K A; Burr, R

1999-06-01

Incubator thermal environments produced by skin versus air servo-control were compared. Infant abdominal skin and incubator air temperatures were recorded from 18 infants in skin servo-control and 14 infants in air servo-control (26- to 29-week gestational age, 14 +/- 2 days postnatal age) for 24 hours. Differences in incubator and infant temperature, neutral thermal environment (NTE) maintenance, and infant and incubator circadian rhythm were examined using analysis of variance and scatterplots. Skin servo-control resulted in more variable air temperature, yet more stable infant temperature, and more time within the NTE. Circadian rhythm of both infant and incubator temperature differed by control mode and the relationship between incubator and infant temperature rhythms was a function of control mode. The differences between incubator control modes extend beyond temperature stability and maintenance of NTE. Circadian rhythm of incubator and infant temperatures is influenced by incubator control.

Textural properties of gelling system of low-methoxy pectins produced by demethoxylating reaction of pectin methyl esterase.

PubMed

Kim, Y; Yoo, Y-H; Kim, K-O; Park, J-B; Yoo, S-H

2008-06-01

After deesterification of commercial pectins with a pectin methyl esterase (PME), their gelling properties were characterized using instrumental texture analysis. The final degree of esterification (DE) of the high- and low-methoxy pectins reached approximately 6% after the PME treatment, while deesterification of low-methoxy amidated pectin stopped at 18% DE. Furthermore, DE of high-methoxy pectin was tailored to be 40%, which is equivalent to the DE of commercial low-methoxy pectin. As a result, significant changes in molecular weight (Mw) distribution were observed in the PME-treated pectins. The texture profile analysis showed that PME modification drastically increased hardness, gumminess, and chewiness, while decreasing cohesiveness and adhesiveness of the pectin gels (P < 0.05). The pectin gel with relatively high peak molecular weight (Mp, 3.5 x 10(5)) and low DE (6), which was produced from high-methoxy pectin, exhibited the greatest hardness, gumminess, chewiness, and resilience. The hardness of low-methoxy amidated pectin increased over 300% after PME deesterification, suggesting that the effects of amide substitution could be reinforced when DE is even lower. The partial least square regression analysis indicated that the Mw and DE of the pectin molecule are the most crucial factors for hardness, chewiness, gumminess, and resilience of gel matrix.

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.

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.

Chlorantraniliprole resistance and its biochemical and new molecular target mechanisms in laboratory and field strains of Chilo suppressalis (Walker).

PubMed

Sun, Yang; Xu, Lu; Chen, Qiong; Qin, Wenjing; Huang, Shuijin; Jiang, Ying; Qin, Houguo

2018-06-01

The rice striped stem borer (SSB), Chilo suppressalis (Walker), is one of the most economically important and destructive rice pests in China. To date, the efficiency of conventional insecticides has decreased greatly because of the development of high resistance. Since the introduction of chlorantraniliprole in 2008, SSB has presented resistance issues. In this study, laboratory resistant strains R1 and R2 [resistance ratio (RR) of 38.8 and 110.4, respectively] were established and a field population HR (RR of 249.6) was collected. Synergist assessment and enzyme activity data suggested the potential involvement of P450s and esterases in the resistance mechanism. No target (ryanodine receptor, RyR) mutation was found in R1, but a novel mutation Y4667D was found in R2. At the same position of RyR in HR strain, Y4667D and Y4667C were observed at low frequencies. In addition, the conserved mutation I4758M was found with a frequency of 94.4%. RyR mRNA expression was significantly lower in R1, R2 and HR than in S. When treated with chlorantraniliprole, RyR mRNA expression in all four strains was downregulated to ∼ 50%. A comprehensive analysis, including biochemical, target mutations and target mRNA expression, was conducted in an attempt to interpret the chlorantraniliprole resistance mechanism in both laboratory and field SSB strains. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

SulE, a Sulfonylurea Herbicide De-Esterification Esterase from Hansschlegelia zhihuaiae S113

PubMed Central

Hang, Bao-Jian; Hong, Qing; Xie, Xiang-Ting; Huang, Xing; Wang, Cheng-Hong; Li, Shun-Peng

2012-01-01

De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag+, Cd2+, Zn2+, methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments. PMID:22247165

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.

Pseudonegative thermal expansion and the state of water in graphene oxide layered assemblies.

PubMed

Zhu, Jian; Andres, Christine M; Xu, Jiadi; Ramamoorthy, Ayyalusamy; Tsotsis, Thomas; Kotov, Nicholas A

2012-09-25

Unraveling the complex interplay between thermal properties and hydration is a part of understanding the fundamental properties of many soft materials and very essential for many applications. Here we show that graphene oxide (GO) demonstrates a highly negative thermal expansion (NTE) coefficient owing to unique thermohydration processes related with fast transport of water between the GO sheets, the amphiphilic nature of nanochannels, and close-to-zero intrinsic thermal expansion of GO. The humidity-dependent NTE of GO layered assemblies, or "pseudonegative thermal expansion" (PNTE), differs from that of other hygroscopic materials due to its relatively fast and highly reversible expansion/contraction cycles and occurrence at low humidity levels while bearing similarities to classic NTE. Thermal expansion of polyvinyl alcohol/GO composites is easily tunable with additional intricacy of thermohydration effects. PNTE combined with isotropy, nontoxicity, and mechanical robustness is an asset for applications of actuators, sensors, MEMS devices, and memory materials and crucial for developing methods of thermal/photopatterning of GO devices.

Common and Distant Structural Characteristics of Feruloyl Esterase Families from Aspergillus oryzae

PubMed Central

Udatha, D. B. R. K. Gupta; Mapelli, Valeria; Panagiotou, Gianni; Olsson, Lisbeth

2012-01-01

Background Feruloyl esterases (FAEs) are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic enzymes. FAEs have gained increased attention in the area of biocatalytic transformations for the synthesis of value added compounds with medicinal and nutritional applications. Following the increasing attention on these enzymes, a novel descriptor based classification system has been proposed for FAEs resulting into 12 distinct families and pharmacophore models for three FAE sub-families have been developed. Methodology/Principal Findings The feruloylome of Aspergillus oryzae contains 13 predicted FAEs belonging to six sub-families based on our recently developed descriptor-based classification system. The three-dimensional structures of the 13 FAEs were modeled for structural analysis of the feruloylome. The three genes coding for three enzymes, viz., A.O.2, A.O.8 and A.O.10 from the feruloylome of A. oryzae, representing sub-families with unknown functional features, were heterologously expressed in Pichia pastoris, characterized for substrate specificity and structural characterization through CD spectroscopy. Common feature-based pharamacophore models were developed according to substrate specificity characteristics of the three enzymes. The active site residues were identified for the three expressed FAEs by determining the titration curves of amino acid residues as a function of the pH by applying molecular simulations. Conclusions/Significance Our findings on the structure-function relationships and substrate specificity of the FAEs of A. oryzae will be instrumental for further understanding of the FAE families in the novel classification system. The developed pharmacophore models could be applied for virtual screening of compound databases for short listing the

Leukocyte Esterase as a Biomarker in the Diagnosis of Periprosthetic Joint Infection.

PubMed

Wang, Chi; Li, Rui; Wang, Qi; Duan, Jinyan; Wang, Chengbin

2017-01-21

BACKGROUND Total joint arthroplasty (TJA) has been one of the most rewarding interventions for treating patients suffering from joint disorders. However, periprosthetic joint infection (PJI) is a serious complication that frequently accompanies TJA. Our study aimed to investigate the application of the leukocyte esterase (LE) strip in the diagnosis of PJI. MATERIAL AND METHODS From October 2014 to July 2015, 72 patients who had undergone joint puncture after arthroplasty in our hospital were enrolled in this trial. One drop of synovial fluid from each available patient was applied to the LE strip, and the results were observed after 1-3 min. If the color turned to dark purple, we recognized this as a positive result, while other colors were regarded as negative results. Centrifugation was used when the synovial fluid was mixed with blood. The Musculoskeletal Infection Society (MSIS) definition was used as the standard reference to identify whether PJI was found in patients or not. The results of diagnosis and LE strips test were compared, and indicators reflecting diagnostic value were calculated. Correlation of the LE data with erythrocyte sedimentation rate (ESR), elevated C-reactive protein (CRP), synovial white blood cell (WBC) counts, and polymorphonuclear neutrophil (PMN) percentage was calculated. RESULTS By MSIS criteria, 38 patients were diagnosed with PJI and 34 patients were not infected. Two types of LE strip presented the same results with sensitivity of 84.21% (95% confidence interval [CI]: 68.75~93.98%), specificity of 97.06% (95% CI: 84.67~99.93%), positive predictive value (PPV) of 96.97% (95% CI: 84.24~99.92%), and negative predictive value (NPV) of 84.62% (95% CI: 69.47~94.14%). There were one false-positive case and six false-negative cases in this trial. There is a strong correlation between LE strip and synovial fluid PMN percentage. CONCLUSIONS The sensitivity and specificity of the LE strip in the diagnosis of PJI are quite high, which means

Reducing cell wall feruloylation by expression of a fungal ferulic acid esterase in Festuca arundinacea modifies plant growth, leaf morphology and the turnover of cell wall arabinoxylans

PubMed Central

Iyer, Prashanti R.; Buanafina, M. Fernanda; Shearer, Erica A.

2017-01-01

A feature of cell wall arabinoxylan in grasses is the presence of ferulic acid which upon oxidative coupling by the action of peroxidases forms diferuloyl bridges between formerly separated arabinoxylans. Ferulate cross-linking is suspected of playing various roles in different plant processes. Here we investigate the role of cell wall feruloyaltion in two major processes, that of leaf growth and the turnover of cell wall arabinoxylans on leaf senescence in tall fescue using plants in which the level of cell wall ferulates has been reduced by targeted expression of the Aspergillus niger ferulic acid esterase A (FAEA) to the apoplast or Golgi. Analysis of FAE expressing plants showed that all the lines had shorter and narrower leaves compared to control, which may be a consequence of the overall growth rate being lower and occurring earlier in FAE expressing leaves than in controls. Furthermore, the final length of epidermal cells was shorter than controls, indicating that their expansion was curtailed earlier than in control leaves. This may be due to the observations that the deposition of both ether and ester linked monomeric hydroxycinnamic acids and ferulate dimerization stopped earlier in FAE expressing leaves but at a lower level than controls, and hydroxycinnamic acid deposition started to slow down when peroxidase levels increased. It would appear therefore that one of the possible mechanisms for controlling overall leaf morphology such as leaf length and width in grasses, where leaf morphology is highly variable between species, may be the timing of hydroxycinnamic acid deposition in the expanding cell walls as they emerge from cell division into the elongation zone, controlled partially by the onset of peroxidase activity in this region. PMID:28934356

Blood Neuropathy Target Esterase as Biochemical Marker for Neuropathic Organophosphates Exposure

DTIC Science & Technology

2001-09-01

E-mail gmakh(@ipac.ac.ru2Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119899, Russia;3Research Center for Molecular ... Diagnostics and Therapy, Moscow, 113149, Russia. INTRODUCTION Organophosphate-induced delayed neurotoxicity (OPIDN) is a distal degeneration of sensory and

Molecular simulation study of feruloyl esterase adsorption on charged surfaces: effects of surface charge density and ionic strength.

PubMed

Liu, Jie; Peng, Chunwang; Yu, Gaobo; Zhou, Jian

2015-10-06

The surrounding conditions, such as surface charge density and ionic strength, play an important role in enzyme adsorption. The adsorption of a nonmodular type-A feruloyl esterase from Aspergillus niger (AnFaeA) on charged surfaces was investigated by parallel tempering Monte Carlo (PTMC) and all-atom molecular dynamics (AAMD) simulations at different surface charge densities (±0.05 and ±0.16 C·m(-2)) and ionic strengths (0.007 and 0.154 M). The adsorption energy, orientation, and conformational changes were analyzed. Simulation results show that whether AnFaeA can adsorb onto a charged surface is mainly controlled by electrostatic interactions between AnFaeA and the charged surface. The electrostatic interactions between AnFaeA and charged surfaces are weakened when the ionic strength increases. The positively charged surface at low surface charge density and high ionic strength conditions can maximize the utilization of the immobilized AnFaeA. The counterion layer plays a key role in the adsorption of AnFaeA on the negatively charged COOH-SAM. The native conformation of AnFaeA is well preserved under all of these conditions. The results of this work can be used for the controlled immobilization of AnFaeA.

An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

PubMed

Koseki, Takuya; Miwa, Yozo; Akao, Takeshi; Akita, Osamu; Hashizume, Katsumi

2006-02-10

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

Effects of juvenile hormone (JH) analog insecticides on larval development and JH esterase activity in two spodopterans.

PubMed

El-Sheikh, El-Sayed A; Kamita, Shizuo G; Hammock, Bruce D

2016-03-01

Juvenile hormone analog (JHA) insecticides are biological and structural mimics of JH, a key insect developmental hormone. Toxic and anti-developmental effects of the JHA insecticides methoprene, fenoxycarb, and pyriproxyfen were investigated on the larval and pupal stages of Spodoptera littoralis and Spodoptera frugiperda. Bioassays showed that fenoxycarb has the highest toxicity and fastest speed of kill in 2nd instar S. littoralis. All three JHAs affected the development of 6th instar (i.e., final instar) and pupal S. frugiperda. JH esterase (JHE) is a critical enzyme that helps to regulate JH levels during insect development. JHE activity in the last instar S. littoralis and S. frugiperda was 11 and 23 nmol min(-1) ml(-1) hemolymph, respectively. Methoprene and pyriproxyfen showed poor inhibition of JHE activity from these insects, whereas fenoxycarb showed stronger inhibition. The inhibitory activity of fenoxycarb, however, was more than 1000-fold lower than that of OTFP, a highly potent inhibitor of JHEs. Surprisingly, topical application of methoprene, fenoxycarb or pyriproxyfen on 6th instars of S. littoralis and S. frugiperda prevented the dramatic reduction in JHE activity that was found in control insects. Our findings suggest that JHAs may function as JH agonists that play a disruptive role or a hormonal replacement role in S. littoralis and S. frugiperda. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Varga, Tamas

Despite the fact that all chemical bonds expand on heating, a small class of materials shrinks when heated. These, so called negative thermal expansion (NTE) materials, are a unique class of materials with some exotic properties. The present chapter offers insight into the structural aspects of pressure- (or temperature-) induced phase transformations, and the energetics of those changes in these fascinating materials, in particular NTE compound cubic ZrW2O8, orthorhombic Sc2W3O12 and Sc2Mo3O12, as well as other members of the 'scandium tungstate family'. In subsequent sections, (i) combined in situ high-pressure synchrotron XRD and XAS studies of NTE material ZrW2O8; (ii)more » an in situ high-pressure synchrotron XRD study of Sc2W3O12, Sc2Mo3O12, and Al2W3O12; and (iii) thermochemical studies of the above materials are presented and discussed. In all of these studies, chemical bonds change, sometimes break and new ones form. Correlations between structure, chemistry, and energetics are revealed. It is also shown that (iv) NTE materials are good candidates as precursors to make novel solid state materials, such as the conducting Sc0.67WO4, using high-pressure, high-temperature synthesis, through modification of bonding and electronic structure, and thus provide vast opportunities for scientific exploration.« less

Effects of a long-acting mutant bacterial cocaine esterase on acute cocaine toxicity in rats

PubMed Central

Collins, Gregory T.; Zaks, Matthew E.; Cunningham, Alyssa R.; St. Clair, Carley; Nichols, Joseph; Narasimhan, Diwahar; Ko, Mei-Chuan; Sunahara, Roger K.; Woods, James H.

2011-01-01

Background A longer acting, double mutant bacterial cocaine esterase (CocE T172R/G173Q; DM CocE) has been shown to protect mice from cocaine-induced lethality, inhibit the reinforcing effects of cocaine in rats, and reverse cocaine’s cardiovascular effects in rhesus monkeys. The current studies evaluated the effectiveness of DM CocE to protect against, and reverse cocaine’s cardiovascular, convulsant, and lethal effects in male and female rats. Methods Pretreatment studies were used to determine the effectiveness and in vivo duration of action for DM CocE to protect rats against the occurrence of cardiovascular changes, convulsion and lethality associated with acute cocaine toxicity. Posttreatment studies were used to evaluate the capacity of DM CocE to rescue rats from the cardiovascular and lethal effects of large doses of cocaine. In addition, male and female rats were studied to determine if there were any potential effects of sex on the capacity of DM CocE to protect against, or reverse acute cocaine toxicity in rats. Results Pretreatment with DM CocE dose-dependently protected rats against cocaine-induced cardiovascular changes, convulsion and lethality, with higher doses active for up to 4 hrs, and shifting cocaine-induced lethality at least 10-fold to the right. In addition to dose-dependently recovering rats from an otherwise lethal dose of cocaine, post-treatment with DM CocE also reversed the cardiovascular effects of cocaine. There were no sex-related differences in the effectiveness of DM CocE to protect against, or reverse acute cocaine toxicity. Conclusions Together, these results support the development of DM CocE for the treatment of acute cocaine toxicity. PMID:21481548

Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family*

PubMed Central

Li, Ping-Yi; Chen, Xiu-Lan; Ji, Peng; Li, Chun-Yang; Wang, Peng; Zhang, Yi; Xie, Bin-Bin; Qin, Qi-Long; Su, Hai-Nan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Zhang, Xi-Ying

2015-01-01

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp202 and Phe203 in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability. PMID:25771540

Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

PubMed

Martínez Morcillo, S; Yela, J L; Capowiez, Y; Mazzia, C; Rault, M; Sanchez-Hernandez, Juan C

2013-05-01

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.

Pressure-dependence of the phase transitions and thermal expansion in zirconium and hafnium pyrovanadate

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

Gallington, Leighanne C.; Hester, Brett R.; Kaplan, Benjamin S.

Low or negative thermal expansion (NTE) has been previously observed in members of the ZrP 2O 7 family at temperatures higher than their order-disorder phase transitions. The thermoelastic properties and phase behavior of the low temperature superstructure and high temperature negative thermal expansion phases of ZrV 2O 7 and HfV 2O 7 were explored via in situ variable temperature/pressure powder x-ray diffraction measurements. The phase transition temperatures of ZrV 2O 7 and HfV 2O 7 exhibited a very strong dependence on pressure (~700 K GPa), with moderate compression suppressing the formation of their NTE phases below 513 K. Compression alsomore » reduced the magnitude of the coefficients of thermal expansion in both the positive and negative thermal expansion phases. Additionally, the high temperature NTE phase of ZrV 2O 7 was found to be twice as stiff as the low temperature positive thermal expansion superstructure (24 and 12 GPa respectively).« less

Neutron diffraction study and anomalous negative thermal expansion in non-superconducting PrFe1-xRuxAsO

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

Yiu, Yuen; Garlea, Vasile O; McGuire, Michael A

2012-01-01

Neutron powder diraction has been used to investigate the structural and magnetic behavior of the isoelectronically doped Fe pnictide material PrFe1-xRuxAsO. Substitution of Ru for Fe sup- presses the structural and magnetic phase transitions that occur in the undoped compound PrFeAsO. Contrary to the behavior usually observed in 1111 pnictide materials, the suppression of both the structural and magnetic transitions does not result in the emergence of superconductivity or any other new ground state. Interestingly, PrFeAsO itself shows an unusual negative thermal expansion (NTE) along the c-axis, from 60K down to at least 4K; this does not occur in superconductingmore » samples such as those formed by doping with fluorine on the oxygen site. We nd that NTE is present for all concentrations of PrFe1-xRuxAsO with x ranging from 0.05 to 0.75. These results suggest that the absence of superconductivity in these materials could be related to the presence of NTE.« less

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.

Aboveground Epichloë coenophiala-Grass Associations Do Not Affect Belowground Fungal Symbionts or Associated Plant, Soil Parameters.

PubMed

Slaughter, Lindsey C; McCulley, Rebecca L

2016-10-01

Cool season grasses host multiple fungal symbionts, such as aboveground Epichloë endophytes and belowground arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSEs). Asexual Epichloë endophytes can influence root colonization by AMF, but the type of interaction-whether antagonistic or beneficial-varies. In Schedonorus arundinaceus (tall fescue), Epichloë coenophiala can negatively affect AMF, which may impact soil properties and ecosystem function. Within field plots of S. arundinaceus that were either E. coenophiala-free (E-), infected with the common, mammal-toxic E. coenophiala strain (CTE+), or infected with one of two novel, non-toxic strains (AR542 NTE+ and AR584 NTE+), we hypothesized that (1) CTE+ would decrease AMF and DSE colonization rates and reduce soil extraradical AMF hyphae compared to E- or NTE+, and (2) this would lead to E- and NTE+ plots having greater water stable soil aggregates and C than CTE+. E. coenophiala presence and strain did not significantly alter AMF or DSE colonization, nor did it affect extraradical AMF hypha length, soil aggregates, or aggregate-associated C and N. Soil extraradical AMF hypha length negatively correlated with root AMF colonization. Our results contrast with previous demonstrations that E. coenophiala symbiosis inhibits belowground AMF communities. In our mesic, relatively nutrient-rich grassland, E. coenophiala symbiosis did not antagonize belowground symbionts, regardless of strain. Manipulating E. coenophiala strains within S. arundinaceus may not significantly alter AMF communities and nutrient cycling, yet we must further explore these relationships under different soils and environmental conditions given that symbiont interactions can be important in determining ecosystem response to global change.

Mass Uncertainty and Application For Space Systems

NASA Technical Reports Server (NTRS)

Beech, Geoffrey

2013-01-01

Expected development maturity under contract (spec) should correlate with Project/Program Approved MGA Depletion Schedule in Mass Properties Control Plan. If specification NTE, MGA is inclusive of Actual MGA (A5 & A6). If specification is not an NTE Actual MGA (e.g. nominal), then MGA values are reduced by A5 values and A5 is representative of remaining uncertainty. Basic Mass = Engineering Estimate based on design and construction principles with NO embedded margin MGA Mass = Basic Mass * assessed % from approved MGA schedule. Predicted Mass = Basic + MGA. Aggregate MGA % = (Aggregate Predicted - Aggregate Basic) /Aggregate Basic.

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

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.

A comparative study on the distribution of non-specific esterase amongst the various constituents of retinas of some vertebrates.

PubMed

Tyagi, H R; Tewari, H B

1979-01-01

A wide coverage of the retinae of a large number of animals (Calotes, Varanus, Naja, Athene, Passer, Streptopelia, Psittacula and Funambulus) from the point of view of the histoenzymological distribution of non-specific esterase amongst the various constituents reveals mostly identical patterns. They are as follows: 1. Outer segments - positive in all cases. 2. Outer plexiform layer - equipped with enzymatic activity in all the instances. 3. Inner nuclear layer - thin cytoplasmic rim of the neurons characterized by positive activity; the nuclei of the neurons are completely negative. 4. Inner plexiform layer - this layer is endowed with the enzymatic activity. 5. Ganglion cells - negative in all cases. 6. Nerve fibres of the layer of nerve fibres, situated adjasent to ganglion cells are positive in all the animals; in case of squirrel oligodandroglial cells present in the region have demonstrated activity of a high order. On of the high-lights of the present contribution is demarcation of the inner plexiform layer into three stratified zones, equipped with enzymatic activity in Calotes, Streptopelia, Naja and Funambulus. Such stratifications are not seen in Varanus, Passer and Psittacula. The significance of the various patterns and the equipment of the enzyme in various constituents at various locals have been discussed in relation to the metabolic functions, zone-wise and interzone-wise in visual processes of various animals.

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%).

Biochemical characterization of a halotolerant feruloyl esterase from Actinomyces spp.: refolding and activity following thermal deactivation.

PubMed

Hunt, Cameron J; Tanksale, Akshat; Haritos, Victoria S

2016-02-01

Ferulic acid esterases (FAE, EC. 3.1.1.73) hydrolyse the linkage between hemicellulose and lignin and thus have potential for use in mild enzymatic pretreatment of biomass as an alternative to thermochemical approaches. Here, we report the characterization of a novel FAE (ActOFaeI) obtained from the bacterium, Actinomyces sp. oral which was recombinantly expressed in Escherichia coli BL21 in two forms: with and without its putative signal peptide. The truncated form was found to have <10 % relative activity compared to the full length and was more prone to aggregation after purification. The enzyme with retained peptide demonstrated 2 to 4-fold higher activity against methyl caffeate and methyl p-coumarate, with specific activities of 477.6 and 174.4 U mg(-1) respectively, than the equivalent activities of the benchmark FAE from Aspergillus niger A and B. ActOFaeI retained activity over a broad pH range with a maximum at 9 but >90 % relative activity at pH 6.5 and an optimum reaction temperature of 30 °C. ActOFaeI increased activity by 15% in high salt conditions (1000 mMNaCl) and its thermal unfolding temperature improved from 41.5 °C in standard buffer to 74 °C in the presence of 2500 mM sodium malonate. ActOFaeI also released ferulic acid from destarched wheat bran when combined with a xylanase preparation. After treatment above the thermal denaturation temperature followed by cooling to room temperature, ActOFaeI demonstrated spontaneous refolding into an active state. ActOFaeI displays many useful characteristics for enzymatic pretreatment of lignocellulose and contributes to our understanding of this important family.

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.

Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo*

PubMed Central

Durham, Timothy B.; Toth, James L.; Klimkowski, Valentine J.; Cao, Julia X. C.; Siesky, Angela M.; Alexander-Chacko, Jesline; Wu, Ginger Y.; Dixon, Jeffrey T.; McGee, James E.; Wang, Yong; Guo, Sherry Y.; Cavitt, Rachel Nicole; Schindler, John; Thibodeaux, Stefan J.; Calvert, Nathan A.; Coghlan, Michael J.; Sindelar, Dana K.; Christe, Michael; Kiselyov, Vladislav V.; Michael, M. Dodson; Sloop, Kyle W.

2015-01-01

Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE−/− mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance. PMID:26085101

Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7

PubMed Central

Rangarajan, Erumbi S; Ruane, Karen M; Proteau, Ariane; Schrag, Joseph D; Valladares, Ricardo; Gonzalez, Claudio F; Gilbert, Michel; Yakunin, Alexander F; Cygler, Miroslaw

2011-01-01

There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates. PMID:21557376

Structural and Enzymatic Characterization of NanS (YjhS) a 9-O-Acetyl N-acetylneuraminic Acid Esterase from Escherichia coli O157:H7

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

E Rangarajan; K Ruane; A Proteau

2011-12-31

There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into twomore » subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.« less

Negative thermal expansion and magnetoelastic coupling in the breathing pyrochlore lattice material LiGaCr4S8

NASA Astrophysics Data System (ADS)

Pokharel, G.; May, A. F.; Parker, D. S.; Calder, S.; Ehlers, G.; Huq, A.; Kimber, S. A. J.; Arachchige, H. Suriya; Poudel, L.; McGuire, M. A.; Mandrus, D.; Christianson, A. D.

2018-04-01

The physical properties of the spinel LiGaCr4S8 have been studied with neutron diffraction, x-ray diffraction, magnetic susceptibility, and heat capacity measurements. The neutron diffraction and synchrotron x-ray diffraction data reveal negative thermal expansion (NTE) below 111(4) K. The magnetic susceptibility deviates from Curie-Weiss behavior with the onset of NTE. At low temperature a broad peak in the magnetic susceptibility at 10.3(3) K is accompanied by the return of normal thermal expansion. First-principles calculations find a strong coupling between the lattice and the simulated magnetic ground state. These results indicate strong magnetoelastic coupling in LiGaCr4S8 .

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

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.

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

Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant Rats▿

PubMed Central

Lai, Kin Kwan; Lorca, Graciela L.; Gonzalez, Claudio F.

2009-01-01

Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammal's intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being Lactobacillus johnsonii that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (Km = 20 to 60 μM) and chlorogenic acid (Km = 10 to 50 μM) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms. PMID:19502437

Biochemical properties of two cinnamoyl esterases purified from a Lactobacillus johnsonii strain isolated from stool samples of diabetes-resistant rats.

PubMed

Lai, Kin Kwan; Lorca, Graciela L; Gonzalez, Claudio F

2009-08-01

Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammal's intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being Lactobacillus johnsonii that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (K(m) = 20 to 60 microM) and chlorogenic acid (K(m) = 10 to 50 microM) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms.

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.

Probiotic Ferulic Acid Esterase Active Lactobacillus fermentum NCIMB 5221 APA Microcapsules for Oral Delivery: Preparation and in Vitro Characterization.

PubMed

Tomaro-Duchesneau, Catherine; Saha, Shyamali; Malhotra, Meenakshi; Coussa-Charley, Michael; Kahouli, Imen; Jones, Mitchell L; Labbé, Alain; Prakash, Satya

2012-02-16

Probiotics possess potential therapeutic and preventative effects for various diseases and metabolic disorders. One important limitation for the oral delivery of probiotics is the harsh conditions of the upper gastrointestinal tract (GIT) which challenge bacterial viability and activity. One proposed method to surpass this obstacle is the use of microencapsulation to improve the delivery of bacterial cells to the lower GIT. The aim of this study is to use alginate-poly-L-lysine-alginate (APA) microcapsules to encapsulate Lactobacillus fermentum NCIMB 5221 and characterize its enzymatic activity and viability through a simulated GIT. This specific strain, in previous research, was characterized for its inherent ferulic acid esterase (FAE) activity which could prove beneficial in the development of a therapeutic for the treatment and prevention of cancers and metabolic disorders. Our findings demonstrate that the APA microcapsule does not slow the mass transfer of substrate into and that of the FA product out of the microcapsule, while also not impairing bacterial cell viability. The use of simulated gastrointestinal conditions led to a significant 2.5 log difference in viability between the free (1.10 × 104 ± 1.00 × 103 cfu/mL) and the microencapsulated (5.50 × 106 ± 1.00 × 105 cfu/mL) L. fermentum NCIMB 5221 following exposure. The work presented here suggests that APA microencapsulation can be used as an effective oral delivery method for L. fermentum NCIMB 5221, a FAE-active probiotic strain.

Expression of a Petunia inflata pectin methyl esterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution.

PubMed

Pilling, J; Willmitzer, L; Fisahn, J

2000-02-01

Transgenic potato (Solanum tuberosum L.) plants were constructed with a Petunia inflata-derived cDNA encoding a pectin methyl esterase (PME; EC 3.1.1.11) in sense orientation under the control of the cauliflower mosaic virus 35S promoter. The PME activity was elevated in leaves and tubers of the transgenic lines but slightly reduced in apical segments of stems from mature plants. Stem segments from the base of juvenile PME-overexpressing plants did not differ in PME activity from the control, whereas in apical parts PME was less active than in the wild-type. During the early stages of development stems of these transgenic plants elongated more rapidly than those of the wild-type. Further evidence that overexpression of a plant-derived PME has an impact on plant development is based on modifications of tuber yield, which was reduced in the transgenic lines. Cell walls from transgenic tubers showed significant differences in their cation-binding properties in comparison with the wild-type. In particular, cell walls displayed increased affinity for sodium and calcium, while potassium binding was constant. Furthermore, the total ion content of transgenic potatoes was modified. Indications of PME-mediated differences in the distribution of ions in transgenic plants were also obtained by monitoring relaxations of the membrane potential of roots subsequent to changes in the ionic composition of the bathing solution. However, no effects on the chemical structure of pectin from tuber cell walls could be detected.

Probiotic Ferulic Acid Esterase Active Lactobacillus fermentum NCIMB 5221 APA Microcapsules for Oral Delivery: Preparation and in Vitro Characterization

PubMed Central

Tomaro-Duchesneau, Catherine; Saha, Shyamali; Malhotra, Meenakshi; Coussa-Charley, Michael; Kahouli, Imen; Jones, Mitchell L.; Labbé, Alain; Prakash, Satya

2012-01-01

Probiotics possess potential therapeutic and preventative effects for various diseases and metabolic disorders. One important limitation for the oral delivery of probiotics is the harsh conditions of the upper gastrointestinal tract (GIT) which challenge bacterial viability and activity. One proposed method to surpass this obstacle is the use of microencapsulation to improve the delivery of bacterial cells to the lower GIT. The aim of this study is to use alginate-poly-L-lysine-alginate (APA) microcapsules to encapsulate Lactobacillus fermentum NCIMB 5221 and characterize its enzymatic activity and viability through a simulated GIT. This specific strain, in previous research, was characterized for its inherent ferulic acid esterase (FAE) activity which could prove beneficial in the development of a therapeutic for the treatment and prevention of cancers and metabolic disorders. Our findings demonstrate that the APA microcapsule does not slow the mass transfer of substrate into and that of the FA product out of the microcapsule, while also not impairing bacterial cell viability. The use of simulated gastrointestinal conditions led to a significant 2.5 log difference in viability between the free (1.10 × 104 ± 1.00 × 103 cfu/mL) and the microencapsulated (5.50 × 106 ± 1.00 × 105 cfu/mL) L. fermentum NCIMB 5221 following exposure. The work presented here suggests that APA microencapsulation can be used as an effective oral delivery method for L. fermentum NCIMB 5221, a FAE-active probiotic strain. PMID:24288090

In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat.

PubMed

Killi, Uday K; Wsol, Vladimir; Soukup, Ondrej; Kuca, Kamil; Winder, Michael; Tobin, Gunnar

2014-02-01

Obidoxime, a weak acetylcholine-esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration-dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90-290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80-450%). Physostigmine concentration-dependently increased methacholine-evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor-evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime. © 2013 Wiley Publishing Asia Pty Ltd.

Composition, response to pressure, and negative thermal expansion in M IIB IVF 6 (M = Ca, Mg; B = Zr, Nb) [Composition, response to pressure, and negative thermal expansion in A IIB IVF 6; A - Ca, Mg, B - Zr, Nb

DOE PAGES

Hester, Brett R.; Hancock, Justin C.; Lapidus, Saul H.; ...

2016-12-27

CaZrF 6 has recently been shown to combine strong negative thermal expansion (NTE) over a very wide temperature range (at least 10–1000 K) with optical transparency from mid-IR into the UV range. Variable-temperature and high-pressure diffraction has been used to determine how the replacement of calcium by magnesium and zirconium by niobium(IV) modifies the phase behavior and physical properties of the compound. Similar to CaZrF 6, CaNbF 6 retains a cubic ReO 3-type structure down to 10 K and displays NTE up until at least 900 K. It undergoes a reconstructive phase transition upon compression to ~400 MPa at room temperature and pressure-induced amorphization above ~4 GPa. Prior to the first transition, it displays very strong pressure-induced softening. MgZrF 6 adopts a cubic ( Fmmore » $$\\bar{3}$$m) structure at 300 K and undergoes a symmetry-lowering phase transition involving octahedral tilts at ~100 K. Immediately above this transition, it shows modest NTE. Its’ thermal expansion increases upon heating, crossing through zero at ~500 K. Unlike CaZrF 6 and CaNbF 6, it undergoes an octahedral tilting transition upon compression (~370 MPa) prior to a reconstructive transition at ~1 GPa. Cubic MgZrF 6 displays both pressure-induced softening and stiffening upon heating. MgNbF 6 is cubic ( Fm$$\\bar{3}$$m) at room temperature, but it undergoes a symmetry-lowering octahedral tilting transition at ~280 K. It does not display NTE within the investigated temperature range (100–950 K). Furthermore the replacement of Zr(IV) by Nb(IV) leads to minor changes in phase behavior and properties, the replacement of the calcium by the smaller and more polarizing magnesium leads to large changes in both phase behavior and thermal expansion.« less

Composition, response to pressure, and negative thermal expansion in M IIB IVF 6 (M = Ca, Mg; B = Zr, Nb) [Composition, response to pressure, and negative thermal expansion in A IIB IVF 6; A - Ca, Mg, B - Zr, Nb

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

Hester, Brett R.; Hancock, Justin C.; Lapidus, Saul H.

CaZrF 6 has recently been shown to combine strong negative thermal expansion (NTE) over a very wide temperature range (at least 10–1000 K) with optical transparency from mid-IR into the UV range. Variable-temperature and high-pressure diffraction has been used to determine how the replacement of calcium by magnesium and zirconium by niobium(IV) modifies the phase behavior and physical properties of the compound. Similar to CaZrF 6, CaNbF 6 retains a cubic ReO 3-type structure down to 10 K and displays NTE up until at least 900 K. It undergoes a reconstructive phase transition upon compression to ~400 MPa at room temperature and pressure-induced amorphization above ~4 GPa. Prior to the first transition, it displays very strong pressure-induced softening. MgZrF 6 adopts a cubic ( Fmmore » $$\\bar{3}$$m) structure at 300 K and undergoes a symmetry-lowering phase transition involving octahedral tilts at ~100 K. Immediately above this transition, it shows modest NTE. Its’ thermal expansion increases upon heating, crossing through zero at ~500 K. Unlike CaZrF 6 and CaNbF 6, it undergoes an octahedral tilting transition upon compression (~370 MPa) prior to a reconstructive transition at ~1 GPa. Cubic MgZrF 6 displays both pressure-induced softening and stiffening upon heating. MgNbF 6 is cubic ( Fm$$\\bar{3}$$m) at room temperature, but it undergoes a symmetry-lowering octahedral tilting transition at ~280 K. It does not display NTE within the investigated temperature range (100–950 K). Furthermore the replacement of Zr(IV) by Nb(IV) leads to minor changes in phase behavior and properties, the replacement of the calcium by the smaller and more polarizing magnesium leads to large changes in both phase behavior and thermal expansion.« less

Assessing exchange-correlation functionals for elasticity and thermodynamics of α -ZrW2O8 : A density functional perturbation theory study

NASA Astrophysics Data System (ADS)

Weck, Philippe F.; Kim, Eunja; Greathouse, Jeffery A.; Gordon, Margaret E.; Bryan, Charles R.

2018-04-01

Elastic and thermodynamic properties of negative thermal expansion (NTE) α -ZrW2O8 have been calculated using PBEsol and PBE exchange-correlation functionals within the framework of density functional perturbation theory (DFPT). Measured elastic constants are reproduced within ∼ 2 % with PBEsol and ∼ 6 % with PBE. The thermal evolution of the Grüneisen parameter computed within the quasi-harmonic approximation exhibits negative values below the Debye temperature, consistent with observation. The standard molar heat capacity is predicted to be CP0 = 192.2 and 193.8 J mol-1K-1 with PBEsol and PBE, respectively. These results suggest superior accuracy of DFPT/PBEsol for studying the lattice dynamics, elasticity and thermodynamics of NTE materials.

Interagency Coordination Group of Inspectors General for Guam Realignment Annual Report

DTIC Science & Technology

2015-01-30

rse n A FB 6/1 8/2 01 0 1, 17 3,5 57 .70 1, 17 3,4 89 .21 15...DO T FH W A - G ov er nm en t o f Gu am C on tra ct Ro ute 1 an d 3 I nte rse cti on Im pr ov em en ts GU -N H- 00 01 (1 25 ) 27 XX 66 00...01 12 57 3P 0 = $5 70 ,74 0 2 7X X6 60 00 11 25 73 V0 = $3 0,2 32 Co ns ult an t fo r Ro ute 1/ 3 I nte rse cti on 6/8 /20 11 60

Negative Thermal Expansion over a Wide Temperature Range in Fe-doped MnNiGe Composites

NASA Astrophysics Data System (ADS)

Zhao, Wenjun; Sun, Ying; Liu, Yufei; Shi, Kewen; Lu, Huiqing; Song, Ping; Wang, Lei; Han, Huimin; Yuan, Xiuliang; Wang, Cong

2018-02-01

Fe-doped MnNiGe alloys were successfully synthesized by solid-state reaction. Giant negative thermal expansion (NTE) behaviors with the coefficients of thermal expansion (CTE) of -285.23×10-6 K-1 (192-305 K) and -1167.09×10-6 K-1 (246-305 K) have been obtained in Mn0.90Fe0.10NiGe and MnNi0.90Fe0.10Ge, respectively. Furthermore, these materials were combined with Cu in order to control the NTE properties. The results indicate that the absolute value of CTE gradually decreases with increasing Cu contents. In Mn0.92Fe0.08NiGe/x%Cu, the CTE gradually changes from -64.92×10-6 K-1 (125-274 K) to -4.73×10-6 K-1 (173-229 K) with increasing value of x from 15 to 70. The magnetic measurements reveal that the NTE behaviors in this work are strongly correlated with the process of the magnetic phase transition and the introduction of Fe atoms could also change the spiral anti-ferromagnetic (s-AFM) state into ferromagnetic (FM) state at low temperature. Our study launches a new candidate for controlling thermal expansion properties of metal matrix materials which could have potential application in variable temperature environment.

Positional linker effects in haptens for cocaine immunopharmacotherapy.

PubMed

Ino, Akira; Dickerson, Tobin J; Janda, Kim D

2007-08-01

Cocaine use remains a serious problem, despite intensive efforts to curb abuse. Given the lack of effective pharmacotherapeutics for the treatment of cocaine addiction, research groups have targeted immunopharmacotherapy in which the drug user's immune system is trained to recognize and remove cocaine prior to entry into the central nervous system. Antibody cocaine esterases and simple binders have been procured, however, rates and/or affinities still need improvement before clinical trials are warranted. Herein, we report the synthesis and testing of two new haptens for the procurement of cocaine binding antibodies and cocaine esterase catalytic antibodies. Central in the design of these haptens was the placement of the linker functionality distal from the anticipated cocaine epitopes in an attempt to bury the hapten deep within an antibody combining site to gain possible entropic and enthalpic advantages.

An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

PubMed

Chertemps, Thomas; Younus, Faisal; Steiner, Claudia; Durand, Nicolas; Coppin, Chris W; Pandey, Gunjan; Oakeshott, John G; Maïbèche, Martine

2015-01-01

Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.

Concomitant use of acetylcholine esterase inhibitors and urinary antispasmodics among Finnish community-dwelling persons with Alzheimer disease.

PubMed

Torvinen-Kiiskinen, Sanna; Taipale, Heidi; Tanskanen, Antti; Tiihonen, Jari; Hartikainen, Sirpa

2014-12-01

Concomitant use of acetylcholine esterase inhibitors (AChEIs) and anticholinergic drugs, such as urinary antispasmodics (UA), is generally considered as inappropriate because of their opposite pharmacological actions. However, prevalence and the duration or factors associated with concomitant use have not been previously studied among community-dwelling persons with Alzheimer disease (AD). The aim of this study was to examine the prevalence and duration of concomitant use of AChEIs and UAs among community-dwelling persons with AD and factors associated with concomitant use. Register-based data of the MEDALZ-2005 Study included all community-dwelling persons with clinically diagnosed AD at the end of year 2005 in Finland. Persons using AChEI drugs during the 4-year follow-up (2006-2009) were included in the present study (n = 20,442). Among AChEI users, 1576 persons used UA during the follow-up. Prevalence of concomitant use of AChEIs and UAs was 7.3% (n = 1491) during the 4-year follow-up. The median duration of concomitant use was 236 days. Factors associated with concomitant use were age younger than 80 years (odds ratio [OR], 1.20; 95% confidence interval [CI], 1.08-1.34), male sex (OR, 1.16; 95% CI, 1.04-1.30), Parkinson disease (OR, 1.98; 95% CI, 1.55-2.52), diabetes (OR, 1.25; 95% CI, 1.08-1.45), and prostatic cancer (OR, 1.54; 95% CI, 1.13-2.09). Despite their antagonizing action, concomitant use of AChEIs and UAs was quite common among Finnish community-dwelling persons with AD. In addition, duration of concomitant use was comparatively long. It is recommended to consider some other options than UAs to treat urinary incontinence among persons with AD.

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

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium Thalassospira sp.

PubMed

De Santi, Concetta; Leiros, Hanna-Kirsti S; Di Scala, Alessia; de Pascale, Donatella; Altermark, Bjørn; Willassen, Nils-Peder

2016-05-01

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.

Neuropathy Target Esterase in Brain Function and Deterioration Caused by Cholinesterase Inhibiting Chemicals

DTIC Science & Technology

2005-08-01

hyperactivity. Invited speaker at 2004 International Meeting for Autism Research – Autism , Genes and Environment Session. Sacramento, California. • Patents...Clinical Findings from Medical Examinations of U.S. Gulf War Veterans Vaccines Linked to Gulf War Syndrome 03.25.03 email to a friend As

Cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse Peyer's patch M cells.

PubMed

Owen, R L; Bhalla, D K

1983-10-01

M cells in Peyer's patch follicle epithelium endocytose and transport luminal materials to intraepithelial lymphocytes. We examined (1) enzymatic characteristics of the epithelium covering mouse and rat Peyer's patches by using cytochemical techniques, (2) distribution of lectin-binding sites by peroxidase-labeled lectins, and (3) anionic site distribution by using cationized ferritin to develop a profile of M cell surface properties. Alkaline phosphatase activity resulted in deposits of dense reaction product over follicle surfaces but was markedly reduced over M cells, unlike esterase which formed equivalent or greater product over M cells. Concanavalin A, ricinus communis agglutinin, wheat germ agglutinin and peanut agglutinin reacted equally with M cells and with surrounding enterocytes over follicle surfaces. Cationized ferritin distributed in a random fashion along microvillus membranes of both M cells and enterocytes, indicating equivalent anionic site distribution. Staining for alkaline phosphatase activity provides a new approach for distinguishing M cells from enterocytes at the light microscopic level. Identical binding of lectins indicates that M cells and enterocytes share common glycoconjugates even though molecular groupings may differ. Lectin binding and anionic charge similarities of M cells and enterocytes may facilitate antigen sampling by M cells of particles and compounds that adhere to intestinal surfaces in non-Peyer's patch areas.

Negative thermal expansion and magnetoelastic coupling in the breathing pyrochlore lattice material LiGaCr 4 S 8

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

Pokharel, G.; May, A. F.; Parker, D. S.

In this paper, the physical properties of the spinel LiGaCr 4S 8 have been studied with neutron diffraction, x-ray diffraction, magnetic susceptibility, and heat capacity measurements. The neutron diffraction and synchrotron x-ray diffraction data reveal negative thermal expansion (NTE) below 111(4) K. The magnetic susceptibility deviates from Curie-Weiss behavior with the onset of NTE. At low temperature a broad peak in the magnetic susceptibility at 10.3(3) K is accompanied by the return of normal thermal expansion. First-principles calculations find a strong coupling between the lattice and the simulated magnetic ground state. Finally, these results indicate strong magnetoelastic coupling in LiGaCrmore » 4S 8.« less

Assessing exchange-correlation functionals for elasticity and thermodynamics of α - ZrW 2 O 8 : A density functional perturbation theory study

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

Weck, Philippe F.; Kim, Eunja; Greathouse, Jeffery A.

Elastic and thermodynamic properties of negative thermal expansion (NTE) αα-ZrW2O8 have been calculated using PBEsol and PBE exchange-correlation functionals within the framework of density functional perturbation theory (DFPT). Measured elastic constants are reproduced within ~2% with PBEsol and 6% with PBE. The thermal evolution of the Grüneisen parameter computed within the quasi-harmonic approximation exhibits negative values below the Debye temperature, consistent with observation. The standard molar heat capacity is predicted to be Cmore » $$O\\atop{P}$$=192.2 and 193.8 J mol -1K -1 with PBEsol and PBE, respectively. These results suggest superior accuracy of DFPT/PBEsol for studying the lattice dynamics, elasticity and thermodynamics of NTE materials.« less

Negative thermal expansion and magnetoelastic coupling in the breathing pyrochlore lattice material LiGaCr 4 S 8

DOE PAGES

Pokharel, G.; May, A. F.; Parker, D. S.; ...

2018-04-30

In this paper, the physical properties of the spinel LiGaCr 4S 8 have been studied with neutron diffraction, x-ray diffraction, magnetic susceptibility, and heat capacity measurements. The neutron diffraction and synchrotron x-ray diffraction data reveal negative thermal expansion (NTE) below 111(4) K. The magnetic susceptibility deviates from Curie-Weiss behavior with the onset of NTE. At low temperature a broad peak in the magnetic susceptibility at 10.3(3) K is accompanied by the return of normal thermal expansion. First-principles calculations find a strong coupling between the lattice and the simulated magnetic ground state. Finally, these results indicate strong magnetoelastic coupling in LiGaCrmore » 4S 8.« less

Assessing exchange-correlation functionals for elasticity and thermodynamics of α - ZrW 2 O 8 : A density functional perturbation theory study

DOE PAGES

Weck, Philippe F.; Kim, Eunja; Greathouse, Jeffery A.; ...

2018-03-15

Elastic and thermodynamic properties of negative thermal expansion (NTE) αα-ZrW2O8 have been calculated using PBEsol and PBE exchange-correlation functionals within the framework of density functional perturbation theory (DFPT). Measured elastic constants are reproduced within ~2% with PBEsol and 6% with PBE. The thermal evolution of the Grüneisen parameter computed within the quasi-harmonic approximation exhibits negative values below the Debye temperature, consistent with observation. The standard molar heat capacity is predicted to be Cmore » $$O\\atop{P}$$=192.2 and 193.8 J mol -1K -1 with PBEsol and PBE, respectively. These results suggest superior accuracy of DFPT/PBEsol for studying the lattice dynamics, elasticity and thermodynamics of NTE materials.« less

Impact of combined C1 esterase inhibitor/coagulation factor XIII or N-acetylcysteine/tirilazad mesylate administration on leucocyte adherence and cytokine release in experimental endotoxaemia.

PubMed

Birnbaum, J; Klotz, E; Spies, C D; Mueller, J; Vargas Hein, O; Feller, J; Lehmann, C

2008-01-01

We determined the effects of combinations of C1 esterase inhibitor (C1-INH) with factor XIII and of N-acetylcysteine (NAC) with tirilazad mesylate (TM) during lipo-polysaccharide (LPS)-induced endotoxaemia in rats. Forty Wistar rats were divided into four groups: the control (CON) group received no LPS; the LPS, C1-INH + factor XIII and NAC + TM groups received endotoxin infusions (5 mg/kg per h). After 30 min of endotoxaemia, 100 U/kg C1-INH + 50 U/kg factor XIII was administered to the C1-INH + factor XIII group, and 150 mg/kg NAC + 10 mg/kg TM was administered in the NAC + TM group. Administration of C1-INH + factor XIII and NAC + TM both resulted in reduced leucocyte adherence and reduced levels of interleukin-1beta (IL-1beta). The LPS-induced increase in IL-6 levels was amplified by both drug combinations. There was no significant effect on mesenteric plasma extravasation. In conclusion, the administration of C1-INH + factor XIII and NAC + TM reduced endothelial leucocyte adherence and IL-1beta plasma levels, but increased IL-6 levels.

Antioxidant and Cholesterol Esterase Inhibitory Properties of Supplementation with Coconut Water in Submerged Cultivation of the Medicinal Chinese Caterpillar Mushroom, Ophiocordyceps sinensis CS1197 (Ascomycetes).

PubMed

Shashidhar, G M; Kumar, S Sravan; Giridhar, P; Manohar, B

2017-01-01

We investigated the potential use of coconut water to supplement potato dextrose broth (PDB) in the production of Ophiocordyceps sinensis CS1197 by submerged cultivation. The basal PDB medium was modified by supplementation with tender coconut water (TCW) and mature coconut water (MCW) at 10% and 5% (v/v), respectively; these mixtures were cultured at 28°C for 14 days, with a pH of 7 and an inoculum volume of 10%. The addition of optimized levels of TCW and MCW improved the biomass yield by 2.2- and 2.5-fold, respectively, and adenosine, cordycepin, and polysaccharide content by 58% and 69%, 50% and 55%, and 19% and 27%, respectively. Antioxidant and cholesterol esterase (CE) inhibitory activities of the aqueous extract from O. sinensis CS1197 mycelia supplemented with TCW and MCW were high compared with those of the control, indicating that coconut water has a positive correlation with the enhanced antioxidant and CE inhibitory activities. These antioxidant and CE inhibitory responses were dependent on concentration, and the larger amounts of bioactives in O. sinensis CS1197 are beneficial in pharmaceutical formulations.