Science.gov

Sample records for acetyl xylan esterases

  1. Hydrolysis of wheat arabinoxylan by two acetyl xylan esterases from Chaetomium thermophilum.

    PubMed

    Tong, Xiaoxue; Lange, Lene; Grell, Morten Nedergaard; Busk, Peter Kamp

    2015-01-01

    The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 3.1.1.72) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has been sequenced, its carbohydrate esterases are not annotated yet. We applied peptide pattern recognition (PPR) tool for sequence analysis of the C. thermophilum genome, and 11 carbohydrate esterase genes were discovered. Furthermore, we cloned and heterologously expressed two putative acetyl xylan esterase genes, CtAxeA and CtAxeB, in Pichia pastoris. The recombinant proteins, rCtAxeA and rCtAxeB, released acetic acids from p-nitrophenyl acetate and water-insoluble wheat arabinoxylan. These results indicate that CtAxeA and CtAxeB are true acetyl xylan esterases. For both recombinant esterases, over 93 % of the initial activity was retained after 24 h of incubation at temperatures up to 60 °C, and over 90 % of the initial activity was retained after 24 h of incubation in different buffers from pH 4.0 to 9.0 at 4 and 50 °C. The overall xylose yield from wheat arabinoxylan hydrolysis was 8 % with xylanase treatment and increased to 34 % when xylanase was combined with rCtAxeA and rCtAxeB. In sum, the present study first report the biochemical characterization of two acetyl xylan esterases from C. thermophilum, which are efficient in hydrolyzing hemicellulose with potential application in biomass bioconversion to high value chemicals or biofuels. PMID:25369895

  2. Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2

    SciTech Connect

    Blum, D.L.; Li, X.L.; Chen, H.; Ljungdahl, L.G.

    1999-09-01

    A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K{sub m} of 0.9 mM and a V{sub max} of 785 {micro}mol min{sup {minus}} mg{sup {minus}1}. It had temperature and pH optima of 30 C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum.

  3. Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.

    PubMed

    Pawar, Prashant Mohan-Anupama; Derba-Maceluch, Marta; Chong, Sun-Li; Gómez, Leonardo D; Miedes, Eva; Banasiak, Alicja; Ratke, Christine; Gaertner, Cyril; Mouille, Grégory; McQueen-Mason, Simon J; Molina, Antonio; Sellstedt, Anita; Tenkanen, Maija; Mellerowicz, Ewa J

    2016-01-01

    Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production. PMID:25960248

  4. Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea.

    PubMed

    Ding, Shaojun; Cao, Jie; Zhou, Rui; Zheng, Fei

    2007-09-01

    A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose. PMID:17623028

  5. Acetyl xylan esterase of Aspergillus ficcum catalyzed the synthesis of peracetic acid from ethyl acetate and hydrogen peroxide.

    PubMed

    Park, Seung-Moon

    2011-11-01

    Recombinant acetyl xylan esterase (rAXE) of Aspergillus ficcum catalyzed the synthesis of peracetic acid (PAA) from ethyl acetate and hydrogen peroxide. Ten micrograms of rAXE catalyzed the synthesis of 1.34 mM of PAA, which can be used for the pretreatment of cellulosic biomass in situ. PMID:21824816

  6. Molecular characterization of a new acetyl xylan esterase (AXEII) from edible straw mushroom Volvariella volvacea with both de-O-acetylation and de-N-acetylation activity.

    PubMed

    Liu, Xiufeng; Ding, Shaojun

    2009-06-01

    A new Volvariella volvacea gene encoding a carbohydrate esterase (CE) family 4 acetyl xylan esterase (AXE) (designated as VvaxeII) was cloned and characterized. The coded polypeptide had 253 amino acid residues, with the first 19 serving as a secretion signal peptide. The VvaxeII transcript levels were high when the fungus was grown on oat spelt xylan, cellobiose, microcrystalline cellulose, carboxymethyl-cellulose, lactose, galactose, and chitin from crab as carbon sources. The recombinant VvAXEII produced by expression of VvaxeII in Pichia pastoris exhibited activity toward acetylated oat spelt xylan and various chitinous substrates, but was totally inactive against artificial aromatic acetates such as beta-nitrophenyl, 4-methylumbelliferyl, and alpha-naphthyl acetates. Enzyme-catalyzed hydrolysis was maximal at pH 7.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 1.42 mg mL(-1) and a V(max) value of 833 IU micromol(-1) protein using glycol chitin as a substrate. The recombinant VvAXEII requires activation by bivalent cations such as Co2+ and Mg2+. Interestingly, the recombinant VvAXEII showed no deacetylation activity to fully acetylated monosaccharides such as xylose tetraacetate. PMID:19473250

  7. Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module.

    PubMed

    Mai-Gisondi, Galina; Turunen, Ossi; Pastinen, Ossi; Pahimanolis, Nikolaos; Master, Emma R

    2015-11-01

    The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE-CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE-CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates. PMID:26320711

  8. Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85▿ †

    PubMed Central

    Yoshida, Shosuke; Mackie, Roderick I.; Cann, Isaac K. O.

    2010-01-01

    Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser44, His273, Glu194, and Asp270, with both Glu194 and Asp270 functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis. PMID:19897648

  9. A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase.

    PubMed

    Zheng, Fei; Huang, Jingxuan; Yin, Yuhao; Ding, Shaojun

    2013-10-01

    A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0-10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K(m) value towards beechwood xylan was 0.548 mg ml⁻¹, and the k(cat)/K(m) ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg⁻¹ s⁻¹ at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries. PMID:23903903

  10. A combined approach for improving alkaline acetyl xylan esterase production in Pichia pastoris, and effects of glycosylation on enzyme secretion, activity and stability.

    PubMed

    Tian, Bin; Chen, Yan; Ding, Shaojun

    2012-09-01

    High level expression of axe1, a gene previously cloned from Volvariella volvacea that encodes an acetyl xylan esterase with two potential N-linked glycosylation sites, has been achieved in Pichia pastoris using a codon-optimized axe1 synthesized by the primer extension PCR procedure. The GC content of the codon-optimized axe1 was 48.62% compared with 55.49% in the native gene. Using the codon-optimized construct, AXE1 expression in P. pastoris was increased from an undetectable level to 136.45 U/ml six days after induction of yeast cultures grown in BMMY medium. A further increase (to 463 U/ml) was achieved when conditions for yeast culture were optimized as follows: 2.8% methanol, 0.63% casamino acids, and pH 8.0. This latter value represented a 3.4-fold and 246-fold increase in the enzyme levels recorded in non-optimized P. pastoris cultures and in rice straw-grown cultures of V. volvacea, respectively. N-linked glycosylation played an essential role in AXE1 secretion but had only a slight effect on the catalytic activity and stability of the recombinant enzyme. PMID:22750674

  11. Alterations of the degree of xylan acetylation in Arabidopsis xylan mutants

    PubMed Central

    Lee, Chanhui; Teng, Quincy; Zhong, Ruiqin; Ye, Zheng-Hua

    2014-01-01

    Xylan is the second most abundant polysaccharide in secondary walls of dicot plants and one of its structural features is the high degree of acetylation of xylosyl residues. In Arabidopsis, about 60% of xylosyl residues in xylan are acetylated and the biochemical mechanisms controlling xylan acetylation are largely unknown. A recent report by Yuan et al. (2013) revealed the essential role of a DUF231 domain-containing protein, ESKIMO1 (ESK1), in xylan acetylation in Arabidopsis as the esk1 mutation caused specific reductions in the degree of xylan 2-O or 3-O-monoacetylation and in the activity of xylan acetyltransferase. Interestingly, the esk1 mutation also resulted in an elevation of glucuronic acid (GlcA) substitutions in xylan. Since GlcA substitutions in xylan occur at the O-2 position of xylosyl residues, it is plausible that the increase in GlcA substitutions in the esk1 mutant is attributed to the reduction in acetylation at O-2 of xylosyl residues, which renders more O-2 positions available for GlcA substitutions. Here, we investigated the effect of removal of GlcA substitutions on the degree of xylan acetylation. We found that a complete loss of GlcA substitutions in the xylan of the gux1/2/3 triple mutant led to a significant increase in the degree of xylan acetylation, indicating that xylan acetyltransferases and glucuronyltransferases compete with each other for xylosyl residues for their acetylation or GlcA substitutions in planta. In addition, detailed structure analysis of xylan from the rwa1/2/3/4 quadruple mutant revealed that it had a uniform reduction of acetyl substitutions at different positions of the xylosyl residues, which is consistent with the proposed role of RWAs as acetyl coenzyme A transporters. The significance of these findings is discussed. PMID:24518588

  12. Two Arabidopsis Proteins Synthesize Acetylated Xylan in Vitro

    PubMed Central

    Urbanowicz, Breeanna R.; Peña, Maria J.; Moniz, Heather A.; Moremen, Kelley W.; York, William S.

    2014-01-01

    SUMMARY Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far-reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally due to collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis. However, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10-L (IRX10-L) and ESKIMO1/ TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase, we have resolved two long-standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways utilized by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties. PMID:25141999

  13. Acetylation of bleached Kraft pulp: effect of xylan content on properties of acetylated compounds.

    PubMed

    Peredo, Karol; Reyes, Herna; Escobar, Danilo; Vega-Lara, Johana; Berg, Alex; Pereira, Miguel

    2015-03-01

    Bleached Kraft pulp (BKP) from Eucalyptus globulus and cotton xylan blends (CXB) was acetylated. The effects of xylan content on cellulose acetylation and the properties of the acetylated material were studied. An increase in xylan content caused a slight decrease in the degree of substitution (2.98 to 2.68 for CXB; 2.93 to 2.84 for BKP). Thermal analysis showed that the melting temperature also decreases from 268.0 to 188.8 °C for CXB and from 221.4 to 212.8 °C for BKP. Moreover, the solubility decreased due to the partial dissolution of acetylated xylans. The presence of xylans during Kraft pulp acetylation does not have a significant negative effect on the physical properties of the acetylated material, but the decrease in melting temperature was beneficial for the application of acetylated polymer as a natural internal plasticizer. This is considered to be an important argument for BKP utilization in the cellulose acetate manufacturing process. PMID:25498729

  14. Roles of Arabidopsis TBL34 and TBL35 in xylan acetylation and plant growth.

    PubMed

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Ye, Zheng-Hua

    2016-02-01

    Xylan is one of the major polymers in lignocellulosic biomass and about 60% of its xylosyl residues are acetylated at O-2 and/or O-3. Because acetylation of cell wall polymers contributes to biomass recalcitrance for biofuel production, it is important to investigate the biochemical mechanism underlying xylan acetylation, the knowledge of which could be applied to custom-design biomass composition tailored for biofuel production. In this report, we investigated the functions of Arabidopsis TRICHOME BIREFRINGENCE-LIKE 34 (TBL34) and TBL35, two DUF231-containing proteins, in xylan acetylation. The TBL34 gene was found to be specifically expressed in xylem cells in stems and root-hypocotyls, and both TBL34 and TBL35 were shown to be localized in the Golgi, where xylan biosynthesis occurs. Chemical analysis revealed that simultaneous mutations of TBL34 and TBL35 caused a mild decrease in xylan acetyl content and a specific reduction in xylan 3-O-monoacetylation and 2,3-di-O-acetylation. Furthermore, simultaneous mutations of TBL34, TBL35 and ESKIMO1 (ESK1) resulted in severely collapsed xylem vessels with altered secondary wall structure, and an extremely retarded plant growth. These findings indicate that TBL34 and TBL35 are putative acetyltransferases required for xylan 3-O-monoacetylation and 2,3-di-O-acetylation and that xylan acetylation is essential for normal secondary wall deposition and plant growth. PMID:26795157

  15. Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene, ae1, encoding the acetyl esterase (Ae1) of Hypocrea jecorina was identified by amino terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino ...

  16. Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition

    PubMed Central

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng-Hua

    2016-01-01

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls. PMID:26745802

  17. Mutations of Arabidopsis TBL32 and TBL33 affect xylan acetylation and secondary wall deposition

    DOE PAGESBeta

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng -Hua; Zhang, Jin -Song

    2016-01-08

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be monoand di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reductionmore » in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-Omonoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. Furthermore, these results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.« less

  18. Polypeptide having acetyl xylan esterase activity and uses thereof

    SciTech Connect

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2015-10-20

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  19. The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana

    PubMed Central

    Busse-Wicher, Marta; Gomes, Thiago C F; Tryfona, Theodora; Nikolovski, Nino; Stott, Katherine; Grantham, Nicholas J; Bolam, David N; Skaf, Munir S; Dupree, Paul

    2014-01-01

    The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy. PMID:24889696

  20. Impact of Cell Wall Acetylation on Corn Stover Hydrolysis by Cellulolytic and Xylanolytic Enzymes

    SciTech Connect

    Selig, M. J.; Adney, W. S.; Himmel, M. E.; Decker, S. R.

    2009-01-01

    Analysis of variously pretreated corn stover samples showed neutral to mildly acidic pretreatments were more effective at removing xylan from corn stover and more likely to maintain the acetyl to xylopyranosyl ratios present in untreated material than were alkaline treatments. Retention of acetyl groups in the residual solids resulted in greater resistance to hydrolysis by endoxylanase alone, although the synergistic combination of endoxylanase and acetyl xylan esterase enzymes permitted higher xylan conversions to be observed. Acetyl xylan esterase alone did little to improve hydrolysis by cellulolytic enzymes, although a direct relationship was observed between the enzymatic removal of acetyl groups and improvements in the enzymatic conversion of xylan present in substrates. In all cases, effective xylan conversions were found to significantly improve glucan conversions achievable by cellulolytic enzymes. Additionally, acetyl and xylan removal not only enhanced the respective initial rates of xylan and glucan conversion, but also the overall extents of conversion. This work emphasizes the necessity for xylanolytic enzymes during saccharification processes and specifically for the optimization of acetyl esterase and xylanase synergies when biomass processes include milder pretreatments, such as hot water or sulfite steam explosion.

  1. Crystal structure of an acetyl esterase complexed with acetate ion provides insights into the catalytic mechanism.

    PubMed

    Uechi, Keiko; Kamachi, Saori; Akita, Hironaga; Mine, Shouhei; Watanabe, Masahiro

    2016-08-26

    We previously reported the crystal structure of an acetyl esterase (TcAE206) belonging to carbohydrate esterase family 3 from Talaromyces cellulolyticus. In this study, we solved the crystal structure of an S10A mutant of TcAE206 complexed with an acetate ion. The acetate ion was stabilized by three hydrogen bonds in the oxyanion hole instead of a water molecule as in the structure of wild-type TcAE206. Furthermore, the catalytic triad residue His182 moved 0.8 Å toward the acetate ion upon substrate entering the active site, suggesting that this movement is necessary for completion of the catalytic reaction. PMID:27329813

  2. Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937.

    PubMed

    Shevchik, V E; Hugouvieux-Cotte-Pattat, N

    1997-06-01

    Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of pae

  3. Crystal structure of Thermotoga maritima acetyl esterase complex with a substrate analog: Insights into the distinctive substrate specificity in the CE7 carbohydrate esterase family.

    PubMed

    Singh, Mrityunjay K; Manoj, Narayanan

    2016-07-22

    The carbohydrate esterase family 7 (CE7) members are acetyl esterases that possess unusual substrate specificity for cephalosporin C and 7-amino-cephalosporanic acid. This family containing the α/β hydrolase fold has a distinctive substrate profile that allows it to carry out hydrolysis of esters containing diverse alcohol moieties while maintaining narrow specificity for an acetate ester. Here we investigate the structural basis of this preference for small acyl groups using the crystal structure of the thermostable Thermotoga maritima CE7 acetyl esterase (TmAcE) complexed with a non-cognate substrate analog. The structure determined at 1.86 Å resolution provides direct evidence for the location of the largely hydrophobic and rigid substrate binding pocket in this family. Furthermore, a three-helix insertion domain near the catalytic machinery shapes the substrate binding site. The structure reveals two residues (Pro228 and Ile276) which constitute a hydrophobic rigid binding surface for the acyl group of the ester and thus restricts the size of the acyl group that be accommodated. In combination with previous literature on kinetic properties of the enzyme, our studies suggest that these residues determine the unique specificity of the TmAcE for short straight chain esters. The structure provides a template for focused attempts to engineer the CE7 enzymes for enhanced stability, selectivity or activity for biocatalytic applications. PMID:27181355

  4. The Four Arabidopsis Reduced Wall Acetylation Genes are Expressed in Secondary Wall-Containing Cells and Required for the Acetylation of Xylan

    EPA Science Inventory

    Xylan is one of the major polysaccharides in cellulosic biomass, and understanding the mechanisms underlying xylan biosynthesis will potentially help us design strategies to produce cellulosic biomass better suited for biofuel production. Although a number of genes have been show...

  5. MODE OF ACTION OF ACETYLXYLAN ESTERASE FROM STREPTOMYCES LIVIDANS: A STUDY WITH DEOXY AND DEOXY-FLUORO ANALOGUES OF ACETYLATED METHYL BETA-D-XYLOPYRANOSIDE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The action of Streptomyces lividans acetylxylan esterase on methyl 2,4-di-O-acetyl- and 3,4-di-O-acetyl beta-D-xylopyranoside was compared with its action on the 2- and 3-deoxy and 2- and 3-deoxy-fluoro-analogues of the two diacetates in order to elucidate the role of the free hydroxyl group in the ...

  6. A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.

    PubMed

    Alalouf, Onit; Balazs, Yael; Volkinshtein, Margarita; Grimpel, Yael; Shoham, Gil; Shoham, Yuval

    2011-12-01

    Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for k(cat) and k(cat)/K(m) suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-β-d-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family. PMID:21994937

  7. Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms: Implications for Xylan Interaction with Cellulose.

    PubMed

    Busse-Wicher, Marta; Li, An; Silveira, Rodrigo L; Pereira, Caroline S; Tryfona, Theodora; Gomes, Thiago C F; Skaf, Munir S; Dupree, Paul

    2016-08-01

    The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls. PMID:27325663

  8. Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms: Implications for Xylan Interaction with Cellulose1[OPEN

    PubMed Central

    Li, An; Gomes, Thiago C.F.

    2016-01-01

    The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls. PMID:27325663

  9. Molecular Docking and Pharmacological Investigations of Rivastigmine-Fluoxetine and Coumarin–Tacrine hybrids against Acetyl Choline Esterase

    PubMed Central

    Babitha, Pallikkara Pulikkal; Sahila, Mohammed Marunnan; Bandaru, Srinivas; Nayarisseri, Anuraj; Sureshkumar, Sivanpillai

    2015-01-01

    The present AChE inhibitors have been successful in the treatment of Alzheimer׳s Diseases however suffers serious side effects. Therefore in this view, the present study was sought to identify compounds with appreciable pharmacological profile targeting AChE. Analogue of Rivastigmine and Fluoxetine hybrid synthesized by Toda et al, 2003 (dataset1), and Coumarin−Tacrine hybrids synthesized by Qi Sun et al (dataset2) formed the test compounds for the present pharmacological evaluation. p-cholorophenyl substituted Rivastigmine and Fluoxetine hybrid compound (26d) from dataset 1 and −OCH3 substitute Coumarin−Tacrine hybrids (1h) from dataset 2 demonstrated superior pharmacological profile. 26 d showed superior pharmacological profile comparison to the entire compounds in either dataset owing to its better electrostatic interactions and hydrogen bonding patterns. In order to identify still better compound with pharmacological profile than 26 d and 1h, virtual screening was performed. The best docked compound (PubCId: PubCid: 68874404) showed better affinity than its parent 26 d, however showed poor ADME profile and AMES toxicity. CHEMBL2391475 (PubCid: 71699632) similar to 1h had reduced affinity in comparison to its parent compound 1h. From, our extensive analysis involving binding affinity analysis, ADMET properties predictions and pharmacophoric mappings, we report p-cholorophenyl substituted rivastigmine and fluoxetine hybrid (26d) to be a potential candidate for AcHE inhibition which in addition can overcome narrow therapeutic window of present AChE inhibitors in clinical treatment of Alzheimer׳s disease. Abbreviations AD - Alzheimer׳s Disease, AChE - Acetyl Choline Estarase, OPLS - Optimized Potentials for Liquid Simulations, PDB - Protein Data Bank. PMID:26420918

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

    SciTech Connect

    E Rangarajan; K Ruane; A Proteau; J Schrag; R Valladares; C Gonzalez; M Gilbert; A Yakunin; M Cygler

    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 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.

  11. Evolutionary Conservation of Xylan Biosynthetic Genes in Selaginella moellendorffii and Physcomitrella patens.

    PubMed

    Haghighat, Marziyeh; Teng, Quincy; Zhong, Ruiqin; Ye, Zheng-Hua

    2016-08-01

    Xylan is a major cross-linking hemicellulose in secondary walls of vascular tissues, and the recruitment of xylan as a secondary wall component was suggested to be a pivotal event for the evolution of vascular tissues. To decipher the evolution of xylan structure and xylan biosynthetic genes, we analyzed xylan substitution patterns and characterized genes mediating methylation of glucuronic acid (GlcA) side chains in xylan of the model seedless vascular plant, Selaginella moellendorffii, and investigated GT43 genes from S. moellendorffii and the model non-vascular plant, Physcomitrella patens, for their roles in xylan biosynthesis. Using nuclear magentic resonance spectroscopy, we have demonstrated that S. moellendorffii xylan consists of β-1,4-linked xylosyl residues subsituted solely with methylated GlcA residues and that xylans from both S. moellendorffii and P. patens are acetylated at O-2 and O-3. To investigate genes responsible for GlcA methylation of xylan, we identified two DUF579 genes in the S. moellendorffii genome and showed that one of them, SmGXM, encodes a glucuronoxylan methyltransferase capable of adding the methyl group onto the GlcA side chain of xylooligomers. Furthermore, we revealed that the two GT43 genes in S. moellendorffii, SmGT43A and SmGT43B, are functional orthologs of the Arabidopsis xylan backbone biosynthetic genes IRX9 and IRX14, respectively, indicating the evolutionary conservation of the involvement of two functionally non-redundant groups of GT43 genes in xylan backbone biosynthesis between seedless and seed vascular plants. Among the five GT43 genes in P. patens, PpGT43A was found to be a functional ortholog of Arabidopsis IRX9, suggesting that the recruitment of GT43 genes in xylan backbone biosynthesis occurred when non-vascular plants appeared on land. PMID:27345025

  12. Investigation of the nature of semisynthetic esterases. Annual progress report, September 15, 1982-September 14, 1983

    SciTech Connect

    Keyes, M.H.

    1983-12-01

    Two semisynthetic esterases, an acid-esterase with a pH optimum of 6.0 and a neutral-esterase with a pH optimum of 7.5, were generated from bovine pancreatic ribonuclease. The method involved perturbation of ribonuclease at pH 3.0, subsequent conformational modification with indole propionic acid, and crosslinking the modified protein with glutaraldehyde. The two esterases generated by this procedure were separated and partially purified by ammonium sulfate fractionation. The neutral-esterase activity was predominantly precipitated at 40% ammonium sulfate saturation, and the acid-esterase at 70 to 90% ammonium sulfate saturation. Nearly 4 fold purification of the esterases was achieved by this step. The two esterases were further purified by gel filtration of the above ammonium sulfate fractions on Biogel P0-30. Nearly 100 fold purification of the esterases over the starting modified RNase has been achieved by the above two purification steps. Kinetic studies with the purified acid-esterase indicated that this semisynthetic esterase hydrolyzed several aminoacid ethyl esters, but preferred ester containing an aromatic residue. The acid-esterase was competitively inhibited by L-tryptophan and also had low amidase activity towards benzoylarginine p-nitroanilide. Neutral-esterase showed a high degree of specificity toward L-TrEE and acetyl tryptophan ethyl ester. Moreover, this esterase had significant amidase activity toward N-acetyltryptophanamide (NATA). Neutral esterase was not inhibited by tryptophan.

  13. Cloning, Overexpression in Escherichia coli, and Characterization of a Thermostable Fungal Acetylxylan Esterase from Talaromyces emersonii

    PubMed Central

    Murray, Patrick G.; Miki, Yuta; Martínez, Angel T.; Tuohy, Maria G.; Faulds, Craig B.

    2012-01-01

    The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels. PMID:22407679

  14. Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii.

    PubMed

    Waters, Deborah M; Murray, Patrick G; Miki, Yuta; Martínez, Angel T; Tuohy, Maria G; Faulds, Craig B

    2012-05-01

    The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels. PMID:22407679

  15. Delignification outperforms alkaline extraction for xylan fingerprinting of oil palm empty fruit bunch.

    PubMed

    Murciano Martínez, Patricia; Kabel, Mirjam A; Gruppen, Harry

    2016-11-20

    Enzyme hydrolysed (hemi-)celluloses from oil palm empty fruit bunches (EFBs) are a source for production of bio-fuels or chemicals. In this study, after either peracetic acid delignification or alkaline extraction, EFB hemicellulose structures were described, aided by xylanase hydrolysis. Delignification of EFB facilitated the hydrolysis of EFB-xylan by a pure endo-β-1,4-xylanase. Up to 91% (w/w) of the non-extracted xylan in the delignified EFB was hydrolysed compared to less than 4% (w/w) of that in untreated EFB. Alkaline extraction of EFB, without prior delignification, yielded only 50% of the xylan. The xylan obtained was hydrolysed only for 40% by the endo-xylanase used. Hence, delignification alone outperformed alkaline extraction as pretreatment for enzymatic fingerprinting of EFB xylans. From the analysis of the oligosaccharide-fingerprint of the delignified endo-xylanase hydrolysed EFB xylan, the structure was proposed as acetylated 4-O-methylglucuronoarabinoxylan. PMID:27561506

  16. Reducing the heterogeneity of xylan through processing.

    PubMed

    Zhang, Wei; Johnson, Amanda M; Barone, Justin R; Renneckar, Scott

    2016-10-01

    Glycerol thermal processing (GTP) of hardwood biomass at temperatures between 200 and 240°C facilitated stepwise biopolymer fractionation, while limiting significant degradation of the major hemicellulose, glucuronoxylan, into water-extractable oligosaccharides. After GTP pretreatment and sequential water and organic solvent extraction, up to 80% of the initial xylan remained in the pretreated biomass. The majority of the xylan from GTP pretreated and water/solvent extracted biomass was removed using a mild alkali extraction and the composition was compared to xylan directly isolated from untreated hardwood. The precipitated xylan from the neutralized alkaline filtrate was isolated as a water insoluble xylan portion (WIX). The residual xylan dissolved in the neutralized filtrate was precipitated in cold methanol and recovered as the water soluble xylan portion (WSX). Results showed that xylan in WIX was in a polymeric form with a number average degree of polymerization (DP) over 100, whereas the WSX had a much lower average DP of 27 (ca) and contained more substitution. As the processing severity increased during GTP pretreatment, the proportion of WIX increased and the purity of the xylan within the WIX sample reached 84% based on compositional analysis. FT-IR analysis of WIX revealed that xylan isolated after GTP contained peaks related to a reduced carbonyl signal compared to the control. Furthermore, crude WSX contained less xylan with more lignin contamination at severe GTP conditions. The recovery of the xylan in two portions facilitated a preferential purification strategy resulting in WIX with an extremely narrow polydispersity index between 1.1 and 1.25, dependent upon the GTP severity. This study provided insight into fractionating higher molecular weight xylan that may serve value-added applications such as healthcare materials and advanced packaging. PMID:27312636

  17. Leukocyte esterase urine test

    MedlinePlus

    ... the urine. This may mean you have a urinary tract infection . If this test is positive, the urine should ... Results Mean An abnormal result indicates a possible urinary tract infection. Alternative Names WBC esterase Images Male urinary system ...

  18. Mechanism of action of Neisseria gonorrhoeae O-acetylpeptidoglycan esterase, an SGNH serine esterase.

    PubMed

    Pfeffer, John M; Weadge, Joel T; Clarke, Anthony J

    2013-01-25

    O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted α/β-hydrolase fold containing a central twisted four-stranded parallel β-sheet flanked by six α-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding. PMID:23209280

  19. Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase*

    PubMed Central

    Pfeffer, John M.; Weadge, Joel T.; Clarke, Anthony J.

    2013-01-01

    O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted α/β-hydrolase fold containing a central twisted four-stranded parallel β-sheet flanked by six α-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding. PMID:23209280

  20. Probing Xylan-Specific Raman Bands for Label-Free Imaging Xylan in Plant Cell Wall

    SciTech Connect

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; Tucker, Melvin P.; Vinzant, Todd; Himmel, Michael E.

    2015-06-15

    Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike lignin and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.

  1. Hydrolyzability of xylan after adsorption on cellulose: Exploration of xylan limitation on enzymatic hydrolysis of cellulose.

    PubMed

    Wang, Xiao; Li, Kena; Yang, Ming; Zhang, Junhua

    2016-09-01

    During pretreatment of lignocellulosic materials, the dissolved xylan would re-adsorb on cellulose, and then inhibits the cellulose hydrolysis by cellulases. However, the hydrolyzability of xylan adsorbed on cellulose is not clear. In this work, the adsorption behavior of xylans on celluloses and the hydrolysis of adsorbed xylan by xylanase (XYL) were investigated. The results indicated that the adsorption of beechwood xylan (BWX) and oat spelt xylan (OSX) on Avicel was conformed to Langmuir-type adsorption isotherm. Higher ion strength increased the adsorption of BWX on Avicel, but not that of OSX. Both BWX and OSX adsorbed on Avicel and corn stover after dilute acid pretreatment (CS-DA) could be hydrolyzed by XYL. Compared to OSX, BWX adsorbed on cellulosic materials could be more easily hydrolyzed by XYL. Thus, supplementation of XYL could hydrolyze the xylan adsorbed on cellulose and potentially improved hydrolysis efficiency of lignocelluloses. PMID:27185150

  2. Comparison of fungal carbohydrate esterases of family CE16 on artificial and natural substrates.

    PubMed

    Puchart, Vladimír; Agger, Jane W; Berrin, Jean-Guy; Várnai, Anikó; Westereng, Bjørge; Biely, Peter

    2016-09-10

    The enzymatic conversion of acetylated hardwood glucuronoxylan to functional food oligomers, biochemicals or fermentable monomers requires besides glycoside hydrolases enzymes liberating acetic acid esterifying position 2 and/or 3 in xylopyranosyl (Xylp) residues. The 3-O-acetyl group at internal Xylp residues substituted by MeGlcA is the only acetyl group of hardwood acetylglucuronoxylan and its fragments not attacked by acetylxylan esterases of carbohydrate esterase (CE) families 1, 4, 5 and 6 and by hemicellulolytic acetyl esterases classified in CE family 16. Monoacetylated aldotetraouronic acid 3″-Ac(3)MeGlcA(3)Xyl3, generated from the polysaccharide by GH10 endoxylanases, appears to be one of the most resistant fragments. The presence of the two substituents on the non-reducing-end Xylp residue prevents liberation of MeGlcA by α-glucuronidase of family GH67 and blocks the action of acetylxylan esterases. The Ac(3)MeGlcA(3)Xyl3 was isolated from an enzymatic hydrolysate of birchwood acetylglucuronoxylan and characterized by (1)H NMR spectroscopy as a mixture of two positional isomers, 3″-Ac(3)MeGlcA(3)Xyl3 and 4″-Ac(3)MeGlcA(3)Xyl3, the latter being the result of acetyl group migration. The mixture was used as a substrate for three members of CE16 family of fungal origin. Trichoderma reesei CE16 esterase, inactive on polymeric substrate, deacetylated both isomers. Podospora anserina and Aspergillus niger esterases, active on acetylglucuronoxylan, deesterified effectively only the 4″-isomer. The results indicate catalytic diversity among CE16 enzymes, but also their common and unifying catalytic ability to exo-deacetylate positions 3 and 4 on non-reducing-end Xylp residues, which is an important step in plant hemicellulose saccharification. PMID:27439201

  3. 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

  4. 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. PMID:27452816

  5. Enzymatic deconstruction of xylan for biofuel production

    PubMed Central

    DODD, DYLAN; CANN, ISAAC K. O.

    2010-01-01

    The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction. PMID:20431716

  6. Measurement of xylanase activity with insoluble xylan substrate.

    PubMed Central

    Nummi, M; Perrin, J M; Niku-Paavola, M L; Enari, T M

    1985-01-01

    Insoluble xylan was prepared from ground birch (Betula pubescens) pulp by alkali extraction and precipitation with ethanol. The only sugar detected after acid hydrolysis of the preparation was xylose. The insoluble xylan was used as substrate in a nephelometric assay to determine the xylanase (EC 3.2.1.8, 1,4-beta-D-xylan xylanohydrolase and EC 3.2.1.37, 1,4-beta-D-xylan xylohydrolase) activities of Aspergillus and Trichoderma enzymes. The nephelometric method is reliable in evaluating xylanase hydrolysis of insoluble xylan. PMID:3922355

  7. Maillard reaction products from chitosan-xylan ionic liquid solution.

    PubMed

    Luo, Yuqiong; Ling, Yunzhi; Wang, Xiaoying; Han, Yang; Zeng, Xianjie; Sun, Runcang

    2013-10-15

    A facile method is reported to prepare Maillard reaction products (MRPs) from chitosan and xylan in co-solvent ionic liquid. UV absorbance and fluorescence changes were regarded as indicators of the occurrence of Maillard reaction. FT-IR, NMR, XRD and TG were used to investigate the structure of chitosan-xylan conjugate. The results revealed that when chitosan reacted with xylan in ionic liquid, the hydrogen bonds in chitosan were destroyed, the facts resulted in the formation of chitosan-xylan MRPs. Moreover, when the mass ratio of chitosan to xylan was 1:1, the Maillard reaction proceeded easily. In addition, relatively high antioxidant property was also noted for the chitosan-xylan conjugate with mass ratio 1:1. So the obtained chitosan-xylan MRP is a promising antioxidant agent for food industry. PMID:23987419

  8. A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds.

    PubMed

    Arnling Bååth, Jenny; Giummarella, Nicola; Klaubauf, Sylvia; Lawoko, Martin; Olsson, Lisbeth

    2016-08-01

    The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and (31) P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood. PMID:27397104

  9. 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. PMID:26712478

  10. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  11. Degradation mechanism of monosaccharides and xylan under pyrolytic conditions with theoretic modeling on the energy profiles.

    PubMed

    Wang, Shurong; Ru, Bin; Lin, Haizhou; Luo, Zhongyang

    2013-09-01

    Xylan and three monosaccharides (mannose, galactose, and arabinose) were selected as model compounds to investigate the mechanism of hemicellulose pyrolysis. The evolution of several typical pyrolysis products were observed by thermogravimetric analysis coupled to Fourier transform infrared spectroscopy. Monosaccharides underwent similar pyrolysis routes involving ring opening and secondary decomposition. Breakage of the O-acetyl groups and 4-O-methylglucuronic acid units in xylan branches resulted in its different pyrolysis behavior for the formation of acetic acid, CO2, and CO. The detailed reaction pathways of the monosaccharides were studied using density functional theory calculations. Furfural formation was more favorable than the formation of 1-hydroxy-2-propanone and 4-hydroxydihydrofuran-2(3H)-one during xylose degradation. However, in the pyrolysis of mannose and galactose, formation of 5-hydroxymethyl-2-furaldehyde was preferred because of the high energy barrier of the dissociation of the hydroxymethyl group. Meanwhile, the breakage of O-acetyl groups leading to acetic acid formation easily occurred because of its lower energy barrier. PMID:23819973

  12. Esterase zymograms of Proteus and Providencia.

    PubMed

    Goullet, P

    1975-03-01

    The intracellular esterases of 80 strains of Proteus and Providencia were analysed by the acrylamide-agarose zymogram technique using several synthetic substrates. The esterase bands were classified in five main groups. The alphaA-esterase bands hydrolysed alpha-naphthyl acetate and were resistant or relatively insensitive to di-isofluoropropyl phosphate (DFP). The alphaB-esterase band hydrolysed both alpha-naphthyl acetate and alpha-naphthyl butyrate and were very sensitive to DFP. Both groups of esterase bands were inactivated by heat. The betaA- and betaB-esterase bands hydrolysed beta-naphthyl acetate and were sensitive to DFP; these were distinguishable by the difference in their relative activity towards beta-naphthyl butyrate and in their relative stability to heat. The alpha-beta-esterase bands hydrolysed alpha- and beta-naphthyl acetates and alpha- and beta-naphthyl butyrates; they were inactivated by heat and were sensitive to DFP. The distribution of these esterase bands among the strains of Proteus and Providencia and their electrophoretic patterns established esterase profile types which correlate with the classification based on traditional bacteriological tests. The degree of inter-strain similarity in esterase pattern varied highly among species. The homogeneity of Proteus mirabilis and especially of Providencia stuartii contrasted with the heterogeneity of other species. This disparity suggests that the bacteria of the tribe Proteae have not the same degree of intra-specific differentiation in physico-chemical properties of esterases. PMID:48538

  13. 2-Hydroxypropyltrimethylammonium xylan adsorption onto rod-like cellulose nanocrystal.

    PubMed

    Sim, Jae Hyun; Dong, Shuping; Röemhild, Katrin; Kaya, Abdulaziz; Sohn, Daewon; Tanaka, Keiji; Roman, Maren; Heinze, Thomas; Esker, Alan R

    2015-02-15

    Chemical incompatibility and relatively weak interaction between lignocellulosic fibers and synthetic polymers have made studies of wood fiber-thermoplastic composite more challenging. In this study, adsorption of 2-hydroxypropyltrimethylammonium xylans onto rod-like cellulose nanocrystals are investigated by zeta-potential measurements, and polarized and depolarized dynamic light scattering as a factor for better understanding of lignocellulosic fibers and cellulose nanocrystals. Zeta-potential measurements show xylan derivative adsorption onto cellulose nanocrystals. Decay time distributions of the ternary system and binary system from dynamic light scattering show that aggregates exist in the binary system and they disappear in the ternary system. At low 2-hydroxypropyltrimethylammonium xylan concentrations relative to that of cellulose nanocrystal, xylan derivatives adsorbed onto some of the cellulose nanocrystal. Hence, more xylan derivatives adsorbed onto cellulose nanocrystal increased with increasing xylan derivative concentration. Also, the concentration dependence of the ratio of the rotational diffusion coefficient to the translational diffusion coefficient revealed a strong adsorptive interaction between xylan derivatives and the cellulose nanocrystals. PMID:25460697

  14. Xylan-Degrading Catalytic Flagellar Nanorods.

    PubMed

    Klein, Ágnes; Szabó, Veronika; Kovács, Mátyás; Patkó, Dániel; Tóth, Balázs; Vonderviszt, Ferenc

    2015-09-01

    Flagellin, the main component of flagellar filaments, is a protein possessing polymerization ability. In this work, a novel fusion construct of xylanase A from B. subtilis and Salmonella flagellin was created which is applicable to build xylan-degrading catalytic nanorods of high stability. The FliC-XynA chimera when overexpressed in a flagellin deficient Salmonella host strain was secreted into the culture medium by the flagellum-specific export machinery allowing easy purification. Filamentous assemblies displaying high surface density of catalytic sites were produced by ammonium sulfate-induced polymerization. FliC-XynA nanorods were resistant to proteolytic degradation and preserved their enzymatic activity for a long period of time. Furnishing enzymes with self-assembling ability to build catalytic nanorods offers a promising alternative approach to enzyme immobilization onto nanostructured synthetic scaffolds. PMID:25966869

  15. Chemical depolymerization of switchgrass xylan with oligosaccharide product analysis by HPAEC-PAD and mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose and consequently pretreatment conditions are in part optimized for maximal xylan depolymerization or displacement. Xylan consists of a lon...

  16. Hydrolysis of xylan by an immobilized xylanase from Aureobasidium pullulans

    SciTech Connect

    Allenza, P.; Scherl, D.S.; Detroy, R.W.; Leathers, T.D.; Scott, C.D. .

    1986-01-01

    The beta-(1,4)-linked xylose residues that comprise the backbone of the abundant plant polymer xylan can be released by enzymic hydrolysis. Xylanase, which is produced in exceptionally high levels by the color-variant strain Y-2311-1 of A. pullulans, was immobilized onto a macroporous ceramic carrier. Despite a low coupling efficiency, it was possible to run the reactor under a wide range of conditions with flow rates of 3-10 bed volumes/minute of 1% soluble xylan with no detectable leaching of enzyme. The size distribution of products and rate of xylan hydrolysis were very similar for the immobilized and soluble enzymes. (Refs. 13).

  17. Hydrolysis of xylan by an immobilized xylanase from Aureobasidium pullanans

    SciTech Connect

    Allenza, P.; Scherl, D.S.; Detroy, R.W.; Leathers, T.D.; Scott, C.D.

    1986-01-01

    The beta-(1,4)-linked xylose residues that comprise the backbone of the abundant plant polymer xylan can be released by enzymic hydrolysis. Xylanase, which is produced in exceptionally high levels by the color-variant strain of A. pullulans, was immobilized onto a macroporous ceramic carrier. Despite a low coupling efficiency, it was possible to run the reactor under a wide range of conditions with flow rates of 3-10 bed volumes/minute of 1% soluble xylan with no detectable leaching of enzyme. The size distribution of products and rate of xylan hydrolysis were very similar for the immobilized and soluble enzymes. (Refs. 13).

  18. Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorte...

  19. Lignin profiling in extracted xylans by size-exclusion chromatography.

    PubMed

    Hutterer, Christian; Schild, Gabriele; Kliba, Gerhard; Potthast, Antje

    2016-10-20

    Utilization of the polymeric parts of lignocellulose is expected to gain increasing importance in future biorefinery scenarios. In that respect, a particular focus is placed on hemicelluloses from different wood species gained from an industrially feasible upgrading step in the production of dissolving pulps from paper pulps. During alkaline post-extractions for hemicellulose removal, residual lignins are extracted as well. They are either covalently linked to the extracted hardwood xylans or simply co-dissolved in the alkaline lye. In order to better describe the lignin in xylan containing lyes, a method for lignin profiling was set up by hyphenating size-exclusion chromatography of xylans with UV detection which facilitates visualization of the residual lignin distribution. Simultaneous lignin quantification was achieved with lignin standards prepared from Kraft cooking liquors. The setup presented may serve as advanced characterization for novel xylan products. PMID:27474629

  20. Butyrivibrio spp. and other xylanolytic microorganisms from the rumen have cinnamoyl esterase activity.

    PubMed

    McSweeney, C S; Dulieu, A; Bunch, R

    1998-02-01

    High concentrations of hydroxycinnamic acids in the hemicellulosic fraction of dry season tropical grasses may influence the rate of microbial degradation of arabinoxylans by ruminant animals. The ability of 22 strains of Butyrivibrio fibrisolvens, other ruminal bacteria (Ruminococcus albus SY3, Ruminococcus flavefaciens RF1,Prevotella ruminicola AR20) and the ruminal phycomycete Neocallimastix patriciarum CX to digest the tropical grass Heteropogon contortus(spear grass) and hydrolyse esterified ferulic and p-coumaric acid was examined. Significant digestion (8-36%) of spear grass occurred with the B. fibrisolvens strains H17c, A38, LP92-1-1, 49,R. albus SY3 and N. patriciarum. Hydrolysis of ester-linked ferulic and p-coumaric acid occurred with all organisms except B. fibrisolvens strains GS113, OB156 and LP1028 and P. ruminicola AR20. The ratio of ferulic to p-coumaric acid hydrolysed by different strains of Butyrivibrio spp. varied markedly from 0.96 for AR 51 to 0.16 for A38. Butyrivibrios which were fibrolytic (H17c and A38) had higher extracellular cinnamoyl esterase activity than bacteria that did not digest spear grass fibre (LP 91-4-1 and AR 20) which had low activities or only produced cell associated enzyme. Cell associated and extracellular esterase activity were induced when Butyrivibrio spp. strains H17c, A38 and E14 and the Ruminococcus spp. were grown on birchwood xylan but induction did not occur to the same extent with N. patriciarum. This is the first reported observation of cinnamoyl esterase activity in the genus Ruminococcus. The fungus N. patriciarum had significantly higher digestibility of spear grass and solubilisation of phenolic acids than the bacteria. The study shows that high levels of extracellular cinnamoyl esterases are characteristic of a selection of fibre-degrading ruminal bacteria and fungi which probably indicates that these enzymes are common amongst xylanolytic ruminal microorganisms. PMID:16887624

  1. [Variability in esterases of Metarhizium anisopliae].

    PubMed

    Estrada-Martínez, M E; Piñón, D R; Capote, M C

    1997-03-01

    The variability in esterases of the entomogenous fungus Metarhizium anisopliae was determined electrophoretically on 8.5% polyacrylamide gel. Ten isolates from diverse taxonomic groups of insects were analyzed. The electrophoretic analysis showed differences and similarities between these isolates and it was possible to distinguish six different patterns. The results obtained show a great polymorphism for the esterase system of M. anisopliae. PMID:15482022

  2. Laccase catalysed grafting of phenolic onto xylan to improve its applicability in films

    NASA Astrophysics Data System (ADS)

    Pei, Jicheng; Wang, Bing; Zhang, Fangdong; Li, Zhongyang; Yin, Yunbei; Zhang, Dongxu

    2015-07-01

    Xylan can be tailored for various value-added applications. However, its use in aqueous systems is hampered by its complex structure, and small molecular weight. This research aimed at improving the xylan molecular weight and changing its structure. Laccase-catalysed oxidation of 4-coumaric acid (PCA), ferulic acid (FA), syringaldehyde (SD), and vanillin (VA) onto xylan was grafted to study the changes in its structure, tensile properties, and antibacterial activities. A Fourier transform infrared (FTIR) spectrum analyser was used to observe the changes in functional groups of xylan. The results showed a band at 1635 cm-1 corresponding to the stretching vibration of conjugated carbonyl carboxy hemoglobin and a benzene ring structure were strengthened; the appearance of a new band between 1200 cm-1 and 1270 cm-1 corresponding to alkyl ethers on the aryl C-O stretching vibration was due to the fact that during the grafting process, the number of benzene ring structures increased and covalent connections occurred between phenols and xylan. The reaction mechanism for the laccase-catalysed oxidation of phenol compounds onto xylan was preliminary explored by 13C-NMR. The results showed that PCA-xylan, FA-xylan graft poly onto xylan by Cγ ester bond, SD-xylan graft poly onto xylan by ether bond and an ester bond, and VD-xylan graft poly onto xylan by ether bond. The film strength of xylan derivatives has been significantly increased, especially for the PCA-xylan derivative. The increases in tensile stress at break, tensile strength, tensile yield stress, and Young's modulus were: 24.04%, 31.30%, 55.56%, and 28.21%, respectively. After laccase/phenolics were modified, xylan had a good antibacterial effect to E. coli, Corynebacterium glutamicum, and Bacillus subtilis. The SD-xylan, FA-xylan, and PCA-xylan showed a greater efficacy against E. coli, Corynebacterium glutamicum, and Bacillus subtilis, respectively.

  3. Structural basis for ligand and substrate recognition by torovirus hemagglutinin esterases

    PubMed Central

    Langereis, Martijn A.; Zeng, Qinghong; Gerwig, Gerrit J.; Frey, Barbara; von Itzstein, Mark; Kamerling, Johannis P.; de Groot, Raoul J.; Huizinga, Eric G.

    2009-01-01

    Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona- and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-O-acetylesterase domains almost identical to corresponding domains in orthomyxo- and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine–Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptor-binding site seems to have been designed to use 9-mono- and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins. PMID:19721004

  4. Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

    PubMed

    Tao, Weixin; Lee, Myung Hwan; Yoon, Mi-Young; Kim, Jin-Cheol; Malhotra, Shweta; Wu, Jing; Hwang, Eul Chul; Lee, Seon-Woo

    2011-12-01

    Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria. PMID:22210605

  5. BLT-esterase in infectious mononucleosis.

    PubMed Central

    Wagner, L; Wiesholzer, M; Worman, C P; Lang, G; Base, W

    1995-01-01

    Peripheral blood lymphocytes of three patients suffering from infectious mononucleosis due to Epstein-Barr virus (EBV) infection were analysed for BLT-esterase expression in peripheral blood lymphocytes by a well established cytochemical staining method. During the acute phase of disease with presence of clinical symptoms a very high level of up to 90% BLT-esterase-expressing lymphocytes were detected. The increased percentage of lymphocytes expressing BLT-esterase coincided with the time of greatest symptoms and the peak elevation of hepatocellular enzymes. The still moderately elevated level only gradually decreased to normal during the further recovery period of 2 months during which the patients described episodes of weakness. Peripheral blood lymphocyte phenotype analysis revealed a marked CD8 lymphocytosis, a CD4/CD8 ratio of about 0.2, low number of CD19+ B cells, and a high level of DR+ CD3+ lymphocytes. Reduction of BLT esterase expression during the recovery period coincided with reduction of CD8+ DR+ lymphocytes. By a combination of BLT-esterase staining with immunocytochemical phenotype analysis, 95% of CD8+ lymphocytes were found to be BLT-esterase-positive. BLT-esterase might be involved in the immunodefence against EBV in infectious mononucleosis by inducing apoptosis in EBV-transformed B cells. Images Fig. 2 PMID:7743659

  6. Esterase profile of human masseter muscle.

    PubMed Central

    Kirkeby, S; Moe, D; Vilmann, H

    1988-01-01

    The esterase profile of fresh human masseter muscle was investigated by use of histochemistry and electrophoresis. The histochemical methods included reactions for alpha-naphthyl esterase, myofibrillar ATPase, reverse myofibrillar ATPase and succinic dehydrogenase. In frozen sections of the muscle the coloured reaction product for esterases was present both as a diffuse sarcoplasmic coloration and as distinct granules. The intensity of diffuse reaction was used to classify the muscle fibres as strongly, moderately and weakly reacting. The fibres with strong esterase activity belonged to Type I and iiC. iM and Type II A fibres showed a moderate esterase reaction and Type II B fibres had a low activity. The electrophoretic gels stained for esterase activity showed that the human masseter muscle possesses a slow migrating double band with high enzyme activity and a cascade of faster migrating isoenzymes. In isoelectric focused gels the major esterases showed isoelectric points around pH 5. Images Fig. 1 Fig. 2 Figs. 3-5 Figs. 6-8 Figs. 9-11 Figs. 12-14 Figs. 15-16 Fig. 17 PMID:3198486

  7. Insights into the mechanism of enzymatic hydrolysis of xylan.

    PubMed

    Moreira, L R S; Filho, E X F

    2016-06-01

    Hemicelluloses are a vast group of complex, non-cellulosic heteropolysaccharides that are classified according to the principal monosaccharides present in its structure. Xylan is the most abundant hemicellulose found in lignocellulosic biomass. In the current trend of a more effective utilization of lignocellulosic biomass and developments of environmentally friendly industrial processes, increasing research activities have been directed to a practical application of the xylan component of plants and plant residues as biopolymer resources. A variety of enzymes, including main- and side-chain acting enzymes, are responsible for xylan breakdown. Xylanase is a main-chain enzyme that randomly cleaves the β-1,4 linkages between the xylopyranosyl residues in xylan backbone. This enzyme presents varying folds, mechanisms of action, substrate specificities, hydrolytic activities, and physicochemical characteristics. This review pays particular attention to different aspects of the mechanisms of action of xylan-degrading enzymes and their contribution to improve the production of bioproducts from plant biomass. Furthermore, the influence of phenolic compounds on xylanase activity is also discussed. PMID:27112349

  8. Hydrolysis kinetics of tulip tree xylan in hot compressed water.

    PubMed

    Yoon, Junho; Lee, Hun Wook; Sim, Seungjae; Myint, Aye Aye; Park, Hee Jeong; Lee, Youn-Woo

    2016-08-01

    Lignocellulosic biomass, a promising renewable resource, can be converted into numerous valuable chemicals post enzymatic saccharification. However, the efficacy of enzymatic saccharification of lignocellulosic biomass is low; therefore, pretreatment is necessary to improve the efficiency. Here, a kinetic analysis was carried out on xylan hydrolysis, after hot compressed water pretreatment of the lignocellulosic biomass conducted at 180-220°C for 5-30min, and on subsequent xylooligosaccharide hydrolysis. The weight ratio of fast-reacting xylan to slow-reacting xylan was 5.25 in tulip tree. Our kinetic results were applied to three different reaction systems to improve the pretreatment efficiency. We found that semi-continuous reactor is promising. Lower reaction temperatures and shorter space times in semi-continuous reactor are recommended for improving xylan conversion and xylooligosaccharide yield. In the theoretical calculation, 95% of xylooligosaccharide yield and xylan conversion were achieved simultaneously with high selectivity (desired product/undesired product) of 100 or more. PMID:27208738

  9. The psychrophilic bacterium Pseudoalteromonas halosplanktis TAC125 possesses a gene coding for a cold-adapted feruloyl esterase activity that shares homology with esterase enzymes from gamma-proteobacteria and yeast.

    PubMed

    Aurilia, Vincenzo; Parracino, Antonietta; Saviano, Michele; Rossi, Mose'; D'Auria, Sabato

    2007-08-01

    The complete genome of the psychrophilic bacteria Pseudoalteromonas haloplanktis TAC 125, recently published, owns a gene coding for a putative esterase activity corresponding to the ORF PSHAa1385, also classified in the Carbohydrate Active Enzymes database (CAZY) belonging to family 1 of carbohydrate esterase proteins. This ORF is 843 bp in length and codes for a protein of 280 amino acid residues. In this study we characterized and cloned the PSHAa1385 gene in Escherichia coli. We also characterized the recombinant protein by biochemical and biophysical methodologies. The PSHAa1385 gene sequence showed a significant homology with several carboxyl-esterase and acetyl-esterase genes from gamma-proteobacteria genera and yeast. The recombinant protein exhibited a significant activity towards pNP-acetate, alpha-and beta-naphthyl acetate as generic substrates, and 4-methylumbelliferyl p-trimethylammonio cinnamate chloride (MUTMAC) as a specific substrate, indicating that the protein exhibits a feruloyl esterase activity that it is displayed by similar enzymes present in other organisms. Finally, a three-dimensional model of the protein was built and the amino acid residues involved in the catalytic function of the protein were identified. PMID:17543477

  10. Effects of cationic xylan from annual plants on the mechanical properties of paper.

    PubMed

    Deutschle, Alexander L; Römhild, Katrin; Meister, Frank; Janzon, Ron; Riegert, Christiane; Saake, Bodo

    2014-02-15

    Xylan from oat spelt and wheat was used as an additive to enhance the dry strength of paper. The absorption of xylan by the cellulose fibers was increased by cationization to different degrees of substitution. Paper hand sheets with different doses of xylan and industrial cationic starch were produced, and the mechanical properties were determined. Absorption measurements of cationic oat spelt xylan on pulp fibers explained the differing influences of low and high cationized xylan addition on paper strength. The addition of cationic oat spelt xylan with a degree of substitution of 0.1 at a 4% dose provided the largest improvement in the tensile-index (67%), burst-index (105%) and tear-index (77%). Compared to cationic starch, cationic oat spelt xylan additives led to similar paper strength values, excepting the tear strength. The structural differences and protein impurities made the wheat xylan unsuitable as a strength additive for paper pulp. PMID:24507328

  11. Preparation of xylan citrate--a potential adsorbent for industrial wastewater treatment.

    PubMed

    Shuaiyang, Wang; Huiling, Li; Junli, Ren; Chuanfu, Liu; Feng, Peng; Runcang, Sun

    2013-02-15

    The novel and degradable xylan citrate was prepared by the environmental-friendly semi-dry oven method. Xylan reacted with citric acid (CA) to yield xylan citrate at high temperature. The influence of the different weight ratios of CA and xylan on the product yield, the carboxyl group content and degree of esterification were comparatively discussed. The results showed that there were higher carboxyl group content and degree of esterification in modified xylan than native xylan. The product yield of 128.2%, the carboxyl group content of 1174.3 meq/100 g and degree of esterification of 33.1% were achieved at the CA/xylan weight ratio of 2.4 in the absence of catalyst. Furthermore, the adsorption capacity of xylan after modification was improved greatly. These materials with better properties can enhance their water affinity, and improve their adsorption of copper ions and methyl orange in aqueous solution due to carboxyl groups. PMID:23399244

  12. Clinical significance of esterases in man.

    PubMed

    Williams, F M

    1985-01-01

    Esterases, hydrolases which split ester bonds, hydrolyse a number of compounds used as drugs in humans. The enzymes involved are classified broadly as cholinesterases (including acetylcholinesterase), carboxylesterases, and arylesterases, but apart from acetylcholinesterase, their biological function is unknown. The acetylcholinesterase present in nerve endings involved in neurotransmission is inhibited by anticholinesterase drugs, e.g. neostigmine, and by organophosphorous compounds (mainly insecticides). Cholinesterases are primarily involved in drug hydrolysis in the plasma, arylesterases in the plasma and red blood cells, and carboxylesterases in the liver, gut and other tissues. The esterases exhibit specificities for certain substrates and inhibitors but a drug is often hydrolysed by more than one esterase at different sites. Aspirin (acetylsalicylic acid), for example, is hydrolysed to salicylate by carboxylesterases in the liver during the first-pass. Only 60% of an oral dose reaches the systemic circulation where it is hydrolysed by plasma cholinesterases and albumin and red blood cell arylesterases. Thus, the concentration of aspirin relative to salicylate in the circulation may be affected by individual variation in esterase levels and the relative roles of the different esterases, and this may influence the overall pharmacological effect. Other drugs have been less extensively investigated than aspirin and these include heroin (diacetylmorphine), suxamethonium (succinylcholine), clofibrate, carbimazole, procaine and other local anaesthetics. Ester prodrugs are widely used to improve absorption of drugs and in depot preparations. The active drug is released by hydrolysis by tissue carboxylesterases. Individual differences in esterase activity may be genetically determined, as is the case with atypical cholinesterases and the polymorphic distribution of serum paraoxonase and red blood cell esterase D. Disease states may also alter esterase activity. PMID

  13. Identification of a novel carbohydrate esterase from Bjerkandera adusta: structural and function predictions through bioinformatics analysis and molecular modeling.

    PubMed

    Cuervo-Soto, Laura I; Valdés-García, Gilberto; Batista-García, Ramón; del Rayo Sánchez-Carbente, María; Balcázar-López, Edgar; Lira-Ruan, Verónica; Pastor, Nina; Folch-Mallol, Jorge Luis

    2015-03-01

    A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg(-1) protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin. PMID:25586442

  14. Designer xylanosomes: protein nanostructures for enhanced xylan hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work is the first report of the successful design, construction, and application of multi-functional, self-assembling biocatalysts for targeted xylan hydrolysis, termed xylanosomes. Using the architecture of cellulosomes found in some anaerobic cellulolytic microbes, four different xylanosomes...

  15. Molecular Dissection of Xylan Biosynthesis During Wood Formation in Poplar

    EPA Science Inventory

    Xylan, being the second most abundant polysaccharide in dicot wood, is considered to be one of the factors contributing to wood biomass recalcitrance for biofuel production. To better utilize wood as biofuel feedstock, it is crucial to functionally characterize all the genes invo...

  16. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis1[C][W][OPEN

    PubMed Central

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha; Harholt, Jesper; Chong, Sun-Li; Pawar, Prashant Mohan-Anupama; Mellerowicz, Ewa J.; Tenkanen, Maija; Cheng, Kun; Pauly, Markus; Scheller, Henrik Vibe

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls. PMID:24019426

  17. Selective chemical oxidation and depolymerization of switchgrass (Panicum virgatum L.) xylan with oligosaccharide product analysis by mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose and consequently pretreatment conditions are in part optimized for maximal xylan depolymerization or displacement. Xylan consists of a long ...

  18. Esterase isozyme polymorphism, specific and nonspecific esterase, syngenic lines development and natural occurrence of a thermostable esterase in the tropical silkworm Bombyx mori L.

    PubMed

    Chattopadhyay, G K; Sengupta, A K; Verma, A K; Sen, S K; Saratchandra, B

    2001-11-01

    Esterase isozyme polymorphism was documented for digestive juice and haemolymph of the tropical multivoltine silkworm, Bombyx mori L., breed CB5 (GP) and its syngenic lines (CB5Lm(e)-1, CB5Lm-2 and CB5Lm-5) using alpha- and beta-naphthylacetate separately as nonspecific substrates (Ogita, Z., Kasai, T., 1965. Genetico-biochemical analysis of specific esterases in Musca domestica. Jpn. J. Genet. 40, 173-184). Polymorphism existed in the isozyme pattern of alpha-esterase with two or three bands in digestive juice and three to five bands in haemolymph. No polymorphism was observed in beta-esterase isozyme pattern having four bands in digestive juice and two bands in haemolymph. During the course of esterase isozyme studies, the presence of some specific alpha-esterase bands (Est-1, 4 and 5) in haemolymph and beta-esterase bands (Est-1, 2 and 3) in digestive juice were observed. But both alpha- and beta-esterase bands Est-3 and 4 in digestive juice and Est-2 and 3 in haemolymph were found to be nonspecific. Nonspecific beta-esterase band (Est-3) in haemolymph of CB5 (GP) and its syngenic lines withstood a temperature up to 80+/-1 degrees C for 10 min. No thermostable band was observed in the isozyme zymogram of alpha-esterase in digestive juice and haemolymph or beta-esterase in digestive juice. Overall, this study discusses the presence of esterase heterogeneity in the CB5 (GP) genepool, syngenic lines development, occurrence of specific alpha- and beta-esterase bands in digestive juice and haemolymph and thermostable beta-esterase band Est-3 in haemolymph in tropical silkworm Bombyx mori L. PMID:11583932

  19. Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.

    PubMed

    Su, Xiaoyun; Han, Yejun; Dodd, Dylan; Moon, Young Hwan; Yoshida, Shosuke; Mackie, Roderick I; Cann, Isaac K O

    2013-03-01

    Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k(cat) values of ∼8,000 and ∼4,500 s(-1), respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures. PMID:23263957

  20. Reconstitution of a Thermostable Xylan-Degrading Enzyme Mixture from the Bacterium Caldicellulosiruptor bescii

    PubMed Central

    Su, Xiaoyun; Han, Yejun; Dodd, Dylan; Moon, Young Hwan; Yoshida, Shosuke; Mackie, Roderick I.

    2013-01-01

    Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated kcat values of ∼8,000 and ∼4,500 s−1, respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures. PMID:23263957

  1. Genetics of a tissue esterase polymorphism (Est-6) in the rabbit (Oryctolagus cuniculus).

    PubMed

    van Zutphen, L F; den Bieman, M G; von Deimling, O; Fox, R R

    1987-06-01

    Genetic analysis of a polymorphic tissue esterase revealed a new locus (Est-6) with two alleles (Est-6a and Est-6b) on linkage group VI of the rabbit. Est-6 is closely linked to the Est-1,2,4 cluster. Esterase of Est-6 is found in many organs, particularly in liver and small intestine, but not in erythrocytes and serum. Est-6 esterase hydrolyzes alpha-naphthyl acetate and butyrate, naphthol AS-D acetate, indoxyl acetate, and butyrate as well as 5-bromoindoxyl acetate, N-acetyl-L-alanine-alpha-naphthyl ester but not 4-methylumbelliferyl acetate and fluorescein diacetate. The enzyme is inhibited by bis-p-nitrophenyl phosphate and eserine but not by p-chloromercuribenzoate. It was classified as a carboxylesterase (EC 3.1.1.1). Based on chromosomal localization, tissue distribution, substrate specificity, inhibitor sensitivity, and range of pI's, rabbit Est-6 is assumed to be homologous with mouse Es-7. PMID:3619880

  2. [3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

    NASA Technical Reports Server (NTRS)

    Hall, P. J.; Bandurski, R. S.

    1986-01-01

    [3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

  3. 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.

  4. Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

    PubMed Central

    Juhl, P Benjamin; Trodler, Peter; Tyagi, Sadhna; Pleiss, Jürgen

    2009-01-01

    Background Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. Results Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i) enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii) enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii) substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. Conclusion The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand), although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria. PMID:19493341

  5. Synthesis and characterization of carboxymethylated xylan and its application as a dispersant.

    PubMed

    Konduri, Mohan K R; Fatehi, Pedram

    2016-08-01

    In this study, carboxymethylated beechwood xylan was produced under alkali conditions using sodium chloroacetate. Taguchi orthogonal design was used to explore the influence of the process parameters, i.e. NaOH concentration, time, temperature, the molar ratio of sodium chloroacetate to xylan and the concentration of the reaction medium on the charge density and degree of substitution (DS) of xylan. Carboxymethylated xylan (CMX) with the maximum charge density of 1.62meq/g and DS of 0.21 was produced under the optimal conditions of 0.75M NaOH concentration, 1.0mol/mol sodium chloroacetate (SCA)/xylan ratio, 2h reaction time, 70°C and 15g/L xylan concentration. The carboxylate group of the product was 1.48mmol/g. The attachment of the carboxymethylated group to xylan was confirmed by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance ((1)H NMR) spectroscopy. The molecular weight of xylan increased and its thermal stability was improved via carboxymethylation. The dispersion performance of the carboxymethylated xylan in clay suspensions was determined by photometric dispersion analyzer (PDA). CMX showed better performance than sodium carbonate-polyacrylic acid (Na2CO3-PAA) in dispersing the clay suspension. The unmodified xylan did not show any dispersion performance. PMID:27112847

  6. Identification of Genes Encoding Microbial Glucuronoyl Esterases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One type of covalent linkages connecting lignin and hemicellulose in plant cell walls is the ester linkage between 4-0-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. An enzyme that could hydrolyze such linkages, named glucuronoyl esterase, occurs in the cellulolytic system of the w...

  7. New Extremophilic Lipases and Esterases from Metagenomics

    PubMed Central

    López-López, Olalla; Cerdán, Maria E; González Siso, Maria I

    2014-01-01

    Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications. PMID:24588890

  8. Production of cutinolytic esterase by filamentous bacteria.

    PubMed

    Fett, W F; Wijey, C; Moreau, R A; Osman, S F

    2000-07-01

    Thirty-eight strains of filamentous bacteria, many of which are thermophilic or thermotolerant and commonly found in composts and mouldy fodders, were examined for their ability to produce cutinolytic esterase (cutinase) in culture media supplemented with cutin, suberin or cutin-containing agricultural by-products. Initially, the ability of culture supernatants to hydrolyse the artificial substrate p-nitrophenyl butyrate was determined by spectrophotometric assays. Only one bacterium, Thermoactinomyces vulgaris NRRL B-16117, exhibited cutinolytic esterase production. The enzyme was highly inducible, was repressed by the presence of glucose in the medium and hydrolysed both apple and tomato cutins. Inducers included apple cutin, apple pomace, tomato peel, potato suberin and commercial cork. Unlike similar fungal enzymes, the T. vulgaris cutinolytic esterase was not inducible by cutin hydrolysate. The cutinolytic esterase exhibited a half-life of over 60 min at 70 degrees C and a pH optimum of >/= 11.0. This study indicates that thermophylic filamentous bacteria may be excellent commercial sources of heat-stable cutin-degrading enzymes that can be produced by fermentation of low cost feedstocks. PMID:10886609

  9. Phenol esterase activity of porcine skin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  10. 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. PMID:25425346

  11. Molecular mechanisms associated with xylan degradation by Xanthomonas plant pathogens.

    PubMed

    Santos, Camila Ramos; Hoffmam, Zaira Bruna; de Matos Martins, Vanesa Peixoto; Zanphorlin, Leticia Maria; de Paula Assis, Leandro Henrique; Honorato, Rodrigo Vargas; Lopes de Oliveira, Paulo Sérgio; Ruller, Roberto; Murakami, Mario Tyago

    2014-11-14

    Xanthomonas pathogens attack a variety of economically relevant plants, and their xylan CUT system (carbohydrate utilization with TonB-dependent outer membrane transporter system) contains two major xylanase-related genes, xynA and xynB, which influence biofilm formation and virulence by molecular mechanisms that are still elusive. Herein, we demonstrated that XynA is a rare reducing end xylose-releasing exo-oligoxylanase and not an endo-β-1,4-xylanase as predicted. Structural analysis revealed that an insertion in the β7-α7 loop induces dimerization and promotes a physical barrier at the +2 subsite conferring this unique mode of action within the GH10 family. A single mutation that impaired dimerization became XynA active against xylan, and high endolytic activity was achieved when this loop was tailored to match a canonical sequence of endo-β-1,4-xylanases, supporting our mechanistic model. On the other hand, the divergent XynB proved to be a classical endo-β-1,4-xylanase, despite the low sequence similarity to characterized GH10 xylanases. Interestingly, this enzyme contains a calcium ion bound nearby to the glycone-binding region, which is required for catalytic activity and structural stability. These results shed light on the molecular basis for xylan degradation by Xanthomonas and suggest how these enzymes synergistically assist infection and pathogenesis. Our findings indicate that XynB contributes to breach the plant cell wall barrier, providing nutrients and facilitating the translocation of effector molecules, whereas the exo-oligoxylanase XynA possibly participates in the suppression of oligosaccharide-induced immune responses. PMID:25266726

  12. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    PubMed

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

    2015-03-01

    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. PMID:25684099

  13. The role of the plant-specific ALTERED XYLOGLUCAN9 protein in Arabidopsis cell wall polysaccharide O-acetylation.

    PubMed

    Schultink, Alex; Naylor, Dan; Dama, Murali; Pauly, Markus

    2015-04-01

    A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway. PMID:25681330

  14. Arabidopsis thaliana IRX10 and two related proteins from psyllium and Physcomitrella patens are xylan xylosyltransferases.

    PubMed

    Jensen, Jacob Krüger; Johnson, Nathan Robert; Wilkerson, Curtis Gene

    2014-10-01

    The enzymatic mechanism that governs the synthesis of the xylan backbone polymer, a linear chain of xylose residues connected by β-1,4 glycosidic linkages, has remained elusive. Xylan is a major constituent of many kinds of plant cell walls, and genetic studies have identified multiple genes that affect xylan formation. In this study, we investigate several homologs of one of these previously identified xylan-related genes, IRX10 from Arabidopsis thaliana, by heterologous expression and in vitro xylan xylosyltransferase assay. We find that an IRX10 homolog from the moss Physcomitrella patens displays robust activity, and we show that the xylosidic linkage formed is a β-1,4 linkage, establishing this protein as a xylan β-1,4-xylosyltransferase. We also find lower but reproducible xylan xylosyltransferase activity with A. thaliana IRX10 and with a homolog from the dicot plant Plantago ovata, showing that xylan xylosyltransferase activity is conserved over large evolutionary distance for these proteins. PMID:25139408

  15. Ring-opening graft polymerization of propylene carbonate onto xylan in an ionic liquid.

    PubMed

    Zhang, Xueqin; Chen, Mingjie; Liu, Chuanfu; Zhang, Aiping; Sun, Runcang

    2015-01-01

    The amidine organocatalyst 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is an effective nucleophilic catalyst. Biocomposites with tuneable properties were successfully synthesized by ring-opening graft polymerization (ROGP) of propylene carbonate (PC) onto xylan using DBU as a catalyst in the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([Amim]Cl). The effects of reaction temperature, reaction time and the molar ratio of PC to anhydroxylose units (AXU) in xylan were investigated. The physico-chemical properties of xylan-graft-poly(propylene carbonate) (xylan-g-PPC) copolymers were characterised by FT-IR, NMR, TGA/DTG, AFM and tensile analysis. The FT-IR and NMR results indicated the successful attachment of PPC onto xylan. TGA/DTG suggested the increased thermal stability of xylan after the attachment of PPC side chains. AFM analysis revealed details about the molecular aggregation of xylan-g-PPC films. The results also showed that with the increased DS of xylan-g-PPC copolymers, the tensile strength and Young's modulus of the films decreased, while the elongation at break increased. PMID:25853319

  16. Redistribution of Xylan in Maize Cell Walls During Dilute Acid Pretreatment

    SciTech Connect

    Brunecky, R.; Vinzant, T. B.; Porter, S. E.; Donohoe, B. S.; Johnson, D. K.; Himmel, M. E.

    2009-04-15

    Developing processes for the conversion of biomass for use in transportation fuels production is becoming a critically important economic and engineering challenge. Dilute acid pretreatment is a promising technology for increasing the enzymatic digestibility of lignocellulosic biomass. However, a deeper understanding of the pretreatability of biomass is needed so that the rate of formation and yields of sugars can be increased. Xylan is an important hemicellulosic component of the plant cell wall and acts as a barrier to cellulose, essentially blocking cellulase action. To better understand xylan hydrolysis in corn stover, we have studied changes in the distribution of xylan caused by dilute acid pretreatment using correlative microscopy. A dramatic loss of xylan antibody signal from the center of the cell wall and an increase or retention of xylan at the plasma membrane interface and middle lamella of the cell were observed by confocal laser scanning microscopy (CLSM). We also observed a reduction in xylan fluorescence signal by CLSM that is generally consistent with the decrease in xylan content measured experimentally in the bulk sample, however, the compartmentalization of this xylan retention was not anticipated.

  17. Novel choline esterase based sensor for monitoring of organophosphorus pollutants

    SciTech Connect

    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.

  18. High throughput determination of glucan and xylan fractions in lignocelluloses.

    PubMed

    Selig, Michael J; Tucker, Melvin P; Law, Cody; Doeppke, Crissa; Himmel, Michael E; Decker, Stephen R

    2011-05-01

    The analysis of structural glucan and xylan in lignocellulose was scaled down from original two-stage sulfuric acid hydrolysis methods (Moore WE and Johnson DB 1967 Procedures for the chemical analysis of wood and wood products. U.S. Forest Products Laboratory, U.S. Department of Agriculture., Madison, WI) and integrated into a recently-developed, high throughput pretreatment and enzymatic saccharification system. Novel 96×1.8 ml-well Hastelloy reactor plates (128×86×51 mm) based on previously described 96-well pretreatment reactor plates were paired with custom aluminum filler plates (128×86×18 mm) for use in Symyx Powdernium solids dispensing systems. The incorporation of glucose oxidase and xylose dehydrogenase linked assays to speed post-hydrolysis sugar analysis dramatically reduced the time for analysis of large lignocellulosic sample sets. The current system permits the determination of the glucan and xylan content of 96 replicates (per reactor plate) in under 6 h and parallel plate processing increases the analysis throughput substantially. PMID:21287235

  19. Catalytic upgrading of xylan over mesoporous Y catalyst.

    PubMed

    Kim, Seong-Soo; Jun, Bo Ram; Park, Sung Hoon; Jeon, Jong-Ki; Suh, Dong Jin; Kim, Tae-Wan; Park, Young-Kwon

    2014-04-01

    In-situ catalytic cracking of xylan, a model compound of hemicellulose, was carried out using pyrolysis-gas chromatography/mass spectrometry over mesoporous Y for the first time. Experiments were conducted at three different temperatures, 400 degrees C, 450 degrees C, and 500 degrees C, to investigate the effect of reaction temperature. Three different biomass-to-catalyst ratios, 1:1, 1:2, and 1:3, were tested at 500 degrees C to examine the effect of catalyst dose. In addition, the catalytic activity of mesoporous Y was compared with that of Al-MCM-41. The catalysts used were characterized by N2 adsorption-desorption, temperature programmed desorption of NH3, and X-ray diffraction. The main pyrolysis products of xylan were acids, hydrocarbons, phenolics, oxygenates, aromatics, and polycyclic aromatic hydrocarbons. Mesoporous Y, which has acid sites with larger quantity and stronger acidity than those of Al-MCM-41, was shown to enhance the quality of bio-oil to a larger extent, producing a larger quantity of high-value-added products, such as aromatics and furans. PMID:24734712

  20. Xylan hemicellulose improves chitosan hydrogel for bone tissue regeneration

    PubMed Central

    Bush, Joshua R.; Liang, Haixiang; Dickinson, Molly; Botchwey, Edward A.

    2016-01-01

    The hemicellulose xylan, which has immunomodulatory effects, has been combined with chitosan to form a composite hydrogel to improve the healing of bone fractures. This thermally responsive and injectable hydrogel, which is liquid at room temperature and gels at physiological temperature, improves the response of animal host tissue compared with similar pure chitosan hydrogels in tissue engineering models. The composite hydrogel was placed in a subcutaneous model where the composite hydrogel is replaced by host tissue within 1 week, much earlier than chitosan hydrogels. A tibia fracture model in mice showed that the composite encourages major remodeling of the fracture callus in less than 4 weeks. A non-union fracture model in rat femurs was used to demonstrate that the composite hydrogel allows bone regeneration and healing of defects that with no treatment are unhealed after 6 weeks. These results suggest that the xylan/chitosan composite hydrogel is a suitable bone graft substitute able to aid in the repair of large bone defects.

  1. From plant biomass to bio-based chemicals: latest developments in xylan research.

    PubMed

    Deutschmann, Rudolf; Dekker, Robert F H

    2012-01-01

    For a hundred years or more, oil and natural gas has supplied fuel and other raw chemicals to support economic growth. In the last decades their shrinking reservoirs and the increasing cost of production has become obvious, leading researchers to look for alternative substitutes of all the chemical materials presently derived from oil and gas. This review is focused on xylan, the second most abundant plant polysaccharide on our planet. Some xylan-derived products have already found commercial applications (ethanol, xylitol, xylo-oligosaccharides) while others could have a great future in a wide range of industries. The chemical and structural variations of xylans produced by different plants, and the concentration of xylan in various plant resources are summarized. This review discusses the latest research developments in extraction and purification methodologies, and chemical modification, as well as the analytical methods necessary for xylan related research. PMID:22776161

  2. Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila.

    PubMed

    Katsimpouras, Constantinos; Bénarouche, Anaïs; Navarro, David; Karpusas, Michael; Dimarogona, Maria; Berrin, Jean-Guy; Christakopoulos, Paul; Topakas, Evangelos

    2014-06-01

    Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl D-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three D-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-D-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs. PMID:24531271

  3. XAX1 from glycosyltransferase family 61 mediates xylosyltransfer to rice xylan

    PubMed Central

    Chiniquy, Dawn; Sharma, Vaishali; Schultink, Alex; Baidoo, Edward E.; Rautengarten, Carsten; Cheng, Kun; Carroll, Andrew; Ulvskov, Peter; Harholt, Jesper; Keasling, Jay D.; Pauly, Markus; Scheller, Henrik V.; Ronald, Pamela C.

    2012-01-01

    Xylan is the second most abundant polysaccharide on Earth and represents an immense quantity of stored energy for biofuel production. Despite its importance, most of the enzymes that synthesize xylan have yet to be identified. Xylans have a backbone of β-1,4–linked xylose residues with substitutions that include α-(1→2)–linked glucuronosyl, 4-O-methyl glucuronosyl, and α-1,2- and α-1,3-arabinofuranosyl residues. The substitutions are structurally diverse and vary by taxonomy, with grass xylan representing a unique composition distinct from dicots and other monocots. To date, no enzyme has yet been identified that is specific to grass xylan synthesis. We identified a xylose-deficient loss-of-function rice mutant in Os02g22380, a putative glycosyltransferase in a grass-specific subfamily of family GT61. We designate the mutant xax1 for xylosyl arabinosyl substitution of xylan 1. Enzymatic fingerprinting of xylan showed the specific absence in the mutant of a peak, which was isolated and determined by 1H-NMR to be (β-1,4-Xyl)4 with a β-Xylp-(1→2)-α-Araf-(1→3). Rice xax1 mutant plants are deficient in ferulic and coumaric acid, aromatic compounds known to be attached to arabinosyl residues in xylan substituted with xylosyl residues. The xax1 mutant plants exhibit an increased extractability of xylan and increased saccharification, probably reflecting a lower degree of diferulic cross-links. Activity assays with microsomes isolated from tobacco plants transiently expressing XAX1 demonstrated xylosyltransferase activity onto endogenous acceptors. Our results provide insight into grass xylan synthesis and how substitutions may be modified for increased saccharification for biofuel generation. PMID:23027943

  4. Improvement in the thermostability of a type A feruloyl esterase, AuFaeA, from Aspergillus usamii by iterative saturation mutagenesis.

    PubMed

    Yin, Xin; Li, Jian-Fang; Wang, Chun-Juan; Hu, Die; Wu, Qin; Gu, Ying; Wu, Min-Chen

    2015-12-01

    Feruloyl or ferulic acid esterase (Fae, EC 3.1.1.73) catalyzes the hydrolysis of ester bonds between polysaccharides and phenolic acid compounds in xylan side chain. In this study, the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii was increased by iterative saturation mutagenesis (ISM). Two amino acids, Ser33 and Asn92, were selected for saturation mutagenesis according to the B-factors analyzed by B-FITTER software and ΔΔG values predicted by PoPMuSiC algorithm. After screening the saturation mutagenesis libraries constructed in Pichia pastoris, 15 promising variants were obtained. The best variant S33E/N92-4 (S33E/N92R) produced a T m value of 44.5 °C, the half-lives (t1/2) of 35 and 198 min at 55 and 50 °C, respectively, corresponding to a 4.7 °C, 2.33- and 3.96-fold improvement compared to the wild type. Additionally, the best S33 variant S33-6 (S33E) was thermostable at 50 °C with a t1/2 of 82 min, which was 32 min longer than that of the wild type. All the screened S33E/N92 variants were more thermostable than the best S33 variant S33-6 (S33E). This work would contribute to the further studies on higher thermostability modification of type A feruloyl esterases, especially those from fungi. The thermostable feruloyl esterase variants were expected to be potential candidates for industrial application in prompting the enzymic degradation of plant biomass materials at elevated temperatures. PMID:26266754

  5. O-acetylated N-acetylneuraminic acid as a novel target for therapy in human pre-B acute lymphoblastic leukemia

    PubMed Central

    Parameswaran, Reshmi; Lim, Min; Arutyunyan, Anna; Abdel-Azim, Hisham; Hurtz, Christian; Lau, Kam; Müschen, Markus; Yu, Robert K.; von Itzstein, Mark; Groffen, John

    2013-01-01

    The development of resistance to chemotherapy is a major cause of relapse in acute lymphoblastic leukemia (ALL). Though several mechanisms associated with drug resistance have been studied in detail, the role of carbohydrate modification remains unexplored. Here, we investigated the contribution of 9-O-acetylated N-acetylneuraminic acid (Neu5Ac) to survival and drug resistance development in ALL cells. A strong induction of 9-O-acetylated Neu5Ac including 9-O-acetyl GD3 was detected in ALL cells that developed resistance against vincristine or nilotinib, drugs with distinct cytotoxic mechanisms. Removal of 9-O-acetyl residues from Neu5Ac on the cell surface by an O-acetylesterase made ALL cells more vulnerable to such drugs. Moreover, removal of intracellular and cell surface–resident 9-O-acetyl Neu5Ac by lentiviral transduction of the esterase was lethal to ALL cells in vitro even in the presence of stromal protection. Interestingly, expression of the esterase in normal fibroblasts or endothelial cells had no effect on their survival. Transplanted mice induced for expression of the O-acetylesterase in the ALL cells exhibited a reduction of leukemia to minimal cell numbers and significantly increased survival. This demonstrates that Neu5Ac 9-O-acetylation is essential for survival of these cells and suggests that Neu5Ac de-O-acetylation could be used as therapy to eradicate drug-resistant ALL cells. PMID:23478187

  6. Non-specific esterases and esterproteases in masticatory muscles from the muscular dystrophic mouse.

    PubMed

    Kirkeby, S; Moe, D; Vilmann, H

    1989-03-01

    With the aid of histochemical and electrophoretic techniques activities for esterase and esterprotease were investigated in the digastric and masseter muscles from normal and dystrophic mice. The substrates used were alpha-naphthyl acetate and N-acetyl-L-alanine alpha-naphthyl ester. According to the microscopic observations of the dystrophic muscles the histopathological changes in the masseter muscle were much more pronounced than in the digastric muscle. The connective tissue surrounding the myofibers of the dystrophic masseter contained a large number of cells with pronounced enzyme activity. Among them were mast cells that were strongly stained for esterprotease. The connective tissue of the dystrophic digastricus was much less infiltrated with cellular elements reacting for esterprotease. In zymograms the normal digastricus, the dystrophic masseter and the dystrophic digastricus showed a strong activity for certain isoenzymes that were absent or weakly expressed in the normal masseter. PMID:2657470

  7. IN VITRO COMPARISON OF RAT AND CHICKEN BRAIN NEUROTOXIC ESTERASE

    EPA Science Inventory

    A systematic comparison was undertaken to characterize neurotoxic esterase (NTE) from rat and chicken brain in terms of inhibitor sensitivities, pH optima, and molecular weights. Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterased showed that rat esterases we...

  8. The Xylan Delignification Process for biomass conversion to ethanol

    SciTech Connect

    Dale, M.C.; Zhao, C.; Lei, S.; Tyson, G.

    1995-10-01

    An extrusion process melded with alkaline peroxide chemical pretreatements allows the lignin and hemicellulose in biomass to be solublibzed, and the cellulose component to be made available for enzymatic breakdown. This process is called the Xylan Delignification Process (XDP). In this paper, some results of the XDP on promoting enzymatic breakdown and SSF of corn stalks switch grass and straw are reported. It was found that the XDP process allowed quick (6 hour) and reasonably complete (85--88%) hydrolysis of the cellulose fraction of cornstalks, but was less effective in allowing utilization of the switch grass with 76% yeild noted in 24 hours. Solubilization of the lignin and hemicellulose were not acheived on a first set of corn stalk, switch grass, and straw samples, but was noted on a second straw sample.

  9. 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. PMID:26216109

  10. Structural features determining thermal adaptation of esterases.

    PubMed

    Kovacic, Filip; Mandrysch, Agathe; Poojari, Chetan; Strodel, Birgit; Jaeger, Karl-Erich

    2016-02-01

    The adaptation of microorganisms to extreme living temperatures requires the evolution of enzymes with a high catalytic efficiency under these conditions. Such extremophilic enzymes represent valuable tools to study the relationship between protein stability, dynamics and function. Nevertheless, the multiple effects of temperature on the structure and function of enzymes are still poorly understood at the molecular level. Our analysis of four homologous esterases isolated from bacteria living at temperatures ranging from 10°C to 70°C suggested an adaptation route for the modulation of protein thermal properties through the optimization of local flexibility at the protein surface. While the biochemical properties of the recombinant esterases are conserved, their thermal properties have evolved to resemble those of the respective bacterial habitats. Molecular dynamics simulations at temperatures around the optimal temperatures for enzyme catalysis revealed temperature-dependent flexibility of four surface-exposed loops. While the flexibility of some loops increased with raising the temperature and decreased with lowering the temperature, as expected for those loops contributing to the protein stability, other loops showed an increment of flexibility upon lowering and raising the temperature. Preserved flexibility in these regions seems to be important for proper enzyme function. The structural differences of these four loops, distant from the active site, are substantially larger than for the overall protein structure, indicating that amino acid exchanges within these loops occurred more frequently thereby allowing the bacteria to tune atomic interactions for different temperature requirements without interfering with the overall enzyme function. PMID:26647400

  11. [Role of Human Orphan Esterases in Drug-induced Toxicity].

    PubMed

    Fukami, Tatsuki

    2015-01-01

    Esterases hydrolyze compounds containing ester, amide, and thioester bonds, causing prodrug activation or detoxification. Among esterases, carboxylesterases have been studied in depth due to their ability to hydrolyze a variety of drugs. However, there are several drugs for which the involved esterase(s) is unknown. We found that flutamide, phenacetin, rifamycins (rifampicin, rifabutin, and rifapentine), and indiplon are hydrolyzed by arylacetamide deacetylase (AADAC), which is highly expressed in human liver and gastrointestinal tissues. Flutamide hydrolysis is considered associated with hepatotoxicity. Phenacetin, a prodrug of acetaminophen, was withdrawn due to side effects such as methemoglobinemia and renal failure. It was demonstrated in vitro and in vivo using mice that AADAC is responsible for phenacetin hydrolysis, which leads to methemoglobinemia. In addition, it was shown that AADAC-mediated hydrolysis attenuates the cytotoxicity of rifamycins. Thus AADAC plays critical roles in drug-induced toxicity. Another orphan esterase, α/β hydrolase domain containing 10 (ABHD10), was found responsible for deglucuronidation of acyl-glucuronides including mycophenolic acid acyl-glucuronide and probenecid acyl-glucuronide. Because acyl-glucuronides appear associated with toxicity, ABHD10 would function as a detoxification enzyme. The roles of orphan esterases are becoming increasingly understood. Further studies will facilitate our knowledge of the pharmacologic and toxicological significance of orphan esterases in drug therapy. PMID:26521872

  12. Accumulation of recalcitrant xylan in mushroom-compost is due to a lack of xylan substituent removing enzyme activities of Agaricus bisporus.

    PubMed

    Jurak, Edita; Patyshakuliyeva, Aleksandrina; Kapsokalyvas, Dimitris; Xing, Lia; van Zandvoort, Marc A M J; de Vries, Ronald P; Gruppen, Harry; Kabel, Mirjam A

    2015-11-01

    The ability of Agaricus bisporus to degrade xylan in wheat straw based compost during mushroom formation is unclear. In this paper, xylan was extracted from the compost with water, 1M and 4M alkali. Over the phases analyzed, the remaining xylan was increasingly substituted with (4-O-methyl-)glucuronic acid and arabinosyl residues, both one and two arabinosyl residues per xylosyl residue remained. In the 1M and 4M KOH soluble solids of spent compost, 33 and 49 out of 100 xylosyl residues, respectively, were substituted. The accumulation of glucuronic acid substituents matched with the analysis that the two A. bisporus genes encoding for α-glucuronidase activity (both GH115) were not expressed in the A. bisporus mycelium in the compost during fruiting. Also, in a maximum likelihood tree it was shown that it is not likely that A. bisporus possesses genes encoding for the activity to remove arabinose from xylosyl residues having two arabinosyl residues. PMID:26256360

  13. Modular modification of xylan with UV-initiated thiol-ene reaction.

    PubMed

    Nurmi, Leena; Salminen, Reeta; Setälä, Harri

    2015-03-01

    Birch xylan was functionalized with various thiols through UV initiated radical thiol-ene reaction under mild conditions. Xylan was allylated through etherification with allyl glycidyl ether under alkaline conditions. The allylated xylan was then reacted with thiols containing varying functional groups: trimethylbenzyl mercaptan, dodecanethiol, thioglycolic acid, L-cysteine and cysteamine hydrochloride. The reactions were conducted under homogeneous conditions at room temperature, either in water (hydrophilic thiols) or in DMF (hydrophobic thiols). The effect of reaction parameters to the functionalization efficiency was studied, including, for example, thiol excess, thiol character, initiator amount and reaction mixture concentration. The reactions were fast and 100% conversion of allyl groups was reached in most cases, sometimes already within 10 min. Water as solvent resulted generally in faster reactions when compared to DMF, and it was possible to conduct the aqueous reaction even without added UV initiator. It was also possible to incorporate two functionalities simultaneously during one reaction into the xylan structure. PMID:25665780

  14. Hydrolysis by commercial enzyme mixtures of AFEX-treated corn fiber and isolated xylans

    SciTech Connect

    Hespell, R.B.; O`Bryan, P.J.; Bothast, R.J.; Moniruzzaman, M.

    1997-01-01

    Corn fiber is a coproduct produced during the corn wet-milling process and is similar to other high hemicellulose/cellulose-containing biomass such as grasses, straws, or bagasse, all of which represent potential fermentation feedstock for conversion into biofuels or other products. Corn fiber was subjected to ammonia-explosion (AFEX) treatment to increase degradability and then enzymatically digested with a combined mixture of commercial amylase, xylanase, and cellulose enzyme preparations. Whereas the starch and cellulose components were converted solely to glucose, oligosaccharides represented 30-40% of the xylan degradation products. This enzyme mixture also produced substantial oligosaccharides with xylans purified from corn fiber, corn germ, beech-wood, oatspelt, or wheat germ. Commercial xylan-degrading enzyme preparations containing xylanase, xylosidase, and arabinosidase activities were then used alone or in varying combinations to attempt to maximize degradation of these isolated xylans of differing chemical compositions. 25 refs., 5 tabs.

  15. Protein Acetylation and Acetyl Coenzyme A Metabolism in Budding Yeast

    PubMed Central

    Galdieri, Luciano; Zhang, Tiantian; Rogerson, Daniella; Lleshi, Rron

    2014-01-01

    Cells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation. PMID:25326522

  16. Investigating Mass Transport Limitations on Xylan Hydrolysis During Dilute Acid Pretreatment of Poplar

    SciTech Connect

    Mittal, Ashutosh; Pilath, Heid M.; Parent, Yves; Chatterjee, Siddharth G.; Donohoe, Bryon S.; Yarbrough, John M.; Himmel, Michael E.; Nimlos, Mark R.; Johnson, David K.

    2014-04-28

    Mass transport limitations could be an impediment to achieving high sugar yields during biomass pretreatment and thus be a critical factor in the economics of biofuels production. The objective of this work was to study the mass transfer restrictions imposed by the structure of biomass on the hydrolysis of xylan during dilute acid pretreatment of biomass. Mass transfer effects were studied by pretreating poplar wood at particle sizes ranging from 10 micrometers to 10 mm. This work showed a significant reduction in the rate of xylan hydrolysis in poplar when compared to the intrinsic rate of hydrolysis for isolated xylan that is possible in the absence of mass transfer. In poplar samples we observed no significant difference in the rates of xylan hydrolysis over more than two orders of magnitude in particle size. It appears that no additional mass transport restrictions are introduced by increasing particle size from 10 micrometers to 10 mm. This work suggests that the rates of xylan hydrolysis in biomass particles are limited primarily by the diffusion of hydrolysis products out of plant cell walls. A mathematical description is presented to describe the kinetics of xylan hydrolysis that includes transport of the hydrolysis products through biomass into the bulk solution. The modeling results show that the effective diffusion coefficient of the hydrolysis products in the cell wall is several orders of magnitude smaller than typical values in other applications signifying the role of plant cell walls in offering resistance to diffusion of the hydrolysis products.

  17. Production of cationic xylan-METAC copolymer as a flocculant for textile industry.

    PubMed

    Wang, Shoujuan; Hou, Qingxi; Kong, Fangong; Fatehi, Pedram

    2015-06-25

    Xylan is a part of hemicelluloses of woody materials and can be converted to value-added products such as flocculants for the textile industry. To assess the production of flocculants from hemicelluloses of woody materials, xylan was selected as a model and rendered cationic via copolymerization. In this study, the copolymerization reaction of xylan and [2-(methacryloyloxy) ethyl] trimethylammonium chloride (METAC) was optimized. The optimum parameters were 3mol/mol METAC/xylose, 3h reaction time, 80°C reaction temperature, pH 7 and 25g/L xylan concentration. The copolymer was characterized by a charge density analyzer, viscometer, gel permeation chromatography (GPC), light scattering instrument, Fourier transform infrared spectroscopy (FTIR) and an elemental analyzer. The application of the cationic xylan copolymer as a flocculant to decolorize the simulated reactive orange 16 azo-dye wastewater was evaluated. The results confirmed that, by having 160mg/L xylan-METAC concentration in the dye solution with the concentration of 100mg/L, 97.8% of dye could be removed. PMID:25839816

  18. Genetics of esterase isoenzymes in Malus.

    PubMed

    Manganaris, A G; Alston, F H

    1992-02-01

    Three main zones of esterase activity (EST-I, EST-III, EST-IV) identified in leaf extracts of cultivated apple and Malus species were determined by the genes EST-1, EST-3 and EST-4, respectively. In addition to earlier reported alleles of EST-1 (a, b) three further bands c, d and f were identified in the EST-I zone of which c was found to be determined by an allele, c. Two alleles, a, b, and a null allele were found for both the genes EST-3 and EST-4. Differences in allelic frequency were observed between cultivars, rootstocks and Malus species. Allele EST-1a was rare amongst the rootstocks. The examination of Malus species and derivatives showed a geographical relationship. Allele EST-1c was confined to species of Asian origin, and EST-1d was confined to American species. PMID:24202593

  19. The Role of the Plant-Specific ALTERED XYLOGLUCAN9 Protein in Arabidopsis Cell Wall Polysaccharide O-Acetylation1[OPEN

    PubMed Central

    Schultink, Alex; Naylor, Dan; Dama, Murali; Pauly, Markus

    2015-01-01

    A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway. PMID:25681330

  20. Suppression of Dwarf and irregular xylem Phenotypes Generates Low-Acetylated Biomass Lines in Arabidopsis1[OPEN

    PubMed Central

    Lefebvre, Valérie; Ducamp, Aloïse; Trouverie, Jacques; Fortabat, Marie-Noëlle; Guillebaux, Alexia; Baldy, Aurélie; Naquin, Delphine; Lapierre, Catherine; Mouille, Gregory; Horlow, Christine; Durand-Tardif, Mylène

    2015-01-01

    eskimo1-5 (esk1-5) is a dwarf Arabidopsis (Arabidopsis thaliana) mutant that has a constitutive drought syndrome and collapsed xylem vessels, along with low acetylation levels in xylan and mannan. ESK1 has xylan O-acetyltransferase activity in vitro. We used a suppressor strategy on esk1-5 to screen for variants with wild-type growth and low acetylation levels, a favorable combination for ethanol production. We found a recessive mutation in the KAKTUS (KAK) gene that suppressed dwarfism and the collapsed xylem character, the cause of decreased hydraulic conductivity in the esk1-5 mutant. Backcrosses between esk1-5 and two independent knockout kak mutants confirmed suppression of the esk1-5 effect. kak single mutants showed larger stem diameters than the wild type. The KAK promoter fused with a reporter gene showed activity in the vascular cambium, phloem, and primary xylem in the stem and hypocotyl. However, suppression of the collapsed xylem phenotype in esk1 kak double mutants was not associated with the recovery of cell wall O-acetylation or any major cell wall modifications. Therefore, our results indicate that, in addition to its described activity as a repressor of endoreduplication, KAK may play a role in vascular development. Furthermore, orthologous esk1 kak double mutants may hold promise for ethanol production in crop plants. PMID:25888614

  1. 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

  2. A DIRECT METHOD TO ASSAY NEUROTOXIC ESTERASE ACTIVITY

    EPA Science Inventory

    A direct photometric method for assaying neurotoxic esterase (NTE) activity of chicken brain microsomal preparation has been developed using 4-nitrophenyl esters as substrates. Paired samples of the microsomal preparation were preincubated for 20 min. with paraoxon plus either (a...

  3. Complete genome sequence of the xylan-degrading Mucilaginibacter sp. strain PAMC26640 isolated from an Arctic lichen.

    PubMed

    Oh, Tae-Jin; Han, So-Ra; Kang, Seunghyun; Park, Hyun; Kim, Augustine Yonghwi

    2016-06-10

    Mucilaginibacter sp. PAMC26640 is a xylan-degrading bacterium isolated from the Arctic lichen Stereocaulon sp. Here, we present the first complete genome sequence of Mucilaginibacter sp. strain PAMC26640, which contains several genes involved in xylan utilization. This genome information provides new insights into the genetic basis of its physiology and further analysis of key metabolic genes related to the xylan degradation pathway. PMID:27080447

  4. The catalytic triad of the influenza C virus glycoprotein HEF esterase: characterization by site-directed mutagenesis and functional analysis.

    PubMed

    Pleschka, S; Klenk, H D; Herrler, G

    1995-10-01

    Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-KB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex. PMID:7595356

  5. The world of protein acetylation.

    PubMed

    Drazic, Adrian; Myklebust, Line M; Ree, Rasmus; Arnesen, Thomas

    2016-10-01

    Acetylation is one of the major post-translational protein modifications in the cell, with manifold effects on the protein level as well as on the metabolome level. The acetyl group, donated by the metabolite acetyl-coenzyme A, can be co- or post-translationally attached to either the α-amino group of the N-terminus of proteins or to the ε-amino group of lysine residues. These reactions are catalyzed by various N-terminal and lysine acetyltransferases. In case of lysine acetylation, the reaction is enzymatically reversible via tightly regulated and metabolism-dependent mechanisms. The interplay between acetylation and deacetylation is crucial for many important cellular processes. In recent years, our understanding of protein acetylation has increased significantly by global proteomics analyses and in depth functional studies. This review gives a general overview of protein acetylation and the respective acetyltransferases, and focuses on the regulation of metabolic processes and physiological consequences that come along with protein acetylation. PMID:27296530

  6. Direct xylan conversion into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma antarctica PYCC 5048(T).

    PubMed

    Faria, Nuno Torres; Marques, Susana; Fonseca, César; Ferreira, Frederico Castelo

    2015-04-01

    Mannosylerythritol lipids (MEL) are glycolipid biosurfactants, produced by Pseudozyma spp., with increasing commercial interest. While MEL can be produced from d-glucose and d-xylose, the direct conversion of the respective lignocellulosic polysaccharides, cellulose and xylan, was not reported yet. The ability of Pseudozyma antarctica PYCC 5048(T) and Pseudozyma aphidis PYCC 5535(T) to use cellulose (Avicel(®)) and xylan (beechwood) as carbon and energy source has been assessed along with their capacity of producing cellulolytic and hemicellulolytic enzymes, toward a consolidated bioprocess (CBP) for MEL production. The yeasts assessed were neither able to grow in medium containing Avicel(®) nor produce cellulolytic enzymes under the conditions tested. On contrary, both yeasts were able to efficiently grow in xylan, but MEL production was only detected in P. antarctica PYCC 5048(T) cultures. MEL titers reached 1.3g/l after 10 days in batch cultures with 40g/l xylan, and 2.0g/l in fed-batch cultures with xylan feeding (additional 40g/l) at day 4. High levels of xylanase activities were detected in xylan cultures, reaching 47-62U/ml (31-32U/mg) at 50°C, and still exhibiting more than 10U/ml under physiological temperature (28°C). Total β-xylosidase activities, displayed mainly as wall-bounded and extracellular activity, accounted for 0.154 and 0.176U/ml in P. antarctica PYCC 5048(T) and P. aphidis PYCC 5535(T) cultures, respectively. The present results demonstrate the potential of Pseudozyma spp. for using directly a fraction of lignocellulosic biomass, xylan, and combining in the same bioprocess the production of xylanolytic enzymes with MEL production. PMID:25765311

  7. Xylan derivatives and their application potential - mini-review of own results.

    PubMed

    Petzold-Welcke, Katrin; Schwikal, Katrin; Daus, Stephan; Heinze, Thomas

    2014-01-16

    The chemical modification of xylan is a promising path to new biopolymer ethers and esters with specific properties depending on the functional groups, the degree of substitution, and the substitution pattern. The reaction of 4-O-methylglucuronoxylan (GX) from birch with sodium monochloroacetate and 2,3-epoxypropyltrimethylammonium chloride in aqueous sodium hydroxide/slurry medium is described. The influence of the conditions of activation on product structure and properties are discussed in some detail. Methylation of GX was investigated under completely heterogeneous conditions or starting with dissolved polymer using methyl halides as reagents in the presence of NaOH. An activation of the biopolymer has been carried out before the reaction to enhance the accessibility of the reagents. Furthermore, novel xylan esters were efficiently synthesized by conversion of the hemicellulose with furan- and pyroglutamic acid as well as ibuprofen and N,N'-carbonyldiimidazole as activating agent under homogeneous conditions in dimethyl sulfoxide. This conditions are also appropriate to synthesize novel xylan ester containing xylan-4-[N,N,N-trimethylammonium]butyrate chloride moieties. Homogeneous syntheses of xylan sulfates could be carried out in a N,N-dimethylformamide (DMF)/LiCl as solvent applying sulfur trioxide complexes with DMF or pyridine. Advanced analytical techniques including NMR spectroscopy, HPLC, scanning electron microscopy, rheology, measurements of turbidity and surface tension allow description of structure-property-relationships; selected results will be briefly discussed. Xylan esters may form spherical nanoparticles of a size down to 60 nm and a narrow particle size distribution applying a simple dialysis process and may be used for drug delivery applications. For cationic xylan derivatives a wide range of applications as paper strength additives, flocculation aids, and antimicrobial agents are proposed. PMID:24188841

  8. 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

  9. An esterase gene from Lactobacillus casei cotranscribed with genes encoding a phosphoenolpyruvate:sugar phosphotransferase system and regulated by a LevR-like activator and sigma54 factor.

    PubMed

    Yebra, María J; Viana, Rosa; Monedero, Vicente; Deutscher, Josef; Pérez-Martínez, Gaspar

    2004-01-01

    A new esterase-encoding gene was found in the draft genome sequence of Lactobacillus casei BL23 (CECT5275). It is located in an operon together with genes encoding the EIIA, EIIB, EIIC, and EIID proteins of a mannose class phosphoenolpyruvate:sugar phosphotransferase system. After overproduction in Escherichia coli and purification, the esterase could hydrolyze acetyl sugars, hence the operon was named esu for esterase-sugar uptake genes. Upstream of the genes encoding the EII components (esuABCD) and the esterase (esuE), two genes transcribed in the opposite sense were found which encode a Bacillus subtilis LevR-like transcriptional activator (esuR) and a sigma54-like transcriptional factor (rpoN). As compared with the wild-type strain, elevated fructose phosphorylation was detected in L. casei mutants constitutively expressing the esu operon. However, none of the many sugars tested could induce the esu operon. The fact that EsuE exhibits esterase activity on acetyl sugars suggests that this operon could be involved in the uptake and metabolism of esterified sugars. Expression of the esu operon is similar to that of the B. subtilis lev operon: it contains a -12,-24 consensus promoter typical of sigma54-regulated genes, and EsuR and RpoN are essential for its transcription which is negatively regulated by EIIB(Esu). The esuABCDE transcription unit represents the first sigma54-regulated operon in lactobacilli. Furthermore, replacement of His852 in the phosphoenolpyruvate:sugar phosphotransferase system regulation domain II of EsuR with Ala indicated that the transcription activator function of EsuR is inhibited by EIIB(Esu)-mediated phosphorylation at His852. PMID:15925903

  10. Hydro-liquefaction of microcrystalline cellulose, xylan and industrial lignin in different supercritical solvents.

    PubMed

    Li, Qingyin; Liu, Dong; Hou, Xulian; Wu, Pingping; Song, Linhua; Yan, Zifeng

    2016-11-01

    The influences of solvent on hydro-liquefaction of cellulose, xylan, and lignin were investigated using micro-autoclave. The maximum conversion and bio-oil yield obtained from cellulose and xylan liquefaction were achieved in methanol, whereas similar liquefaction characteristics of lignin were observed in methanol and ethanol. The molecular simulation of interactions between solvents and subcomponents indicated that methanol and ethanol were highly miscible with raw materials. GC-MS and FT-ICR MS characterization revealed that the chemical compositions of liquid products highly depended on the utilized feedstocks. Esters, ketones, and aldehydes were mainly produced from cellulose and xylan conversion, whereas aromatic compounds were primarily derived from lignin conversion. EA results showed that methanol favored the hydrogenation and deoxygenation, resulting in the heating value increased. It could be concluded that the oil quality was highly improved in supercritical methanol. PMID:27497089

  11. An esterase from the basidiomycete Pleurotus sapidus hydrolyzes feruloylated saccharides.

    PubMed

    Linke, Diana; Matthes, Rene; Nimtz, Manfred; Zorn, Holger; Bunzel, Mirko; Berger, Ralf G

    2013-08-01

    Investigating the secretion of esterases by the basidiomycetous fungus Pleurotus sapidus in a Tween 80-rich nutrient medium, an enzyme was discovered that hydrolyzed the ester bond of feruloylated saccharides. The enzyme was purified by ion exchange and size exclusion chromatography. Polyacrylamide gel electrophoresis analysis showed a monomeric protein of about 55 kDa. The complete coding sequence with an open reading frame of 1,665 bp encoded a protein (Est1) consisting of 554 amino acids. The enzyme showed no significant homology to any published feruloyl esterase sequences, but possessed putative conserved domains of the lipase/esterase superfamily. Substrate specificity studies classified the new enzyme as type-A feruloyl esterase, hydrolyzing methyl ferulate, methyl sinapate, and methyl p-coumarate but no methyl caffeate. The enzyme had a pH optimum of 6 and a temperature optimum at 50 °C. Ferulic acid was efficiently released from ferulated saccharides, and the feruloyl esterase exhibited moderate stability in biphasic systems (50 % toluene or tert-butylmethyl ether). PMID:23203636

  12. Direct conversion of xylan to butanol by a wild-type Clostridium species strain G117.

    PubMed

    Yan, Yu; Basu, Anindya; Li, Tinggang; He, Jianzhong

    2016-08-01

    Lignocellulosic biomass has great potential for use as a carbon source for the production of second-generation biofuels by solventogenic bacteria. Here we describe the production of butanol by a newly discovered wild-type Clostridium species strain G117 with xylan as the sole carbon source for fermentation. Strain G117 produced 0.86 ± 0.07 g/L butanol and 53.4 ± 0.05 mL hydrogen directly from 60 g/L xylan provided that had undergone no prior enzymatic hydrolysis. After process optimization, the amount of butanol produced from xylan was increased to 1.24 ± 0.37 g/L. In contrast to traditional acetone-butanol-ethanol (ABE) solventogenic fermentation, xylan supported fermentation in strain G117 and negligible amount of acetone was produced. The expression of genes normally associated with acetone production (adc and ctfB2) were down-regulated compared to xylose fed cultures. This lack of acetone production may greatly simplify downstream separation process. Moreover, higher amount of butanol (2.94 g/L) was produced from 16.99 g/L xylo-oligosaccharides, suggesting a major role for strain G117 in butanol production from xylan and its oligosaccharides. The unique ability of strain G117 to produce a considerable amount of butanol directly from xylan without producing undesirable fermentation byproducts opens the door to the possibility of cost-effective biofuels production in a single step. Biotechnol. Bioeng. 2016;113: 1702-1710. © 2016 Wiley Periodicals, Inc. PMID:26803924

  13. Role of UDP-Glucuronic Acid Decarboxylase in Xylan Biosynthesis in Arabidopsis.

    PubMed

    Kuang, Beiqing; Zhao, Xianhai; Zhou, Chun; Zeng, Wei; Ren, Junli; Ebert, Berit; Beahan, Cherie T; Deng, Xiaomei; Zeng, Qingyin; Zhou, Gongke; Doblin, Monika S; Heazlewood, Joshua L; Bacic, Antony; Chen, Xiaoyang; Wu, Ai-Min

    2016-08-01

    UDP-xylose (UDP-Xyl) is the Xyl donor used in the synthesis of major plant cell-wall polysaccharides such as xylan (as a backbone-chain monosaccharide) and xyloglucan (as a branching monosaccharide). The biosynthesis of UDP-Xyl from UDP-glucuronic acid (UDP-GlcA) is irreversibly catalyzed by UDP-glucuronic acid decarboxylase (UXS). Until now, little has been known about the physiological roles of UXS in plants. Here, we report that AtUXS1, AtUXS2, and AtUXS4 are located in the Golgi apparatus whereas AtUXS3, AtUXS5, and AtUXS6 are located in the cytosol. Although all six single AtUXS T-DNA mutants and the uxs1 usx2 uxs4 triple mutant show no obvious phenotype, the uxs3 uxs5 uxs6 triple mutant has an irregular xylem phenotype. Monosaccharide analysis showed that Xyl levels decreased in uxs3 uxs5 uxs6 and linkage analysis confirmed that the xylan content in uxs3 xus5 uxs6 declined, indicating that UDP-Xyl from cytosol AtUXS participates in xylan synthesis. Gel-permeation chromatography showed that the molecular weight of non-cellulosic polysaccharides in the triple mutants, mainly composed of xylans, is lower than that in the wild type, suggesting an effect on the elongation of the xylan backbone. Upon saccharification treatment stems of the uxs3 uxs5 uxs6 triple mutants released monosaccharides with a higher efficiency than those of the wild type. Taken together, our results indicate that the cytosol UXS plays a more important role than the Golgi-localized UXS in xylan biosynthesis. PMID:27179920

  14. Transcriptomic Analysis of Xylan Utilization Systems in Paenibacillus sp. Strain JDR-2

    PubMed Central

    Sawhney, Neha; Crooks, Casey; St. John, Franz

    2014-01-01

    Xylans, including methylglucuronoxylans (MeGXn) and methylglucuronoarabinoxylans (MeGAXn), are the predominant polysaccharides in hemicellulose fractions of dicots and monocots available for conversion to biofuels and chemicals. Paenibacillus sp. strain JDR-2 (Pjdr2) efficiently depolymerizes MeGXn and MeGAXn and assimilates the generated oligosaccharides, resulting in efficient saccharification and subsequent metabolism of these polysaccharides. A xylan utilization regulon encoding a cell-associated GH10 (glycoside hydrolase family 10) endoxylanase, transcriptional regulators, ABC (ATP binding cassette) transporters, an intracellular GH67 α-glucuronidase, and other glycoside hydrolases contributes to complete metabolism. This GH10/GH67 system has been proposed to account for preferential utilization of xylans compared to free oligo- and monosaccharides. To identify additional genes contributing to MeGXn and MeGAXn utilization, the transcriptome of Pjdr2 has been sequenced following growth on each of these substrates as well as xylose and arabinose. Increased expression of genes with different substrates identified pathways common or unique to the utilization of MeGXn or MeGAXn. Coordinate upregulation of genes comprising the GH10/GH67 xylan utilization regulon is accompanied with upregulation of genes encoding a GH11 endoxylanase and a GH115 α-glucuronidase, providing evidence for a novel complementary pathway for processing xylans. Elevated expression of genes encoding a GH43 arabinoxylan arabinofuranohydrolase and an arabinose ABC transporter on MeGAXn but not on MeGXn supports a process in which arabinose may be removed extracellularly followed by its rapid assimilation. Further development of Pjdr2 for direct conversion of xylans to targeted products or introduction of these systems into fermentative strains of related bacteria may lead to biocatalysts for consolidated bioprocessing of hemicelluloses released from lignocellulose. PMID:25527555

  15. NEUROPATHY TARGET ESTERASE INHIBITION BY ORGANOPHOSPHORUS ESTERS IN HUMAN NEUROBLASTOMA CELLS

    EPA Science Inventory

    Certain organophosphorus compounds (OPs) produce a delayed neuropathy (OPIDN) in man and some animal species. apability to cause OPIDN is generally predicted in animal models by early and irreversible inhibition of neuropathy target esterase (NTE, neurotoxic esterase) . In this s...

  16. In Vitro Antioxidant, Anticoagulant and Antimicrobial Activity and in Inhibition of Cancer Cell Proliferation by Xylan Extracted from Corn Cobs

    PubMed Central

    Melo-Silveira, Raniere Fagundes; Fidelis, Gabriel Pereira; Costa, Mariana Santana Santos Pereira; Telles, Cinthia Beatrice Silva; Dantas-Santos, Nednaldo; de Oliveira Elias, Susana; Ribeiro, Vanessa Bley; Barth, Afonso Luis; Macedo, Alexandre José; Leite, Edda Lisboa; Rocha, Hugo Alexandre Oliveira

    2012-01-01

    Xylan is one of most abundant polymer after cellulose. However, its potential has yet to be completely recognized. Corn cobs contain a considerable reservoir of xylan. The aim of this work was to study some of the biological activities of xylan obtained from corn cobs after alkaline extraction enhanced by ultrasonication. Physical chemistry and infrared analyses showed 130 kDa heteroxylan containing mainly xylose:arabinose: galactose:glucose (5.0:1.5:2.0:1.2). Xylan obtained exhibited total antioxidant activity corresponding to 48.5 mg of ascorbic acid equivalent/g of xylan. Furthermore, xylan displayed high ferric chelating activity (70%) at 2 mg/mL. Xylan also showed anticoagulant activity in aPTT test. In antimicrobial assay, the polysaccharide significantly inhibited bacterial growth of Klebsiella pneumoniae. In a test with normal and tumor human cells, after 72 h, only HeLa tumor cell proliferation was inhibited (p < 0.05) in a dose-dependent manner by xylan, reaching saturation at around 2 mg/mL, whereas 3T3 normal cell proliferation was not affected. The results suggest that it has potential clinical applications as antioxidant, anticoagulant, antimicrobial and antiproliferative compounds. PMID:22312261

  17. Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass (Panicum virgatum, L.) is a potential renewable source of carbohydrates for use in microbial conversion to biofuels. Xylan comprises approximately 30% of the switchgrass cell wall. To understand the limitations of commercial enzyme mixtures, alkali-extracted, isolated switchgrass xylan w...

  18. Three-dimensional structure of homodimeric cholesterol esterase-ligand complex at 1.4 Å resolution

    SciTech Connect

    Pletnev, V.; Addlagatta, A.; Wawrzak, Z.; Duax, W.

    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 and 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.

  19. Differential effect of the serine protease inhibitor phenyl methyl sulfonyl fluoride on cytochemically detectable esterases in human leucocytes and platelets.

    PubMed

    Dufer, J; Trentesaux, C; Desplaces, A

    1984-01-01

    Esterases of human leucocytes and platelets were studied by cytochemical methods. The aim of the study was to clarify the cellular distribution and possible nature of esterases types differing in their substrate specificity and/or their inhibitor sensitivity. 3 substrates (alpha-naphthyl acetate: ANA; naphthol AS-D chloroacetate: NASDCA; and N-acetyl DL-alanine alpha-naphthyl ester: NACALA) were used and the effects of 2 inhibitors (sodium fluoride and the serine protease inhibitor phenyl methyl sulfonyl fluoride: PMSF) were evaluated. 4 enzyme types were described: Type I, present in granulocytes, was detected using NASDCA and NACALA and was resistant to fluoride but sensitive to PMSF. Other types were detected using ANA as substrate. Type II, present in monocytes, was inhibited by both fluoride and PMSF. Type III, present in platelets and plasma cells, was inhibited by fluoride but resistant to PMSF. Type IV, present in lymphocytes, was resistant to both fluoride and PMSF. The specific aims and possible areas for application of these results are discussed. PMID:6364322

  20. 2,2-Disubstituted 4-acylthio-3-oxobutyl groups as esterase- and thermolabile protecting groups of phosphodiesters.

    PubMed

    Kiuru, Emilia; Ahmed, Zafar; Lönnberg, Harri; Beigelman, Leonid; Ora, Mikko

    2013-02-01

    Five different 2,2-disubstituted 4-acylthio-3-oxobutyl groups have been introduced as esterase-labile phosphodiester protecting groups that additionally are thermolabile. The phosphotriesters 1-3 were prepared to determine the rate of the enzymatic and nonenzymatic removal of such groups at 37 °C and pH 7.5 by HPLC-ESI-MS. Additionally, (1)H NMR spectroscopic monitoring was used for structural characterization of the intermediates and products. When treated with hog liver esterase, these groups were removed by enzymatic deacylation followed by rapid chemical cyclization to 4,4-disubstituted dihydrothiophen-3(2H)-one. The rate of the enzymatic deprotection could be tuned by the nature of the 4-acylthio substituent, the benzoyl group and acetyl groups being removed 50 and 5 times as fast as the pivaloyl group. No alkylation of glutathione could be observed upon the enzymatic deprotection. The half-life for the nonenzymatic deprotection varied from 0.57 to 35 h depending on the electronegativity of the 2-substituents and the size of the acylthio group. The acyl group evidently migrates from the sulfur atom to C3-gem-diol obtained by hydration of the keto group and the exposed mercapto group attacks on C1 resulting in departure of the protecting group as 4,4-disubstituted 3-acyloxy-4,5-dihydrothiophene with concomitant release of the desired phosphodiester. PMID:23272806

  1. Pregastric esterase in milk sham fed to adult jersey steers.

    PubMed

    Leidy, R B; Russell, R W; Wise, G H

    1975-04-01

    Pregastric esterase activity was detected in reconstituted nonfat milk sham fed from a nipple pail to two 4-yr-old rumen-fistualted steers. Lipolytic activity, determined in a medium containing 5% tri-n-butyrin, averaged 8.6 plus or minus .4 lipase units. Further assays, in which activitiy was measured by free fatty acids released from a condensed milk substrate, averaged 166.9 plus or minus 9.2 mumol. These values are higher than those noted for young calves, indicating that secretion of pregastric esterase may persist in cattle beyond calfhood. Esterase activity in one of the steers fed whole milk until he was 2 yr of age showed no marked residual effect of earlier intake of milk fat. PMID:1127162

  2. Facile fabrication and selective detection for cysteine of xylan/Au nanoparticles composite.

    PubMed

    Luo, Yuqiong; Shen, Zuguang; Liu, Pai; Zhao, Lihong; Wang, Xiaoying

    2016-04-20

    This work reported a facile and green method to prepare highly stable and uniformly distributed Au nanoparticles (AuNPs), using biopolymer xylan as stabilizing and reducing agent. Full characterizations were performed and the results revealed that AuNPs were well dispersed with the diameters of 10-30nm. The optimal condition was as follows: the ratio of xylan to HAuCl4 was 150mg:15mg, reaction temperature was 80°C and reaction time was 40min. The xylan/AuNPs composite exhibited highly selective and sensitive sensing of cysteine in aqueous solution, it could distinguish cysteine among dozens kinds of amino acids, and the limit of detection (LOD) for cysteine was calculated as 0.57μM. Besides, the xylan/AuNPs composite was applied for Cys detection in human serum. This study provides a new way for high-value utilization of the rich biomass resource and a cheap, rapid and simple method for Cys detection in real biological samples. PMID:26876835

  3. Arabidopsis GUX Proteins Are Glucuronyltransferases Responsible for the Addition of Glucuronic Acid Side Chains onto Xylan

    EPA Science Inventory

    Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of β-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that muta...

  4. Complete genome sequence of the xylan-degrading subseafloor bacterium Microcella alkaliphila JAM-AC0309.

    PubMed

    Kurata, Atsushi; Hirose, Yuu; Misawa, Naomi; Wakazuki, Sachiko; Kishimoto, Noriaki; Kobayashi, Tohru

    2016-03-10

    Here we report the complete genome sequence of Microcella alkaliphila JAM-AC0309, which was newly isolated from the deep subseafloor core sediment from offshore of the Shimokita Peninsula of Japan. An array of genes related to utilization of xylan in this bacterium was identified by whole genome analysis. PMID:26808869

  5. In situ enzyme aided adsorption of soluble xylan biopolymers onto cellulosic material.

    PubMed

    Chimphango, Annie F A; Görgens, J F; van Zyl, W H

    2016-06-01

    The functional properties of cellulose fibers can be modified by adsorption of xylan biopolymers. The adsorption is improved when the degree of biopolymers substitution with arabinose and 4-O-methyl-glucuronic acid (MeGlcA) side groups, is reduced. α-l-Arabinofuranosidase (AbfB) and α-d-glucuronidase (AguA) enzymes were applied for side group removal, to increase adsorption of xylan from sugarcane (Saccharum officinarum L) bagasse (BH), bamboo (Bambusa balcooa) (BM), Pinus patula (PP) and Eucalyptus grandis (EH) onto cotton lint. The AguA treatment increased the adsorption of all xylans by up to 334%, whereas, the AbfB increased the adsorption of the BM and PP by 31% and 44%, respectively. A combination of AguA and AbfB treatment increased the adsorption, but to a lesser extent than achieved with AguA treatment. This indicated that the removal of the glucuronic acid side groups provided the most significant increase in xylan adsorption to cellulose, in particular through enzymatic treatment. PMID:27083357

  6. Digestive and physiological effects of a wheat bran extract, arabino-xylan-oligosaccharide, in breakfast cereal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We assessed whether a wheat bran extract containing arabino-xylan-oligosaccharide (AXOS) elicited a prebiotic effect and influenced other physiologic parameters when consumed in ready-to-eat cereal at two dose levels. This double-blind, randomized, controlled, crossover trial evaluated the effects o...

  7. Synthesis and characterization of carboxymethyl xylan-g-poly(propylene oxide) and its application in films.

    PubMed

    Peng, Pai; Zhai, Meizhi; She, Diao; Gao, Yuefang

    2015-11-20

    Carboxymethyl xylan-g-poly(propylene oxide) (CMX-g-PPO) was successfully synthesized by grafting poly(propylene oxide) chains onto xylan from bamboo using the Al(Oi-Pr)3 initiated ring-opening polymerization of propylene oxides, followed by carboxymethylation with sodium chloroacetate under microwave irradiation. The synthesized CMX-g-PPO was well characterized by FT-IR, (13)C NMR, and AFM. The AFM imaging showed that the average sizes of xylan were 422.1×67.4×1.2nm, while the average sizes of grafting branches PPO were 128.0×38.5×5.1nm, which firstly provided an irrefutable and visual evidence for the structure of grafted xylan at single molecular level. Subsequently, a serial of CMX-g-PPO/CS films were prepared without addition of any plasticizers. The surface morphologies, wettability, water vapor barrier properties, mechanical properties, and thermal stabilities of the obtained films were characterized and compared with those of the control films by AFM, contact angle, WVP, tensile testing, and TGA, respectively. PMID:26344263

  8. Xylan-based temperature/pH sensitive hydrogels for drug controlled release.

    PubMed

    Gao, Cundian; Ren, Junli; Zhao, Cui; Kong, Weiqing; Dai, Qingqing; Chen, Qifeng; Liu, Chuanfu; Sun, Runcang

    2016-10-20

    Xylan-based temperature/pH sensitive hydrogels were prepared by the crosslinking copolymerization of xylan with N-isopropylacrylamide (NIPAm) and acrylic acid (AA) using N,Ń-methylenebis-acrylamide (MBA) as a cross-linker and 2,2-dimethoxy-2-phenylacetophenone as a photoinitiator via ultraviolet irradiation. The influence of the NIPAm, AA and MBA amount on properties of xylan-based hydrogels was discussed. The morphology and interactions of hydrogels were characterized by SEM and FTIR. The lower critical solution temperature (LCST) of hydrogels was investigated by DSC. The results indicated that the LCST of hydrogels emerged at around 34°C and increased with increasing the AA content. The drug encapsulation efficiency of as-prepared hydrogels reached to 97.60% and the cumulative release rate of acetylsalicylic acid was 90.12% and 26.35% in the intestinal and gastric fluid, respectively. Xylan-based hydrogels were proved to be biocompatible with NIH3T3 cell by MTT assay and showed the promising application as drug carriers for the intestinal-targeted oral drug delivery. PMID:27474557

  9. Acetyl transfer in arylamine metabolism

    PubMed Central

    Booth, J.

    1966-01-01

    1. N-Hydroxyacetamidoaryl compounds (hydroxamic acids) are metabolites of arylamides, and an enzyme that transfers the acetyl group from these derivatives to arylamines has been found in rat tissues. The reaction products were identified by thin-layer chromatography and a spectrophotometric method, with 4-amino-azobenzene as acetyl acceptor, was used to measure enzyme activity. 2. The acetyltransferase was in the soluble fraction of rat liver, required a thiol for maximum activity and had a pH optimum between 6·0 and 7·5. 3. The soluble fractions of various rat tissues showed decreasing activity in the following order: liver, adrenal, kidney, lung, spleen, testis, heart; brain was inactive. 4. With the exception of aniline and aniline derivatives all the arylamines tested were effective as acetyl acceptors but aromatic compounds with side-chain amino groups were inactive. 5. The N-hydroxyacetamido derivatives of 2-naphthylamine, 4-amino-biphenyl and 2-aminofluorene were active acetyl donors but N-hydroxyacetanilide showed only slight activity. Acetyl-CoA was not a donor. 6. Some properties of the enzyme are compared with those of other acetyltransferases. PMID:5969287

  10. Fatal Intoxication with Acetyl Fentanyl.

    PubMed

    Cunningham, Susan M; Haikal, Nabila A; Kraner, James C

    2016-01-01

    Among the new psychoactive substances encountered in forensic investigations is the opioid, acetyl fentanyl. The death of a 28-year-old man from recreational use of this compound is reported. The decedent was found in the bathroom of his residence with a tourniquet secured around his arm and a syringe nearby. Postmortem examination findings included marked pulmonary and cerebral edema and needle track marks. Toxicological analysis revealed acetyl fentanyl in subclavian blood, liver, vitreous fluid, and urine at concentrations of 235 ng/mL, 2400 ng/g, 131 ng/mL, and 234 ng/mL, respectively. Acetyl fentanyl was also detected in the accompanying syringe. Death was attributed to recreational acetyl fentanyl abuse, likely through intravenous administration. The blood acetyl fentanyl concentration is considerably higher than typically found in fatal fentanyl intoxications. Analysis of this case underscores the need for consideration of a wide range of compounds with potential opioid-agonist activity when investigating apparent recreational drug-related deaths. PMID:26389815

  11. Insight into Glycoside Hydrolases for Debranched Xylan Degradation from Extremely Thermophilic Bacterium Caldicellulosiruptor lactoaceticus

    PubMed Central

    Jia, Xiaojing; Mi, Shuofu; Wang, Jinzhi; Qiao, Weibo; Peng, Xiaowei; Han, Yejun

    2014-01-01

    Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 α-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80°C and pH 6.5, as 75°C and pH 6.5 for Agu67A. Xyn10A had good stability at 75°C, 80°C, and pH 4.5–8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus. PMID:25184498

  12. Birch pulp xylan works as a food hydrocolloid in acid milk gels and is fermented slowly in vitro.

    PubMed

    Rosa-Sibakov, Natalia; Hakala, Terhi K; Sözer, Nesli; Nordlund, Emilia; Poutanen, Kaisa; Aura, Anna-Marja

    2016-12-10

    The objective was to evaluate the potential of birch xylan as a food hydrocolloid and dietary fibre. High-molecular weight xylan was isolated from birch kraft pulp by alkaline extraction, and enzymatically hydrolysed. Fermentability of xylans was evaluated using an in vitro colon model and performance as a hydrocolloid was studied in low-fat acid milk gels (1.5% and 3% w/w). Texture of the gels and water holding capacity of xylans were compared with inulin, fructooligosaccharide and xylooligosaccharide. Xylans showed slower fermentation rate by faecal microbiota than the references. Xylan-enriched acid milk gels (3% w/w) had improved water holding capacity (over 2-fold) and showed lower spontaneous syneresis, firmness and elasticity when compared to control (no hydrocolloids) or to references. In conclusion, birch xylan improved texture of low-fat acid milk gel applications, and the slow in vitro fermentation rate predicts lower incidence of intestinal discomfort in comparison to the commercial references. PMID:27577922

  13. A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A.

    PubMed Central

    Boraston, A B; Tomme, P; Amandoron, E A; Kilburn, D G

    2000-01-01

    The C-terminal module of xylanase 10A from Streptomyces lividans is a family 13 carbohydrate-binding module (CBM13). CBM13 binds mono- and oligo-saccharides with association constants of approximately 1x10(2) M(-1)-1x10(3) M(-1). It appears to be specific only for pyranose sugars. CBM13 binds insoluble and soluble xylan, holocellulose, pachyman, lichenan, arabinogalactan and laminarin. The association constant for binding to soluble xylan is (6.2+/-0. 6)x10(3)/mol of xylan polymer. Site-directed mutation indicates the involvement of three functional sites on CBM13 in binding to soluble xylan. The sites are similar in sequence, and are predicted to have similar structures, to the alpha, beta and gamma sites of ricin toxin B-chain, which is also in family 13. The affinity of a single binding site on CBM13 for soluble xylan is only approximately (0. 5+/-0.1)x10(3)/mol of xylan. The binding of CBM13 to soluble xylan involves additive and co-operative interactions between the three binding sites. This mechanism of binding has not previously been reported for CBMs binding polysaccharides. CBM13 is the first bacterial module from family 13 to be described in detail. PMID:10970811

  14. Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium saccharolyticum through Targeted Gene Deletions

    PubMed Central

    Podkaminer, Kara K.; Guss, Adam M.; Trajano, Heather L.; Hogsett, David A.

    2012-01-01

    The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C5 and C6 sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of β-xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum. PMID:23023741

  15. Characterization of xylan utilization and discovery of a new endoxylanase in Thermoanaerobacterium saccharolyticum through targeted gene deletions

    SciTech Connect

    Podkaminer, Kara K; Guss, Adam M; McKenzie, Heather; Hogsett, David; Lynd, Lee R

    2012-01-01

    The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C(5) and C(6) sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of -xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.

  16. 9-O-Acetylation of sialomucins: a novel marker of murine CD4 T cells that is regulated during maturation and activation.

    PubMed

    Krishna, M; Varki, A

    1997-06-01

    Terminal sialic acids on cell surface glycoconjugates can carry 9-O-acetyl esters. For technical reasons, it has previously been difficult to determine their precise distribution on different cell types. Using a recombinant soluble form of the Influenza C virus hemagglutinin-esterase as a probe for 9-O-acetylated sialic acids, we demonstrate here their preferential expression on the CD4 T cell lineage in normal B10.A mouse lymphoid organs. Of total thymocytes, 8-10% carry 9-O-acetylation; the great majority of these are the more mature PNA-, HSA-, and TCRhi medullary cells. While low levels of 9-O-acetylation are seen on some CD4/CD8 double positive (DP) and CD8 single positive (SP) cells, high levels are present primarily on 80- 85% of CD4 SP cells. Correlation with CD4 and CD8 levels suggests that 9-O-acetylation appears as an early differentiation marker as cells mature from the DP to the CD4 SP phenotype. This high degree of 9-O-acetylation is also present on 90-95% of peripheral spleen and lymph node CD4 T cells. In contrast, only a small minority of CD8 T cells and B cells show such levels of 9-O-acetylation. Among mature peripheral CD4 T lymphocytes, the highly O-acetylated cells are Mel 14(hi), CD44(lo), and CD45R(exon B)hi, features typical of naive cells. Digestions with trypsin and O-sialoglycoprotease (OSGPase) and ELISA studies of lipid extracts indicate that the 9-O-acetylated sialic acids on peripheral CD4 T cells are predominantly on O-linked mucintype glycoproteins and to a lesser degree, on sialylated glycolipids (gangliosides). In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells. The 9-O-acetylated gangliosides on mouse T cells are not bound by CD60 antibodies, which recognize O-acetylated gangliosides in human T cells. Tethering 9-O-acetylated mucins with the Influenza C probe with

  17. 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.

  18. Characterization of a feruloyl esterase B from Talaromyces cellulolyticus.

    PubMed

    Watanabe, Masahiro; Yoshida, Erika; Fukada, Hiroaki; Inoue, Hiroyuki; Tokura, Mitsunori; Ishikawa, Kazuhiko

    2015-01-01

    A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification. PMID:26110915

  19. In vitro comparison of rat and chicken brain neurotoxic esterase

    SciTech Connect

    Novak, R.; Padilla, S.

    1986-04-01

    A systematic comparison was undertaken to characterize neurotoxic esterase (NTE) from rat and chicken brain in terms of inhibitor sensitivities, pH optima, and molecular weights. Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterases showed that rat esterases were more sensitive than chicken to paraoxon inhibition at concentrations less than or equal to microM and superimposable with chicken esterases at concentrations of 2.5-1000 microM. Mipafox titration of the paraoxon-resistant esterases at a fixed paraoxon concentration of 100 microM (mipafox concentration: 0-1000 microM) resulted in a mipafox I50 of 7.3 microM for chicken brain NTE and 11.6 microM for rat brain NTE. NTE (i.e., paraoxon-resistant, mipafox-sensitive esterase activity) comprised 80% of chicken and 60% of rat brain paraoxon-resistant activity with the specific activity of chicken brain NTE approximately twice that of rat brain NTE. The pH maxima for NTE from both species was similar showing broad, slightly alkaline optima from pH 7.9 to 8.6. (/sup 3/H)Diisopropyl phosphorofluoridate (DFP)-labeled NTE from the brains of both species had an apparent mol wt of 160,000 measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In conclusion, NTE from both species was very similar, with the mipafox I50 for rat NTE within the range of reported values for chicken and human NTE, and the inhibitor parameters of the chicken NTE assay were applicable for the rat NTE assay.

  20. Bacterial protein acetylation: new discoveries unanswered questions.

    PubMed

    Wolfe, Alan J

    2016-05-01

    Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl-coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering. PMID:26660885

  1. Electrophoretic and densitometric analysis of esterase activity as an indicator of mercury toxicity

    SciTech Connect

    Benton, M.J.; Guttman, S.I.

    1995-12-31

    In an earlier experiment, esterase activity as determined by starch gel electrophoresis was absent in larval caddisflies (Nectopsyche albida) that succumbed to mercury exposure, but was present in control larvae. To test the effects of mercury exposure duration on esterase activity, additional larval N. albida were exposed under conditions identical to those in the earlier experiment, and esterase activity was determined by electrophoresis of several live individuals every 12 hours. To test the effects of mercury concentration on esterase activity, homogenates of unexposed N. albida were electrophoresed, and esterase activity was determined using esterase-specific stains spiked with various concentrations of mercury. Following both experiments, esterase activity was quantified by laser densitometry of stained electrophoresis gels, Results indicate that: (1) inorganic mercury inhibited esterase activity, (2) inhibition increased with exposure time, and (3) inhibition increased with mercury concentration. Esterase inhibition may be a causal factor in mortality related to mercury exposure. Quantification of esterase activity by densitometry of electrophoretic gels may be an alternative method of rapid toxicity assessment.

  2. Structural Basis for the De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine in Gram-positive Bacteria*

    PubMed Central

    Little, Dustin J.; Bamford, Natalie C.; Pokrovskaya, Varvara; Robinson, Howard; Nitz, Mark; Howell, P. Lynne

    2014-01-01

    Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaBAd) has been determined to 1.7 Å resolution. The structure of IcaBAd reveals a (β/α)7 barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaBAd is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni2+, Co2+, and Zn2+. From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci. PMID:25359777

  3. Utilization of xylan by yeasts and its conversion to ethanol by Pichia stipitis strains. [Cryptococcus; Pichia stipitis; Candida shehatae

    SciTech Connect

    Lee, H.; Biely, P.; Latta, R.K.; Barbosa, M.F.S.; Schneider, H.

    1986-08-01

    Yeasts able to grow on D-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains. 2. P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with about 60% of a theoretical yield computed on the basis of the amount of D-xylose present originally that could be released by acid hydrolysis.

  4. Digestion of cellulose and xylan by symbiotic bacteria in the intestine of the Indian flying fox (Pteropus giganteus).

    PubMed

    Prem Anand, A Alwin; Sripathi, K

    2004-09-01

    Bats (Order Chiroptera) are a widely distributed group of mammals. Pteropus giganteus belongs to the Suborder Megachiroptera. This bat consumes fruits and leaves as their major food. Cellulose and xylan are the major composition of leaves. As they consume leaves in their diet, their digestive tract must contain cellulolytic and xylanolytic bacteria which help in the digestion of cellulose and xylan. The cellulolytic and xylanolytic bacteria were isolated and screened on Berg's agar containing cellulose and xylan. The bacteria isolated were characterized biochemically and found to be Proteus vulgaris, Proteus mirabilis, Citrobacter freundii, Serratia liquefaciens and Klebsiella oxytoca. These bacteria help in digestion of cellulose and xylan in the diet of the bat, P. giganteus. Here we show that leaves are also used as a carbohydrate source by these bats. An insectivorous bat, Hipposideros fulvus, was used as a control and does not possess cellulolytic and xylanolytic bacteria. PMID:15471682

  5. Improving biofuel feedstocks by modifying xylan biosynthesis (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

    SciTech Connect

    Lau, Jane

    2013-03-01

    Jane Lau of the Joint BioEnergy Institute on "Improving biofuel feedstocks by modifying xylan biosynthesis" at the 8th Annual Genomics of Energy & Environment Meeting on March 28, 2013 in Walnut Creek, Calif.

  6. Mode of action of Bacillus licheniformis pectin methylesterase on highly methylesterified and acetylated pectins.

    PubMed

    Remoroza, Connie; Wagenknecht, Martin; Buchholt, Hans Christian; Moerschbacher, Bruno M; Gruppen, Harry; Schols, Henk A

    2015-01-22

    A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme (BliPME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50°C. BliPME is able to release up to 100% of the methylesters from lime pectin (DM 34-76→DM 0) and up to 73% of all methylesters from SBPs (DM 30-73→DM 14). BliPME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the BliPME-modified pectins using HILIC-MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of BliPME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME. PMID:25439930

  7. Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds.

    PubMed

    Voiniciuc, Cătălin; Günl, Markus; Schmidt, Maximilian Heinrich-Wilhelm; Usadel, Björn

    2015-12-01

    All cells of terrestrial plants are fortified by walls composed of crystalline cellulose microfibrils and a variety of matrix polymers. Xylans are the second most abundant type of polysaccharides on Earth. Previous studies of Arabidopsis (Arabidopsis thaliana) irregular xylem (irx) mutants, with collapsed xylem vessels and dwarfed stature, highlighted the importance of this cell wall component and revealed multiple players required for its synthesis. Nevertheless, xylan elongation and substitution are complex processes that remain poorly understood. Recently, seed coat epidermal cells were shown to provide an excellent system for deciphering hemicellulose production. Using a coexpression and sequence-based strategy, we predicted several MUCILAGE-RELATED (MUCI) genes that encode glycosyltransferases (GTs) involved in the production of xylan. We now show that MUCI21, a member of an uncharacterized clade of the GT61 family, and IRX14 (GT43 protein) are essential for the synthesis of highly branched xylan in seed coat epidermal cells. Our results reveal that xylan is the most abundant xylose-rich component in Arabidopsis seed mucilage and is required to maintain its architecture. Characterization of muci21 and irx14 single and double mutants indicates that MUCI21 is a Golgi-localized protein that likely facilitates the addition of xylose residues directly to the xylan backbone. These unique branches seem to be necessary for pectin attachment to the seed surface, while the xylan backbone maintains cellulose distribution. Evaluation of muci21 and irx14 alongside mutants that disrupt other wall components suggests that mucilage adherence is maintained by complex interactions between several polymers: cellulose, xylans, pectins, and glycoproteins. PMID:26482889

  8. Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds1[OPEN

    PubMed Central

    Günl, Markus; Usadel, Björn

    2015-01-01

    All cells of terrestrial plants are fortified by walls composed of crystalline cellulose microfibrils and a variety of matrix polymers. Xylans are the second most abundant type of polysaccharides on Earth. Previous studies of Arabidopsis (Arabidopsis thaliana) irregular xylem (irx) mutants, with collapsed xylem vessels and dwarfed stature, highlighted the importance of this cell wall component and revealed multiple players required for its synthesis. Nevertheless, xylan elongation and substitution are complex processes that remain poorly understood. Recently, seed coat epidermal cells were shown to provide an excellent system for deciphering hemicellulose production. Using a coexpression and sequence-based strategy, we predicted several MUCILAGE-RELATED (MUCI) genes that encode glycosyltransferases (GTs) involved in the production of xylan. We now show that MUCI21, a member of an uncharacterized clade of the GT61 family, and IRX14 (GT43 protein) are essential for the synthesis of highly branched xylan in seed coat epidermal cells. Our results reveal that xylan is the most abundant xylose-rich component in Arabidopsis seed mucilage and is required to maintain its architecture. Characterization of muci21 and irx14 single and double mutants indicates that MUCI21 is a Golgi-localized protein that likely facilitates the addition of xylose residues directly to the xylan backbone. These unique branches seem to be necessary for pectin attachment to the seed surface, while the xylan backbone maintains cellulose distribution. Evaluation of muci21 and irx14 alongside mutants that disrupt other wall components suggests that mucilage adherence is maintained by complex interactions between several polymers: cellulose, xylans, pectins, and glycoproteins. PMID:26482889

  9. An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

    SciTech Connect

    Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan; Tryfona, Theodora; Sorieul, Mathias; Ng, Yao Z.; Zhang, Zhinong; Stott, Katherine; Anders, Nadine; Dupree, Paul

    2015-06-04

    Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.

  10. An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

    DOE PAGESBeta

    Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan; Tryfona, Theodora; Sorieul, Mathias; Ng, Yao Z.; Zhang, Zhinong; Stott, Katherine; Anders, Nadine; Dupree, Paul

    2015-06-04

    Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays,more » and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.« less

  11. An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

    PubMed Central

    Mortimer, Jenny C; Faria-Blanc, Nuno; Yu, Xiaolan; Tryfona, Theodora; Sorieul, Mathias; Ng, Yao Z; Zhang, Zhinong; Stott, Katherine; Anders, Nadine; Dupree, Paul

    2015-01-01

    Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components. PMID:26043357

  12. The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis.

    PubMed

    Jensen, Jacob K; Kim, Hoon; Cocuron, Jean-Christophe; Orler, Robert; Ralph, John; Wilkerson, Curtis G

    2011-05-01

    Xylan is the principal hemicellulose in the secondary cell walls of eudicots and in the primary and secondary cell walls of grasses and cereals. The biosynthesis of this important cell wall component has yet to be fully determined although a number of proteins have been shown to be required for xylan synthesis. To discover new genes involved in xylan biosynthesis we explored the psyllium (Plantago ovata Forsk) seed mucilaginous layer through EST profiling. This tissue synthesizes large amounts of a complex heteroxylan over a short period of time. By comparing abundant transcripts in this tissue with abundant transcripts specifically present during secondary cell wall formation in Arabidopsis thaliana, where glucuronoxylan biosynthesis is pronounced, we identified two Arabidopsis genes likely involved in xylan biosynthesis. These genes encode proteins containing a Domain of Unknown Function (DUF) 579 and were designated IRREGULAR XYLEM (IRX) 15 and IRX15-LIKE (IRX15-L). We obtained Arabidopsis T-DNA knockout lines for the two genes and analyzed their lower stems for changes in neutral monosaccharide composition. No changes were observed in each of these mutants, although the irx15 irx15-L double mutant displayed a moderate reduction in stem xylose. Further characterization of the irx15 irx15-L mutant revealed irregular secondary cell wall margins in fiber cells and a lower xylan degree of polymerization. Through these studies we conclude that IRX15 and IRX15-L function in a redundant manner and are involved in xylan biosynthesis. PMID:21288268

  13. Distinct roles of residual xylan and lignin in limiting enzymatic hydrolysis of organosolv pretreated loblolly pine and sweetgum.

    PubMed

    Li, Mi; Tu, Maobing; Cao, Dongxu; Bass, Patrick; Adhikari, Sushil

    2013-01-23

    The interactions between xylan/lignin and cellulase enzymes play a key role in the effective hydrolysis of lignocellulosic biomass. Organosolv pretreated loblolly pine (OPLP) and sweetgum (OPSG) were used to quantitatively elucidate the distinct roles of residual xylan and lignin on enzymatic hydrolysis, based on the initial hydrolysis rates and the final hydrolysis yields. The initial hydrolysis rates of OPLP and OPSG were 1.45 (glucose) and 1.19 g/L/h (glucose), respectively, under the enzyme loading of 20 FPU/g glucan. The final glucan hydrolysis yields of OPLP and OPSG at 72 h were 76.4 and 98.9%, respectively. By correlating the amount of residual lignin and xylan to the initial hydrolysis rate and the final hydrolysis yield in OPLP and OPSG, a more accurate fundamental understanding of the roles of xylan and lignin in limiting the enzymatic hydrolysis has been developed. The higher amount of residual xylan (9.7%) in OPSG resulted in lower initial hydrolysis rate (1.19 g/L/h). The higher amount of residual lignin in OPLP (18.6%) resulted in lower final hydrolysis yield of glucan (76.4%). In addition, we observed in the simultaneous saccharification and fermentation (SSF) that ethyl xyloside was produced by the enzymatic catalysis of xylose/xylan and ethanol. PMID:23270516

  14. Effects of Impurities in Alkali-Extracted Xylan on Its Enzymatic Hydrolysis to Produce Xylo-Oligosaccharides.

    PubMed

    Shen, Rui; Li, Hong-Qiang; Zhang, Jie; Xu, Jian

    2016-07-01

    As the second abundant natural carbohydrate, xylan is normally prepared through alkaline extraction and then used for xylo-oligosaccharides (XOS) production. However, the extracted xylan inevitably contains salt, ethanol, and pigment. In order to investigate the effects of these impurities on XOS production, the alkaline-extracted xylan with different kinds and concentrations of impurities was made and then hydrolyzed using alkaline xylanase (EC 3.2.1.8) to produce XOS. The results showed that a certain concentration of salt (NaCl) promoted the XOS production, while ethanol and pigment inhibited the enzymatic hydrolysis process significantly. The color value mainly ascribed to the phenolic compounds binding to xylan was a key restriction factor in the enzymatic hydrolysis later stage. Using optimal xylan sample (with 10 mg/mL NaCl, color value of 4.6 × 10(5), without ethanol) as substrate, the highest XOS yield of 58.58 % was obtained. As the substrate of XOS production, prepared xylan should contain colored materials and ethanol as less as possible, however, retains appropriate salt. PMID:26922729

  15. Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a heteroxylan containing mucilaginous tissue.

    PubMed

    Jensen, Jacob K; Johnson, Nathan; Wilkerson, Curtis G

    2013-01-01

    The exact biochemical steps of xylan backbone synthesis remain elusive. In Arabidopsis, three non-redundant genes from two glycosyltransferase (GT) families, IRX9 and IRX14 from GT43 and IRX10 from GT47, are candidates for forming the xylan backbone. In other plants, evidence exists that different tissues express these three genes at widely different levels, which suggests that diversity in the makeup of the xylan synthase complex exists. Recently we have profiled the transcripts present in the developing mucilaginous tissue of psyllium (Plantago ovata Forsk). This tissue was found to have high expression levels of an IRX10 homolog, but very low levels of the two GT43 family members. This contrasts with recent wheat endosperm tissue profiling that found a relatively high abundance of the GT43 family members. We have performed an in-depth analysis of all GTs genes expressed in four developmental stages of the psyllium mucilagenous layer and in a single stage of the psyllium stem using RNA-Seq. This analysis revealed several IRX10 homologs, an expansion in GT61 (homologs of At3g18170/At3g18180), and several GTs from other GT families that are highly abundant and specifically expressed in the mucilaginous tissue. Our current hypothesis is that the four IRX10 genes present in the mucilagenous tissues have evolved to function without the GT43 genes. These four genes represent some of the most divergent IRX10 genes identified to date. Conversely, those present in the psyllium stem are very similar to those in other eudicots. This suggests these genes are under selective pressure, likely due to the synthesis of the various xylan structures present in mucilage that has a different biochemical role than that present in secondary walls. The numerous GT61 family members also show a wide sequence diversity and may be responsible for the larger number of side chain structures present in the psyllium mucilage. PMID:23761806

  16. Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a heteroxylan containing mucilaginous tissue

    PubMed Central

    Jensen, Jacob K.; Johnson, Nathan; Wilkerson, Curtis G.

    2013-01-01

    The exact biochemical steps of xylan backbone synthesis remain elusive. In Arabidopsis, three non-redundant genes from two glycosyltransferase (GT) families, IRX9 and IRX14 from GT43 and IRX10 from GT47, are candidates for forming the xylan backbone. In other plants, evidence exists that different tissues express these three genes at widely different levels, which suggests that diversity in the makeup of the xylan synthase complex exists. Recently we have profiled the transcripts present in the developing mucilaginous tissue of psyllium (Plantago ovata Forsk). This tissue was found to have high expression levels of an IRX10 homolog, but very low levels of the two GT43 family members. This contrasts with recent wheat endosperm tissue profiling that found a relatively high abundance of the GT43 family members. We have performed an in-depth analysis of all GTs genes expressed in four developmental stages of the psyllium mucilagenous layer and in a single stage of the psyllium stem using RNA-Seq. This analysis revealed several IRX10 homologs, an expansion in GT61 (homologs of At3g18170/At3g18180), and several GTs from other GT families that are highly abundant and specifically expressed in the mucilaginous tissue. Our current hypothesis is that the four IRX10 genes present in the mucilagenous tissues have evolved to function without the GT43 genes. These four genes represent some of the most divergent IRX10 genes identified to date. Conversely, those present in the psyllium stem are very similar to those in other eudicots. This suggests these genes are under selective pressure, likely due to the synthesis of the various xylan structures present in mucilage that has a different biochemical role than that present in secondary walls. The numerous GT61 family members also show a wide sequence diversity and may be responsible for the larger number of side chain structures present in the psyllium mucilage. PMID:23761806

  17. Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex1[OPEN

    PubMed Central

    Jiang, Nan; Wiemels, Richard E.; Soya, Aaron; Whitley, Rebekah; Held, Michael; Faik, Ahmed

    2016-01-01

    Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH2-terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants. PMID:26917684

  18. Overexpression of esterase D in kidney from trisomy 13 fetuses

    SciTech Connect

    Loughna, S.; Moore, G. ); Gau, G.; Blunt, S. ); Nicolaides, K. )

    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 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. 34 refs., 3 figs., 2 tabs.

  19. Production of xylooligosaccharides from corncob xylan by fungal xylanase and their utilization by probiotics.

    PubMed

    Chapla, Digantkumar; Pandit, Pratima; Shah, Amita

    2012-07-01

    The selective production of xylooligosaccharides (XOS) was carried out using partially purified xylanase from Aspergillus foetidus MTCC 4898. Corncob xylan was extracted using a mild alkali treatment which yielded 178.73±5.8 g of xylan/kg of corncobs. Partially purified β-xylosidase free xylanase was found efficient in releasing xylooligosaccharides from corncob xylan. Maximum yield of xylooligosaccharides was 6.73±0.23 mg/ml after 8 h of reaction time using 20 U of xylanase at 45°C. Purification of XOS was done using activated charcoal column chromatography. The purified XOS preparation contained mainly xylobiose and xylotriose. XOS mixture was found suitable for food industry looking at its high thermal stability at low pH. Prebiotic effect of XOS was evaluated by in vitro fermentation of XOS using known probiotic strains viz. Bifidobacterium adolescentis, Bifidobacterium bifidum, Lactobacillus fermentum, Lactobacillus acidophilus. The results of this study revealed better growth of Bifidobacterium spp. on XOS than Lactobacillus spp. PMID:22100233

  20. A molecular modeling approach to understand the structure and conformation relationship of (GlcpA)Xylan.

    PubMed

    Guo, Qingbin; Kang, Ji; Wu, Yan; Cui, Steve W; Hu, Xinzhong; Yada, Rickey Y

    2015-12-10

    The structure and conformation relationships of a heteropolysaccharide (GlcpA)Xylan in terms of various molecular weights, Xylp/GlcpA ratio and the distribution of GlcpA along xylan chain were investigated using computer modeling. The adiabatic contour maps of xylobiose, XylpXylp(GlcpA) and (GlcpA)XylpXylp(GlcpA) indicated that the insertion of the side group (GlcpA) influenced the accessible conformational space of xylobiose molecule. RIS-Metropolis Monte Carlo method indicated that insertion of GlcpA side chain induced a lowering effect of the calculated chain extension at low GlcpA:Xylp ratio (GlcpA:Xylp = 1:3). The chain, however, became extended when the ratio of GlcpA:Xylp above 2/3. It was also shown that the spatial extension of the polymer chains was dependent on the distribution of side chain: the random distribution demonstrated the most flexible structure compared to block and alternative distribution. The present studies provide a unique insight into the dependence of both side chain ratio and distribution on the stiffness and flexibility of various (GlcpA)Xylan molecules. PMID:26428114

  1. Enhancing enzymatic hydrolysis of xylan by adding sodium lignosulfonate and long-chain fatty alcohols.

    PubMed

    Lou, Hongming; Yuan, Long; Qiu, Xueqing; Qiu, Kexian; Fu, Jinguo; Pang, Yuxia; Huang, Jinhao

    2016-01-01

    Sodium lignosulfonate (SXSL) and long-chain fatty alcohols (LFAs) could enhance the enzymatic hydrolysis of xylan, and the compound of SXSL and LFAs have synergies on the enzymatic hydrolysis. SXSL shows a strong enhancement in buffer pH range from 4.0 to 6.0. The enhancement increased with the SXSL dosage and the xylanase loading. The cellulose and lignin in corncob substrate could not only adsorb xylanase nonproductively, but also seriously reduce the accessibility of xylanase on xylan to impede the enzymatic hydrolysis of xylan. Cellulase could break the plant cell wall structure of corncob and make additives work better. The xylose yield of corncob at 72h increased from 59.4% to 73.7% by adding the compound of 5g/L SXSL and 0.01% (v/v) n-decanol, which was higher than that without cellulase and additives by 30.7%. Meanwhile, the glucose yield at 72h of corncob increased from 45.8% to 62.3%. PMID:26476164

  2. Production and Properties of Xylan-Degrading Enzymes from Cellulomonas uda

    PubMed Central

    Rapp, Peter; Wagner, Fritz

    1986-01-01

    Xylan degradation and production of β-xylanase and β-xylosidase activities were studied in cultures of Cellulomonas uda grown on purified xylan from birchwood. β-Xylanase activity was found to be associated with the cells, although in various degrees. The formation of β-xylanase activity was induced by xylotriose and repressed by xylose. β-Xylosidase activity was cell bound. Both constitutive and inducible β-xylosidase activities were suggested. β-Xylanase and β-xylosidase activities were inhibited competitively by xylose. β-Xylanase activity had a pronounced optimum pH of 5.8, whereas the optimum pH of β-xylosidase activity ranged from 5.4 to 6.1. The major products of xylan degradation by a crude preparation of β-xylanase activity, in decreasing order of amount, were xylobiose, xylotriose, xylose, and small amounts of xylotetraose. This pattern suggests that β-xylanase activity secreted by C. uda is of the endosplitting type. Supernatants of cultures grown on cellulose showed not only β-glucanase but also β-xylanase activity. The latter could be attributed to an endo-1,4-β-glucanase activity which had a low β-xylanase activity. PMID:16347038

  3. Kinetic study of hydrolysis of xylan and agricultural wastes with hot liquid water.

    PubMed

    Zhuang, Xinshu; Yuan, Zhenhong; Ma, Longlong; Wu, Chuangzhi; Xu, Mingzhong; Xu, Jingliang; Zhu, Shunni; Qi, Wei

    2009-01-01

    We investigated the kinetics of hot liquid water (HLW) hydrolysis over a 60-min period using a self-designed setup. The reaction was performed within the range 160-220 degrees C, under reaction conditions of 4.0 MPa, a 1:20 solid:liquid ratio (g/mL), at 500 rpm stirring speed. Xylan was chosen as a model compound for hemicelluloses, and two kinds of agricultural wastes-rice straw and palm shell-were used as typical feedstocks representative of herbaceous and woody biomasses, respectively. The hydrolysis reactions for the three kinds of materials followed a first-order sequential kinetic model, and the hydrolysis activation energies were 65.58 kJ/mol for xylan, 68.76 kJ/mol for rice straw, and 95.19 kJ/mol for palm shell. The activation energies of sugar degradation were 147.21 kJ/mol for xylan, 47.08 kJ/mol for rice straw and 79.74 kJ/mol for palm shell. These differences may be due to differences in the composition and construction of the three kinds of materials. In order to reduce the decomposition of sugars, the hydrolysis time of biomasses such as rice straw and palm shell should be strictly controlled. PMID:19397989

  4. Toxicological implications of esterases-From molecular structures to functions

    SciTech Connect

    Satoh, Tetsuo . E-mail: satohbri@peach.ifnet.or.jp

    2005-09-01

    This article reports on a keynote lecture at the 10th International Congress of Toxicology sponsored by the International Union of Toxicology and held on July 2004. Current developments in molecular-based studies into the structure and function of cholinesterases, carboxylesterases, and paraoxonases are described. This article covers mechanisms of regulation of gene expression of the various esterases by developmental factors and xenobiotics, as well as the interplay between physiological and chemical regulation of the enzyme activity.

  5. Preparation and Properties of Novel Dentin Adhesives with Esterase Resistance

    PubMed Central

    Park, Jong-Gu; Ye, Qiang; Topp, Elizabeth M.; Kostoryz, Elisabet L.; Wang, Yong; Kieweg, Sarah L.; Spencer, Paulette

    2012-01-01

    A new methacrylate monomer, trimethylolpropane mono allyl ether dimethacrylate (TMPEDMA), was synthesized and evaluated. This branched methacrylate was designed to increase esterase-resistance when incorporated into conventional HEMA (2-hydroxyethyl methacrylate)/BisGMA (2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane) dental adhesives. The new adhesives, HEMA/BisGMA/TMPEDMA in a 45/30/25 (w/w) ratio were formulated with H2O at 0 (A0T) and 8 wt % water (A8T) and compared with control adhesives (HEMA/BisGMA, 45/55 (w/w), at 0 (A0) and 8 wt % (A8) water). Camphoroquinone (CQ), 2-(dimethylamino) ethyl methacrylate and diphenyliodonium hexafluorophosphate were used as photoinitiators. The new adhesives showed a degree of conversion comparable with the control and improved modulus and glass transition temperature (Tg). Exposure of photopolymerized discs to porcine liver esterase for up to eight days showed that the net cumulative methacrylic acid (MAA) release in adhesives formulated with the new monomer and 8% water (A8T: 182 μg/mL) was dramatically (P < 0.05) decreased in comparison to the control (A8: 361.6 μg/mL). The results demonstrate that adhesives made with the new monomer and cured in water to simulate wet bonding are more resistant to esterase than conventional HEMA/BisGMA adhesive. PMID:22919119

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

    SciTech Connect

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

    1986-05-01

    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.. pyrone 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.

  7. The effect of age and frailty upon blood esterase activities and their response to dietary supplementation.

    PubMed Central

    Summerbell, J; Wynne, H; Hankey, C R; Williams, F M

    1993-01-01

    1. The aims of this study were two-fold. First, to define ranges of blood esterase activities in three groups, namely young subjects, fit community dwelling elderly and frail, chronically hospitalised elderly subjects, and second, to determine whether low blood esterase activities in the frail patients could be altered by increasing their nutritional intake. 2. Plasma cholinesterase, aspirin esterase, paraoxonase and phenylacetate esterase activities were all significantly lower in the frail elderly compared with the young and fit elderly volunteers. The activity of red blood cell esterase was not different in the frail elderly. 3. Fourteen frail elderly patients were randomly assigned to receive either hospital meal provision plus supplemental feeding with Build-up (Nestle) and Maxijul (SHS Ltd) or hospital provision alone for 8 weeks. Dietary intake was measured for all patients at the start of the study and at week 8. Measurements of blood esterase (cholinesterase, phenylacetate esterase, paraoxonase, aspirin esterase and red blood cell esterase), albumin and anthropometric indices (weight, triceps skinfold thickness and mid arm circumference) were made before the study and repeated at week 4 and 8. 4. There was a significant increase in plasma cholinesterase at week 4 (P < 0.05) but this was not statistically significant at week 8. There were no significant changes in any of the other esterase activities or anthropometric measurements. 5. We conclude that the lower esterase activities of the frail chronically hospitalised elderly do not respond to dietary supplementation for a period of 8 weeks with routinely available products. The hypothesis that lower esterase activities are the direct result of undernutrition which would be corrected by dietary supplementation has not been supported by this study. PMID:12959286

  8. Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants

    PubMed Central

    2012-01-01

    Background Cost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production. Results Xylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx) mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter. Conclusions Spatial and temporal deposition of xylan in the secondary cell wall of Arabidopsis can be manipulated by

  9. The effect of liver esterases and temperature on remifentanil degradation in vitro.

    PubMed

    Piazza, Ornella; Cascone, Sara; Sessa, Linda; De Robertis, Edoardo; Lamberti, Gaetano

    2016-08-20

    Remifentanil is a potent opioid metabolized by serum and tissue esterases; it is routinely administered to patients with liver failure as anaesthetic and analgo-sedative without variation in doses, even if prolonged clinical effects and respiratory depression have been observed in these patients. The aim of this study was to determine remifentanil enzymatic degradation kinetics bearing in mind the effect of liver esterases in order to trace a more accurate pharmacokinetic profile of the drug. Solution samples were taken over time and analysed to measure remifentanil concentration by HPLC. We reproduced the physiological settings, varying temperature and pH in vitro and evaluated the kinetics of degradation of remifentanil in the presence of Rhizopus Oryzae esterases, equine liver esterases and porcine liver esterases. Remifentanil kinetics of degradation was accelerated by porcine liver esterases. Remifentanil in vitro half-life decreases with increasing temperatures in the presence of porcine liver esterases. A drug model simulation considering the effect of temperature in the presence of liver esterases was developed. Remifentanil in vitro half-life decreases with increasing temperatures when porcine liver esterases are present. In this paper we propose a model for describing remifentanil degradation kinetics at various temperatures. PMID:27370912

  10. Isozymic variations in specific and nonspecific esterase and its thermostability in silkworm, Bombyx mori L.

    PubMed

    Patnaik, Bharat Bhusan; Biswas, Tapati Datta; Nayak, Sandeepta Kumar; Saha, A K; Majumdar, M K

    2012-09-01

    Esterase isozymic variations were documented in the haemolymph of developed multivoltine and bivoltine silkworm breeds during unfavorable seed crop seasons of May - September using á- and â- napthylacetate separately to identify specific and nonspecific esterase having thermotolerant potentiality. Variations existed in the isozyme pattern with three bands (Est-2, 3 and 4) in pure Nistari race and other developed multivoltine and bivoltine breeds. Est-2 and Est-3 were non-specific esterases as they were observed when both á- and â-napthylacetate was used as substrates separately. Est-4 band was observed only with á-napthylacetate as substrate and was therefore confirmed to be specific á-esterase band in the haemolymph of silkworm, Bombyx mori L. Zymograms showed that the non-specific esterase band (Est-3) with R1 of 0.43 and specific á-esterase band (Est-4) with R(f) of 0.32 predominately withstood a temperature of 70 +/- 2 degrees C for a duration of 10 min and were confirmed as thermostable esterases in haemolymph of silkworm, Bombyx mori L. This also categorized the presence of thermostable esterases in developed multivoltine and bivoltine breeds of silkworm, even though the qualitative activity was more in the former than the latter. The qualitative presence of thermostable esterases and their activity could be adopted as an indicative biochemical marker in relation to thermotolerance in silkworm. PMID:23734447

  11. Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals.

    PubMed

    Andreasen, M F; Kroon, P A; Williamson, G; Garcia-Conesa, M T

    2001-11-01

    Hydroxycinnamic acids are effective antioxidants and are abundant components of plant cell walls, especially in cereal bran. For example, wheat and rye brans are rich sources of the hydroxycinnamates ferulic acid, sinapic acid, and p-coumaric acid. These phenolics are part of human and animal diets and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary hydroxycinnamates are distributed throughout the intestinal tract of mammals. In rats, the cinnamoyl esterase activity in the small intestine is derived mainly from the mucosa, whereas in the large intestine the esterase activity was found predominantly in the luminal microflora. Mucosa cell-free extracts obtained from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye and wheat brans. Hydrolysis by intestinal esterase(s) is very likely the major route for release of antioxidant hydroxycinnamic acids in vivo. PMID:11714377

  12. Hemicellulases from anaerobic thermophiles. Progress report

    SciTech Connect

    Wiegel, J.

    1994-05-01

    The longterm goal of this research effort is to obtain an anaerobic thermophilic bacterium that efficiently converts various hemicellulose-containing biomass to ethanol over a broad pH range. The strategy is to modify the outfit and regulation of the rate-limiting xylanases, glycosidases and xylan esterases in the ethanologenic, anaerobic thermophile Thermoanaerobacter ethanolicus, which grows between pH 4.5 and 9.5. Although it utilizes xylans, the xylanase, acetyl(xylan) esterase and O-methylglucuronidase activities in T. ethanolicus are barely measurable and regarded as the rate limiting steps in its xylan utilization. Thus, and also due to the presently limited knowledge of hemicellulases in anaerobic thermophiles, we characterize the hemicellulolytic enzymes from this and other anaerobic thermophiles as enzyme donors. Beside the active xylosidase/arabinosidase from T. ethanolicus, exhibiting the two different activities, we characterized 2 xylosidases, two acetyl(xylan) esterases, and an O-methylglucuronidase from Thermoanaerobacterium spec. We will continue with the characterization of xylanases from novel isolated slightly acidophilic, neutrophilic and slightly alkalophilic thermophiles. We have cloned, subcloned and partially sequenced the 165,000 Da (2 x 85,000) xylosidase/arabinosidase from T. ethanolicus and started with the cloning of the esterases from Thermoanaerobacterium spec. Consequently, we will develop a shuttle vector and continue to apply electroporation of autoplasts as a method for cloning into T. ethanolicus.

  13. An extended loop in CE7 carbohydrate esterase family is dispensable for oligomerization but required for activity and thermostability.

    PubMed

    Singh, Mrityunjay K; Manoj, Narayanan

    2016-06-01

    The carbohydrate esterase family 7 (CE7) belonging to the α/β hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical α/β hydrolase fold. This element called the β-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75Å and 1.76Å, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the β-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability. PMID:27085421

  14. Biosensor analysis of blood esterases for organophosphorus compounds exposure assessment: approaches to simultaneous determination of several esterases.

    PubMed

    Sigolaeva, Larisa; Makhaeva, Galina; Rudakova, Elena; Boltneva, Natalia; Porus, Marya; Dubacheva, Galina; Eremenko, Arkadi; Kurochkin, Ilya; Richardson, Rudy J

    2010-09-01

    This paper reviews our previously published data and presents new results on biosensor assay of blood esterases. Tyrosinase and choline oxidase biosensors based on nanostructured polyelectrolyte films were developed for these purposes. Experiments were performed on the quantitative determination of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), and neuropathy target esterase (NTE) in samples of whole blood of rats, mice, and humans. Good agreement was found between biosensor and spectrophotometric assays for AChE, BChE, and CaE. No direct comparison could be made for NTE because its activity cannot be measured spectrophotometrically in whole blood. A new method of simultaneous quantitative determination of AChE and BChE in test mixtures is also described. This method represents a bifunctional biosensor for the simultaneous analysis of choline and phenol based on integration of individual sensors. Algorithms for calculation of separate concentrations of AChE and BChE in the mixture were developed. The mean error of calculated component concentrations was approximately 6% for binary test mixtures. The present work provides a foundation for building multiplexed systems for the simultaneous determination of multiple esterases with applications to biomonitoring for exposures to organophosphorus compounds. PMID:20097186

  15. Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    PubMed

    Fan, J; Baeza, J; Denu, J M

    2016-01-01

    Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated. PMID:27423860

  16. 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

  17. Variations in elastaselike esterase activities in human leucocytes during cell maturation.

    PubMed

    Feinstein, G; Janoff, A

    1976-05-01

    Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic sybstrates such as N-acetyl-dl-alanine-alpha-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)2-Pro-AlaCH2Cl (a powerful inactivator of the leucocyte elastase isozymes at 10-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development. PMID:1265076

  18. Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola.

    PubMed

    Roelants, Sophie L K W; Ciesielska, Katarzyna; De Maeseneire, Sofie L; Moens, Helena; Everaert, Bernd; Verweire, Stijn; Denon, Quenten; Vanlerberghe, Brecht; Van Bogaert, Inge N A; Van der Meeren, Paul; Devreese, Bart; Soetaert, Wim

    2016-03-01

    Although sophorolipids (SLs) produced by S. bombicola are a real showcase for the industrialization of microbial biosurfactants, some important drawbacks are associated with this efficient biological process, e.g., the simultaneous production of acidic and lactonic SLs. Depending on the application, there is a requirement for the naturally produced mixture to be manipulated to give defined ratios of the components. Recently, the enzyme responsible for the lactonization of SLs was discovered. The discovery of the gene encoding this lactone esterase (sble) enabled the development of promising S. bombicola strains producing either solely lactonic (using a sble overexpression strain described in this paper: oe sble) or solely acidic SLs (using a sble deletion strain, which was recently described, but not characterized yet: Δsble). The new S. bombicola strains were used to investigate the production processes (fermentation and purification) of either lactonic or acidic SLs. The strains maintain the high inherent productivities of the wild-type or even perform slightly better and thus represent a realistic industrial opportunity. 100% acidic SLs with a mixed acetylation pattern were obtained for the Δsble strain, while the inherent capacity to selectively produce lactonic SLs was significantly increased (+42%) for the oe sble strain (99% lactonic SLs). Moreover, the regulatory effect of citrate on lactone SL formation for the wild-type was absent in this new strain, which indicates that it is more robust and better suited for the industrial production of lactonic SLs. Basic parameters were determined for the purified SLs, which confirm that the two new strains produce molecules with distinctive properties of which the application potential can now easily be investigated independently. PMID:26301720

  19. Mitochondrial Acetylation and Diseases of Aging

    PubMed Central

    Wagner, Gregory R.; Payne, R. Mark

    2011-01-01

    In recent years, protein lysine acetylation has emerged as a prominent and conserved regulatory posttranslational modification that is abundant on numerous enzymes involved in the processes of intermediary metabolism. Well-characterized mitochondrial processes of carbon utilization are enriched in acetyl-lysine modifications. Although seminal discoveries have been made in the basic biology of mitochondrial acetylation, an understanding of how acetylation states influence enzyme function and metabolic reprogramming during pathological states remains largely unknown. This paper will examine our current understanding of eukaryotic acetate metabolism and present recent findings in the field of mitochondrial acetylation biology. The implications of mitochondrial acetylation for the aging process will be discussed, as well as its potential implications for the unique and localized metabolic states that occur during the aging-associated conditions of heart failure and cancer growth. PMID:21437190

  20. A Method to determine lysine acetylation stoichiometries

    SciTech Connect

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  1. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism

    PubMed Central

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F.

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  2. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

    PubMed

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  3. Distribution of cell-wall xylans in bryophytes and tracheophytes: new insights into basal interrelationships of land plants.

    PubMed

    Carafa, Anna; Duckett, Jeffrey G; Knox, J Paul; Ligrone, Roberto

    2005-10-01

    Xylans are known to be major cellulose-linking polysaccharides in secondary cell walls in higher plants. We used two monoclonal antibodies (LM10 and LM11) for a comparative immunocytochemical analysis of tissue and cell distribution of xylans in a number of taxa representative of all major tracheophyte and bryophyte lineages. The results show that xylans containing the epitopes recognized by LM10 and LM11 are ubiquitous components of secondary cell walls in vascular and mechanical tissues in all present-living tracheophytes. In contrast, among the three bryophyte lineages, LM11 binding was detected in specific cell-wall layers in pseudoelaters and spores in the sporophyte of hornworts, while no binding was observed with either antibody in the gametophyte or sporophyte of liverworts and mosses. The ubiquitous occurrence of xylans containing LM10 and LM11 epitopes in tracheophytes suggests that the appearance of these polysaccharides has been a pivotal event for the evolution of highly efficient vascular and mechanical tissues. LM11 binding in the sporophyte of hornworts, indicating the presence of relatively highly substituted xylans (possibly arabinoxylans), separates these from the other bryophytes and is consistent with recent molecular data indicating a sister relationship of the hornworts with tracheophytes. PMID:16159336

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. ASSAY OF CHICKEN BRAIN NEUROTOXIC ESTERASE ACTIVITY USING LEPTOPHOSOXON AS THE SELECTIVE NEUROTOXIC INHIBITOR

    EPA Science Inventory

    Hen brain microsomal preparation has phenyl valeratehydrolyzing activity associated with neurotoxic esterase activity. Part of that activity is due to paraoxon-insensitive esterases and a sub-part of this is sensitive to neurotoxic organophosphates, i.e., mipafox and leptophosoxo...

  10. Structure of the catalytic domain of glucuronoyl esterase Cip2 from Hypocrea jecorina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Esterase phenotyping in human liver in vitro: specificity of carboxylesterase inhibitors.

    PubMed

    Umehara, Ken-Ichi; Zollinger, Markus; Kigondu, Elizabeth; Witschi, Marc; Juif, Claire; Huth, Felix; Schiller, Hilmar; Chibale, Kelly; Camenisch, Gian

    2016-10-01

    1. Esterases may play a major role in the clearance of drugs with functional groups amenable to hydrolysis, particularly in the case of ester prodrugs. To understand the processes involved in the elimination of such drugs, it is necessary to determine the esterases involved. However, the tools currently available for this enzyme phenotyping are relatively scarce. 2. The work was aimed at summarizing the selectivity of esterase inhibitors for carboxylesterases 1 and 2 (CES1 and CES2) in the human liver to clarify their suitability for esterase phenotyping. Eserine, at around 10 μM, was found to be a highly specific CES2 inhibitor, whereas other esterase inhibitors turned out less selective. When used together with tacrine, which inhibits cholinesterases but not CES, and ethylenediaminetetraacetic acid (inhibitor of paraoxonases), the involvement of the hydrolyzing esterases in the hepatic clearance of a drug can be elucidated. 3. The second approach to esterase phenotyping is based on data from recombinant or isolated esterases, together with relative activity factors, which relate their activities to those of the same enzymes in subcellular fractions. 4. These two approaches will help to characterize the hydrolytic metabolism of drug candidates in a similar manner as practiced routinely for the oxidative metabolism by cytochrome P450 enzymes. PMID:26887925

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Xylan oligosaccharides and cellobiohydrolase I (TrCel7A) interaction and effect on activity

    PubMed Central

    2011-01-01

    Background The well-studied cellulase mixture secreted by Trichoderma reesei (anamorph to Hypocrea jecorina) contains two cellobiohydolases (CBHs), cellobiohydrolase I (TrCel7A) and cellobiohydrolase II (TrCeI6A), that are core enzymes for the solubilisation of cellulose. This has attracted significant research interest because of the role of the CBHs in the conversion of biomass to fermentable sugars. However, the CHBs are notoriously slow and susceptible to inhibition, which presents a challenge for the commercial utilisation of biomass. The xylans and xylan fragments that are also present in the biomass have been suggested repeatedly as one cause of the reduced activity of CHBs. Yet, the extent and mechanisms of this inhibition remain poorly elucidated. Therefore, we studied xylan oligosaccharides (XOSs) of variable lengths with respect to their binding and inhibition of both TrCel7A and an enzyme variant without the cellulose-binding domain (CBM). Results We studied the binding of XOSs to TrCel7A by isothermal titration calorimetry. We found that XOSs bind to TrCel7A and that the affinity increases commensurate with XOS length. The CBM, on the other hand, did not affect the affinity significantly, which suggests that XOSs may bind to the active site. Activity assays of TrCel7A clearly demonstrated the negative effect of the presence of XOSs on the turnover number. Conclusions On the basis of these binding data and a comparison of XOS inhibition of the activity of the two enzyme variants towards, respectively, soluble and insoluble substrates, we propose a competitive mechanism for XOS inhibition of TrCel7A with phosphoric swollen cellulose as a substrate. PMID:22035059

  17. Transcriptomic Analyses of Xylan Degradation by Prevotella bryantii and Insights into Energy Acquisition by Xylanolytic Bacteroidetes*

    PubMed Central

    Dodd, Dylan; Moon, Young-Hwan; Swaminathan, Kankshita; Mackie, Roderick I.; Cann, Isaac K. O.

    2010-01-01

    Enzymatic depolymerization of lignocellulose by microbes in the bovine rumen and the human colon is critical to gut health and function within the host. Prevotella bryantii B14 is a rumen bacterium that efficiently degrades soluble xylan. To identify the genes harnessed by this bacterium to degrade xylan, the transcriptomes of P. bryantii cultured on either wheat arabinoxylan or a mixture of its monosaccharide components were compared by DNA microarray and RNA sequencing approaches. The most highly induced genes formed a cluster that contained putative outer membrane proteins analogous to the starch utilization system identified in the prominent human gut symbiont Bacteroides thetaiotaomicron. The arrangement of genes in the cluster was highly conserved in other xylanolytic Bacteroidetes, suggesting that the mechanism employed by xylan utilizers in this phylum is conserved. A number of genes encoding proteins with unassigned function were also induced on wheat arabinoxylan. Among these proteins, a hypothetical protein with low similarity to glycoside hydrolases was shown to possess endoxylanase activity and subsequently assigned to glycoside hydrolase family 5. The enzyme was designated PbXyn5A. Two of the most similar proteins to PbXyn5A were hypothetical proteins from human colonic Bacteroides spp., and when expressed each protein exhibited endoxylanase activity. By using site-directed mutagenesis, we identified two amino acid residues that likely serve as the catalytic acid/base and nucleophile as in other GH5 proteins. This study therefore provides insights into capture of energy by xylanolytic Bacteroidetes and the application of their enzymes as a resource in the biofuel industry. PMID:20622018

  18. Esterase activity of BSA-ZnO nanoparticle complex

    NASA Astrophysics Data System (ADS)

    Bhogale, A.; Nair, A.; Patel, N.; Miotello, A.; Kothari, D. C.

    2014-04-01

    The effect of Zinc Oxide Nanoparticles (ZnO NPs) on functional properties of Bovine Serum Albumin (BSA) protein was studied. ZnO NPs were synthesized with average size of ˜7.5 nm as obtained from TEM analysis. The catalytic conversion of p-nitrophenylacetate (PNPA) to p-nitrophenol in the presence of BSA attached with ZnO NPs was examined by UV-Vis spectroscopy at room temperature. The result suggests that esterase activity of BSA is significantly enhanced (6 times) due to the ground state BSA-ZnO complex formation.

  19. Assays for the classification of two types of esterases: carboxylic ester hydrolases and phosphoric triester hydrolases.

    PubMed

    Anspaugh, Douglas D; Roe, R Michael

    2002-11-01

    Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolase and Phosphoric Triester Hydrolase (Douglas D. Anspaugh and Michael Roe, North Carolina State University, Raleigh, North Carolina). This unit describes assays that quantitate two types of esterase the carboxylic ester hydrolases and the phosphoric triester hydrolases. Carboxylic ester hydrolases include the B-esterases, which are inhibited by organophosphorus compounds. Among the phosphoric triester hydrolases is aryldialkylphosphatase, which has been called A-esterase or paraoxonase due to its ability to oxidize paraoxon and other organophosphates. These assays are colorimetric and miniaturized for rapid simultaneous testing of multiple, small-volume samples in a microtiter plate format. There is also a discussion of the history of esterase nomenclature and the reasons why this large group of enzymes is so difficult to classify. PMID:20945297

  20. 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

  1. A comparison of multiple esterases as biomarkers of organophosphate exposure and effect in two earthworm species.

    PubMed

    Henson-Ramsey, Heather; Schneider, Ashley; Stoskopf, Michael K

    2011-04-01

    Two different earthworm species, Eisenia fetida and Lumbricus terrestris, were exposed to 5 μg/cm(2) of malathion to evaluate their usefulness as sentinels of organophosphate exposure and to assess three different esterases, as biomarkers of malathion exposure and effect. Tissue xenobiotic burdens and esterase activity were determined for each species and each esterase in order to assess variability. E. fetida exhibited 4-fold less variability in tissue burdens than did L. terrestris and had less variable basal esterase activities. An attempt was made to correlate malathion and malaoxon tissue burdens with esterase activity post-exposure. There was no malaoxon present in the earthworm tissues. No significant correlations were determined by comparing acetylcholinesterase, butyrylcholinesterase, nor carboxylesterase activities with malathion burdens. PMID:21404045

  2. An advanced understanding of the specific effects of xylan and surface lignin contents on enzymatic hydrolysis of lignocellulosic biomass

    SciTech Connect

    Ju, Xiaohui; Engelhard, Mark H.; Zhang, Xiao

    2013-01-17

    A deep understanding of biomass recalcitrance has been hampered by the intricate and heterogeneous nature of pretreated biomass substrates obtained from random deconstruction methods. In this study, we established a unique methodology based on chemical pulping principles to create "reference substrates" with intact cellulose fibers and controlled morphological and chemical properties that enable us to investigate the individual effect of xylan, bulk, and surface lignin content on enzymatic hydrolysis. We also developed and demonstrated an X-ray photoelectron spectroscopy (XPS) technique for quantifying surface lignin content on biomass substrates. The results from this study show that, apart from its hindrance effect, xylan can facilitate cellulose fibril swelling and thus create more accessible surface area, which improves enzyme and substrate interactions. Surface lignin has a significant impact on enzyme adsorption kinetics and hydrolysis rate. Advanced understanding of xylan, bulk, and surface lignin effects provides critical information for an effective biomass conversion process.

  3. Green synthesis of silver nanoparticles in xylan solution via Tollens reaction and their detection for Hg2+

    NASA Astrophysics Data System (ADS)

    Luo, Yuqiong; Shen, Suqin; Luo, Jiwen; Wang, Xiaoying; Sun, Runcang

    2014-12-01

    This work reported a facile and green method to prepare highly stable and uniformly distributed Ag nanoparticles (AgNPs), in which a biopolymer xylan was used as the stabilizing and reducing agent via the Tollens reaction under microwave irradiation. Different variables were evaluated to optimize the reaction conditions. Complete characterization was performed using UV-Vis, XRD, TEM, size distribution analysis and XPS. The results revealed that AgNPs were well dispersed with diameters of 20-35 nm due to the packing of xylan. The optimal conditions were as follows: microwave irradiation temperature was 60-70 °C, microwave power was 800 W, microwave time was 30 min, the ratio of xylan to AgNO3 was 50 mg: 0.13 mmol, and ammonia concentration was 2%. In addition, the AgNPs were collected via high-speed centrifugal separation, and the supernatant was tested by HPAEC, GPC, FT-IR, and NMR. By comparing the structure of xylan before and after the reaction, the reaction mechanism was discussed. It was noted that the xylan-AgNPs composites showed high selectivity and sensitivity for Hg2+ detection. The other 15 metal ions used had no obvious effect on the detection of Hg2+, and the limit of detection (LOD) was 4.6 nM, which is lower than the allowed maximum level of 30 nM for drinking water by WHO. In addition, the xylan-AgNPs composites can be applied for Hg2+ detection in real water samples. This study provides a novel way for the high-value utilization of a rich biomass resource, and a green method for the synthesis of AgNPs for the selective and sensitive detection of harmful heavy metals.This work reported a facile and green method to prepare highly stable and uniformly distributed Ag nanoparticles (AgNPs), in which a biopolymer xylan was used as the stabilizing and reducing agent via the Tollens reaction under microwave irradiation. Different variables were evaluated to optimize the reaction conditions. Complete characterization was performed using UV-Vis, XRD, TEM

  4. 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

  5. Green synthesis of silver nanoparticles in xylan solution via Tollens reaction and their detection for Hg(2+).

    PubMed

    Luo, Yuqiong; Shen, Suqin; Luo, Jiwen; Wang, Xiaoying; Sun, Runcang

    2015-01-14

    This work reported a facile and green method to prepare highly stable and uniformly distributed Ag nanoparticles (AgNPs), in which a biopolymer xylan was used as the stabilizing and reducing agent via the Tollens reaction under microwave irradiation. Different variables were evaluated to optimize the reaction conditions. Complete characterization was performed using UV-Vis, XRD, TEM, size distribution analysis and XPS. The results revealed that AgNPs were well dispersed with diameters of 20-35 nm due to the packing of xylan. The optimal conditions were as follows: microwave irradiation temperature was 60-70 °C, microwave power was 800 W, microwave time was 30 min, the ratio of xylan to AgNO3 was 50 mg: 0.13 mmol, and ammonia concentration was 2%. In addition, the AgNPs were collected via high-speed centrifugal separation, and the supernatant was tested by HPAEC, GPC, FT-IR, and NMR. By comparing the structure of xylan before and after the reaction, the reaction mechanism was discussed. It was noted that the xylan-AgNPs composites showed high selectivity and sensitivity for Hg(2+) detection. The other 15 metal ions used had no obvious effect on the detection of Hg(2+), and the limit of detection (LOD) was 4.6 nM, which is lower than the allowed maximum level of 30 nM for drinking water by WHO. In addition, the xylan-AgNPs composites can be applied for Hg(2+) detection in real water samples. This study provides a novel way for the high-value utilization of a rich biomass resource, and a green method for the synthesis of AgNPs for the selective and sensitive detection of harmful heavy metals. PMID:25429650

  6. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    PubMed

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  7. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGESBeta

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong-Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; et al

    2014-01-01

    Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

  8. Comparative analysis of end point enzymatic digests of arabino-xylan isolated from switchgrass (Panicum virgatum L) of varying maturities using LC-MS(n)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass (Panicum virgatum L., SG) is a perennial grass presently used for forage and being developed as a bioenergy crop for conversion of cell wall carbohydrates to biofuels. Up to 50% of the cell wall associated carbohydrates are xylan. SG was analyzed for xylan structural features at variab...

  9. Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

    NASA Astrophysics Data System (ADS)

    Risveden, Klas; Dick, Kimberly A.; Bhand, Sunil; Rydberg, Patrik; Samuelson, Lars; Danielsson, Bengt

    2010-02-01

    A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiNx-covered wafers. Two different reactors are shown: one with simple, one-dimensional nanorods and one with branched nanorod structures (nanotrees). Significantly higher enzymatic activity is found for the nanotree reactors than for the nanorod reactors, most likely due to the increased gold surface area and thereby higher enzyme binding capacity. A theoretical calculation is included to show how the enzyme kinetics and hence the sensitivity can be influenced and increased by the control of electrical fields in relation to the active sites of enzymes in an electronic biosensor. The possible effects of electrical fields employed in the RISFET on the function of acetylcholine esterase is investigated using quantum chemical methods, which show that the small electric field strengths used are unlikely to affect enzyme kinetics. Acetylcholine esterase activity is determined using choline oxidase and peroxidase by measuring the amount of choline formed using the chemiluminescent luminol reaction.

  10. A halotolerant type A feruloyl esterase from Pleurotus eryngii.

    PubMed

    Nieter, Annabel; Haase-Aschoff, Paul; Linke, Diana; Nimtz, Manfred; Berger, Ralf G

    2014-03-01

    An extracellular feruloyl esterase (PeFaeA) from the culture supernatant of Pleurotus eryngii was purified to homogeneity using cation exchange, hydrophobic interaction, and size exclusion chromatography. The length of the complete coding sequence of PeFaeA was determined to 1668 bp corresponding to a protein of 555 amino acids. The catalytic triad of Ser-Glu-His demonstrated the uniqueness of the enzyme compared to previously published FAEs. The purified PeFaeA was a monomer with an estimated molecular mass of 67 kDa. Maximum feruloyl esterase (FAE) activity was observed at pH 5.0 and 50 °C, respectively. Metal ions (5 mM), except Hg(2+), had no significant influence on the enzyme activity. Substrate specificity profiling characterized the enzyme as a type A FAE preferring bulky natural substrates, such as feruloylated saccharides, rather than small synthetic ones. Km and kcat of the purified enzyme for methyl ferulate were 0.15 mM and 0.85 s(-1). In the presence of 3 M NaCl activity of the enzyme increased by 28 %. PeFaeA alone released only little ferulic acid from destarched wheat bran (DSWB), whereas after addition of Trichoderma viride xylanase the concentration increased more than 20 fold. PMID:24607359

  11. Improved thermostability of a Bacillus subtilis esterase by domain exchange.

    PubMed

    Gall, Markus G; Nobili, Alberto; Pavlidis, Ioannis V; Bornscheuer, Uwe T

    2014-02-01

    A moderately thermostable esterase from Geobacillus stearothermophilus (BsteE) and its homolog from Bacillus subtilis (BsubE) show a high structural similarity with more than 95% homology and 74% amino acid identity. Interestingly, their thermal stability differs significantly by 30 °C in their melting temperature. In order to identify the positions that are responsible for this difference, most of the flexible amino acids assumed to confer instability were found to be in the cap region. For this reason, a 30 amino acid long cap domain fragment containing ten differing positions derived from BsteE was incorporated into the homologous gene encoding for the more labile BsubE by spliced overlap-extension PCR. The melting temperature of the two wild-type esterases and the mutant was evaluated by circular dichroism spectroscopy, while the kinetic parameters and the stability were determined with a photometric assay. The cap domain mutant maintained its activity, with a catalytic efficiency more similar to BsteE, while it exhibited an increase of the melting temperature by 4 °C compared to BsubE. Additional point mutations based on the differences of the parent enzymes gave a further increase of the thermostability up to 11 °C compared to BsubE; however, a significant reduction in activity was observed. PMID:23812333

  12. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  13. Histone Acetylation in Fungal Pathogens of Plants

    PubMed Central

    Jeon, Junhyun; Kwon, Seomun; Lee, Yong-Hwan

    2014-01-01

    Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed. PMID:25288980

  14. Direct Conversion of Xylan to Ethanol by Recombinant Saccharomyces cerevisiae Strains Displaying an Engineered Minihemicellulosome

    PubMed Central

    Sun, Jie; Wen, Fei; Si, Tong; Xu, Jian-He

    2012-01-01

    Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant Saccharomyces cerevisiae strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/CipA1) and up to three chimeric enzymes. The miniscaffoldin derived from Clostridium thermocellum contained one or three cohesin modules and was tethered to the cell surface through the S. cerevisiae a-agglutinin adhesion receptor. Up to three types of hemicellulases, an endoxylanase (XynII), an arabinofuranosidase (AbfB), and a β-xylosidase (XlnD), each bearing a C-terminal dockerin, were assembled onto the miniscaffoldin by high-affinity cohesin-dockerin interactions. Compared to uni- and bifunctional minihemicellulosomes, the resulting quaternary trifunctional complexes exhibited an enhanced rate of hydrolysis of arabinoxylan. Furthermore, with an integrated d-xylose-utilizing pathway, the recombinant yeast displaying the bifunctional minihemicellulosome CipA3-XynII-XlnD could simultaneously hydrolyze and ferment birchwood xylan to ethanol with a yield of 0.31 g per g of sugar consumed. PMID:22447594

  15. Acid-catalyzed conversion of xylose, xylan and straw into furfural by microwave-assisted reaction.

    PubMed

    Yemiş, Oktay; Mazza, Giuseppe

    2011-08-01

    Furfural is a biomass derived-chemical that can be used to replace petrochemicals. In this study, the acid-catalyzed conversion of xylose and xylan to furfural by microwave-assisted reaction was investigated at selected ranges of temperature (140-190°C), time (1-30 min), substrate concentration (1:5-1:200 solid:liquid ratio), and pH (2-0.13). We found that a temperature of 180°C, a solid:liquid ratio of 1:200, a residence time of 20 min, and a pH of 1.12 gave the best furfural yields. The effect of different Brønsted acids on the conversion efficiency of xylose and xylan was also evaluated, with hydrochloric acid being found to be the most effective catalyst. The microwave-assisted process provides highly efficient conversion: furfural yields obtained from wheat straw, triticale straw, and flax shives were 48.4%, 45.7%, and 72.1%, respectively. PMID:21620690

  16. Kinetic analysis of two-phase enzymatic hydrolysis of hemicellulose of xylan type.

    PubMed

    Dutta, Sajal Kanti; Chakraborty, Saikat

    2015-12-01

    We present a coupled experimental and theoretical framework for quantifying the kinetics of two-phase enzymatic hydrolysis of hemicellulose. For xylan loading of 1-5mg/ml, the nature of inhibition by the product xylose (non-competitive), the kinetic constants (Km=3.93 mg/ml, Vmax=0.0252 mg/ml/min) and the xylose inhibition constant (Kx=0.122 mg/ml) are experimentally determined. Our multi-step two-phase kinetic model incorporating enzyme adsorption to the solid substrate and non-competitive product inhibition is simulated using our kinetic data and validated against our experimentally measured temporal dynamics of xylose and reducing sugars. Further experiments show that higher substrate loading reduces the specific adsorption of the endoxylanase to the solid xylan and the enzyme's solid-liquid distribution ratio, which decelerates the solid hydrolysis and accelerates the liquid phase reactions. Thus, the xylose yield increases with substrate loading, which increases product inhibition and decreases reducing sugar yields. An operating cost analysis gives 3mg/ml as the optimal substrate loading. PMID:26433789

  17. Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

    PubMed Central

    de Vries, Ronald P.; Gruppen, Harry; Kabel, Mirjam A.

    2015-01-01

    The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments. PMID:26237450

  18. Glucuronic acid in Arabidopsis thaliana xylans carries a novel pentose substituent.

    PubMed

    Chong, Sun-Li; Koutaniemi, Sanna; Juvonen, Minna; Derba-Maceluch, Marta; Mellerowicz, Ewa J; Tenkanen, Maija

    2015-08-01

    Glucuronic acids in Arabidopsis thaliana xylans exist in 4-O-methylated (MeGlcA) and non-methylated (GlcA) forms at a ratio of about 3:2. The matrix-assisted laser desorption/ionization mass spectrometry analysis of the endoxylanase liberated acidic oligosaccharides from the Arabidopsis inflorescence stem showed that two peaks with GlcA (GlcA-Xyl4Ac1 and GlcA-Xyl5Ac2) had abnormally high intensities, as well as different tandem mass spectra, than their 4-O-methylated counterparts. These peaks were interestingly enriched in the xylan biosynthesis mutant irx7 and irx9-1. Multi-stages fragmentation analysis using negative ion electrospray-ion trap mass spectrometry indicated that this GlcA was further carrying a pentose residue in the glucuronoxylan-derived oligosaccharide from irx9-1. The structure was also identified in Arabidopsis wild type. The results prove evidence of a new pentose substitution on the GlcA residue of Arabidopsis GX, which is likely present in the primary walls. PMID:26047894

  19. Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca

    SciTech Connect

    Burchhardt, G.; Ingram, L.O. )

    1992-04-01

    A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism. The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals. This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555). Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production. Cells containing xylanase for saccharification. After cooling, the hydrolysate was fermented to ethanol with the same organism (30C), thereby replenishing the supply of xylanase for a subsequent saccharification. Recombinant E. coli metabolized only xylose, while recombinant K. oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose. Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis. By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process. The yield appeared to be limited by the digestability of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity. In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers.

  20. The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine*

    PubMed Central

    Little, Dustin J.; Poloczek, Joanna; Whitney, John C.; Robinson, Howard; Nitz, Mark; Howell, P. Lynne

    2012-01-01

    Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni2+ and Fe3+ have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)4 oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co2+ and Ni2+ under aerobic conditions, and Co2+, Ni2+ and Fe2+ under anaerobic conditions, but decreased activity with Zn2+. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms. PMID:22810235

  1. An organic-solvent-tolerant esterase from thermophilic Bacillus licheniformis S-86.

    PubMed

    Torres, Sebastián; Martínez, M Alejandra; Pandey, Ashok; Castro, Guillermo R

    2009-01-01

    A thermophile, halotolerant and organic-solvent-tolerant esterase producer Bacillus sp. S-86 strain previously isolated was found to belong to Bacillus licheniformis species through morphological, biochemical, 16S rRNA gene sequence analyses and rDNA intergenic spacers amplification (ITS-PCR). The strain can grow at 55 degrees C in presence of C2-C7 alkanols (log P=-0.86 to 2.39), and NaCl concentrations up to 15% (w/v). This bacterium showed optimal growth and esterase production at 50 degrees C. Two different molecular weight esterase activities were detected in zymographic assays. PMSF inhibited type I esterase activity, showing no inhibitory effect on type II esterase activity. B. licheniformis S-86 was able to grow in presence of hydroxylic organic-solvents like propan-2-ol, butan-1-ol and 3-methylbutan-1-ol. At a sub-lethal concentration of these solvents (392 mmoll(-1) propan-2-ol; 99 mmol l(-1) butan-1-ol, 37 mmol l(-1) 3-methylbutan-1-ol), adequate to produce 50% cell growth inhibition at 50 degrees C, an increment between 1.9 and 2.3 times was observed in type I esterase production, and between 2.2 and 3.1 times in type II esterase production. PMID:18723341

  2. Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli.

    PubMed

    Donaghy, J; Kelly, P F; McKay, A M

    1998-08-01

    The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agarplate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 degrees C respectively. PMID:9763694

  3. Angiooedema due to acquired deficiency of C1-esterase inhibitor associated with leucocytoclastic vasculitis.

    PubMed

    Farkas, H; Szongoth, M; Bély, M; Varga, L; Fekete, B; Karádi, I; Füst, G

    2001-01-01

    A hereditary and an acquired type of C1-esterase inhibitor deficiency have been described. Manifestations characteristic of both forms include recurrent subcutaneous and submucosal angiooedema. Acquired C1-esterase inhibitor deficiency has been observed in association with lymphoproliferative disorders, malignancy, autoimmune diseases and infections. We report on a case with the acquired form of the disease accompanied by leucocytoclastic vasculitis. Treatment with antimalarial agents resulted in complete resolution of symptoms and signs. Furthermore, C1-esterase inhibitor concentration and activity, as well as C1 levels, all returned to normal. PMID:11720182

  4. Acetylation phenotypes in patients with bladder carcinoma.

    PubMed

    Bicho, M P; Breitenfeld, L; Carvalho, A A; Manso, C F

    1988-01-01

    The present study was done to evaluate the possible association of bladder carcinoma with the slow acetylator phenotype in a portuguese population. 49 patients with bladder carcinoma were compared to a normal control group of 84 individuals. No statistically significant association was detected. But when subdividing the group of slow acetylators it is found that in the subgroup with 12-36% acetylation there is a higher percentage of patients, which is statistically significant. These results are in agreement with two other studies, using populations of similar ethnic origin. PMID:3265609

  5. Biological Activities of xylooligosaccharides generated from garlic straw xylan by purified xylanase from Bacillus mojavensis UEB-FK.

    PubMed

    Kallel, Fatma; Driss, Dorra; Chaabouni, Semia Ellouz; Ghorbel, Raoudha

    2015-01-01

    A newly isolated Bacterium strain named UEB-FK was selected from Tunisian Sahara, exhibiting the highest clear zone on agar plates containing oat spelt xylan by staining with Congo red. On the basis of 16S rDNA sequence analysis, this strain was identified as Bacillus mojavensis. This strain produced extracellular xylanase. Xylanase from the strain was purified to homogeneity and had an apparent molecular weight of 14 kDa. The K m and V max values of the purified xylanase on oat spelt xylan were 3.85 mg/mL and 250.02 U/mg, respectively. The optimum pH and temperature for the enzyme were found to be 4.0 and 50 °C, respectively, and the enzyme exhibited significant heat stability. In addition, the enzyme was found to be stable in a wide range of pH (3-9). The main hydrolysis products yielded from garlic straw-extracted xylan were xylobiose and xylotriose. The antioxidant and antibacterial activities of xylan oligosaccharide (XOS) were investigated. As regards to the in vitro antioxidant activities, the XOS showed a important DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (IC50 = 0.45 mg/mL) and a high β-carotene bleaching (IC50 = 2.2 mg/mL). Furthermore, XOS had a high antimicrobial activity against Klebsiella pneumoniae, Enterococcus faecalis, Bacillus thuringiensis, and Pseudomonas aeruginosa. PMID:25351626

  6. Paenibacillus tundrae sp. nov. and Paenibacillus xylanexedens sp. nov., Psychrotolerant, Xylan-Degrading, Bacteria from Alaskan Tundra

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Psychrotolerant, xylan-degrading, strains of bacteria were isolated from soil beneath moist non-acidic and acidic tundra in northern Alaska. Phylogenetic analysis based on 16S rRNA gene sequences revealed that each strain belonged to the genus Paenibacillus. The highest levels of 16S rRNA gene sim...

  7. Expression of Aeromonas punctata ME-1 exo-xylanase X in E. coli for efficient hydrolysis of xylan to xylose.

    PubMed

    Juturu, Veeresh; Teh, Tong Mei; Wu, Jin Chuan

    2014-12-01

    exo-Xylanase X from Aeromonas punctata ME-1 was functionally expressed in Escherichia coli with a carboxy terminal His tag (6×) and a molecular mass of 39.42 kDa, which is in agreement with the prediction from its amino acid composition. The recombinant exo-xylanase reached 186 mg l(-1) after induction by isopropyl β-D-1-thiogalactopyranoside. Its optimal temperature and pH were 50 °C and 6, respectively. The enzyme showed not only an exo-xylanase activity with K m of 3.90 mg ml(-1) and V max of 12.9 U μg(-1) for hydrolysis of Remazol Brilliant Blue-xylan but also a considerable exo-glucanase activity (27.9 U mg(-1)) on P-nitrophenyl β-D-cellobioside. It hydrolyzed xylan predominantly to xylobiose, xylotriose, xylotetraose, and xylose. An enzyme mixture of exo-xylanase and endo-xylanase (50 μg ml(-1) each) yielded a larger amount (330 mg l(-1)) of xylose from beechwood xylan than the controls (270 and 150 mg l(-1)) using them alone at 100 μg ml(-1), indicating a synergistic action between the two xylanases favoring the hydrolysis of beechwood xylan to release more xylose. PMID:25213085

  8. Deconstruction of lignin linked p-coumarates, ferulates and xylan by NaOH enhances the enzymatic conversion of glucan.

    PubMed

    Murciano Martínez, Patricia; Punt, Arjen M; Kabel, Mirjam A; Gruppen, Harry

    2016-09-01

    Thermo-assisted NaOH pretreatment to deconstruct xylan and lignin in sugar cane bagasse (SCB) is poorly understood. Hence, in this research it is was aimed to study the effect of NaOH pretreatment on the insoluble remaining lignin structures. Hereto, SCB milled fibres were pretreated using different dosages of NaOH at different temperatures and residence times. Of untreated SCB about 63% of the lignin compounds were assigned as p-coumarates and ferulates, analysed by pyrolysis-GC/MS as 4-vinyl phenol and 4-vinyl guaiacol, and designated as non-core lignin (NCL) compounds. More severe NaOH pretreatments resulted in lower xylan and lower lignin recoveries in the insoluble residues. Especially, the relative abundance of NCL decreased and this decrease followed a linear trend with the decrease in xylan. Core lignin compounds, analysed as phenol, guaiacol and syringol, accumulated in the residues. The decrease in residual xylan and NCL correlated positively with the enzymatic hydrolysis of the residual glucan. PMID:27233096

  9. Complete genome sequence of the marine, cellulose and xylan degrading bacterium Glaciecola sp. 4H-3-7+YE-5

    SciTech Connect

    Klippel, Dr Barbara; Bruce, David; Davenport, Karen W.; Goodwin, Lynne A.; Han, James; Han, Shunsheng; Land, Miriam L; Mikhailova, Natalia; Nolan, Matt; Pennacchio, Len; Pitluck, Sam; Tapia, Roxanne; Woyke, Tanja; Wiebusch, Sigrid; Basner, Alexander; Abe, Fumiyoshi; Horikoshi, Koki; Antranikian, Garabed

    2011-01-01

    Glaciecola sp. 4H-3-7+YE-5 was isolated from deep sea sediments at Suruga Bay in Japan and is capable of efficiently hydrolyzing cellulose and xylan. The complete genome sequence of Glaciecola sp. 4H-3-7+YE-5 revealed several genes encoding putatively novel glycoside hydrolases associated with plant biomass degradation.

  10. Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood.

    PubMed

    Derba-Maceluch, Marta; Awano, Tatsuya; Takahashi, Junko; Lucenius, Jessica; Ratke, Christine; Kontro, Inkeri; Busse-Wicher, Marta; Kosik, Ondrej; Tanaka, Ryo; Winzéll, Anders; Kallas, Åsa; Leśniewska, Joanna; Berthold, Fredrik; Immerzeel, Peter; Teeri, Tuula T; Ezcurra, Ines; Dupree, Paul; Serimaa, Ritva; Mellerowicz, Ewa J

    2015-01-01

    Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development. PMID:25307149

  11. 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.

  12. A new approach for determination of neuropathy target esterase activity.

    PubMed

    Sigolaeva, L V; Eremenko, A V; Makower, A; Makhaeva, G F; Malygin, V V; Kurochkin, I N

    1999-05-14

    Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE. PMID:10421495

  13. Heterologous Expression of Two Ferulic Acid Esterases from Penicillium Funiculosum

    SciTech Connect

    Knoshaug, E. P.; Selig, M. J.; Baker, J. O.; Decker, S. R.; Himmel, M. E.; Adney, W. S.

    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 their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

  14. Impact of acetylation on tumor metabolism

    PubMed Central

    Zhao, Di; Li, Fu-Long; Cheng, Zhou-Li; Lei, Qun-Ying

    2014-01-01

    Acetylation of protein lysine residues is a reversible and dynamic process that is controlled by histone acetyltransferases (HATs) and deacetylases (HDACs and SIRTs). Recent studies have revealed that acetylation modulates not only nuclear proteins but also cytoplasmic or mitochondrial proteins, including many metabolic enzymes. In tumors, cellular metabolism is reprogrammed to provide intermediates for biosynthesis such as nucleotides, fatty acids, and amino acids, and thereby favor the rapid proliferation of cancer cells and tumor development. An increasing number of investigations have indicated that acetylation plays an important role in tumor metabolism. Here, we summarize the substrates that are modified by acetylation, especially oncogenes, tumor suppressor genes, and enzymes that are implicated in tumor metabolism. PMID:27308346

  15. Acetylator phenotypes in Papua New Guinea

    PubMed Central

    Penketh, R J A; Gibney, S F A; Nurse, G T; Hopkinson, D A

    1983-01-01

    Acetylator phenotypes have been determined in 139 unrelated subjects from the hitherto untested populations of Papua New Guinea, and their relevance to current antituberculous isoniazid chemotherapy is discussed. PMID:6842533

  16. Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart.

    PubMed

    Kerner, Janos; Yohannes, Elizabeth; Lee, Kwangwon; Virmani, Ashraf; Koverech, Aleardo; Cavazza, Claudio; Chance, Mark R; Hoppel, Charles

    2015-01-01

    Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein, we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitine's effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial β-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. Two-dimensional fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a two-fold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins. PMID:25660059

  17. Levels of histone acetylation in thyroid tumors.

    PubMed

    Puppin, Cinzia; Passon, Nadia; Lavarone, Elisa; Di Loreto, Carla; Frasca, Francesco; Vella, Veronica; Vigneri, Riccardo; Damante, Giuseppe

    2011-08-12

    Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells. PMID:21763277

  18. Diagnostic assays based on esterase-mediated resistance mechanisms in western corn rootworms (Coleoptera: Chrysomelidae).

    PubMed

    Zhou, Xuguo; Scharf, Michael E; Parimi, Srinivas; Meinke, Lance J; Wright, Robert J; Chandler, Laurence D; Siegfried, Blair D

    2002-12-01

    Resistance to methyl-parathion among Nebraska western corn rootworm, Diabrotica virgifera virgifera LeConte, populations is associated with increased hydrolytic metabolism of an organophosphate insecticide substrate. An electrophoretic method to identify resistant individuals based on the staining intensity of esterase isozymes on nondenaturing polyacrylamide gels was developed. Three groups of esterases (I, II, and III) were visible on the gels, but only group II esterase isozymes were intensified in resistant populations. A total of 26 and 31 field populations of western corn rootworms from Nebraska (in 1998 and 1999, respectively) were assessed with nondenaturing polyacrylamide gel electrophoresis (PAGE) assays and diagnostic concentration bioassays. Significant correlations were observed between the two diagnostic assays. Group II esterase isozymes provide a reliable biochemical marker for detection of methyl-parathion resistance in individual western corn rootworms and a tool for monitoring the frequency of resistant individuals in field populations. PMID:12539840

  19. Fungal genomes mining to discover novel sterol esterases and lipases as catalysts

    PubMed Central

    2013-01-01

    Background Sterol esterases and lipases are enzymes able to efficiently catalyze synthesis and hydrolysis reactions of both sterol esters and triglycerides and due to their versatility could be widely used in different industrial applications. Lipases with this ability have been reported in the yeast Candida rugosa that secretes several extracellular enzymes with a high level of sequence identity, although different substrate specificity. This versatility has also been found in the sterol esterases from the ascomycetes Ophiostoma piceae and Melanocarpus albomyces. Results In this work we present an in silico search of new sterol esterase and lipase sequences from the genomes of environmental fungi. The strategy followed included identification and search of conserved domains from these versatile enzymes, phylogenetic studies, sequence analysis and 3D modeling of the selected candidates. Conclusions Six potential putative enzymes were selected and their kinetic properties and substrate selectivity are discussed on the basis of their similarity with previously characterized sterol esterases/lipases with known structures. PMID:24138290

  20. TRIPHENYL PHOSPHITE: IN VIVO AND IN VITRO INHIBITION OF RAT NEUROTOXIC ESTERASE (JOURNAL VERSION)

    EPA Science Inventory

    Organophosphorus compounds which, after acute administration, inhibit neurotoxic esterase (NTE) by > or = 65% and undergo a subsequent 'aging' reaction, produce a delayed neuropathy characterized by degeneration of large and long nerve fibers. The present studies examine in detai...

  1. CORRELATION BETWEEN NEUROTOXIC ESTERASE INHIBITION AND MIPAFOX-INDUCED NEUROPATHIC DAMAGE IN RATS

    EPA Science Inventory

    The correlation between neuropathic damage and inhibition of neurotoxic esterase or neuropathy target enzyme (NTE) was examined in rats acutely exposed to Mipafox (N, N'-diisopropylphosphorodiamidofluoridate), a neurotoxic organophospate. Brain and spinal cord NTE activities were...

  2. RELATIONSHIP OF NEUROPATHY TARGET ESTERASE INHIBITION TO NEUROPATHOLOGY AND ATAXIA IN HENS GIVEN ORGANOPHOSPHORUS ESTERS

    EPA Science Inventory

    Adult WhiteLeghorn hens were acutely exposed to 3 dosages of the following organophosphorus esters: mipafox, tri-ortho-tolyl phosphate (TOTP), penyl saligenin phosphate, diisppropylophosphoro-fluoridate (DFP), malathion and dichlorvos. europathy target esterase (NTE) activity was...

  3. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis. PMID:26503676

  4. Esterase inhibition by grapefruit juice flavonoids leading to a new drug interaction.

    PubMed

    Li, Ping; Callery, Patrick S; Gan, Liang-Shang; Balani, Suresh K

    2007-07-01

    Our previous studies described a newly identified potential of grapefruit juice (GFJ) in mediating pharmacokinetic drug interactions due to its capability of esterase inhibition. The current study identifies the active components in GFJ responsible for its esterase-inhibitory effect. The esterase-inhibitory potential of 10 constitutive flavonoids and furanocoumarins toward p-nitrophenylacetate (PNPA) hydrolysis was investigated. The furanocoumarins bergamottin, 6',7'-dihydroxybergamottin, and bergapten, and the glycoside flavonoids naringin and hesperidin, at concentrations found in GFJ or higher, did not inhibit the hydrolysis of PNPA by purified porcine esterase and human liver microsomes. However, the flavonoid aglycones morin, galangin, kaempferol, quercetin, and naringenin showed appreciable inhibition of PNPA hydrolysis in purified porcine esterase, and human and rat liver systems. In Caco-2 cells, demonstrated to contain minimal CYP3A activity, the permeability coefficient of the prodrugs lovastatin and enalapril was increased in the presence of the active flavonoids kaempferol and naringenin, consistent with inhibition of esterase activity. In rats, oral coadministration of kaempferol and naringenin with these prodrugs led to significant increases in plasma exposure to the active acids. In addition, in portal vein-cannulated rats, coadministration of lovastatin with kaempferol (10 mg/kg) led to a 154% and a 113% increase in the portal plasma exposure to the prodrug and active acid, respectively, compared with coadministration with water. The contribution of CYP3A inhibition was demonstrated to be minimal. Overall, a series of flavonoids present in GFJ are identified as esterase inhibitors, of which kaempferol and naringenin are shown to mediate pharmacokinetic drug interaction with the prodrugs lovastatin and enalapril due to their capability of esterase inhibition. PMID:17452418

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

    SciTech Connect

    Moretto, A. . E-mail: angelo.moretto@icps.it; Nicolli, A.; Lotti, M.

    2007-03-15

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

  6. Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima.

    PubMed

    Levisson, Mark; van der Oost, John; Kengen, Servé W M

    2007-06-01

    A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family. PMID:17466017

  7. Esterase in imported fire ants, Solenopsis invicta and S. richteri (Hymenoptera: Formicidae): activity, kinetics and variation.

    PubMed

    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

  8. Production and purification of a solvent-resistant esterase from Bacillus licheniformis S-86.

    PubMed

    Torres, Sebastián; Baigorí, Mario D; Pandey, Ashok; Castro, Guillermo R

    2008-12-01

    New thermophilic and organic-solvent-tolerant Bacillus licheniformis S-86 strain is able to produce two active and solvent-stable esterases. Production of type I and II esterases was substantially enhanced when oils and surfactants were supplied as carbon sources. Grape oil (0.1% v/v) and Tween 20 to 60 (0.1% v/v) had enhanced enzyme production between 1.6- and 2.2-folds. Type II esterase was purified to homogeneity in a five-step procedure. This esterase was purified 76.7-fold with a specific activity of 135 U mg(-1). Molecular mass of the enzyme was estimated to be 38.4 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Type II esterase was active mostly on esters with short acyl chains, which allowed to classify the enzyme as a carboxylesterase with a K (m) of 80.2 mmol l(-1) and a V (max) of 256.4 micromol min(-1) mg(-1) for p-nitrophenyl acetate. Also, B. licheniformis S-86 type II esterase displayed activity in presence of water-miscible organic solvents at 50% concentration and stability after 1-h incubation. PMID:18543118

  9. Differences in Esterase Activity to Aspirin and p-Nitrophenyl Acetate among Human Serum Albumin Preparations.

    PubMed

    Tatsumi, Akitoshi; Okada, Masaya; Inagaki, Yoshihiro; Inoue, Sachiyo; Hamaguchi, Tsuneo; Iwakawa, Seigo

    2016-01-01

    Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and also hydrolyzes some compounds at both sites. In the present study, we investigated differences in esterase activity among HSA preparations, and also the effects of warfarin, indomethacin, and naproxen on the hydrolytic activities of HSA to aspirin and p-nitrophenyl acetate. The esterase activities of HSA to aspirin or p-nitrophenyl acetate were measured from the pseudo-first-order formation rate constant (kobs) of salicylic acid or p-nitrophenol by HSA. Inter-lot variations were observed in the esterase activities of HSA to aspirin and p-nitrophenyl acetate; however, the esterase activity of HSA to aspirin did not correlate with that to p-nitrophenyl acetate. The inhibitory effects of warfarin and indomethacin on the esterase activity of HSA to aspirin were stronger than that of naproxen. In contrast, the inhibitory effect of naproxen on the esterase activity of HSA to p-nitrophenyl acetate was stronger than those of warfarin and indomethacin. These results suggest that the administration of different commercial HSA preparations and the co-administration with site I or II high-affinity binding drugs may change the pharmacokinetic profiles of drugs that are hydrolyzed by HSA. PMID:27476944

  10. Identification of a cocaine esterase in a strain of Pseudomonas maltophilia.

    PubMed Central

    Britt, A J; Bruce, N C; Lowe, C R

    1992-01-01

    A strain of Pseudomonas maltophilia (termed MB11L) which was capable of using cocaine as its sole carbon and energy source was isolated by selective enrichment. An inducible esterase catalyzing the hydrolysis of cocaine to ecgonine methyl ester and benzoic acid was identified and purified 22-fold. In the presence of the solubilizing agent cholate, cocaine esterase had a native Mr of 110,000 and was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a monomer. In the absence of cholate, cocaine esterase had a native Mr of 410,000 and probably existed as a tetramer. The pH optimum of the enzyme was 8.0, and the Km values for cocaine, ethyl benzoate, and ethyl 2-hydroxybenzoate were 0.36, 1.89, and 1.75 mM, respectively. Inhibition studies indicated that the enzyme was a serine esterase, possibly possessing a cation-binding site similar to those of mammalian acetylcholinesterase and the atropine esterase of Pseudomonas putida PMBL-1. The cocaine esterase of P. maltophilia MB11L showed no activity with atropine, despite the structural similarity of cocaine and atropine. PMID:1551831

  11. Switching catalysis from hydrolysis to perhydrolysis in Pseudomonas fluorescens esterase.

    PubMed

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

    2010-03-01

    Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids 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. (2005) Angew. Chem., Int. Ed. 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 epsilon-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 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), 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 epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access

  12. Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase

    SciTech Connect

    Yin, D.; Bernhardt, P; Morley, K; Jiang, Y; Cheeseman, J; Purpero, V; Schrag, J; Kazlauskas, R

    2010-01-01

    Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids 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. (2005) Angew. Chem., Int. Ed. 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 {var_epsilon}-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 2-fold higher k{sub cat}, but K{sub m} also increased so the specificity constant, k{sub cat}/K{sub m}, 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 {var_epsilon}-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the

  13. Histone Acetylation Regulates Intracellular pH

    PubMed Central

    McBrian, Matthew A.; Behbahan, Iman Saramipoor; Ferrari, Roberto; Su, Trent; Huang, Ta-Wei; Li, Kunwu; Hong, Candice S.; Christofk, Heather R.; Vogelauer, Maria; Seligson, David B.; Kurdistani, Siavash K.

    2014-01-01

    SUMMARY Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  14. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus.

    PubMed

    Lee, Dong-Woo; Kim, Dooil; Lee, Yong-Jik; Kim, Jung-Ae; Choi, Ji Young; Kang, Sunghyun; Pan, Jae-Gu

    2013-08-01

    Recent analysis of prokaryotic N(ε)-lysine-acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N(ε)-lysine-acetylated proteome of gram-positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl-lysine-specific antibodies followed by LC-MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism. PMID:23696451

  15. Solid acids as catalysts for the conversion of D-xylose, xylan and lignocellulosics into furfural in ionic liquid.

    PubMed

    Zhang, Luxin; Yu, Hongbing; Wang, Pan

    2013-05-01

    With the aim to develop an ecologically viable catalytic pathway for furfural production without the use of inorganic acids, H3PW12O40, Amberlyst-5 and NKC-9 (macroporous styrene-based sulfonic acid resin) were used as catalysts for producing furfural from xylose, xylan and lignocellulosic biomass in [BMIM]Cl under microwave irradiation at atmospheric pressure. A surprisingly high furfural yield of 93.7% from xylan was obtained by H3PW12O40 at 160 °C in 10 min. The degradation of furfural affected by single addition of [BMIM]Cl and solid acids was also investigated. The IL could be easily recycled and reused with stable solvent capacity for multiple runs (5×) after the product furfural was extracted with ethyl acetate. PMID:23567725

  16. Esterase inhibition attribute of grapefruit juice leading to a new drug interaction.

    PubMed

    Li, Ping; Callery, Patrick S; Gan, Liang-Shang; Balani, Suresh K

    2007-07-01

    This report describes a newly identified potential of grapefruit juice (GFJ) in mediating pharmacokinetic drug interactions due to its capability to inhibit esterase. The study demonstrates that GFJ inhibits purified porcine esterase activity toward p-nitrophenyl acetate and the prodrugs lovastatin and enalapril. In rat and human hepatic or gut S9 fractions and rat gut lumen, GFJ inhibited the hydrolysis of enalapril and lovastatin, which are known to be metabolized principally by esterases, lovastatin being metabolized also by CYP3A. In Caco-2 cells, with minimal CYP3A activity, permeability of these prodrugs was increased in the presence of GFJ. In rats, oral coadministration of GFJ or an esterase inhibitor, bis-(p-nitrophenylphosphate), with the prodrugs led to respective increases in plasma area under the curve by 70% or 57% for enalaprilat and 279% or 141% for lovastatin acid. In addition, portal vein-cannulated rats pretreated with GFJ at -15 and -2 h before lovastatin administration (10 mg/kg p.o.) as a solution, 1) in water and 2) in GFJ, showed, respectively, a 49% increase (CYP3A-inhibited) and a 116% increase (both CYP3A and gut esterase-inhibited) in the portal plasma exposure to the active acid, compared with a non-GFJ pretreatment group. Overall, along with the CYP3A inactivation by GFJ, the decreased esterase activity also played a significant role in increasing the metabolic stability and permeability of esters leading to enhancement of exposure to the active drugs in rats. These new esterase inhibition findings indicate that the potential of drug interaction between ester prodrugs and GFJ should also be considered in the clinic. PMID:17392396

  17. 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

  18. Structural analysis of thermostabilizing mutations of cocaine esterase

    SciTech Connect

    Narasimhan, Diwahar; Nance, Mark R.; Gao, Daquan; Ko, Mei-Chuan; Macdonald, Joanne; Tamburi, Patricia; Yoon, Dan; Landry, Donald M.; Woods, James H.; Zhan, Chang-Guo; Tesmer, John J.G.; Sunahara, Roger K.

    2010-09-03

    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 unstable 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.

  19. Genetic variability for esterase enzyme in Onobrychis species.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G; Burke, J J; Nunna, R R

    1990-10-01

    Understanding polymorphism at the enzyme level is basic to its use in population and genetic studies. However, no such information is available on the variability among different sainfoin (Onobrychis) species. Therefore, our objective was to study the existence of genetic polymorphism for esterase in 17 Onobrychis species and three cultivars of O. viciifolia Scop. Three regions of banding were observed in all the materials tested, with the number of bands varying from 0 to 3, 3 to 14, and 1 to 2 bands in each of these zones, which have been designated EST1, EST2, and EST3 respectively. All the materials studied had unique banding patterns, the only common feature being that all of them, except one species, had isozyme 1. Identification was possible only for four species (O. iberica, O. kachetica, O. transcaucasica, and O. bieberstenii) and one cultivar ('Nova') based on the banding patterns. Large diversity was evident from the wide range of percent similarity values (0%-79%). Subsequent studies should be directed in using these isozyme banding patterns as markers to the desirable agronomic and quality traits of different germplasm lines. PMID:24220999

  20. Inhibition of monocyte esterase activity by organophosphate insecticides.

    PubMed

    Lee, M J; Waters, H C

    1977-11-01

    Organophosphate insecticides, such as Vapona, Naled, and Rabon, are highly potent inhibitors of an enzyme found in human monocytes. The enzyme, a specific monocyte esterase, could be inhibited by Vapona in blood samples via airborne contamination at levels easily achieved from commercial slow-release insecticide strips. Fifty percent inhibition (I50)--as measured on the Hemalog D (Technicon Corp.)--occurred at solution concentrations of 0.22, 1.5, and 2.6 X 10(-6) g/liter for Vapona, Rabon, and Naled, respectively. Parathion (a thiophosphate) and Baygon (a carbamate) were less potent, with I50 values of 3.7 X 10(-5) and 1.5 X 10(-4) g/liter, respectively. Dursban (another thiophosphate) and Carbaryl (a carbamate) showed only marginal inhibition. Eserine, malathion, nicotine and pyrethrum had no inhibitory effect up to 0.5 g/liter. The occurrence of this effect in vivo has not yet been shown, nor is it clear what the implications of such an effect would be. The inhibition of this enzyme by airborne contaminants, however, may interfere with the proper functioning of the Hemalog D. PMID:907842

  1. Hydrolysis of synthetic polyesters by Clostridium botulinum esterases.

    PubMed

    Perz, Veronika; Baumschlager, Armin; Bleymaier, Klaus; Zitzenbacher, Sabine; Hromic, Altijana; Steinkellner, Georg; Pairitsch, Andris; Łyskowski, Andrzej; Gruber, Karl; Sinkel, Carsten; Küper, Ulf; Ribitsch, Doris; Guebitz, Georg M

    2016-05-01

    Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site. Biotechnol. Bioeng. 2016;113: 1024-1034. © 2015 Wiley Periodicals, Inc. PMID:26524601

  2. Identification of petrogenic produced water components as acetylcholine esterase inhibitors.

    PubMed

    Froment, Jean; Langford, Katherine; Tollefsen, Knut Erik; Bråte, Inger Lise N; Brooks, Steven J; Thomas, Kevin V

    2016-08-01

    Effect-directed analysis (EDA) was applied to identify acetylcholine esterase (AChE) inhibitors in produced water. Common produced water components from oil production activities, such as polycyclic aromatic hydrocarbons (PAHs), alkylphenols, and naphthenic acids were tested for AChE inhibition using a simple mixture of PAHs and naphthenic acids. Produced water samples collected from two offshore platforms in the Norwegian sector of the North Sea were extracted by solid phase extraction and fractionated by open-column liquid solid chromatography and high-performance liquid chromatography (HPLC) before being tested using a high-throughput and automated AChE assay. The HPLC fractions causing the strongest AChE inhibition were analysed by gas chromatography coupled to a high-resolution time-of-flight mass spectrometry (GC-HR-ToF-MS). Butylated hydroxytoluene and 4-phenyl-1,2-dihydronaphthalene were identified as two produced water components capable of inhibiting AChE at low concentrations. In order to assess the potential presence of such compounds discharged into aquatic ecosystems, AChE activity in fish tissues was measured. Saithe (Pollachius virens) caught near two offshore platforms showed lower enzymatic activity than those collected from a reference location. Target analysis of saithe did not detected the presence of these two putative AChE inhibitors and suggest that additional compounds such as PAHs, naphthenic acids and yet un-identified compounds may also contribute to the purported AChE inhibition observed in saithe. PMID:27176761

  3. Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

    PubMed Central

    Jia, Yangyang; Wilkins, David; Lu, Hongyuan; Cai, Mingwei

    2015-01-01

    Cellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genus Methanobacterium) and bacterial (order Clostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions. PMID:26712547

  4. Direct transformation of xylan-type hemicelluloses to furfural via SnCl₄ catalysts in aqueous and biphasic systems.

    PubMed

    Wang, Wenju; Ren, Junli; Li, Huiling; Deng, Aojie; Sun, Runcang

    2015-05-01

    Direct catalytic transformation of xylan-type hemicelluloses to furfural in the aqueous system and the biphasic system were comparatively investigated under mild conditions. Screening of several promising chlorides for conversion of beech xylan in the aqueous system revealed the Lewis acid SnCl4 was the most effective catalyst. Comparing to the single aqueous system, the bio-based 2-methyltetrahydrofuran (2-MTHF)/H2O biphasic system was more conducive to the synthesis of furfural, in which the highest furfural yield of 78.1% was achieved by using SnCl4 as catalysts under the optimized reaction conditions (150°C, 120 min). Additionally, the influences of xylan-type hemicelluloses with different chemical and structural features from beech, corncob and bagasse on the furfural production were studied. It was found that furfural yield to some extent was determined by the xylose content in hemicelluloses and also had relationships with the molecular weight of hemicelluloses and the degree of crystallization. PMID:25742750

  5. Assembly of Xylanases into Designer Cellulosomes Promotes Efficient Hydrolysis of the Xylan Component of a Natural Recalcitrant Cellulosic Substrate

    PubMed Central

    Moraïs, Sarah; Barak, Yoav; Hadar, Yitzhak; Wilson, David B.; Shoham, Yuval; Lamed, Raphael; Bayer, Edward A.

    2011-01-01

    ABSTRACT In nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose component of the plant cell wall is embedded in a colloidal blend of hemicelluloses, a major component of which is xylan. In order to enhance enzymatic degradation of the xylan component of a natural complex substrate (wheat straw) and to study the synergistic action among different xylanases, we have employed a variation of the designer cellulosome approach by fabricating a tetravalent complex that includes the three endoxylanases of Thermobifida fusca (Xyn10A, Xyn10B, and Xyn11A) and an Xyl43A β-xylosidase from the same bacterium. Here, we describe the conversion of Xyn10A and Xyl43A to the cellulosomal mode. The incorporation of the Xyl43A enzyme together with the three endoxylanases into a common designer cellulosome served to enhance the level of reducing sugars produced during wheat straw degradation. The enhanced synergistic action of the four xylanases reflected their immediate juxtaposition in the complex, and these tetravalent xylanolytic designer cellulosomes succeeded in degrading significant (~25%) levels of the total xylan component of the wheat straw substrate. The results suggest that the incorporation of xylanases into cellulosome complexes is advantageous for efficient decomposition of recalcitrant cellulosic substrates—a distinction previously reserved for cellulose-degrading enzymes. PMID:22086489

  6. Facilitating the enzymatic saccharification of pulped bamboo residues by degrading the remained xylan and lignin-carbohydrates complexes.

    PubMed

    Huang, Caoxing; He, Juan; Li, Xin; Min, Douyong; Yong, Qiang

    2015-09-01

    Kraft pulping was performed on bamboo residues and its impact on the chemical compositions and the enzymatic digestibility of the samples were investigated. To improve the digestibility of sample by degrading the xylan and lignin-carbohydrates complexes (LCCs), xylanase and α-L-arabinofuranosidase (AF) were supplemented with cellulase. The results showed more carbohydrates were remained in the samples pulped with low effective alkali (EA) charge, compared to conventional kraft pulping. When 120 IU/g xylanase and 15 IU/g AF were supplemented with 20 FPU/g cellulase, the xylan degradation yield of the sample pulped with 12% EA charge increased from 68.20% to 88.35%, resulting in an increased enzymatic saccharification efficiency from 58.98% to 83.23%. The amount of LCCs in this sample decreased from 8.63/100C9 to 2.99/100C9 after saccharification with these enzymes. The results indicated that degrading the remained xylan and LCCs in the pulp could improve its enzymatic digestibility. PMID:26080104

  7. Development of strains of the thermotolerant yeast Hansenula polymorpha capable of alcoholic fermentation of starch and xylan.

    PubMed

    Voronovsky, Andriy Y; Rohulya, Olha V; Abbas, Charles A; Sibirny, Andriy A

    2009-01-01

    The thermotolerant yeast Hansenula polymorpha ferments glucose and xylose to ethanol at high temperatures. However, H. polymorpha cannot utilize starchy materials or xylans. Heterologous amylolytic and xylanolytic enzymes have to be expressed in this yeast to provide for utilization and growth on starch and xylan. Genes SWA2 and GAM1 from the yeast Schwanniomyces occidentalis, encoding alpha-amylase and glucoamylase, respectively, were expressed in H. polymorpha. The expression was achieved by integration of the SWA2 and GAM1 genes under the strong constitutive promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP) into H. polymorpha genome. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source. Ethanol production at high-temperature fermentation from starch by the recombinant strains was up to 10 g/L. The XYN2 gene encoding endoxylanase of the fungus Trichoderma reseei was expressed in H. polymorpha. Co-expression of xlnD gene coding for beta-xylosidase of the fungus Aspergillus niger and the XYN2 gene in H. polymorpha was achieved by integration of these genes under control of the HpGAP promoter. Resulting transformants were capable of growth and alcoholic fermentation on a minimal medium supplemented with birchwood xylan as a sole carbon source at 48 degrees C. PMID:19379821

  8. Non-enzymatic protein acetylation detected by NAPPA protein arrays*

    PubMed Central

    Olia, Adam S.; Barker, Kristi; McCullough, Cheryl E.; Tang, Hsin-Yao; Speicher, David W.; Qiu, Ji; LaBaer, Joshua; Marmorstein, Ronen

    2015-01-01

    Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here we address the possibility that non-enzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the −7 to −3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria, and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated, and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation. PMID:26083674

  9. Nucleosome structure incorporated histone acetylation site prediction in arabidopsis thaliana

    PubMed Central

    2010-01-01

    Abstract Background Acetylation is a crucial post-translational modification for histones, and plays a key role in gene expression regulation. Due to limited data and lack of a clear acetylation consensus sequence, a few researches have focused on prediction of lysine acetylation sites. Several systematic prediction studies have been conducted for human and yeast, but less for Arabidopsis thaliana. Results Concerning the insufficient observation on acetylation site, we analyzed contributions of the peptide-alignment-based distance definition and 3D structure factors in acetylation prediction. We found that traditional structure contributes little to acetylation site prediction. Identified acetylation sites of histones in Arabidopsis thaliana are conserved and cross predictable with that of human by peptide based methods. However, the predicted specificity is overestimated, because of the existence of non-observed acetylable site. Here, by performing a complete exploration on the factors that affect the acetylability of lysines in histones, we focused on the relative position of lysine at nucleosome level, and defined a new structure feature to promote the performance in predicting the acetylability of all the histone lysines in A. thaliana. Conclusion We found a new spacial correlated acetylation factor, and defined a ε-N spacial location based feature, which contains five core spacial ellipsoid wired areas. By incorporating the new feature, the performance of predicting the acetylability of all the histone lysines in A. Thaliana was promoted, in which the previous mispredicted acetylable lysines were corrected by comparing to the peptide-based prediction. PMID:21047388

  10. Xylanase production with xylan rich lignocellulosic wastes by a local soil isolate of Trichoderma viride.

    PubMed

    Goyal, Meenakshi; Kalra, K L; Sareen, V K; Soni, G

    2008-07-01

    In the present study, cultural and nutritional conditions for enhanced production of xylanase by a local soil isolate of Trichoderma viride, using various lignocellulosic substrates in submerged culture fermentation have been optimized. Of the lignocellulosics used, maize straw was the best inducer followed by jowar straw for xylanase production. The highest activity achieved was between 14 to 17 days of fermentation. A continuous increase in xylanase production was observed with increasing level of lignocellulosics in the medium and highest activity was observed with maize straw at 5% level. Xylanase production with higher levels of lignocellulosics (3 to 5%) of maize, jowar and barseem was found to be higher as compared to that with commercial xylan as carbon source. Sodium nitrate was the best nitrogen source among the six sources used. Maximum xylanase production was achieved with initial medium pH of 3.5-4.0 and incubation temperature of 25ºC.The enzyme preparation was effective in bringing about saccharification of different lignocellulosics. The xylanase production could be further improved by using alkali treated straw as carbon source. PMID:24031262

  11. Xylanase production with xylan rich lignocellulosic wastes by a local soil isolate of Trichoderma viride

    PubMed Central

    Goyal, Meenakshi; Kalra, K.L.; Sareen, V.K.; Soni, G.

    2008-01-01

    In the present study, cultural and nutritional conditions for enhanced production of xylanase by a local soil isolate of Trichoderma viride, using various lignocellulosic substrates in submerged culture fermentation have been optimized. Of the lignocellulosics used, maize straw was the best inducer followed by jowar straw for xylanase production. The highest activity achieved was between 14 to 17 days of fermentation. A continuous increase in xylanase production was observed with increasing level of lignocellulosics in the medium and highest activity was observed with maize straw at 5% level. Xylanase production with higher levels of lignocellulosics (3 to 5%) of maize, jowar and barseem was found to be higher as compared to that with commercial xylan as carbon source. Sodium nitrate was the best nitrogen source among the six sources used. Maximum xylanase production was achieved with initial medium pH of 3.5–4.0 and incubation temperature of 25ºC.The enzyme preparation was effective in bringing about saccharification of different lignocellulosics. The xylanase production could be further improved by using alkali treated straw as carbon source. PMID:24031262

  12. Cationic and anionic polyelectrolyte complexes of xylan and chitosan. Interaction with lignocellulosic surfaces.

    PubMed

    Mocchiutti, Paulina; Schnell, Carla N; Rossi, Gerardo D; Peresin, María S; Zanuttini, Miguel A; Galván, María V

    2016-10-01

    Cationic (CatPECs) and anionic (AnPECs) polyelectrolyte complexes from xylan and chitosan were formed, characterized and adsorbed onto unbleached fibers for improving the papermaking properties. They were prepared at a level of 30% of neutralization charge ratio by modifying the order of addition of polyelectrolytes and the ionic strength (0.01N and 0.1N NaCl). The charge density, colloidal stability and particle size of polyelectrolyte complexes (PECs) was measured using polyelectrolyte titration method, Turbiscan and Zetasizer Nano equipments, respectively. All the complexes were stable even after seven days from PEC formation. DRIFT spectra of complexes were also analyzed. The adsorption behavior of them onto cellulose nanofibrils model surfaces was studied using quartz crystal microbalance with dissipation monitoring, and surface plasmon resonance. It was found that the PEC layers were viscoelastic and highly hydrated. Finally, it is shown that the adsorbed PECs onto cellulosic fibers markedly improved the tensile and crushing strengths of paper. PMID:27312617

  13. 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. PMID:25575887

  14. Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165

    PubMed Central

    Shainu, Anju; Pandey, Ashok; Sukumaran, Rajeev K.

    2014-01-01

    Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a Km and Vmax of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol. PMID:24800063

  15. Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165.

    PubMed

    Alex, Deepthy; Shainu, Anju; Pandey, Ashok; Sukumaran, Rajeev K

    2014-01-01

    Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a K m and V max of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol. PMID:24800063

  16. 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. PMID:26369647

  17. Profiling Esterases in Mycobacterium tuberculosis Using Far-Red Fluorogenic Substrates.

    PubMed

    Tallman, Katie R; Levine, Samantha R; Beatty, Kimberly E

    2016-07-15

    Enzyme-activated, fluorogenic probes are powerful tools for studying bacterial pathogens, including Mycobacterium tuberculosis (Mtb). In prior work, we reported two 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO)-derived acetoxymethyl ether probes for esterase and lipase detection. Here, we report four-carbon (C4) and eight-carbon (C8) acyloxymethyl ether derivatives, which are longer-chain fluorogenic substrates. These new probes demonstrate greater stability and lipase reactivity than the two-carbon (C2) acetoxymethyl ether-masked substrates. We used these new C4 and C8 probes to profile esterases and lipases from Mtb. The C8-masked probes revealed a new esterase band in gel-resolved Mtb lysates that was not present in lysates from nonpathogenic M. bovis (bacillus Calmette-Guérin), a close genetic relative. We identified this Mtb-specific enzyme as the secreted esterase Culp1 (Rv1984c). Our C4- and C8-masked probes also produced distinct Mtb banding patterns in lysates from Mtb-infected macrophages, demonstrating the potential of these probes for detecting Mtb esterases that are active during infections. PMID:27177211

  18. Simultaneous determination of N(1)-acetyl sulfisoxazole and its metabolites, and relative bioavailability compare to sulfisoxazole in rats.

    PubMed

    Kim, Eunyoung; Kang, Wonku

    2016-09-10

    N(1)-acetyl sulfisoxazole (N1AS), a dihydropteroate synthase inhibitor is known to be biotransformed primarily to sulfisoxazole, partly to N(4)-acetyl sulfisoxazole (N4AS), and likely also to diacetyl sulfisoxazole (DAS) and other compounds. Although its clinical use has been limited due to urolithiasis, some countries still use the drug in combination with trimethoprim in cattle. A liquid chromatographic method using ultraviolet detection was developed for the simultaneous determination of four substances for the first time. Four analytes and sulfamethoxazole (IS) were separated on a reversed-phase column with gradient elution of a mobile phase. Because DAS and N1AS in plasma were changed very quickly into N4AS and sulfisoxazole, respectively, and esterase inhibitors (sodium fluoride and eserine) could not prevent the transformation, sulfisoxazole and N4AS were monitored in rat plasma following a single oral administration of N1AS and sulfisoxazole in five rats. The relative bioavailability of N1AS to sulfisoxazole was about two, indicating that a half-dose of N1AS would be equivalent to a dose of sulfisoxazole to achieve the same systemic exposure to sulfisoxazole. PMID:27423008

  19. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  20. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  1. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  2. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  3. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has...

  4. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  5. Cellular function of neuropathy target esterase in lysophosphatidylcholine action

    SciTech Connect

    Vose, Sarah C.; Fujioka, Kazutoshi; Gulevich, Alex G.; Lin, Amy Y.; Holland, Nina T.; Casida, John E.

    2008-11-01

    Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.

  6. Extraction and purification of wheat-esterase using aqueous two-phase systems of ionic liquid and salt.

    PubMed

    Jiang, Bin; Feng, Zhibiao; Liu, Chunhong; Xu, Yingcao; Li, Dongmei; Ji, Guo

    2015-05-01

    To explore a new and simple rapid extraction and purification technique for wheat-esterase, an ionic liquids (ILs)-based aqueous two-phase system (ATPS) was developed for the purification of wheat-esterase from wheat extracts. Effects of various process parameters such as the concentrations of [Bmim]BF4, the types and concentrations of phase-forming salt, the system pH and the temperature on partitioning of wheat-esterase were evaluated. The obtained data indicated that wheat-esterase was preferentially partitioned into the ILs-rich phase and the ATPS composed of 20 % [Bmim]BF4 (w/w) and 25 % (w/w) NaH2PO4(pH = 4.8) showed good selectivity on wheat-esterase. Under the optimum conditions, wheat-esterase was purified with an acceptable yield (88.93 %), but produced wheat-esterase was 4.23 times as pure. It was obvious that temperature shows little influence on the purification between 10 and 50 °C. Sephadex G-150FF revealed that the band intensity of contaminating proteins in ATPS fraction almost disappeared. Therefore, ILs-based ATPS was an effective method for partitioning and recovery of wheat-esterase from wheat crude extracts. PMID:25892786

  7. Cutinolytic esterase activity of bacteria isolated from mixed-plant compost and characterization of a cutinase gene from Pseudomonas pseudoalcaligenes.

    PubMed

    Inglis, G D; Yanke, L J; Selinger, L B

    2011-11-01

    The objective of the current study was to examine cutinolytic esterase (i.e., cutinase) activity by pseudomonads and bacteria isolated from mixed-plant compost. Approximately 400 isolates representing 52 taxa recovered from mixed-plant compost using cuticle baits, along with 117 pseudomonad isolates obtained from a culture collection (i.e., non-compost habitats), were evaluated. The ability of isolates to degrade the synthetic cutin polycaprolactone (PCL) was initially measured. Isolates from 23 taxa recovered from the compost degraded PCL. As well, isolates from 13 taxa of pseudomonads cleared PCL. Secondary screening measured esterase activity induced by the presence of apple cuticle using the chromogenic substrate p-nitrophenyl butyrate. Eighteen isolates representing four taxa (Alcaligenes faecalis , Bacillus licheniformis , Bacillus pumilus , and Pseudomonas pseudoalcaligenes) recovered from compost exhibited substantial esterase activity when grown with cuticle. In contrast, none of the pseudomonad isolates from the culture collection produced appreciable esterase activity. Although degradation of PCL was not correlated with esterase activity, isolates that were unable to degrade PCL failed to produce measureable esterase activities. Zymogram analysis indicated that the esterases produced by bacteria from compost ranged in size from 29 to 47 kDa. A gene from P. pseudoalcaligenes (cutA) was found to code for a cutin-induced esterase consisting of 302 amino acids and a theoretical protein size of 32 kDa. The enzyme was unique and was most closely related to other bacterial lipases (≤48% similarity). PMID:22029433

  8. Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a Type C feruloyl esterase, which acted on methyl ferulate, methyl p-coumarate, methyl sinapinate, methyl caffeate, but not diferul...

  9. COMPARISON OF THE RELATIVE INHIBITION OF ACETYLCHOLINESTERASE AND NEUROPATHY TARGET ESTERASE IN RATS AND HENS GIVEN CHOLINESTERASE INHIBITORS

    EPA Science Inventory

    Inhibition of neuropathy target esterase (NTE, neurotoxic esterase) and acetylcholinesterase (AME) activities was compared in brain and spinal cords of adult. hile Leghorn hens and adult male Long Evans rats 4-48 hr after administration of tri-ortho-tolyl phosphate (TOTP po, 50-5...

  10. ROLE OF NEUROTOXIC ESTERASE (NTE) IN THE PREVENTION AND POTENTIATION OF ORGANOPHOSPHORUS-INDUCED DELAYED NEUROTOXICITY (OPIDN)

    EPA Science Inventory

    The first step in the initiation of organophosphorus-induced delayed neuropathy (OPIDN) is proposed to be the phosphorylation of an enzyme found in the nervous system called neurotoxic esterase (neuropathy target esterase, NTE). t has been known for over twenty years that non-neu...

  11. Expression of feruloyl esterase A from Aspergillus terreus and its application in biomass degradation.

    PubMed

    Zhang, Shuai-Bing; Wang, Le; Liu, Yan; Zhai, Huan-Chen; Cai, Jing-Ping; Hu, Yuan-Sen

    2015-11-01

    Feruloyl esterases (FAEs) are key enzymes involved in the complete biodegradation of lignocelluloses, which could hydrolyze the ester bonds between hemicellulose and lignin. The coding sequence of a feruloyl esterase A (AtFaeA) was cloned from Aspergillus terreus and the recombinant AtFaeA was constitutively expressed in Pichia pastoris. The SDS-PAGE analysis of purified AtFaeA showed two protein bands owing to the different extent of glycosylation, and the recombinant AtFaeA had an optimum temperature of 50°C and an optimum pH of 5.0. The substrate utilization and primary sequence identity of AtFaeA demonstrated that it is a type-A feruloyl esterase. The hydrolysis of corn stalk and corncob by xylanase from Aspergillus niger could be significantly improved in concert with recombinant AfFaeA. PMID:26282562

  12. 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.

  13. Transition-State Analysis of 2-O-Acetyl-ADP-Ribose Hydrolysis by Human Macrodomain 1

    PubMed Central

    2015-01-01

    Macrodomains, including the human macrodomain 1 (MacroD1), are erasers of the post-translational modification of monoadenosinediphospho-ribosylation and hydrolytically deacetylate the sirtuin product O-acetyl-ADP-ribose (OAADPr). OAADPr has been reported to play a role in cell signaling based on oocyte microinjection studies, and macrodomains affect an array of cell processes including transcription and response to DNA damage. Here, we investigate human MacroD1 by transition-state (TS) analysis based on kinetic isotope effects (KIEs) from isotopically labeled OAADPr substrates. Competitive radiolabeled-isotope effects and mass spectrometry were used to obtain KIE data to yield intrinsic KIE values. Intrinsic KIEs were matched to a quantum chemical structure of the TS that includes the active site residues Asp184 and Asn174 and a structural water molecule. Transition-state analysis supports a concerted mechanism with an early TS involving simultaneous nucleophilic water attack and leaving group bond cleavage where the breaking C–O ester bond = 1.60 Å and the C–O bond to the attacking water nucleophile = 2.30 Å. The MacroD1 TS provides mechanistic understanding of the OAADPr esterase chemistry. PMID:25051211

  14. Characterization of a novel cold active and salt tolerant esterase from Zunongwangia profunda.

    PubMed

    Rahman, Mohammad Asadur; Culsum, Umma; Tang, Wenhao; Zhang, Shao Wei; Wu, Gaobing; Liu, Ziduo

    2016-04-01

    A novel cold active esterase, EstLiu was cloned from the marine bacterium Zunongwangia profunda, overexpressed in E. coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography. The mature esterase EstLiu sequence encodes a protein of 273 amino acids residues, with a predicted molecular weight of 30KDa and containing the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243. Although, EstLiu showed 64% similarity with the hypothetical esterase from Chryseobacterium sp. StRB126 (WP_045498424), phylogenetic analysis showed it had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family. The purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). EstLiu showed remarkable activity (75%) at 0°Cand the optimal activity at pH 8.0 and 30°C with good thermostability and quickened inactivation above 60°C. EstLiu retained 81, 103, 67 and 78% of its original activity at 50% (v/v) in ethanol, isopropanol, DMSO and ethylene glycol, respectively. In the presence of Tween 20, Tween 80 and Triton X-100, EstLiu showed 88, 100 and 117% of relative activity. It is also co-factor independent. The high activity at low temperature and desirable stability in organic solvents and salts of this novel family esterase represents a good evidence of novel biocatalyst. Overall, this novel enzyme showed better activity than previously reported esterases in extreme reaction conditions and could promote the reaction in both aqueous and non-aqueous conditions, indicating its great potential for industrial applications. PMID:26920474

  15. 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. PMID:25852987

  16. Purified human C1-esterase inhibitor is safe in acute relapses of neuromyelitis optica

    PubMed Central

    Levy, Michael

    2014-01-01

    Objective: To minimize complement-mediated damage in acute relapses of neuromyelitis optica (NMO) by adding treatment with a complement inhibitor, purified C1-esterase inhibitor, to the current standard of care (high-dose glucocorticoids). Method: We conducted an open-label phase 1b safety and proof-of-concept trial in 10 patients with NMO–immunoglobulin G seropositive NMO or NMO spectrum disease (NMOSD) who presented with acute transverse myelitis and/or optic neuritis. In addition to treating with 1 g of daily IV methylprednisolone, we infused 2,000 units of C1-esterase inhibitor daily for 3 days, beginning on day 1 of hospitalization. The primary outcome measure was safety, and the secondary efficacy measure was change in Expanded Disability Status Scale (EDSS) scores. Results: Ten patients with NMO/NMOSD were enrolled, 7 of whom presented with acute transverse myelitis and 3 with acute optic neuritis. C1-esterase inhibitor proved to be safe in all 10 patients, with no serious adverse events recorded. There were no thromboembolic events or related lab abnormalities in any of the subjects. EDSS scores dropped from a median of 4.5 on admission to 4.0 on discharge and then down to 2.5 on 30-day follow-up. All but 1 patient returned to preattack EDSS or better and only 2 patients required escalation to plasmapheresis. Conclusions: C1-esterase inhibitor is a safe add-on therapy for patients with NMO/NMOSD presenting with acute transverse myelitis and optic neuritis. Preliminary evidence suggests a promising benefit with C1-esterase inhibitor in reducing neurologic damage and improving outcomes. A placebo-controlled trial is necessary to confirm these findings. Classification of evidence: This study provides Class IV evidence that for patients with NMO with acute transverse myelitis or optic neuritis, C1-esterase inhibitor is safe and improves disability. PMID:25340061

  17. Esterases immobilized on aminosilane modified magnetic nanoparticles as a catalyst for biotransformation reactions.

    PubMed

    Alex, Deepthy; Mathew, Abraham; Sukumaran, Rajeev K

    2014-09-01

    Magnetite nanoparticles were prepared by reacting ferrous and ferric salts in presence of aqueous ammonia. The magnetic nanoparticles (MNPs) were amino functionalized by treating with 3-aminopropyl triethoxy silane (APTES) and was coupled with glutaraldehyde. A novel solvent tolerant esterase from Pseudozyma sp. NII 08165 was immobilized on the MNPs through covalent bonding to the glutaraldehyde. The magnetite nanoparticles had a size range of 10-100 nm, confirmed by DLS. Lipases immobilized on MNPs were evaluated for biotransformation reactions including synthesis of ethyl acetate and transesterification of vegetable oil for producing biodiesel. The MNP immobilized esterase had prolonged shelf life and there was no loss in enzyme activity. PMID:24968816

  18. Nanocomposite films based on xylan-rich hemicelluloses and cellulose nanofibers with enhanced mechanical properties.

    PubMed

    Peng, Xin-wen; Ren, Jun-li; Zhong, Lin-xin; Sun, Run-cang

    2011-09-12

    Interest in xylan-rich hemicelluloses (XH) film is growing, and efforts have been made to prepare XH films with improved mechanical properties. This work described an effective approach to produce nanocomposite films with enhanced mechanical properties by incorporation of cellulose nanofibers (CNFs) into XH. Aqueous dispersions of XH (64-75 wt %), sorbitol (16-25 wt %), and CNF (0-20 wt %) were cast at a temperature of 23 °C and 50% relative humidity. The surface morphology of the films was revealed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The thermal properties and crystal structure of the films were evaluated by thermal analysis (TG) and X-ray diffraction (XRD). The surface of XH films with and without CNF was composed primarily of nanonodules, and CNFs were embedded in the XH matrix. Freeze-dried XH powder was amorphous, whereas the films with and without CNF showed a distinct peak at around 2θ = 18°, which suggested that XH molecules aggregated or reordered in the casting solution or during water evaporation. Furthermore, the nanocomposite films had improved thermal stability. XH film with 25 wt % plasticizer (sorbitol, based on dry XH weight) showed poor mechanical properties, whereas incorporation of CNF (5-20 wt %, based on the total dry mixture) into the film resulted in enhanced mechanical properties due to the high aspect ratio and mechanical strength of CNF and strong interactions between CNF and XH matrix. This effective method makes it possible to produce hemicellulose-based biomaterials of high quality. PMID:21815695

  19. Xylans Provide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat.

    PubMed

    Ralet, Marie-Christine; Crépeau, Marie-Jeanne; Vigouroux, Jacqueline; Tran, Joseph; Berger, Adeline; Sallé, Christine; Granier, Fabienne; Botran, Lucy; North, Helen M

    2016-05-01

    Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells produce large amounts of mucilage that is released upon imbibition. This mucilage is structured into two domains: an outer diffuse layer that can be easily removed by agitation and an inner layer that remains attached to the outer seed coat. Both layers are composed primarily of pectic rhamnogalacturonan I (RG-I), the inner layer also containing rays of cellulose that extend from the top of each columella. Perturbation in cellulosic ray formation has systematically been associated with a redistribution of pectic mucilage from the inner to the outer layer, in agreement with cellulose-pectin interactions, the nature of which remained unknown. Here, by analyzing the outer layer composition of a series of mutant alleles, a tight proportionality of xylose, galacturonic acid, and rhamnose was evidenced, except for mucilage modified5-1 (mum5-1; a mutant showing a redistribution of mucilage pectin from the inner adherent layer to the outer soluble one), for which the rhamnose-xylose ratio was increased drastically. Biochemical and in vitro binding assay data demonstrated that xylan chains are attached to RG-I chains and mediate the adsorption of mucilage to cellulose microfibrils. mum5-1 mucilage exhibited very weak adsorption to cellulose. MUM5 was identified as a putative xylosyl transferase recently characterized as MUCI21. Together, these findings suggest that the binding affinity of xylose ramifications on RG-I to a cellulose scaffold is one of the factors involved in the formation of the adherent mucilage layer. PMID:26979331

  20. Regulation of JH epoxide hydrolase versus JH esterase activity in the cabbage looper, Trichoplusia ni, by juvenile hormone and xenobiotics.

    PubMed

    Anspaugh, Douglas D; Roe, R Michael

    2005-05-01

    JH III esterase and JH III epoxide hydrolase (EH) in vitro activity was compared in whole body Trichoplusia ni homogenates at each stage of development (egg, larva, pupa and adult). While activity of both enzymes was detected at all ages tested, JH esterase was significantly higher than EH activity except for day three of the fifth (last) stadium (L5D3). For both enzymes, activity was highest in eggs. Adult virgin females had 4.6- and 4.0-fold higher JH esterase and EH activities, respectively, than adult virgin males. JH III metabolic activity also was measured in whole body homogenates of fifth stadium T. ni that were fed a nutritive diet (control) or starved on a non-nutritive diet of alphacel, agar and water. With larvae that were starved for 6, 28 and 52 h, EH activity per insect equivalent was 48%, 5% and 1%, respectively, of the control insects. At the same time points, JH esterase activity levels in starved T. ni were 29%, 4% and 3% of that of insects fed the nutritive diet. Selected insect hormones and xenobiotics were administered topically or orally to fifth stadium larvae for up to 52 h, and the effects on whole body EH and JH esterase activity analyzed. JH III increased the JH III esterase activity as high as 2.2-fold, but not the JH III EH activity. The JH analog, methoprene, increased both JH esterase and EH activity as high as 2.5-fold. The JH esterase inhibitor, 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP), had no impact on EH activity. The epoxides trans- and cis-stilbene oxide (TSO and CSO) in separate experiments increased the EH activity approximately 2.0-fold. TSO did not alter JH esterase levels when topically applied, but oral administration reduced activity to 70% of the control at 28 h, and then increased the activity 1.8-fold at 52 h after the beginning of treatment. CSO had no effect on JH esterase activity. Phenobarbital increased EH activity by 1.9-fold, but did not change JH esterase levels. Clofibrate and cholesterol 5alpha,6alpha

  1. Dynamic Protein Acetylation in Plant–Pathogen Interactions

    PubMed Central

    Song, Gaoyuan; Walley, Justin W.

    2016-01-01

    Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection. PMID:27066055

  2. Xylan synthesized by Irregular Xylem 14 (IRX14) maintains the structure of seed coat mucilage in Arabidopsis

    PubMed Central

    Hu, Ruibo; Li, Junling; Wang, Xiaoyu; Zhao, Xun; Yang, Xuanwen; Tang, Qi; He, Guo; Zhou, Gongke; Kong, Yingzhen

    2016-01-01

    During differentiation, the Arabidopsis seed coat epidermal cells synthesize and secrete large quantities of pectinaceous mucilage into the apoplast, which is then released to encapsulate the seed upon imbibition. In this study, we showed that mutation in Irregular Xylem 14 (IRX14) led to a mucilage cohesiveness defect due to a reduced xylan content. Expression of IRX14 was detected specifically in the seed coat epidermal cells, reaching peak expression at 13 days post-anthesis (DPA) when the accumulation of mucilage polysaccharides has ceased. Sectioning of the irx14-1 seed coat revealed no visible structural change in mucilage secretory cell morphology. Although the total amount of mucilage was comparable with the wild type (WT), the partition between water-soluble and adherent layers was significantly altered in irx14-1, with redistribution from the adherent layer to the water-soluble layer. The monosaccharide composition analysis revealed that xylose content was significantly reduced in irx14-1 water-soluble and adherent mucilage compared with the WT. The macromolecular characteristics of the water-soluble mucilage were modified in irx14-1 with a loss of the larger polymeric components. In accordance, glycome profiling and dot immunoblotting of seed mucilage using antibodies specific for rhamnogalacturonan I (RG I) and xylan confirmed the ultra-structural alterations in the irx14-1 mucilage. Meanwhile, the crystalline cellulose content was reduced in the irx14-1 mucilage. These results demonstrated that IRX14 was required for the biosynthesis of seed mucilage xylan, which plays an essential role in maintaining mucilage architecture potentially through altering the crystallization and organization of cellulose. PMID:26834178

  3. Xylan synthesized by Irregular Xylem 14 (IRX14) maintains the structure of seed coat mucilage in Arabidopsis.

    PubMed

    Hu, Ruibo; Li, Junling; Wang, Xiaoyu; Zhao, Xun; Yang, Xuanwen; Tang, Qi; He, Guo; Zhou, Gongke; Kong, Yingzhen

    2016-03-01

    During differentiation, the Arabidopsis seed coat epidermal cells synthesize and secrete large quantities of pectinaceous mucilage into the apoplast, which is then released to encapsulate the seed upon imbibition. In this study, we showed that mutation in Irregular Xylem 14 (IRX14) led to a mucilage cohesiveness defect due to a reduced xylan content. Expression of IRX14 was detected specifically in the seed coat epidermal cells, reaching peak expression at 13 days post-anthesis (DPA) when the accumulation of mucilage polysaccharides has ceased. Sectioning of the irx14-1 seed coat revealed no visible structural change in mucilage secretory cell morphology. Although the total amount of mucilage was comparable with the wild type (WT), the partition between water-soluble and adherent layers was significantly altered in irx14-1, with redistribution from the adherent layer to the water-soluble layer. The monosaccharide composition analysis revealed that xylose content was significantly reduced in irx14-1 water-soluble and adherent mucilage compared with the WT. The macromolecular characteristics of the water-soluble mucilage were modified in irx14-1 with a loss of the larger polymeric components. In accordance, glycome profiling and dot immunoblotting of seed mucilage using antibodies specific for rhamnogalacturonan I (RG I) and xylan confirmed the ultra-structural alterations in the irx14-1 mucilage. Meanwhile, the crystalline cellulose content was reduced in the irx14-1 mucilage. These results demonstrated that IRX14 was required for the biosynthesis of seed mucilage xylan, which plays an essential role in maintaining mucilage architecture potentially through altering the crystallization and organization of cellulose. PMID:26834178

  4. The neurobiology of acetyl-L-carnitine.

    PubMed

    Traina, Giovanna

    2016-01-01

    A large body of evidence points to the positive effects of dietary supplementation of acetyl-L-carnitine (ALC). Its use has shown health benefits in neuroinflammation, which is a common denominator in a host of neurodegenerative diseases. ALC is the principal acetyl ester of L-Carnitine (LC), and it plays an essential role in intermediary metabolism, acting as a donor of acetyl groups and facilitating the transfer of fatty acids from cytosol to mitochondria during beta-oxidation. Dietary supplementation of ALC exerts neuroprotective, neurotrophic, antidepressive and analgesic effects in painful neuropathies. ALC also has antioxidant and anti-apoptotic activity. Moreover, ALC exhibits positive effects on mitochondrial metabolism, and shows promise in the treatment of aging and neurodegenerative pathologies by slowing the progression of mental deterioration. In addition, ALC plays neuromodulatory effects on both synaptic morphology and synaptic transmission. These effects are likely due to affects of ALC through modulation of gene expression on several targets in the central nervous system. Here, we review the current state of knowledge on effects of ALC in the nervous system. PMID:27100509

  5. Enhancement of lysine acetylation accelerates wound repair

    PubMed Central

    Spallotta, Francesco; Cencioni, Chiara; Straino, Stefania; Sbardella, Gianluca; Castellano, Sabrina; Capogrossi, Maurizio C; Martelli, Fabio; Gaetano, Carlo

    2013-01-01

    In physiopathological conditions, such as diabetes, wound healing is significantly compromised and chronic complications, including ulcers, may occur. In a mouse model of skin repair, we recently reported that wound treatment with Sirtuin activators and class I HDAC inhibitors induced keratinocyte proliferation and enhanced healing via a nitric oxide (NO) dependent mechanism. We observed an increase in total protein acetylation in the wound area, as determined by acetylation of α-tubulin and histone H3 Lysine 9. We reasoned that this process activated cell function as well as regulated gene expression to foster tissue repair. We report here that the direct activation of P300/CBP-associated factor (PCAF) by the histone acetylase activator pentadecylidenemalonate 1b (SPV-106) induced Lysine acetylation in the wound area. This intervention was sufficient to enhance repair process by a NO-independent mechanism. Hence, an impairment of PCAF and/or other GCN5 family acetylases may delay skin repair in physiopathological conditions. PMID:24265859

  6. Fragrance material review on acetyl cedrene.

    PubMed

    Scognamiglio, J; Letizia, C S; Politano, V T; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl cedrene when used as a fragrance ingredient is presented. Acetyl cedrene is a member of the fragrance structural group Alkyl Cyclic Ketones. The generic formula for this group can be represented as (R1)(R2)CO. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl cedrene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, and genotoxicity data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (2013) (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. Submitted with this manuscript.) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. PMID:23907023

  7. 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

  8. 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

  9. Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the present study was to gain new insights into the evolution, homologous recombination and selection pressures imposed on the porcine torovirus (PToV), by examining changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerge...

  10. Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Crystallization and preliminary X-ray diffraction studies of the pneumococcal teichoic acid phosphorylcholine esterase Pce

    SciTech Connect

    Lagartera, Laura; González, Ana; Stelter, Meike; García, Pedro; Kahn, Richard; Menéndez, Margarita; Hermoso, Juan A.

    2005-02-01

    The modular choline-binding protein Pce, the phosphorylcholine esterase from S. pneumoniae, has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a derivative with a gadolinium complex has been collected to 2.7 Å resolution.

  12. 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

  13. 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...

  14. Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase.

    PubMed

    Videira, P A; Fialho, A M; Marques, A R; Coutinho, P M; Sá-Correia, I

    2003-06-01

    The ces10 gene of the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461 was cloned and sequenced. Multi-sequence alignment of the deduced protein indicated that Ces10 belongs to the serine hydrolase family with a potential catalytic triad comprising Ser(153) (within the G-X-S-X-G consensus sequence), His(75) and Asp(125). The mixed block results obtained following pattern search and the low identities detected in a BLAST analysis indicate that Ces10 is significantly different from other characterised bacterial esterases/lipases. Nevertheless, the Ces10 amino acid sequence showed 45% similarity with Rhodococcus sp. heroin esterase and 48% with Bacillus subtilis p-nitrobenzyl esterase. Ces10, with a predicted molecular mass of 30,641 Da, was overproduced in Escherichia coli and purified to homogeneity in a histidine-tagged form. Enzyme assays using p-nitrophenyl-esters (p-NP-esters) with different acyl chain-lengths as the substrate confirmed the anticipated esterase activity. Ces10 exhibited a marked preference for short-chain fatty acids, yielding the highest activity with p-NP-propionate (optimal pH 7.4, optimal temperature 37 degrees C). PMID:12764567

  15. Tissue-specific inhibition and recovery of esterase activities in Lumbricus terrestris experimentally exposed to chlorpyrifos.

    PubMed

    Vejares, Sandra González; Sabat, Pablo; Sanchez-Hernandez, Juan C

    2010-04-01

    Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil. PMID:20045489

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. Total esterase activity in human saliva: Validation of an automated assay, characterization and behaviour after physical stress.

    PubMed

    Tecles, Fernando; Tvarijonaviciute, Asta; De Torre, Carlos; Carrillo, José M; Rubio, Mónica; García, Montserrat; Cugat, Ramón; Cerón, José J

    2016-07-01

    Although saliva has esterase activity, this activity has not been characterized or studied in individuals subjected to physical stress. The aim of this report was to develop and validate an automated spectrophotometric assay for total esterase activity measurement in human saliva, as well as to study the contribution of different enzymes on this activity and its behaviour under physical stress in healthy subjects. The assay used 4-nitrophenyl acetate as substrate and was precise, accurate and provided low limits of detection and quantification. Inhibition with diisopropylfluorophosphate showed that cholinesterase, carboxylesterase and cholesterol esterase contributions not represented more than 20% of total esterase. Addition of standards of lipase and albumin to saliva samples showed that both proteins significantly contributed to esterase activity only when equal or higher than 11.6 IU/L and 250 μg/mL, respectively. Western blot analyses showed absence of paraoxonase-1 and high amount of carbonic anhydrase-VI. The high affinity of purified carbonic anhydrase-VI for the substrate supported a major contribution of this enzyme. Total esterase activity and alpha-amylase was measured in saliva samples from 12 healthy male students before and after participation in an indoor football match. The activity significantly increased after match and positively correlated with salivary alpha-amylase. This method could be used as a biomarker of physical stress in humans, with carbonic anhydrase-VI being the esterase that contributed more to the activity of the assay. PMID:27045801

  18. Engineering of Saccharomyces cerevisiae to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase, xylosidase and a bacterial xylose isomerase.

    PubMed

    Mert, Marlin John; la Grange, Daniël Coenrad; Rose, Shaunita Hellouise; van Zyl, Willem Heber

    2016-04-01

    Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes. PMID:26749525

  19. Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate.

    PubMed Central

    Williams, D G; Johnson, M K

    1981-01-01

    The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1. PMID:7340807

  20. Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.

    PubMed Central

    Borneman, W S; Ljungdahl, L G; Hartley, R D; Akin, D E

    1991-01-01

    An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes. Images PMID:1768103

  1. Association of esterases with insecticide resistance in Culex quinquefasciatus (Diptera: Culicidae).

    PubMed

    Gordon, Jennifer R; Ottea, James

    2012-06-01

    The southern house mosquito, Culex quinquefasciatus Say, is a competent vector of human disease and an important target of mosquito abatement programs. However, these management programs have been compromised by development of insecticide resistance. In the current study, susceptibilities to naled and resmethrin, two adulticides used in mosquito abatement, were monitored using a topical and contact bioassay, respectively, in five field- collected populations of C. quinquefasciatus (MARC, HOOD1, HOOD2, MINLOVE, and THIB). Frequencies of resistance, measured as survival after treatment with discriminating concentrations (i.e., sufficient to kill > 90% of a reference susceptible strain) were high (88.0-96.8%) in all field collections treated with naled, but were variable (3.3-94.2%) with resmethrin. In addition, esterase activities in mosquitoes from these collections were quantified using alpha-naphthyl acetate and ranged from 1.08 to 3.39 micromol alpha-naphthol produced min(-1) mg prot(-1). Heightened activities were associated with decreased insecticide susceptibility in HOOD1, THIB, and MINLOVE but not HOOD2. Esterases were visualized using native polyacrylamide gel electrophoresis, and intra- and interstrain differences in banding patterns were detected. In addition, esterases from MINLOVE mosquitoes were more numerous and intensely staining when compared with those from a laboratory-susceptible strain. Finally, naled synergized the toxicity of resmethrin in populations with decreased insecticide susceptibility and increased esterase activity by 2.5-(MINLOVE) to three-fold (THIB). Results from this study will allow management strategies for populations of C. quinquefasciatus to be optimized, and provide a foundation for further studies exploring use of esterase inhibitors as synergists of pyrethroid toxicity. PMID:22812138

  2. Xylan utilization regulon in Xanthomonas citri pv. citri Strain 306: gene expression and utilization of oligoxylosides.

    PubMed

    Chow, V; Shantharaj, D; Guo, Y; Nong, G; Minsavage, G V; Jones, J B; Preston, J F

    2015-03-01

    Xanthomonas citri pv. citri strain 306 (Xcc306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. In Xcc306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, the agu67 gene, encoding a GH67 α-glucuronidase (Agu67), the xyn43E gene, encoding a putative GH43 α-l-arabinofuranosidase, and the xyn43F gene, encoding a putative β-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4-O-methylglucuronoxylan (MeGXn) to oligoxylosides methylglucuronoxylotriose (MeGX3), xylotriose (X3), and xylobiose (X2). Xcc306 completely utilizes MeGXn predigested with Xyn10A or Xyn10C but shows little utilization of MeGXn. Xcc306 with a deletion in the gene encoding α-glucuronidase (Xcc306 Δagu67) will not utilize MeGX3 for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGXn. Preferential growth on oligoxylosides compared to growth on polymeric MeGXn indicates that GH10 xylanases, either secreted by Xcc306 in planta or produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides of xyn10, agu67, cirA, the tonB receptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action of Xcc306 GH10 xylanases on MeGXn. The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival of Xcc306 and, ultimately, to plant disease. PMID:25595763

  3. Structural characterization (1->2)-beta-xylose-(1->3)-alpha-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan treatment with alpha-arabinofuranosidase and beta-endo-xylanase.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Switchgrass (Panicum virgatum, L.) is a potential dedicated biomass crop for use in biocatalytic conversion systems to biofuels. Nearly 30% of switchgrass cell wall material is xylan. The complete depolymerization of xylan is desirable both as an additional carbon source for microbial fermentation a...

  4. Effects of different levels of gum arabic, low methylated pectin and xylan on in vitro digestibility of beta-lactoglobulin.

    PubMed

    Mouécoucou, J; Sanchez, C; Villaume, C; Marrion, O; Frémont, S; Laurent, F; Méjean, L

    2003-12-01

    Plant hydrocolloids used in the food industry to improve texture and stability of food, such as dairy products, can reduce protein digestibility and, consequently, modify the bioavailability of amino acids. We studied the in vitro hydrolysis at 37 degrees C of beta-lactoglobulin (beta-lg) in mixed dispersions containing either gum arabic or low-methylated pectin or xylan at levels of 0, 1, 10, 20, 30, and 50% weight. Proteolysis used either pepsin alone by progressive reduction of pH during proteolysis or pepsin followed by trypsin and chymotrypsin in two different dialysis bags with a molecular weight (MW) cutoff of 1000 or 8000 Da. Results showed that beta-lg was almost resistant to pepsin digestion and that the three plant hydrocolloids inhibited significantly beta-lg digestibility as determined using dialysis bag with a 1000-Da MW cutoff. Among the three polysaccharides used, xylan showed a digestibility decrease greater than that obtained with gum arabic and low-methylated pectin. On the other hand, no significant effect of polysaccharides on the in vitro beta-lg digestibility was detected using the dialysis bag with an 8000 Da MW cutoff. This mainly suggests that peptides with MW in the range 1000 to 8000 Da may interact with polysaccharides more than peptides and proteins with a greater molecular weight to decrease the protein digestibility, and that the nature of the polysaccharides plays a role in the interaction. PMID:14740820

  5. Acetylation and characterization of spruce (Picea abies) galactoglucomannans.

    PubMed

    Xu, Chunlin; Leppänen, Ann-Sofie; Eklund, Patrik; Holmlund, Peter; Sjöholm, Rainer; Sundberg, Kenneth; Willför, Stefan

    2010-04-19

    Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species and can be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coating polymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, as a classic method, it can be potentially transferred to the industrial scale. The effects of the amount of catalyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated. A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylated GGMs were determined by SEC-MALLS/RI, (1)H and (13)C NMR and FTIR spectroscopy. NMR studies also indicated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. The thermal stability of the products was investigated by DSC-TGA. PMID:20144827

  6. Interfacing protein lysine acetylation and protein phosphorylation

    PubMed Central

    Tran, Hue T.; Uhrig, R. Glen; Nimick, Mhairi; Moorhead, Greg B.

    2012-01-01

    Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature. PMID:22827947

  7. Determination of amphetamine by HPLC after acetylation.

    PubMed

    Veress, T

    2000-01-01

    An analytical procedure has been developed for the HPLC determination of amphetamine by off-line pre-column derivatization. The proposed procedure consists of sample preparation by acetylation of amphetamine with acetic anhydride and a subsequent reversed-phase HPLC separation on an octadecyl silica stationary phase with salt-free mobile phase (tetrahydrofuran, acetonitrile, 0.1% triethylamine in water, 15:15:70 v/v) applying UV-detection. The applicability of the elaborated procedure is demonstrated with results obtained by analysis of real samples seized in the Hungarian black market. PMID:10641931

  8. Comparative study of human intestinal and hepatic esterases as related to enzymatic properties and hydrolizing activity for ester-type drugs.

    PubMed

    Inoue, M; Morikawa, M; Tsuboi, M; Ito, Y; Sugiura, M

    1980-08-01

    In attempts to determine the exact role of intestinal esterase in the body, we purified esterases from human intestinal mucosa and liver, and compared the enzymatic properties and substrate specificities with those of purified esterases. Esterase from human liver was purified 58-fold, by treatment with butanol, DE-52 and DEAE Sephadex A-50 column chromatographies, Sephadex G-200 gel filtration, and isoelectric focusing. The purified preparation showed a single band by polyacylamide gel electrophoresis. The molecular weights of intestinal and hepatic esterases were determined to be 53,000-55,000 and 180,000, respectively, by gel filtration on Sephadex G-200. The activity of the purified intestinal and hepatic esterases was strongly inhibited by diethyl-p-nitrophenyl phosphate and diisopropyl fluorophosphate, and was not inhibited by eserine sulfate and p-chloromercuribenzoate. Moreover, the purified esterases hydrolyzed ester-type drugs such as aspirin, clofibrate, indanyl carbenicillin and procaine. Hepatic esterase had properties similar to those of intestinal esterase with respect to the sensitivity to organophosphate and the substrate specificity. However, the two purified esterases differed in properties such as molecular weight, isoelectric point, thermostability and optimal pH. PMID:7206363

  9. Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    (-)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimiz...

  10. Structure, morphology and functionality of acetylated and oxidised barley starches.

    PubMed

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Pinto, Vânia Zanella; Bartz, Josiane; Radunz, Marjana; Carreño, Neftali Lenin Villarreal; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2015-02-01

    Acetylation and oxidation are chemical modifications which alter the properties of starch. The degree of modification of acetylated and oxidized starches is dependent on the catalyst and active chlorine concentrations, respectively. The objective of this study was to evaluate the effect of acetylation and oxidation on the structural, morphological, physical-chemical, thermal and pasting properties of barley starch. Barley starches were acetylated at different catalyst levels (11%, 17%, and 23% of NaOH solution) and oxidized at different sodium hypochlorite concentrations (1.0%, 1.5%, and 2.0% of active chlorine). Fourier-transformed infrared spectroscopy (FTIR), X-ray diffractograms, thermal, morphological, and pasting properties, swelling power and solubility of starches were evaluated. The degree of substitution (DS) of the acetylated starches increased with the rise in catalyst concentration. The percentage of carbonyl (CO) and carboxyl (COOH) groups in oxidized starches also increased with the rise of active chlorine level. The presence of hydrophobic acetyl groups, carbonyl and carboxyl groups caused a partial disorganization and depolymerization of starch granules. The structural, morphological and functional changes in acetylated and oxidized starches varied according to reaction conditions. Acetylation makes barley starch more hydrophobic by the insertion of acetyl groups. Also the oxidation promotes low retrogradation and viscosity. All these characteristics are important for biodegradable film production. PMID:25172707

  11. Protein lysine acetylation in bacteria: Current state of the art.

    PubMed

    Ouidir, Tassadit; Kentache, Takfarinas; Hardouin, Julie

    2016-01-01

    Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field. PMID:26390373

  12. Determination of Acetylation of the Gli Transcription Factors.

    PubMed

    Coni, Sonia; Di Magno, Laura; Canettieri, Gianluca

    2015-01-01

    The Gli transcription factors (Gli1, Gli2, and Gli3) are the final effectors of the Hedgehog (Hh) signaling and play a key role in development and cancer. The activity of the Gli proteins is finely regulated by covalent modifications, such as phosphorylation, ubiquitination, and acetylation. Both Gli1 and Gli2 are acetylated at a conserved lysine, and this modification causes the inhibition of their transcriptional activity. Thus, the acetylation status of these proteins represents a useful marker to monitor Hh activation in pathophysiological conditions. Herein we describe the techniques utilized to detect in vitro and intracellular acetylation of the Gli transcription factors. PMID:26179046

  13. Probing the acetylation code of histone H4.

    PubMed

    Lang, Diana; Schümann, Michael; Gelato, Kathy; Fischle, Wolfgang; Schwarzer, Dirk; Krause, Eberhard

    2013-10-01

    Histone modifications play crucial roles in genome regulation with lysine acetylation being implicated in transcriptional control. Here we report a proteome-wide investigation of the acetylation-dependent protein-protein interactions of the N-terminal tail of histone H4. Quantitative peptide-based affinity MS experiments using the SILAC approach determined the interactomes of H4 tails monoacetylated at the four known acetylation sites K5, K8, K12, and K16, bis-acetylated at K5/K12, triple-acetylated at K8/12/16 and fully tetra-acetylated. A set of 29 proteins was found enriched on the fully acetylated H4 tail while specific binders of the mono and bis-acetylated tails were barely detectable. These observations are in good agreement with earlier reports indicating that the H4 acetylation state establishes its regulatory effects in a cumulative manner rather than via site-specific recruitment of regulatory proteins. PMID:23970329

  14. Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides

    PubMed Central

    Guan, Kun-Liang; Yu, Wei; Lin, Yan; Xiong, Yue; Zhao, Shimin

    2016-01-01

    Lysine acetylation has emerged as one of the major post-translational modifications, as indicated by its roles in chromatin remodeling, activation of transcription factors and, most recently, regulation of metabolic enzymes. Identification of acetylation sites in a protein is the first essential step for functional characterization of acetylation in physiological regulation. However, the study of the acetylome is hindered by the lack of suitable physical and biochemical properties of the acetyl group and existence of high-abundance acetylated histones in the cell, and needs a robust method to overcome these problems. Here we present protocols for (i) using chemically acetylated ovalbumin and synthetic acetylated peptide to generate a pan-acetyllysine antibody and a site-specific antibody to Lys288-acetylated argininosuccinate lyase, respectively; (ii) using subcellular fractionation to reduce highly abundant acetylated histones; and (iii) using acetyllysine antibody affinity purification and mass spectrometry to characterize acetylome of human liver tissue. The entire characterization procedure takes ~2–3 d to complete. PMID:21085124

  15. Evolution of the Alpha-Esterase Duplication within the Montana Subphylad of the Virilis Species Group of Drosophila

    PubMed Central

    Baker, William K.

    1980-01-01

    Previous studies on linkage disequilibrium involving four tightly linked genes that code for the alpha-esterases of Drosophila montana suggest that these loci arose from a primitive esterase gene by gene duplication, followed by tandem duplication (Roberts and Baker 1973). We have examined the esterase variants in the closely related species, lacicola, flavomontana and borealis. These studies reveal that borealis has only a single esterase locus, and flavomontana may have only two loci. Cytological studies, using aceto-orcein staining and Hoechst fluorescence of squashes of ganglion chromosomes, reveal acrocentric Y chromosomes for all six species of the montana phylad, with the exception of borealis, which has the primitive rod-shaped Y chromosome. These studies provide evidence against the hypothesis (Stone, Guest and Wilson 1960) that borealis and flavomontana are derived from montana, but support Throckmorton's (1978) conclusion of the early divergence of the former two species. This phylogenetic relationship supports our contention that the difference in the number of esterase genes with active alleles between borealis and montana is based on an increase in the number of genes coding for the alpha-esterases, rather than the retention in borealis of three genes with null alleles. PMID:17249016

  16. Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1

    PubMed Central

    Bresler, Matthew M.; Rosser, Susan J.; Basran, Amrik; Bruce, Neil C.

    2000-01-01

    A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an Mr of approximately 65,000. The apparent Km of the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine. PMID:10698749

  17. The role of calcium in the hydrolysis of the organophosphate paraoxon by human serum A-esterase.

    PubMed

    Vitarius, J A; Sultatos, L G

    1995-01-01

    Human serum A-esterase is a calcium-dependent enzyme that hydrolyzes the organophosphate paraoxon by an Ordered Uni Bi kinetic mechanism. Incubation of various concentrations of calcium chloride with human serum A-esterase resulted in corresponding changes in appk3 and appE for the reaction, while appk2 was unaffected. Carboxyglutamic acid (CAG) prevented calcium chloride from altering appk3, but not appE. Similarly CAG reduced the calcium-stimulated nonenzymatic hydrolysis of paraoxon, as well as the calcium-stimulated de-phosphorylation of chymotrypsin phosphorylated by paraoxon. These results suggest that calcium plays two roles in the hydrolysis of paraoxon by A-esterase. Firstly, calcium is required in order to maintain an active site. In this capacity calcium might participate directly in the catalytic reaction, or it might be required in order to maintain the appropriate confirmation of the active site. And secondly, free calcium (or calcium weakly associated with A-esterase) facilitates the removal of diethyl phosphate from A-esterase, probably by polarizing the P = O bond of the diethyl phosphate-A-esterase intermediate, thereby rendering phosphorus more susceptible to nucleophilic attack by hydroxide ions. PMID:7823759

  18. Complete Genome Sequence of the Marine Cellulose- and Xylan-Degrading Bacterium Glaciecolasp. Strain 4H-3-7+YE-5▿

    PubMed Central

    Klippel, Barbara; Lochner, Adriane; Bruce, David C.; Walston Davenport, Karen; Detter, Chris; Goodwin, Lynne A.; Han, James; Han, Shunsheng; Land, Miriam L.; Mikhailova, Natalia; Nolan, Matt; Pennacchio, Len; Pitluck, Sam; Tapia, Roxanne; Woyke, Tanja; Wiebusch, Sigrid; Basner, Alexander; Abe, Fumiyoshi; Horikoshi, Koki; Keller, Martin; Antranikian, Garabed

    2011-01-01

    Glaciecolasp. strain 4H-3-7+YE-5 was isolated from subseafloor sediments at Suruga Bay in Japan and is capable of efficiently hydrolyzing cellulose and xylan. The complete genome sequence of Glaciecolasp. 4H-3-7+YE-5 revealed several genes encoding putatively novel glycoside hydrolases, offering a high potential for plant biomass degradation. PMID:21705587

  19. Remarkable similarity among bacteria isolated from four hosts after eight-week enrichments of feces with cellulose and xylan/pectin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The intestinal microbiota allows mammals to recover energy stored in plant biomass through fermentation of plant cell walls, primarily cellulose and hemicellulose. Bacteria were isolated from 8-week continuous culture enrichments with cellulose and xylan/pectin from cow (n=4), goat (n=4), human (n=4...

  20. The Fasted/Fed Mouse Metabolic Acetylome: N6-Acetylation Differences Suggest Acetylation Coordinates Organ-Specific Fuel Switching

    PubMed Central

    Yang, Li; Vaitheesvaran, Bhavapriya; Hartil, Kirsten; Robinson, Alan J.; Hoopmann, Michael R.; Eng, Jimmy K.; Kurland, Irwin J.; Bruce, James E.

    2011-01-01

    The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the co-substrate for acetylation, acetyl CoA, is at a key metabolic intersection. Our hypothesis was that mitochondrial and cytoplasmic protein acetylation may be part of a fasted/re-fed feedback control system for the regulation of the metabolic network in fuel switching, where acetyl CoA would be provided by fatty acid oxidation, or glycolysis, respectively. To test this we characterized the mitochondrial and cytoplasmic acetylome in various organs that have a high metabolic rate relative to their mass, and/or switch fuels, under fasted and re-fed conditions (brain, kidney, liver, skeletal muscle, heart muscle, white and brown adipose tissues). Using immunoprecipitation, coupled with LC-MSMS label free quantification, we show there is a dramatic variation in global quantitative profiles of acetylated proteins from different organs. In total, 733 acetylated peptides from 337 proteins were identified and quantified, out of which 31 acetylated peptides from the metabolic proteins that may play organ-specific roles were analyzed in detail. Results suggest that fasted/re-fed acetylation changes coordinated by organ-specific (de-)acetylases in insulin-sensitive versus insensitive organs may underlie fuel use and switching. Characterization of the tissue-specific acetylome should increase understanding of metabolic conditions wherein normal fuel switching is disrupted, such as in Type II diabetes. PMID:21728379

  1. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes

    PubMed Central

    Prokesch, A.; Pelzmann, H. J.; Pessentheiner, A. R.; Huber, K.; Madreiter-Sokolowski, C. T.; Drougard, A.; Schittmayer, M.; Kolb, D.; Magnes, C.; Trausinger, G.; Graier, W. F.; Birner-Gruenberger, R.; Pospisilik, J. A.; Bogner-Strauss, J. G.

    2016-01-01

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

  2. A New Strategy for Fluorogenic Esterase Probes Displaying Low Levels of Non-specific Hydrolysis.

    PubMed

    Kim, Sungwoo; Kim, Hyunjin; Choi, Yongdoo; Kim, Youngmi

    2015-06-26

    A new design for fluorescence probes of esterase activity that features a carboxylate-side pro-fluorophore is demonstrated with boron dipyrromethene (BODIPY)-based probes 1 a and 1 b. Because the design relies on the enzyme-catalyzed hydrolysis of an ester group that is not electronically activated, these probes exhibit a stability to background hydrolysis that is far superior to classical alcohol-side profluorophore-based probes, large signal-to-noise ratios, reduced sensitivity to pH variations, and high enzymatic reactivity. The utility of probe 1 a was established with a real-time fluorescence imaging experiment of endogenous esterase activity that does not require washing of the extracellular medium. PMID:26033618

  3. Review on technological and scientific aspects of feruloyl esterases: A versatile enzyme for biorefining of biomass.

    PubMed

    Gopalan, Nishant; Rodríguez-Duran, L V; Saucedo-Castaneda, G; Nampoothiri, K Madhavan

    2015-10-01

    With increasing focus on sustainable energy, bio-refining from lignocellulosic biomass has become a thrust area of research. With most of the works being focused on biofuels, significant efforts are also being directed towards other value added products. Feruloyl esterases (EC. 3.1.1.73) can be used as a tool for bio-refining of lignocellulosic material for the recovery and purification of ferulic acid and related hydroxycinnamic acids ubiquitously found in the plant cell wall. More and more genes coding for feruloyl esterases have been mined out from various sources to allow efficient enzymatic release of ferulic acid and allied hydroxycinnamic acids (HCAs) from plant-based biomass. A sum up on enzymatic extraction of HCAs and its recovery from less explored agro residual by-products is still a missing link and this review brushes up the achieved landmarks so far in this direction and also covers a detailed patent search on this biomass refining enzyme. PMID:26159377

  4. Esterase mediated resistance in deltamethrin resistant reference tick colony of Rhipicephalus (Boophilus) microplus.

    PubMed

    Gupta, Snehil; Ajith Kumar, K G; Sharma, Anil Kumar; Nagar, Gaurav; Kumar, Sachin; Saravanan, B C; Ravikumar, Gandham; Ghosh, Srikant

    2016-06-01

    Monitoring of acaricide resistance is considered as one of the important facets of integrated tick management. In an attempt of development of resistance monitoring indicators, in the present study two reference tick lines of Rhipicephalus (Boophilus) microplus maintained in the Entomology laboratory, Indian Veterinary Research Institute (IVRI), Izatnagar, India, were studied to determine the possible contributing factors involved in development of resistance to deltamethrin. Electrophoretic profiling of esterase enzymes detected high activities of EST-1 in reference resistant tick colony designated as IVRI-IV whereas it was not detectable in reference susceptible IVRI-I line of R. (B.) microplus. Esterases were further characterized as carboxylesterase or acetylcholinesterase based on inhibitor study using PMSF, eserine sulphate, malathion, TPP and copper sulphate. It was concluded that an acetylcholinesterase, EST-1, possibly plays an important role for development of deltamethrin resistance in IVRI-IV colony of R. (B.) microplus. PMID:26979585

  5. Is Esterase-P Encoded by a Cryptic Pseudogene in Drosophila Melanogaster?

    PubMed Central

    Balakirev, E. S.; Ayala, F. J.

    1996-01-01

    We have amplified and sequenced the gene encoding Esterase-P (Est-P) in 10 strains of Drosophila melanogaster. Three premature termination codons occur in the coding region of the gene in two strains. This observation, together with other indirect evidence, leads us to propose that Est-P may be a pseudogene in D. melanogaster. Est-P would be a ``cryptic'' pseudogene, in the sense that it retains intact the coding sequence (without stop codons and other alterations usually observed in pseudogenes) in most D. melanogaster strains. We conjecture that the β-esterase cluster may consist in other Drosophila species of functional and nonfunctional genes. We also conjecture that the rarity of detected pseudogenes in Drosophila may be due to the difficulty of discovering them, because most of them are cryptic. PMID:8978040

  6. Biocatalytic synthesis of poly(δ-valerolactone) using a thermophilic esterase from archaeoglobus fulgidus as catalyst.

    PubMed

    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

  7. Polymorphism of salivary esterase and alpha-amylase in the Greek population.

    PubMed

    Petalopoulos, A; Fousteri, M; Kouvatsi, A; Triantaphyllidis, C

    1993-01-01

    The genetic polymorphism of two salivary enzymes (esterase and alpha-amylase) was studied in individuals from eight districts of Greece. The pooled gene frequencies were: SetS = 0.63, SetF = 0.37, AMY1 = 0.87, AMY2 = 0.10, AMY3 = 0.02, and AMY4 = 0.01. There was no intrapopulation heterogeneity, while there was a significant difference between the Greeks and the few other European populations studied. PMID:7507080

  8. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst)

    PubMed Central

    Martins, Julia M.; DeMarco, Ricardo; Jameson, David M.; Castro, Aline M.; Bossolan, Nelma R. S.; Wallace, B. A.; Araujo, Ana P. U.

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required. PMID:27351338

  9. Genetic diversity analysis of Capsicum spp germplasm bank accessions based on α/β-esterase polymorphism.

    PubMed

    Monteiro, E R; Bronzato, A R; Orasmo, G R; Lopes, A C A; Gomes, R L F; Mangolin, C A; Machado, M F P S

    2013-01-01

    Genetic diversity and structure were analyzed in 10 accessions belonging to Banco Ativo de Germoplasma de Capsicum located at Federal University of Piauí in northwestern Brazil that receives pepper samples grown in community gardens in various regions and Brazilian states. Selections were made from seeds of C. chinense (4 accessions), C. annuum (5 accessions), and C. baccatum (1 accession). Samples consisting of leaves were collected from 4-10 plants of each accession (a total of 85 plants). Native polyacrylamide gel electrophoresis was used to identify α- and β-esterase polymorphisms. Polymorphism was clearly detected in 5 loci. Sixteen alleles were found at 5 α/β-esterase loci of the three Capsicum species. In the C. chinense samples, the highest HO and HE values were 0.3625 and 0.4395, respectively, whereas in C. annuum samples, HO and HE values were 0.2980 and 0.3310, respectively; the estimated HO and HE values in C. chinense samples were higher than those detected in C. annuum samples. A deficit of homozygous individuals was found in C. chinense (FIS = -0.6978) and C. annuum (FIS = 0.7750). Genetic differentiation between C. chinense and C. annuum at these loci was high (FST = 0.1867) indicating that C. chinense and C. annuum are genetically structured species for α/β- esterase isozymes. The esterase analysis showed high genetic diversity among the C. chinense and C. annuum samples and very high genetic differentiation (FST = 0.6321) among the C. chinense and C. annuum samples and the C. baccatum accession. PMID:23661440

  10. Gene cloning and characterization of a cold-adapted esterase from Acinetobacter venetianus V28.

    PubMed

    Kim, Young-Ok; Heo, Yu Li; Kim, Hyung-Kwoun; Nam, Bo-Hye; Kong, Hee Jeong; Kim, Dong-Gyun; Kim, Woo-Jin; Kim, Bong-Seok; Jee, Young-Ju; Lee, Sang-Jun

    2012-09-01

    Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The maximal activity of the purified enzyme was observed at a temperature of 40 degrees C and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at 5 degrees C with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetalloprotein and was active against p-nitrophenyl esters of C4, C8, and C14. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents. PMID:22814499

  11. 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

  12. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

    PubMed

    Lopes, Jose L S; Yoneda, Juliana S; Martins, Julia M; DeMarco, Ricardo; Jameson, David M; Castro, Aline M; Bossolan, Nelma R S; Wallace, B A; Araujo, Ana P U

    2016-01-01

    Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required. PMID:27351338

  13. Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome

    PubMed Central

    De Santi, Concetta; Willassen, Nils Peder

    2016-01-01

    Background 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. Results 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. PMID:27433797

  14. Contrasted enzymatic cocktails reveal the importance of cellulases and hemicellulases activity ratios for the hydrolysis of cellulose in presence of xylans.

    PubMed

    Dondelinger, Eve; Aubry, Nathalie; Ben Chaabane, Fadhel; Cohen, Céline; Tayeb, Jean; Rémond, Caroline

    2016-03-01

    Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery. PMID:27001439

  15. Activity of influenza C virus O-acetylesterase with O-acetyl-containing compounds.

    PubMed

    Garcia-Sastre, A; Villar, E; Manuguerra, J C; Hannoun, C; Cabezas, J A

    1991-01-15

    Influenza C virus (strain C/Johannesburg/1/66) was grown, harvested, purified and used as source for the enzyme O-acetylesterase (N-acyl-O-acetylneuraminate O-acetylhydrolase; EC 3.1.1.53). This activity was studied and characterized with regard to some new substrates. The pH optimum of the enzyme is around 7.6, its stability at different pH values shows a result similar to that of the pH optimum, and its activity is well maintained in the pH range from 7.0 to 8.5 (all these tests were performed with 4-nitrophenyl acetate as substrate). Remarkable differences were found in the values of both Km and Vmax, with the synthetic substrates 4-nitrophenyl acetate, 2-nitrophenyl acetate, 4-methylumbelliferyl acetate, 1-naphthyl acetate and fluorescein diacetate. The use of 4-nitrophenyl acetate, 4-methylumbelliferyl acetate or 1-naphthyl acetate as substrate seems to be convenient for routine work, but it is better to carry out the measurements in parallel with those on bovine submandibular gland mucin (the latter is a natural and commercially available substrate). It was found that 4-acetoxybenzoic acid, as well as the methyl ester of 2-acetoxybenzoic acid, but not 2-acetoxybenzoic acid itself, are cleaved by this enzyme. Triacetin, di-O-acetyladenosine, tri-O-acetyladenosine, and di-O-acetyl-N-acetyladenosine phosphate, hitherto unreported as substrates for this viral esterase, are hydrolysed at different rates by this enzyme. We conclude that the O-acetylesterase from influenza C virus has a broad specificity towards both synthetic and natural non-sialic acid-containing substrates. Zn2+, Mn2+ and Pb2+ (as their chloride salts), N-acetylneuraminic acid, 4-methyl-umbelliferone and 2-acetoxybenzoic acid (acetylsalicylic acid) did not act as inhibitors. PMID:1991039

  16. Purification and characterization of two 1,4-beta-xylan endohydrolases from the rumen fungus Neocallimastix frontalis.

    PubMed Central

    Gomez de Segura, B; Fevre, M

    1993-01-01

    Two beta-endoxylanases produced by Neocallimastix frontalis have been purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. Xylanase I is a nonglycosylated protein with an apparent molecular mass of 45 kDa. Xylanase II is a glycoprotein with an apparent molecular mass of 70 kDa. The pH optima of these enzymes were 5.5 and 6, respectively, and the temperature optimum was 55 degrees C for each enzyme. The endo mode of action of the enzymes was revealed by thin-layer chromatography of xylan hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, confirming the biochemical specificities of the enzymes. Both enzymes exhibited carboxymethyl cellulase activity, and xylanase I was absorbed on crystalline cellulose, indicating that these enzymes might belong to the F family of beta-1,4-glycanases. Images PMID:8285672

  17. Identification and characterization of a novel salt-tolerant esterase from a Tibetan glacier metagenomic library.

    PubMed

    De Santi, Concetta; Ambrosino, Luca; Tedesco, Pietro; Zhai, Lei; Zhou, Cheng; Xue, Yanfen; Ma, Yanhe; de Pascale, Donatella

    2015-01-01

    A salt-tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p-nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three-dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat-resistant features. PMID:25920073

  18. 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.

  19. Quorum-Sensing Mechanisms Mediated by Farnesol in Ophiostoma piceae: Effect on Secretion of Sterol Esterase

    PubMed Central

    de Salas, Felipe

    2015-01-01

    Ophiostoma piceae CECT 20416 is a dimorphic wood-staining fungus able to produce an extracellular sterol-esterase/lipase (OPE) that is of great biotechnological interest. In this work, we have studied the morphological change of this fungus from yeast to hyphae, which is associated with the cell density-related mechanism known as quorum sensing (QS), and how this affects the secretion of OPE. The data presented here confirm that the molecule E,E-farnesol accumulates as the cell number is growing within the population. The exogenous addition of this molecule or spent medium to the cultures increased the extracellular activity of OPE 2.5 times. This fact was related not to an increase in microbial biomass or in the expression of the gene coding for OPE but to a marked morphological transition in the cultures. Moreover, the morphological transition also occurred when a high cell density was inoculated into the medium. The results suggest that E,E-farnesol regulates through QS mechanisms the morphological transition in the dimorphic fungus O. piceae and that it is associated with a higher extracellular esterase activity. Furthermore, identification and transcriptional analysis of genes tup1 and cyr1, which are involved in the response, was carried out. Here we report enhanced production of a sterol-esterase/lipase of biotechnological interest by means of QS mechanisms. These results may be useful in increasing the production of secreted enzymes of other dimorphic fungi of biotechnological interest. PMID:25888179

  20. Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome.

    PubMed

    Berlemont, Renaud; Jacquin, Olivier; Delsaute, Maud; La Salla, Marcello; Georis, Jacques; Verté, Fabienne; Galleni, Moreno; Power, Pablo

    2013-01-01

    An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6-9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm) at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation). In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases. PMID:24832657

  1. Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

    PubMed Central

    Berlemont, Renaud; Jacquin, Olivier; Delsaute, Maud; Salla, Marcello La; Georis, Jacques; Verté, Fabienne; Galleni, Moreno; Power, Pablo

    2013-01-01

    An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the α/β hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6–9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm) at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation). In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/β hydrolases. PMID:24832657

  2. Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases.

    PubMed

    Ali, Yassine Ben; Carrière, Frédéric; Verger, Robert; Petry, Stefan; Muller, Günter; Abousalham, Abdelkarim

    2005-05-01

    Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase. PMID:15716583

  3. 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. PMID:25173466

  4. Hormone-sensitive lipase is a cholesterol esterase of the intestinal mucosa.

    PubMed

    Grober, Jacques; Lucas, Stéphanie; Sörhede-Winzell, Maria; Zaghini, Isabelle; Mairal, Aline; Contreras, Juan-Antonio; Besnard, Philippe; Holm, Cecilia; Langin, Dominique

    2003-02-21

    The identity of the enzymes responsible for lipase and cholesterol esterase activities in the small intestinal mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters, we sought to determine whether HSL could be involved. HSL mRNA and protein were detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in the different segments. Analysis of gut from HSL-null mice showed that diacylglycerol lipase activity was unchanged in the duodenum and reduced in jejunum. Neutral cholesterol esterase activity was totally abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of HSL mRNA structure showed two types of transcripts expressed in equal amounts with alternative 5'-ends transcribed from two exons. This work demonstrates that HSL is expressed in the mucosa of the small intestine. The results also reveal that the enzyme participates in acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester hydrolysis throughout the small intestine. PMID:12482847

  5. Thermochemical characteristics of cellulose acetates with different degrees of acetylation

    NASA Astrophysics Data System (ADS)

    Larina, V. N.; Ur'yash, V. F.; Kushch, D. S.

    2012-12-01

    The standard enthalpies of combustion and formation of cellulose acetates with different degrees of acetylation are determined. It is established that there is a proportional dependence of these thermochemical characteristics vs. the degree of acetylation, weight fraction of bonded acetic acid, and molar mass of the repeating unit of cellulose acetates.

  6. Emerging Functions for N-Terminal Protein Acetylation in Plants.

    PubMed

    Gibbs, Daniel J

    2015-10-01

    N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour. PMID:26319188

  7. Effect of acetaminophen on sulfamethazine acetylation in male volunteers.

    PubMed

    Tahir, I M; Iqbal, T; Saleem, S; Mehboob, H; Akhter, N; Riaz, M

    2016-03-01

    The effect of acetaminophen on sulfamethazine N-acetylation by human N-acetyltrasferase-2 (NAT2) was studied in 19 (n=19) healthy male volunteers in two different phases. In the first phase of the study the volunteers were given an oral dose of sulfamethazine 500 mg alone and blood and urine samples were collected. After the 10-day washout period the same selected volunteers were again administered sulfamethazine 500 mg along with 1000 mg acetaminophen. The acetylation of sulfamethazine by human NAT2 in both phases with and without acetaminophen was determined by HPLC to establish their respective phenotypes. In conclusion obtained statistics of present study revealed that acetaminophen significantly (P<0.0001) decreased sulfamethazine acetylation in plasma of both slow and fast acetylator male volunteers. A highly significant (P<0.0001) decrease in plasma-free and total sulfamethazine concentration was also observed when acetaminophen was co-administered. Urine acetylation status in both phases of the study was found not to be in complete concordance with that of plasma. Acetaminophen significantly (P<0.0001) increased the acetyl, free and total sulfamethazine concentration in urine of both slow and fast acetylators. Urine acetylation analysis has not been found to be a suitable approach for phenotypic studies. PMID:26519524

  8. An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.

    PubMed

    Kim, Sun-Yee; Sim, Choon Kiat; Zhang, Qiongyi; Tang, Hui; Brunmeir, Reinhard; Pan, Hong; Karnani, Neerja; Han, Weiping; Zhang, Kangling; Xu, Feng

    2016-01-01

    Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies. PMID:27606599

  9. Medial temporal N-acetyl aspartate in pediatric major depression

    PubMed Central

    MacMaster, Frank P.; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S. Preeya; Buhagiar, Christian; Rosenberg, David R.

    2008-01-01

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD-case control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  10. Medial temporal N-acetyl-aspartate in pediatric major depression.

    PubMed

    MacMaster, Frank P; Moore, Gregory J; Russell, Aileen; Mirza, Yousha; Taormina, S Preeya; Buhagiar, Christian; Rosenberg, David R

    2008-10-30

    The medial temporal cortex (MTC) has been implicated in the pathogenesis of pediatric major depressive disorder (MDD). Eleven MDD case-control pairs underwent proton magnetic resonance spectroscopic imaging. N-acetyl-aspartate was lower in the left MTC (27%) in MDD patients versus controls. Lower N-acetyl-aspartate concentrations in MDD patients may reflect reduced neuronal viability. PMID:18703320

  11. Global analysis of lysine acetylation in strawberry leaves

    PubMed Central

    Fang, Xianping; Chen, Wenyue; Zhao, Yun; Ruan, Songlin; Zhang, Hengmu; Yan, Chengqi; Jin, Liang; Cao, Lingling; Zhu, Jun; Ma, Huasheng; Cheng, Zhongyi

    2015-01-01

    Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways, and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome in plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plants to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants. PMID:26442052

  12. Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes

    PubMed Central

    Zhang, Meiling; Chekan, Jonathan R.; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K.; Mackie, Roderick I.; Cann, Isaac

    2014-01-01

    Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health. PMID:25136124

  13. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    PubMed

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. PMID:26920270

  14. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

    Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.

  15. SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation.

    PubMed

    Grishina, Inna; Debus, Katherina; García-Limones, Carmen; Schneider, Constanze; Shresta, Amit; García, Carlos; Calzado, Marco A; Schmitz, M Lienhard

    2012-12-01

    Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response. PMID:23044042

  16. Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson's Disease.

    PubMed

    Vázquez-Mayorga, Emmanuel; Díaz-Sánchez, Ángel G; Dagda, Ruben K; Domínguez-Solís, Carlos A; Dagda, Raul Y; Coronado-Ramírez, Cynthia K; Martínez-Martínez, Alejandro

    2016-01-01

    Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein. PMID:27556455

  17. THE CESA (CE3B) CARBOXY-TERMINAL DOMAIN OF RUMINOCOCCUS FLAVEFACIENS 17 HAS GLUCURONOYL ESTERASE ACTIVITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several types of covalent linkages between lignin and xylan in plant cell walls have been shown. One of such linkages could be an ester bond between hydroxyl groups of lignin moieties and the carboxyl group of the 4-O-methyl-D-glucuronic acid (MeGlcA) side groups of glucuronoxylan. Enzymes capable...

  18. Downregulation of GAUT12 in Populus deltoides by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a woody biofuel feedstock

    DOE PAGESBeta

    Biswal, Ajaya K.; Hao, Zhangying; Pattathil, Sivakumar; Yang, Xiaohan; Winkeler, Kim; Collins, Cassandra; Mohanty, Sushree S.; Richardson, Elizabeth A.; Gelineo-Albersheim, Ivana; Hunt, Kimberly; et al

    2015-03-12

    The inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner. The following are the results: GAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of bothmore » xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in

  19. Characterization of O-Acetylation of N-Acetylglucosamine

    PubMed Central

    Bernard, Elvis; Rolain, Thomas; Courtin, Pascal; Guillot, Alain; Langella, Philippe; Hols, Pascal; Chapot-Chartier, Marie-Pierre

    2011-01-01

    Peptidoglycan (PG) N-acetyl muramic acid (MurNAc) O-acetylation is widely spread in Gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-l-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins. PMID:21586574

  20. Chitosan Molecular Structure as a Function of N-Acetylation

    SciTech Connect

    Franca, Eduardo F.; Freitas, Luiz C.; Lins, Roberto D.

    2011-07-01

    Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle-like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N-acetyl groups in the particles. The polysaccharide chains of highly N-deacetylated particles where the N-acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two-fold helix and five-fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two-fold helix with f and w values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N-acetyl groups are unevenly distributed. Systems with 60% or higher N-acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N-acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3- HO3(n) ... O5(n+ 1) hydrogen bond, which in turn controls particle aggregation.

  1. Role of Histone Acetylation in Cell Cycle Regulation.

    PubMed

    Koprinarova, Miglena; Schnekenburger, Michael; Diederich, Marc

    2016-01-01

    Core histone acetylation is a key prerequisite for chromatin decondensation and plays a pivotal role in regulation of chromatin structure, function and dynamics. The addition of acetyl groups disturbs histone/DNA interactions in the nucleosome and alters histone/histone interactions in the same or adjacent nucleosomes. Acetyl groups can also provide binding sites for recruitment of bromodomain (BRD)-containing non-histone readers and regulatory complexes to chromatin allowing them to perform distinct downstream functions. The presence of a particular acetylation pattern influences appearance of other histone modifications in the immediate vicinity forming the "histone code". Although the roles of the acetylation of particular lysine residues for the ongoing chromatin functions is largely studied, the epigenetic inheritance of histone acetylation is a debated issue. The dynamics of local or global histone acetylation is associated with fundamental cellular processes such as gene transcription, DNA replication, DNA repair or chromatin condensation. Therefore, it is an essential part of the epigenetic cell response to processes related to internal and external signals. PMID:26303420

  2. Characterization of general esterases from methyl parathion-resistant and -susceptible populations of western corn rootworm (Coleoptera: Chrysomelidae).

    PubMed

    Zhou, Xuguo; Scharf, Michael E; Meinke, Lance J; Chandler, Laurence D; Siegfried, Blair D

    2003-12-01

    A consistent correlation between elevated esterase activity and methyl parathion resistance among Nebraska western corn rootworm, Diabrotica virgifera virgifera LeConte, populations has previously been documented. Characterization of general esterase activity using naphtholic esters as model substrates indicated that differences between resistant and susceptible strains could be maximized by optimizing assay conditions. The optimal conditions identified here were similar to those reported for other insect species. The majority of general esterase activity was found in the cytosolic fractions of resistant populations, whereas the activity was more evenly distributed between cytosolic and mitochondrial/nuclear fractions in the susceptible population. General esterase activity was predominately located in the adult thorax and abdomen. Although there were significant differences in general esterase activities between resistant and susceptible populations, the differences exhibited in single beetle activity assays did not provide sufficient discrimination to identify resistant individuals. In contrast, single larva activity assays provided greater discrimination and could be considered as an alternative to traditional bioassay techniques. PMID:14977127

  3. Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

    PubMed Central

    Dupont, C; Clarke, A J

    1991-01-01

    O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover. PMID:2066331

  4. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate

    PubMed Central

    Baumann, Anna-Maria T.; Bakkers, Mark J. G.; Buettner, Falk F. R.; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A.; de Groot, Raoul J.; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  5. Application of reverse-phase HPLC to quantify oligopeptide acetylation eliminates interference from unspecific acetyl CoA hydrolysis

    PubMed Central

    Evjenth, Rune; Hole, Kristine; Ziegler, Mathias; Lillehaug, Johan R

    2009-01-01

    Protein acetylation is a common modification that plays a central role in several cellular processes. The most widely used methods to study these modifications are either based on the detection of radioactively acetylated oligopetide products or an enzyme-coupled reaction measuring conversion of the acetyl donor acetyl CoA to the product CoASH. Due to several disadvantages of these methods, we designed a new method to study oligopeptide acetylation. Based on reverse phase HPLC we detect both reaction products in a highly robust and reproducible way. The method reported here is also fully compatible with subsequent product analysis, e.g. by mass spectroscopy. The catalytic subunit, hNaa30p, of the human NatC protein N-acetyltransferase complex was used for N-terminal oligopeptide acetylation. We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis. The method is highly reproducible and enables reliable quantification of both substrates and products. It is therefore well-suited to determine kinetic parameters of acetyltransferases. PMID:19660098

  6. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.

    PubMed

    Baumann, Anna-Maria T; Bakkers, Mark J G; Buettner, Falk F R; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A; de Groot, Raoul J; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1--a previously identified human candidate gene--is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  7. Furfural Production from d-Xylose and Xylan by Using Stable Nafion NR50 and NaCl in a Microwave-Assisted Biphasic Reaction.

    PubMed

    Le Guenic, Sarah; Gergela, David; Ceballos, Claire; Delbecq, Frederic; Len, Christophe

    2016-01-01

    Pentose dehydration and direct transformation of xylan into furfural were performed in a water-cyclopentyl methyl ether (CPME) biphasic system under microwave irradiation. Heated up between 170 and 190 °C in the presence of Nafion NR50 and NaCl, d-xylose, l-arabinose and xylan gave furfural with maximum yields of 80%, 42% and 55%, respectively. The influence of temperature and reaction time on the reaction kinetics was discussed. This study was also completed by the survey of different reactant ratios, such as organic layer-water or catalyst-inorganic salt ratios. The exchange between proton and cation induced by an excess of NaCl was monitored, and a synergetic effect between the remaining protons and the released HCl was also discovered. PMID:27556444

  8. Production of furfural from xylose, xylan and corncob in gamma-valerolactone using FeCl3·6H2O as catalyst.

    PubMed

    Zhang, Luxin; Yu, Hongbing; Wang, Pan; Li, Yong

    2014-01-01

    An efficient and simple one-pot monophasic reaction system with small carbon footprint for converting xylose, xylan and corncob into furfural was developed in gamma-valerolactone (GVL, an ideal sustainable solvent derived from lignocelluloses) by using FeCl3·6H2O as catalyst. Good yields of furfural from xylose were obtained, and the system was shown to work for xylan and corncob as well. A surprisingly high furfural yield of 79.6% from untreated corncob was achieved at 458 K for 100 min. Contrary to what was generally believed, the addition of water, reduced the rate of the reactions, but showed positive effect on preventing the furfural from degradation in GVL. Besides, the C6 sugars (glucose and cellulose) afforded 11.4-24.5% furfural yields when employing this catalytic approach. The reaction system proposed in this manuscript showed great potential for optimizing the catalytic process in furfural production. PMID:24262845

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Kinetic and structural relationships of transition monomeric and oligomeric carboxyl- and choline-esterases.

    PubMed

    Main, A R

    1983-01-01

    The kinetic and structural relationships of eight electrophoretically pure mammalian serum and liver serine carboxylesterases (CE) and cholinesterases (ChE) have been studied. Eight CE's and ChE's, which were fully resolved but only partially purified, provided additional information. Five of the electrophoretically pure esterases were monomeric, and of these, four belonged to a new and widely distributed class. These four monomeric esterases hydrolyzed choline esters, but at widely differing rates. Thus two were termed monomeric butyrylcholinesterases, mBuChE I and II, and two were monomeric CE's (mCE). The rabbit liver mCE was not a subunit of the oligomeric CE (oCE), although the oCE also hydrolyzed choline esters at a very low rate. The complex kinetics of the mCE's, mBuChE's, oCE's, and of the oligomeric BuChE's of horse and human serum could be interpreted according to a single reaction scheme involving an allosteric site and the equation derived from it. Thus activation and inhibition at high substrate concentrations, together with sigmoidal activity versus substrate concentration plots, all of which characterize the reactions of these esterases, could be interpreted by a single scheme and equation. Structural and kinetic comparisons showed a progressive transition of properties from the oCE's through the mCE's to the oBuChE's. One of the purified mCE's was from horse serum, and it exhibited physical and kinetic properties unlike those of the liver mCE's or oCE's. PMID:6339600

  11. B-esterase determination and organophosphate insecticide inhibitory effects in JEG-3 trophoblasts.

    PubMed

    Espinoza, Marlon; Rivero Osimani, Valeria; Sánchez, Victoria; Rosenbaum, Enrique; Guiñazú, Natalia

    2016-04-01

    The placenta and trophoblasts express several B-esterases. This family includes acethylcholinesterase (AChE), carboxylesterase (CES) and butyrylcholinesterase (BChE), which are important targets of organophosphate insecticide (OP) toxicity. To better understand OP effects on trophoblasts, B-esterase basal activity and kinetic behavior were studied in JEG-3 choriocarcinoma cell cultures. Effects of the OP azinphos-methyl (Am) and chlorpyrifos (Cp) on cellular enzyme activity were also evaluated. JEG-3 cells showed measurable activity levels of AChE and CES, while BChE was undetected. Recorded Km for AChE and CES were 0.33 and 0.26mM respectively. Native gel electrophoresis and RT-PCR analysis demonstrated CES1 and CES2 isoform expression. Cells exposed for 4 and 24h to the OP Am or Cp, showed a differential CES and AChE inhibition profiles. Am inhibited CES and AChE at 4h treatment while Cp showed the highest inhibition profile at 24h. Interestingly, both insecticides differentially affected CES1 and CES2 activities. Results demonstrated that JEG-3 trophoblasts express AChE, CES1 and CES2. B-esterase enzymes were inhibited by in vitro OP exposure, indicating that JEG-3 cells metabolization capabilities include phase I enzymes, able to bioactivate OP. In addition, since CES enzymes are important for medicinal drug activation/deactivation, OP exposure may interfere with trophoblast CES metabolization, probably being relevant in a co-exposure scenario during pregnancy. PMID:26790371

  12. Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications.

    PubMed

    Maester, Thaís Carvalho; Pereira, Mariana Rangel; Machado Sierra, E G; Balan, Andrea; de Macedo Lemos, Eliana Gertrudes

    2016-07-01

    Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity against p-nitrophenyl esters of different chain lengths and highest catalytic efficiency against p-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn(+2), Li(+), EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications. PMID:26915995

  13. 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

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

    PubMed

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

    2014-03-01

    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

  15. Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

    PubMed Central

    Yang, Shaoqing; Qin, Zhen; Duan, Xiaojie; Yan, Qiaojuan; Jiang, Zhengqiang

    2015-01-01

    Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 Å and 2.43 Å resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2–C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi.jlr The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases. PMID:26108223

  16. 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

  17. Benzoyl-L-arginine methyl ester (BAME)-esterase activity in human plasma during the gravidic-puerperal cycle.

    PubMed

    Salles Meirelles, R

    1977-01-01

    Benzoyl-L-arginine methyl ester (BAME)-esterase activity of plasma was measured in women going through the gravidic-puerperal cycle and compared with plasma of non-pregnant women. Plasma from women in the 36th to 40th week of pregnancy hydrolyzes BAME two times more rapidly than that from non-pregnant women. During pregnancy, BAME-esterase activity in plasma increases progressively up to the 40th week, decreases during labor, and after delivery reaches the same level as in non-pregnant women. The BAME-esterase activity of plasma was affected by the storage temperature, with differences demonstrable between -20 and -4 C and between pregnant and non-pregnant women. PMID:754510

  18. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    SciTech Connect

    Wood, S. J.; Li, X.-L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.

    2008-04-01

    The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  19. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus1[OPEN

    PubMed Central

    Zeng, Wei; Picard, Kelsey L.; Song, Lili; Wu, Ai-Min; Farion, Isabela M.; Zhao, Jia; Ford, Kris; Bacic, Antony

    2016-01-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana. We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  20. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

    PubMed

    Zeng, Wei; Lampugnani, Edwin R; Picard, Kelsey L; Song, Lili; Wu, Ai-Min; Farion, Isabela M; Zhao, Jia; Ford, Kris; Doblin, Monika S; Bacic, Antony

    2016-05-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  1. Acetylation of C/EBPα inhibits its granulopoietic function

    PubMed Central

    Bararia, Deepak; Kwok, Hui Si; Welner, Robert S.; Numata, Akihiko; Sárosi, Menyhárt B.; Yang, Henry; Wee, Sheena; Tschuri, Sebastian; Ray, Debleena; Weigert, Oliver; Levantini, Elena; Ebralidze, Alexander K.; Gunaratne, Jayantha; Tenen, Daniel G.

    2016-01-01

    CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation. PMID:27005833

  2. Rapid test for acetyl-methyl-carbinol formation by Enterobacteriaceae.

    PubMed Central

    Qadri, S M; Nichols, C W; Qadri, S G; Villarreal, A

    1978-01-01

    A modified Voges-Proskauer test is described which distinguishes within 4 to 8 hours between organisms that can produce acetyl-methyl-carbinol (acetoin) from glucose fermentation and those that cannot. PMID:363745

  3. Acetylation of C/EBPα inhibits its granulopoietic function.

    PubMed

    Bararia, Deepak; Kwok, Hui Si; Welner, Robert S; Numata, Akihiko; Sárosi, Menyhárt B; Yang, Henry; Wee, Sheena; Tschuri, Sebastian; Ray, Debleena; Weigert, Oliver; Levantini, Elena; Ebralidze, Alexander K; Gunaratne, Jayantha; Tenen, Daniel G

    2016-01-01

    CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation. PMID:27005833

  4. Data detailing the platelet acetyl-lysine proteome

    PubMed Central

    Aslan, Joseph E.; David, Larry L.; McCarty, Owen J.T.

    2015-01-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification – mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  5. Data detailing the platelet acetyl-lysine proteome.

    PubMed

    Aslan, Joseph E; David, Larry L; McCarty, Owen J T

    2015-12-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification - mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  6. Partially Acetylated Sugarcane Bagasse For Wicking Oil From Contaminated Wetlands

    EPA Science Inventory

    Sugarcane bagasse was partially acetylated to enhance its oil-wicking ability in saturated environments while holding moisture for hydrocarbon biodegradation. The water sorption capacity of raw bagasse was reduced fourfold after treatment, which indicated considerably increased ...

  7. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  8. Rapid test for acetyl-methyl-carbinol formation by Enterobacteriaceae.

    PubMed

    Qadri, S M; Nichols, C W; Qadri, S G; Villarreal, A

    1978-10-01

    A modified Voges-Proskauer test is described which distinguishes within 4 to 8 hours between organisms that can produce acetyl-methyl-carbinol (acetoin) from glucose fermentation and those that cannot. PMID:363745

  9. p-Nitrophenylacetate hydrolysis by honey bee esterases: kinetics and inhibition.

    PubMed

    Spoonamore, J E; Frohlich, D R; Wells, M A

    1993-03-01

    1. The kinetics and inhibition of p-nitrophenylacetate hydrolysis by cytosolic esterases of 1-day old female honey bees, Apis mellifera L., were studied. 2. The calculated values obtained were Km = 2.27 x 10(-5)M and Vmax = 2.48 x 10(-8) mol/s per mg protein. 3. The inhibition mechanisms examined for four organophosphorus insecticides were highly competitive in nature and based on competitive inhibition coefficients the order of toxicity was naled > dichlorvos > cis-mevinphos = trans-mevinphos. 4. Comparisons are made with the alfalfa leafcutting bee, Megachile rotundata (Fab). PMID:8498090

  10. Effects on operant learning and brain acetylcholine esterase activity in rats following chronic inorganic arsenic intake.

    PubMed

    Nagaraja, T N; Desiraju, T

    1994-05-01

    1. Very young and adult Wistar rats were given As5+, 5 mg arsenic kg-1 body weight day-1 (sodium arsenate). 2. Operant learning was tested in a Skinner box at the end of exposure and, in the case of developing animals, also after a recovery period. 3. Acetylcholine esterase (AChE) activity was estimated in discrete brain regions of these animals. 4. The animals exposed to arsenic took longer to acquire the learned behaviour and to extinguish the operant. AChE activity was inhibited in some regions of the brain. PMID:8043317

  11. Mechanistic insights into the regulation of metabolic enzymes by acetylation

    PubMed Central

    2012-01-01

    The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

  12. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false N-Acetyl-L-methionine. 172.372 Section 172.372 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine....

  13. Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development

    PubMed Central

    Dai, Jinxiang; Bercury, Kathryn K.; Jin, Weilin

    2015-01-01

    The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation. SIGNIFICANCE STATEMENT The nuclear to cytoplasmic translocation of Olig1 protein has been observed during mouse and human brain development and in multiple sclerosis in several studies, but the detailed molecular mechanism of this translocation remains elusive. Here, we provide insight into the mechanism by which acetylation of Olig1 regulates its unique nuclear-cytoplasmic shuttling during oligodendrocyte development and how the acetylation status of Olig1 modulates its distinct function in the nucleus versus the cytoplasm. The current study provides a unique example of a lineage-specific transcription factor that is actively translocated from the nucleus to the cytoplasm as the cell differentiates. Importantly, we demonstrate that this process is tightly controlled by acetylation at a single

  14. Nucleosome Dancing at the Tempo of Histone Tail Acetylation

    PubMed Central

    Galvani, Angélique; Thiriet, Christophe

    2015-01-01

    The impact of histone acetylation on transcription was revealed over 50 years ago by Allfrey and colleagues. However, it took decades for an understanding of the fine mechanism by which this posttranslational modification affects chromatin structure and promotes transcription. Here, we review breakthroughs linking histone tail acetylation, histone dynamics, and transcription. We also discuss the histone exchange during transcription and highlight the important function of a pool of non-chromatinized histones in chromatin dynamics. PMID:26184324

  15. The binding, synergistic and structural characteristics of BsEXLX1 for loosening the main components of lignocellulose: Lignin, xylan, and cellulose.

    PubMed

    Wang, Qun; Chen, Liang; Lin, Hui; Yu, Daobing; Shen, Qi; Wan, Li; Zhao, Yuhua

    2016-10-01

    The bacterial expansin, BsEXLX1, has been studied as a model to understand the synergistic effect of expansins on lignocellulose degradation and the structure-function relationships of expansins. However, the specific mechanism is still poorly understood. In this study, the binding, synergistic and structural characteristics of BsEXLX1 for loosening the main components of lignocellulose: lignin, xylan, and cellulose, were further characterized. Results showed that BsEXLX1 preferentially binds to xylan over lignin or cellulose under various conditions. The binding of BsEXLX1 to all substrates increased linearly with the initial concentration of BsEXLX1. But the changing rate of binding (i.e., slope of the line; k value) varied with the incubation temperature. Interestingly, the binding of BsEXLX1 to substrates did not saturate. Mutating residue-125, 126 or 171 indicated their importance for binding, but they were less important for binding to xylan. Through binding assays and homologous modeling, it was concluded that residue-125 and 171 play more important roles in the structural maintenance of BsEXLX1. By comparing the synergistic activity of BsEXLX1 or its mutants, it was found that synergistic activity is not correlated with specific binding. All these results can lead deeper understand about the mechanism of wall loosening by expansins, and further promote the application of expansins in lignocellulose degradation. PMID:27542746

  16. 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. PMID:25973851

  17. Purification and characterization of an extracellular esterase with organic solvent tolerance from a halotolerant isolate, Salimicrobium sp. LY19

    PubMed Central

    2013-01-01

    Background Halotolerant bacteria are excellent sources for selecting novel enzymes. Being intrinsically stable and active under high salinities, enzymes from these prokaryotes have evolved to function optimally under extreme conditions, making them robust biocatalysts with potential applications in harsh industrial processes. Results A halotolerant strain LY19 showing lipolytic activity was isolated from saline soil of Yuncheng Salt Lake, China. It was identified as belonging to the genus of Salimicrobium by 16S rRNA gene sequence analysis. The extracellular enzyme was purified to homogeneity with molecular mass of 57 kDa by SDS-PAGE. Substrate specificity test revealed that the enzyme preferred short-chain p-nitrophenyl esters and exhibited maximum activity towards p-nitrophenyl butyrate (p-NPB), indicating an esterase activity. The esterase was highly active and stable over broad temperature (20°C-70°C), pH (7.0-10.0) and NaCl concentration (2.5%-25%) ranges, with an optimum at 50°C, pH 7.0 and 5% NaCl. Significant inhibition of the esterase was shown by ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF) and phenylarsine oxide (PAO), which indicated that it was a metalloenzyme with serine and cysteine residues essential for enzyme activity. Moreover, the esterase displayed high activity and stability in the presence of hydrophobic organic solvents with log Pow ≥ 0.88 than in the absence of an organic solvent or in the presence of hydrophilic solvents. Conclusions Results from the present study indicated the novel extracellular esterase from Salimicrobium sp. LY19 exhibited thermostable, alkali-stable, halotolerant and organic solvent-tolerant properties. These features led us to conclude that the esterase may have considerable potential for industrial applications in organic synthesis reactions. PMID:24325447

  18. Functional characterization of a novel microbial esterase identified from the Indian Ocean and its use in the stereoselective preparation of (R)-methyl mandelate

    NASA Astrophysics Data System (ADS)

    Liang, Jiayuan; Sun, Aijun; Zhang, Yun; Deng, Dun; Wang, Yongfei; Ma, Sanmei; Hu, Yunfeng

    2016-03-01

    Genomic mining has identified a novel microbial alkaline esterase from the Indian Ocean. This esterase was overexpressed in E. coli BL21 (DE3) and further functionally characterized. Under optimal conditions (10 mmol/L substrate, pH 6.0, 2 h at 40 °C), this esterase can hydrolyze racemic methyl mandelate to (R)-methyl mandelate with very high optical purity (e.e. >99%) and yield (nearly 90%). Interestingly, the stereoselectivity of this esterase is opposite to that of two previously reported lipases that can generate (S)-methyl mandelate through the hydrolysis of racemic methyl mandelate. No organic solvents or other additives were required to optimize the optical purity and production of the final chiral product (R)-methyl mandelate, which can potentially simplify the production procedure of (R)-methyl mandelate catalyzed by esterase.

  19. An acetylation switch controls TDP-43 function and aggregation propensity.

    PubMed

    Cohen, Todd J; Hwang, Andrew W; Restrepo, Clark R; Yuan, Chao-Xing; Trojanowski, John Q; Lee, Virginia M Y

    2015-01-01

    TDP-43 pathology is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Although a critical role for TDP-43 as an RNA-binding protein has emerged, the regulation of TDP-43 function is poorly understood. Here, we identify lysine acetylation as a novel post-translational modification controlling TDP-43 function and aggregation. We provide evidence that TDP-43 acetylation impairs RNA binding and promotes accumulation of insoluble, hyper-phosphorylated TDP-43 species that largely resemble pathological inclusions in ALS and FTLD-TDP. Moreover, biochemical and cell-based assays identify oxidative stress as a signalling cue that promotes acetylated TDP-43 aggregates that are readily engaged by the cellular defense machinery. Importantly, acetylated TDP-43 lesions are found in ALS patient spinal cord, indicating that aberrant TDP-43 acetylation and loss of RNA binding are linked to TDP-43 proteinopathy. Thus, modulating TDP-43 acetylation represents a plausible strategy to fine-tune TDP-43 activity, which could provide new therapeutic avenues for TDP-43 proteinopathies. PMID:25556531

  20. Effects of peptide acetylation and dimethylation on electrospray ionization efficiency.

    PubMed

    Cho, Kyung-Cho; Kang, Jeong Won; Choi, Yuri; Kim, Tae Woo; Kim, Kwang Pyo

    2016-02-01

    Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N-termini and the ε-amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC-ESI-MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two-fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26889926

  1. Acetyl radical generation in cigarette smoke: Quantification and simulations

    NASA Astrophysics Data System (ADS)

    Hu, Na; Green, Sarah A.

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high-performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commercial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass fiber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acetaldehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke.

  2. An acetylation switch controls TDP-43 function and aggregation propensity

    PubMed Central

    Cohen, Todd J.; Hwang, Andrew W.; Restrepo, Clark R.; Yuan, Chao-Xing; Trojanowski, John Q.; Lee, Virginia M.Y.

    2015-01-01

    TDP-43 pathology is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Although a critical role for TDP-43 as an RNA-binding protein has emerged, the regulation of TDP-43 function is poorly understood. Here we identify lysine acetylation as a novel post-translational modification controlling TDP-43 function and aggregation. We provide evidence that TDP-43 acetylation impairs RNA-binding and promotes accumulation of insoluble, hyper-phosphorylated TDP-43 species that largely resemble pathological inclusions in ALS and FTLD-TDP. Moreover, biochemical and cell-based assays identify oxidative stress as a signaling cue that promotes acetylated TDP-43 aggregates that are readily engaged by the cellular defense machinery. Importantly, acetylated TDP-43 lesions are found in ALS patient spinal cord, indicating that aberrant TDP-43 acetylation and loss of RNA binding are linked to TDP-43 proteinopathy. Thus, modulating TDP-43 acetylation represents a plausible strategy to fine-tune TDP-43 activity, which could provide new therapeutic avenues for TDP-43 proteinopathies. PMID:25556531

  3. Acetyl Radical Generation in Cigarette Smoke: Quantification and Simulations

    PubMed Central

    Hu, Na; Green, Sarah A.

    2014-01-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography–mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10–150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commerial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass filber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acealdehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke. PMID:25253993

  4. Methods to detect NF-κB Acetylation and Methylation

    PubMed Central

    Chen, JinJing; Chen, Lin-Feng

    2015-01-01

    Summary Post-translational modifications of NF-κB, including acetylation and methylation, have emerged as an important regulatory mechanism for determining the duration and strength of NF-κB nuclear activity as well as its transcriptional output. Within the seven NF-κB family proteins, the RelA subunit of NF-κB is the most studied for its regulation by lysine acetylation and methylation. Acetylation or methylation at different lysine residues modulates distinct functions of NF-κB, including DNA binding and transcription activity, protein stability, and its interaction with NF-κB modulators. Here, we describe the experimental methods to monitor the in vitro and in vivo acetylated or methylated forms of NF-κB. These methods include radiolabeling the acetyl- or methyl- groups and immunoblotting with pan or site-specific acetyl- or methyl-lysine antibodies. Radiolabeling is useful in the initial validation of the modifications. Immunoblotting with antibodies provides a rapid and powerful approach to detect and analyze the functions of these modifications in vitro and in vivo. PMID:25736763

  5. Molecular characterization of a cold-active recombinant xylanase from Flavobacterium johnsoniae and its applicability in xylan hydrolysis

    PubMed Central

    Chen, Shicheng; Kaufman, Michael G.; Miazgowicz, Kerri L.; Bagdasarian, Michael; Walker, Edward D.

    2014-01-01

    A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30° C, but Xyn10A retained 50% activity at 4°C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-β-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose. PMID:23196234

  6. Agar-agar entrapment increases the stability of endo-β-1,4-xylanase for repeated biodegradation of xylan.

    PubMed

    Bibi, Zainab; Shahid, Faiza; Ul Qader, Shah Ali; Aman, Afsheen

    2015-04-01

    Microbial xylanases, specially endo-β-1,4-xylanase catalyzes the hydrolysis of xylan, is considered one of the most significant hydrolases. It has numerous applications but most extensively is utilized in paper and pulp industry as a bio-bleaching agent. Immobilization technique is comprehensively studied with the expectation of modifying and improving enzyme stability and characteristics for commercial purposes. Currently, matrix entrapment technique is applied to immobilize endo-β-1,4-xylanase within agar-agar gel beads produced by Geobacillus stearothermophilus KIBGE-IB29. Maximal enzyme immobilization yield was achieved at 2.5% of agar-agar concentration. Optimized conditions demonstrated an increase in the optimal reaction time from 05 min to 30 min and incubation temperature from 50 °C to 60 °C with reference to free enzyme whereas; no effect was observed for optimum pH. Entrapment technique uniquely changed the kinetic parameters of immobilized endo-β-1,4-xylanase (Km: 0.5074 mg min(-1) to 0.5230 mg min(-1) and Vmax: 4773 U min(-1) to 968 U min(-1)) as compared to free enzyme. However, immobilized enzyme displayed broad thermal stability and retained 79.0% of its initial activity at 80 °C up to 30 min whereas; free enzyme completely lost its activity at this temperature. With respect to economic feasibility, the immobilized enzyme showed impressive recycling efficiency up to six reaction cycles. PMID:25603143

  7. Rapid selection and identification of Miscanthus genotypes with enhanced glucan and xylan yields from hydrothermal pretreatment followed by enzymatic hydrolysis

    PubMed Central

    2012-01-01

    Background Because many Miscanthus genotypes can be cultivated with relatively high productivity and carbohydrate content, Miscanthus has great potential as an energy crop that can support large scale biological production of biofuels. Results In this study, batch hydrothermal pretreatment at 180°C for 35 min followed by enzymatic hydrolysis was shown to give the highest total sugar yields for Miscanthus x giganteus cv. Illinois planted in Illinois. High throughput pretreatment at 180°C for 35 min and 17.5 min followed by co-hydrolysis in a multi-well batch reactor identified two varieties out of 80 that had significantly higher sugar yields from pretreatment and enzymatic hydrolysis than others. The differences in performance were then related to compositions of the 80 varieties to provide insights into desirable traits for Miscanthus that enhance sugar yields. Conclusions High throughput pretreatment and co-hydrolysis (HTPH) rapidly identified promising genotypes from a wide range of Miscanthus genotypes, including hybrids of Miscanthus sacchariflorus/M. sinensis and Miscanthus lutarioriparius, differentiating the more commercially promising species from the rest. The total glucan plus xylan content in Miscanthus appeared to influence both mass and theoretical yields, while lignin and ash contents did not have a predictable influence on performance. PMID:22863302

  8. Crystal structures of Ophiostoma piceae sterol esterase: structural insights into activation mechanism and product release.

    PubMed

    Gutiérrez-Fernández, Javier; Vaquero, María Eugenia; Prieto, Alicia; Barriuso, Jorge; Martínez, María Jesús; Hermoso, Juan A

    2014-09-01

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16-α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30 Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications. PMID:25108239

  9. Use of esterase activities for the detection of chemical neurotoxic agents.

    PubMed

    Manco, Giuseppe; Nucci, Roberto; Febbraio, Ferdinando

    2009-01-01

    The quest for a quick and easy detection of the neurotoxin levels in the environment has fostered the search for systems alternative to currently employed analytical methods such as spectrophotometer, gas-liquid chromatography, thin-layer chromatography, and more recently mass spectrometry. These drawbacks lead to intense research efforts to develop biosensor devices for the determination of these compounds. In this review, we present an overview of the actual development of research in neurotoxin detection by using enzymatic biosensors based on esterase activity, in particular cholinesterases, and carboxylesterases. Detection by enzymatic activity could be carried out measuring the hydrolysis products or the residual enzymatic activity after inhibition, using a transducer system that makes possible the correlation between the determined activity and the analyte concentration. Several transducer systems were adopted for the neurotoxins identification using esterases, including electrochemical, optical, conductimetric and piezoelectric procedures. The differences in the used transducer determine the final sensitivity and specificity of the biosensor. Moreover, a brief description of immobilization procedure, that is an important step in the biosensor development and could affect the final characteristic of biosensor (sensibility, stability, response time and reproducibility), was accomplished. Final considerations on advantages and problems, related to actual development of these technologies, and its prospective were discussed. PMID:19508179

  10. An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

    PubMed

    Perz, Veronika; Hromic, Altijana; Baumschlager, Armin; Steinkellner, Georg; Pavkov-Keller, Tea; Gruber, Karl; Bleymaier, Klaus; Zitzenbacher, Sabine; Zankel, Armin; Mayrhofer, Claudia; Sinkel, Carsten; Kueper, Ulf; Schlegel, Katharina; Ribitsch, Doris; Guebitz, Georg M

    2016-03-15

    Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/β-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT. PMID:26878094

  11. Purification and characterization of a pregastric esterase from a hygienized kid rennet paste.

    PubMed

    Calvo, M V; Fontecha, J

    2004-05-01

    Rennet pastes obtained by maceration of gastric tissues from suckling kids are used traditionally to produce some artisanal cheeses in Spain. Besides milk-clotting function, rennet pastes provide proteolytic activity and lipolytic system, essentially pregastric, necessary in the development of piquant flavor typical of these cheeses. A simple and reproducible procedure allows us to obtain a standardized rennet paste that posses the desired activity and is of good microbiological quality. Concomitantly, a kid pregastric esterase (KPGE) was purified to homogeneity. The purification procedure was based on an aqueous extract of hygienized rennet paste (HRP), which was chromatographed on DEAE-Sepharose Fast Flow then adsorbed on phenyl superose followed by a re-chromatography on the same column. The final enzymatic preparation, where the overall activity recovery was 3%, showed a molecular mass of 53 kDa. The highest activity was determined on p-nitrophenyl butyrate, but marked hydrolysis was also detected on beta-naphthyl caprylate. In contrast, low activity on tributyrin (substrate under emulsion form) was detected, thus confirming the esterase character of purified enzyme. PMID:15290959

  12. Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

    SciTech Connect

    Hiraki, Toshiki; Shibayama, Naoya; Yoon, Young-Ho; Yun, Kyung-Mook; Hamamoto, Toshiro; Tame, Jeremy R. H.; Park, Sam-Yong

    2007-09-01

    Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

  13. Electrophoretic survey of seedling esterases in wheats in relation to their phylogeny.

    PubMed

    Jaaska, V

    1980-11-01

    Evolutionary and ontogenetic variation of six seedling esterases of independent genetic control is studied in polyploid wheats and their diploid relatives by means of polyacrylamide gel electrophoresis. Four of them are shown to be controlled by homoeoallelic genes in chromosomes of third, sixth and seventh homoeologous groups.The isoesterase electrophoretic data are considered supporting a monophyletic origin of both the primitive tetraploid and the primitive hexaploid wheat from which contemporary taxa of polyploid wheats have emerged polyphyletically and polytopically through recurrent introgressive hybridization and accumulation of mutations. Ancestral diploids belonging or closely related to Triticum boeoticum, T. urartu, Aegilops speltoides and Ae. tauschii ssp. strangulata are genetically the most suitable genome donors of polyploid wheats. Diploids of the Emarginata subsection of the section Sitopsis, Aegilops longissima s.str., Ae. sharonensis, Ae. searsii and Ae. bicornis, are unsuitable for the role of the wheat B genome donors, being all fixed for the esterase B and D electromorphs different from those of tetraploid wheats. PMID:24305916

  14. Dynamic mechanical analysis and esterase degradation of dentin adhesives containing a branched methacrylate

    PubMed Central

    Park, Jong-Gu; Ye, Qiang; Topp, Elizabeth M.; Lee, Chi H.; Kostoryz, Elisabet L.; Misra, Anil; Spencer, Paulette

    2010-01-01

    A study of the dynamic mechanical properties and the enzymatic degradation of new dentin adhesives containing a multifunctional methacrylate are described. Adhesives contained 2-hydroxyethyl methacrylate (HEMA), 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy) phenyl]-propane (BisGMA), and a new multifunctional methacrylate with a branched side chain-trimethylolpropane mono allyl ether dimethacrylate (TMPEDMA). Adhesives were photopolymerized in the presence of 0, 8 and 16 wt% water to simulate wet bonding conditions in the mouth and compared to control adhesives. The degree of conversion as a function of irradiation time was comparable for experimental and control adhesives. In dynamic mechanical analysis (DMA), broad tan δ peaks were obtained for all samples, indicating that the polymerized networks are heterogeneous; comparison of the full-width-at-half-maximum values obtained from the tan δ curves indicated increased heterogeneity for samples cured in the presence of water and/or containing TMPEDMA. The experimental adhesive showed higher Tg and higher rubbery modulus indicating increased crosslink density as compared to the control. The improvement in esterase resistance afforded by adhesives containing the TMPEDMA is greater when this material is photopolymerized in the presence of water, suggesting better performance in the moist environment of the mouth. The improved esterase resistance of the new adhesive could be explained in terms of the densely crosslinked network structure and/or the steric hindrance of branched alkyl side chains. PMID:19358261

  15. Functional Analysis of Esterase TCE2 Gene from Tetranychus cinnabarinus (Boisduval) involved in Acaricide Resistance

    PubMed Central

    Shi, Li; Wei, Peng; Wang, Xiangzun; Shen, Guangmao; Zhang, Jiao; Xiao, Wei; Xu, Zhifeng; Xu, Qiang; He, Lin

    2016-01-01

    The carmine spider mite, Tetranychus cinnabarinus is an important pest of crops and vegetables worldwide, and it has the ability to develop resistance against acaricides rapidly. Our previous study identified an esterase gene (designated TCE2) over-expressed in resistant mites. To investigate this gene’s function in resistance, the expression levels of TCE2 in susceptible, abamectin-, fenpropathrin-, and cyflumetofen-resistant strains were knocked down (65.02%, 63.14%, 57.82%, and 63.99%, respectively) via RNA interference. The bioassay data showed that the resistant levels to three acaricides were significantly decreased after the down-regulation of TCE2, indicating a correlation between the expression of TCE2 and the acaricide-resistance in T. cinnabarinus. TCE2 gene was then re-engineered for heterologous expression in Escherichia coli. The recombinant TCE2 exhibited α-naphthyl acetate activity (483.3 ± 71.8 nmol/mg pro. min−1), and the activity of this enzyme could be inhibited by abamectin, fenpropathrin, and cyflumetofen, respectively. HPLC and GC results showed that 10 μg of the recombinant TCE2 could effectively decompose 21.23% fenpropathrin and 49.70% cyflumetofen within 2 hours. This is the first report of a successful heterologous expression of an esterase gene from mites. This study provides direct evidence that TCE2 is a functional gene involved in acaricide resistance in T. cinnabarinus. PMID:26725309

  16. Aliphatic esters as targets of esterase activity in the parsnip webworm (Depressaria pastinacella).

    PubMed

    Zangerl, Arthur R; Liao, Ling-Hsiu; Jogesh, Tania; Berenbaum, May R

    2012-02-01

    As a specialist on the reproductive structures of Pastinaca sativa and species in the related genus Heracleum, the parsnip webworm (Depressaria pastinacella) routinely encounters a distinctive suite of phytochemicals in hostplant tissues. Little is known, however, about the detoxification mechanisms upon which this species relies to metabolize these compounds. In this study, larval guts containing hostplant tissues were homogenized, and metabolism was determined by incubating reactions with and without NADPH and analyzing for substrate disappearance and product appearance by gas chromatography-mass spectrometry. Using this approach, we found indications of carboxylesterase activity, in the form of appropriate alcohol metabolites for three aliphatic esters in hostplant tissues-octyl acetate, octyl butyrate, and hexyl butyrate. Involvement of webworm esterases in hostplant detoxification subsequently was confirmed with metabolism assays with pure compounds. This study is the first to implicate esterases in lepidopteran larval midgut metabolism of aliphatic esters, ubiquitous constituents of flowers and fruits. In addition, this method confirmed that webworms detoxify furanocoumarins and myristicin in their hostplants via cytochrome P450-mediated metabolism, and demonstrated that these enzymes also metabolize the coumarin osthol and the fatty acid derivative palmitolactone. PMID:22350520

  17. Esterase activity of lactic acid bacteria isolated from malolactic fermentation of red wines.

    PubMed

    Pérez-Martín, Fátima; Seseña, Susana; Izquierdo, Pedro Miguel; Palop, María Llanos

    2013-05-15

    The goal of this study was to examine the esterase activity of 243 lactic acid bacteria (LAB) strains from wines of different red grape varieties, belonging to the genera Oenococcus, Lactobacillus, Pediococcus and Enterococcus. p-Nitrophenyl octanoate was used as substrate. All strains presented esterase activity in the first screening, but only those showing higher activity were used in subsequent studies to determine the cellular location of this activity, the influence of pH, temperature and the presence of ethanol and the substrate specificity. For the thirteen selected strains, the highest activity was observed in the intracellular fraction. Responses to pH, temperature and ethanol were strain-dependent, but for all the strains, a marked decrease in activity in presence of ethanol was observed. When the influence of pH and ethanol acting together was studied at 25 °C and 37 °C, temperature-dependent differences were not observed for any of the strains except for Oen6. In the substrate specificity assay, the majority of strains of all genera displayed a trend to more readily hydrolyse ester substrates from C8 and longer. PMID:23558198

  18. An essential role of caffeoyl shikimate esterase in monolignol biosynthesis in Medicago truncatula.

    PubMed

    Ha, Chan Man; Escamilla-Trevino, Luis; Yarce, Juan Carlos Serrani; Kim, Hoon; Ralph, John; Chen, Fang; Dixon, Richard A

    2016-06-01

    Biochemical and genetic analyses have previously identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana, although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncatula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only a weak esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species. PMID:27037613

  19. A self-calibrating PARACEST MRI contrast agent that detects esterase enzyme activity

    PubMed Central

    Li, Yuguo; Sheth, Vipul R.; Liu, Guanshu; Pagel, Mark D.

    2016-01-01

    The CEST effect of many PARACEST MRI contrast agents changes in response to a molecular biomarker. However, other molecular biomarkers or environmental factors can influence CEST, so that a change in CEST is not conclusive proof for detecting the biomarker. To overcome this problem, a second control CEST effect may be included in the same PARACEST agent, which is responsive to all factors that alter the first CEST effect except for the biomarker to be measured. To investigate this approach, a PARACEST MRI contrast agent was developed with one CEST effect that is responsive to esterase enzyme activity and a second control CEST effect. The ratio of the two CEST effects was independent of concentration and T1 relaxation, so that this agent was self-calibrating with respect to these factors. This ratiometric method was dependent on temperature and was influenced by MR coalescence as the chemical exchange rates approached the chemical shifts of the exchangable protons as temperature was increased. The two CEST effects also showed evidence of having different pH dependencies, so that this agent was not self-calibrating with respect to pH. Therefore, a self-calibrating PARACEST MRI contrast agent can more accurately detect a molecular biomarker such as esterase enzyme activity, as long as temperature and pH are within an acceptable physiological range and remain constant. PMID:21861282

  20. A SUMO-acetyl switch in PXR biology.

    PubMed

    Cui, Wenqi; Sun, Mengxi; Zhang, Shupei; Shen, Xunan; Galeva, Nadezhda; Williams, Todd D; Staudinger, Jeff L

    2016-09-01

    Post-translational modification (PTM) of nuclear receptor superfamily members regulates various aspects of their biology to include sub-cellular localization, the repertoire of protein-binding partners, as well as their stability and mode of degradation. The nuclear receptor pregnane X receptor (PXR, NR1I2) is a master-regulator of the drug-inducible gene expression in liver and intestine. The PXR-mediated gene activation program is primarily recognized to increase drug metabolism, drug transport, and drug efflux pathways in these tissues. The activation of PXR also has important implications in significant human diseases including inflammatory bowel disease and cancer. Our recent investigations reveal that PXR is modified by multiple PTMs to include phosphorylation, SUMOylation, and ubiquitination. Using both primary cultures of hepatocytes and cell-based assays, we show here that PXR is modified through acetylation on lysine residues. Further, we show that increased acetylation of PXR stimulates its increased SUMO-modification to support active transcriptional suppression. Pharmacologic inhibition of lysine de-acetylation using trichostatin A (TSA) alters the sub-cellular localization of PXR in cultured hepatocytes, and also has a profound impact upon PXR transactivation capacity. Both the acetylation and SUMOylation status of the PXR protein is affected by its ability to associate with the lysine de-acetylating enzyme histone de-acetylase (HDAC)3 in a complex with silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Taken together, our data support a model in which a SUMO-acetyl 'switch' occurs such that acetylation of PXR likely stimulates SUMO-modification of PXR to promote the active repression of PXR-target gene expression. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. PMID:26883953

  1. Identification of cellular factors binding to acetylated HIV-1 integrase.

    PubMed

    Allouch, Awatef; Cereseto, Anna

    2011-11-01

    The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle. PMID:20016921

  2. Crystallization and Preliminary X-ray Diffraction Analysis of the Glucuronoyl Esterase Catalytic Domain from Hypocrea jecorina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was over-expressed, purified, and crystallized by sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. Crystals had space group P212121 and X-ray diffraction data were...

  3. Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 ...

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

    SciTech Connect

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

    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 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 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.

  5. Juvenile Hormone (JH) Esterase of the Mosquito Culex quinquefasciatus Is Not a Target of the JH Analog Insecticide Methoprene

    PubMed Central

    Kamita, Shizuo G.; Samra, Aman I.; Liu, Jun-Yan; Cornel, Anthony J.; Hammock, Bruce D.

    2011-01-01

    Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (kcat/KM ratio) and Vmax values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE. PMID:22174797

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Novel feruloyl esterase from gram-positive lactic acid bacteria and analysis of the recombinant enzyme produced in Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Biocatalytic Resolution of Rac-α-Ethyl-2-Oxo-Pyrrolidineacetic Acid Methyl Ester by Immobilized Recombinant Bacillus cereus Esterase.

    PubMed

    Zheng, Jian-Yong; Liu, Yin-Yan; Luo, Wei-Feng; Zheng, Ren-Chao; Ying, Xiang-Xian; Wang, Zhao

    2016-04-01

    A new esterase-producing strain (Bacillus cereus WZZ001) which exhibiting high hydrolytic activity and excellent enantioselectivity on rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester (R, S-1) has been isolated from soil sample by our laboratory. In this study, the stereoselective hydrolysis of (R, S-1) was performed using the recombinant Bacillus cereus esterase which expressed in Escherichia coli BL21 (DE3). Under the optimized conditions of pH 8.0, 35 °C, and concentration of substrate 400 mM, a successful enzymatic resolution was achieved with an e.e. s of 99.5 % and conversion of 49 %. Immobilization considerably increased the reusability of the recombinant esterase; the immobilized enzyme showed excellent reusability during 6 cycles of repeated 2 h reactions at 35 °C. Thereby, it makes the recombinant B. cereus esterase a usable biocatalyst for industrial application. PMID:26695776

  9. 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

  10. Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni▿ †

    PubMed Central

    Sumby, Krista M.; Matthews, Angela H.; Grbin, Paul R.; Jiranek, Vladimir

    2009-01-01

    We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4 pNP-linked substrates. PMID:19734337

  11. Statistical optimization of medium components and physicochemical parameters to simultaneously enhance bacterial growth and esterase production by Bacillus thuringiensis.

    PubMed

    Mazzucotelli, Cintia Anabela; Moreira, María del Rosario; Ansorena, María Roberta

    2016-01-01

    Bacillus thuringiensis is a genus extensively studied because of its high potential for biotechnological application, principally in biocontrol techniques. However, the optimization of esterase production by this strain has been scarcely studied. The aim of this work was to select and optimize the physicochemical and nutritional parameters that significantly influence the growth and esterase production of B. thuringiensis. To this purpose, 6 nutritional factors and 2 physicochemical parameters were evaluated using a Plackett-Burman design. Significant variables were optimized using a Box-Behnken design and through the desirability function to select the levels of the variables that simultaneously maximize microbial growth and esterase production. The optimum conditions resulting from simultaneous optimization of the responses under study were found to be 1 g/L glucose, 15 g/L peptone, and 3.25 g/L NaCl. Under these optimal conditions, it was possible to achieve a 2.5 log CFU/mL increase in bacterial growth and a 113-fold increase in esterase productivity, compared with minimal medium without agitation. PMID:26529589

  12. 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...

  13. 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. PMID:25820516

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  15. Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

    PubMed Central

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D. Ramesh; Alfonso, Lloyd F.; Marimuthu, Srinivasan; Bhat, G. Jayarama

    2016-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH. PMID:27356773

  16. Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development.

    PubMed

    Dai, Jinxiang; Bercury, Kathryn K; Jin, Weilin; Macklin, Wendy B

    2015-12-01

    The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation. PMID:26631469

  17. A genomic search approach to identify esterases in Propionibacterium freudenreichii involved in the formation of flavour in Emmental cheese

    PubMed Central

    Dherbécourt, Julien; Falentin, Hélène; Canaan, Stéphane; Thierry, Anne

    2008-01-01

    Background Lipolysis is an important process of cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027T was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. Results The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027T predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. Conclusion We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii

  18. Bacillus coreaensis sp. nov.: a xylan-hydrolyzing bacterium isolated from the soil of Jeju Island, Republic of Korea.

    PubMed

    Chi, Won-Jae; Youn, Young Sang; Park, Jae-Seon; Hong, Soon-Kwang

    2015-07-01

    A xylan-degrading bacterium, designated as MS5(T) strain, was isolated from soil collected from the Jeju Island, Republic of Korea. Strain MS5(T) was Gram-stain-positive, aerobic, and motile by polar flagellum. The major fatty acids identified in this bacterium were iso-C15:0 (32.3%), C16:0 (27.3%), and anteiso-C15:0 (10.2%). A similarity search based on the 16S rRNA gene sequence revealed that the strain belongs to the class Bacilli and shared the highest similarity with the type strains Bacillus beringensis BR035(T) (98.7%) and Bacillus korlensis ZLC-26(T) (98.6%) which form a coherent cluster in a neighbor-joining phylogenetic tree. The DNA G+C content of strain MS5(T) was 43.0 mol%. The major menaquinone was MK-7 and the diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The DNADNA relatedness values between strain MS5(T) and two closely related species, B. beringensis BR035(T) and B. korlensis ZLC-26(T), were less than 70%. DNA-DNA relatedness analysis and 16S rRNA sequence similarity, as well as phenotypic and chemotaxonomic characteristics suggest that the strain MS5(T) constitutes a novel Bacillus species, for which the name Bacillus coreaensis sp. nov. is proposed. The type strain is MS5(T) (=DSM25506(T) =KCTC13895(T)). PMID:26115993

  19. Effect of xylan oligosaccharides generated from corncobs on food acceptability, growth performance, haematology and immunological parameters of Dicentrarchus labrax fingerlings.

    PubMed

    Abdelmalek, Baha Eddine; Driss, Dorra; Kallel, Fatma; Guargouri, Molka; Missaoui, Hechmi; Chaabouni, Semia Ellouz; Ayadi, Mohamed Ali; Bougatef, Ali

    2015-12-01

    The objective of this study was to determine the effect of two levels of inclusion of xylan oligosaccharides (XOS) extracted from corncob on growth, feed utilization, immune status and disease resistance of Mediterranean sea bass (Dicentrarchus labrax) fingerlings. Specimens of 4.75 ± 0.69 g at initial density of 2.7 ± 0.13 kg/m(3) were fed during 12 weeks at 0 g kg(-1) diet, 5 g kg(-1) diet and 10 g kg(-1) diet, dietary XOS level of inclusion in a commercial sea bass diet. Feeding the fish at both XOS dietary inclusion levels significantly increased weight gain, protein efficiency ratio and feed conversion ratio. Feeding of supplemented diets to fish led to reducing mortalities after challenging with A. hydrophila. The haematological and immunological parameters were assayed in both pre-challenged and post-challenged groups. There was an increased trend in red blood corpuscles, white blood corpuscles, pack cell volume, haemoglobin (Hb %) and serum protein content in treated groups over the control as time elapsed with the feeding trials. The serum immunoglobulin level and lysozyme activity showed an increased trend in the fed groups. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients supported with the morphometric data. In conclusion, XOS added at a designated dose (5 g kg(-1) diet) in the diet improves growth and stimulates the immunity and makes D. labrax fingerlings more resistant to infection by A. hydrophila. PMID:26242460

  20. Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

    PubMed Central

    Santo-Orihuela, Pablo Luis; Carvajal, Guillermo; Picollo, María Inés; Vassena, Claudia Viviana

    2013-01-01

    The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia) and from domiciliary areas, including El Palmar (Bolivia) and La Pista (Argentina). Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL) were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR) value (44.90) compared to the susceptible reference strain (NFS), whereas the Veinte de Octubre samples had the lowest value (0.50). Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP) on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein) and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively). The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP). PMID:24402155

  1. Leucocyte esterase, glucose and C-reactive protein in the diagnosis of prosthetic joint infections: a prospective study.

    PubMed

    De Vecchi, E; Villa, F; Bortolin, M; Toscano, M; Tacchini, L; Romanò, C L; Drago, L

    2016-06-01

    Analysis of joint fluid is of paramount importance for the diagnosis of prosthetic joint infections. Different markers of inflammation and/or infection in joint fluid have been proposed for diagnosis of these infections. In this study we evaluated the performance of leucocyte esterase, C-reactive protein (CRP) and glucose assays in synovial fluids from 129 patients with septic (n = 27) or aseptic (n = 102) prosthetic joint failure. Samples were collected in serum tubes and centrifuged to limit the presence of corpuscle interfering with the assays. Determinations of leucocyte esterase and glucose were carried out by means of enzymatic colorimetric reactions performed on strips for urine analysis. Tests were considered positive when graded + or ++ whereas traces or absence of colour were considered negative. CRP was measured using an automated turbidimetric method and considered suggestive for infections when >10 mg/L. Leucocyte esterase was positive in 25/27 infected patients and negative in 99/102 not infected patients (sensitivity 92.6%, specificity 97.0%). CRP was higher than the threshold in 22/27 infected patients and in 6/102 not infected patients (sensitivity: 81.5%; specificity: 94.1%) whereas glucose showed the lowest sensitivity (77.8%) and specificity (81.4%), being negative in 21/27 and 19/102 infected and not infected patients, respectively. CRP led to a correct diagnosis in 19 of 22 patients with discordant esterase and glucose results. In conclusion, evaluation of leucocyte esterase, glucose and CRP may represent a useful tool for rapid diagnosis of prosthetic joint infections. PMID:27040804

  2. Esterase detoxication of acetylcholinesterase inhibitors using human liver samples in vitro.

    PubMed

    Moser, Virginia C; Padilla, Stephanie

    2016-04-15

    Organophosphorus (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxication can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are considered factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxication of AChE-inhibiting pesticides mediated via these esterases. Recombinant human AChE was used as a bioassay of inhibitor concentration following incubation with detoxifying tissue: liver plus Ca(+2) (to stimulate PON1s, measuring activity of both esterases) or EGTA (to inhibit PON1s, thereby measuring CaE activity). AChE inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methylparaoxon were incubated with liver homogenates from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxication was defined as the difference in inhibition produced by the pesticide alone and inhibition measured in combination with liver plus Ca(+2) or liver plus EGTA. Generally, rat liver produced more detoxication than did the human samples. There were large detoxication differences across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos); for the most part these differences did not correlate with age or sex. Chlorpyrifos oxon was fully detoxified only in the presence of Ca(+2) in both rat and human livers. Detoxication of paraoxon and methylparaoxon in rat liver was greater with Ca(+2), but humans showed less differentiation than rats between Ca(+2) and EGTA conditions. This suggests the importance of PON1 detoxication for these three OPs in the rat, but mostly only for chlorpyrifos oxon in human samples. Malaoxon was detoxified similarly with Ca(+2) or EGTA, and the differences across humans correlated with metabolism of p

  3. KINETICS AND MECHANISM OF INHIBITION OF SERINE ESTERASES BY FLUORINATED AMINOPHOSPHONATES

    PubMed Central

    Makhaeva, G.F.; Aksinenko, A.Y.; Sokolov, V.B.; Baskin, I.I.; Palyulin, V.A.; Zefirov, N.S.; Hein, N.D.; Kampf, J.W.; Wijeyesakere, S.J.; Richardson, R.J.

    2016-01-01

    This paper reviews previously published data and presents new results to address the hypothesis that fluorinated aminophosphonates (FAPs), (RO)2P(O)C(CF3)2NHS(O)2C6H5, R = alkyl, inhibit serine esterases by scission of the P-C bond. Kinetics studies demonstrated that FAPs are progressive irreversible inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7.), butyrylcholinesterase (BChE, EC 3.1.1.8.), carboxylesterase (CaE, EC 3.1.1.1.), and neuropathy target esterase (NTE, EC 3.1.1.5.), consistent with P-C bond breakage. Chemical reactivity experiments showed that diMe-FAP and diEt-FAP react with water to yield the corresponding dialkylphosphates and (CF3)2CHNHS(O)2C6H5, indicating lability of the P-C bond. X-ray crystallography of diEt-FAP revealed an elongated (and therefore weaker) P-C bond (1.8797 (13) Å) compared to P-C bonds in dialkylphosphonates lacking α-CF3 groups (1.805–1.822 Å). Semi-empirical and non-empirical molecular modeling of diEt-FAP and (EtO)2P(O)C(CH3)2NHS(O)2C6H5 (diEt-AP), which lacks CF3 groups, indicated lengthening and destabilization of the P-C bond in diEt-FAP compared to diEt-AP. Active site peptide adducts formed by reacting diEt-FAP with BChE and diBu-FAP with NTE catalytic domain (NEST) were identified using peptide mass mapping with mass spectrometry (MS). Mass shifts (mean ± SE, average mass) for peaks corresponding to active site peptides with diethylphosphoryl and monoethylphosphoryl adducts on BChE were 136.1 ± 0.1 and 108.0 ± 0.1 Da, respectively. Corresponding mass shifts for dibutylphosphoryl and monobutylphosphoryl adducts on NEST were 191.8 ± 0.2 and 135.5 ± 0.1 Da, respectively. Each of these values was statistically identical to the theoretical mass shift for each dialkylphosphoryl and monoalkylphosphoryl species. The MS results demonstrate that inhibition of BChE and NEST by FAPs yields dialkylphosphoryl and monoalkylphosphoryl adducts, consistent with phosphorylation via P-C bond cleavage and aging by net

  4. Determination of NAT2 acetylation status in the Greenlandic population.

    PubMed

    Geller, Frank; Soborg, Bolette; Koch, Anders; Michelsen, Sascha Wilk; Bjorn-Mortensen, Karen; Carstensen, Lisbeth; Birch, Emilie; Nordholm, Anne Christine; Johansen, Marie Mila Broby; Børresen, Malene Landbo; Feenstra, Bjarke; Melbye, Mads

    2016-04-01

    N-acetyltransferase 2 (NAT2) is a well-studied phase II xenobiotic metabolizing enzyme relevant in drug metabolism and cancerogenesis. NAT2 activity is largely determined by genetic polymorphisms in the coding region of the corresponding gene. We investigated NAT2 acetylation status in 1556 individuals from Greenland based on four different single nucleotide polymorphism (SNP) panels and the tagging SNP rs1495741. There was good concordance between the NAT2 status inferred by the different SNP combinations. Overall, the fraction of slow acetylators was low with 17.5 % and varied depending on the degree of Inuit ancestry; in individuals with <50 % Inuit ancestry, we observed more than 25 % slow acetylators reflecting European ancestry. Greenland has a high incidence of tuberculosis, and individual dosing of isoniazid according to NAT2 status has been shown to improve treatment and reduce side effects. Our findings could be a first step in pharmacogenetics-based tuberculosis therapy in Greenland. PMID:25794903

  5. Synthetic biology for engineering acetyl coenzyme A metabolism in yeast.

    PubMed

    Nielsen, Jens

    2014-01-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl-CoA can be formed from glucose in high yield. Here the perspective of generating yeast platform strains that have such properties is discussed in the context of a major breakthrough with expression of a functional pyruvate dehydrogenase complex in the cytosol. PMID:25370498

  6. Synthesis of polyrotaxanes from acetyl-β-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Ristić, I. S.; Nikolić, L.; Nikolić, V.; Ilić, D.; Budinski-Simendić, J.

    2011-12-01

    Polyrotaxanes are intermediary products in the synthesis of topological gels. They are created by inclusion complex formation of hydrophobic linear macromolecules with cyclodextrins or their derivatives. Then, pairs of cyclodextrin molecules with covalently linkage were practically forming the nodes of the semi-flexible polymer network. Such gels are called topological gels and they can absorb huge quantities of water due to the net flexibility allowing the poly(ethylene oxide) chains to slide through the cyclodextrin cavities, without being pulled out altogether. For polyrotaxane formation poly(ethylene oxide) was used like linear macromolecules. There are hydroxyl groups at poly(ethylene oxide) chains, whereby the linking of the voluminous molecules should be made. To avoid the reaction of cyclodextrin OH groups with stoppers, they should be protected by, e.g., acetylation. In this work, the acetylation of the OH groups of β-cyclodextrin was performed by acetic acid anhydride with iodine as the catalyst. The acetylation reaction was assessed by the FTIR and HPLC method. By the HPLC analysis was found that the acetylation was completed in 20 minutes. Inserting of poly(ethylene oxide) with 4000 g/mol molecule mass into acetyl-β-cyclodextrin with 2:1 poly(ethylene oxide) monomer unit to acetyl-β-cyclodextrin ratio was also monitored by FTIR, and it was found that the process was completed in 12 h at the temperature of 10°C. If the process is performed at temperatures above 10°C, or for periods longer than 12 hours, the process of uncontrolled hydrolysis of acetate groups was initiated.

  7. Interaction of RNA polymerase II with acetylated nucleosomal core particles

    SciTech Connect

    Pineiro, M.; Gonzalez, P.J.; Hernandez, F.; Palacian, E. )

    1991-05-31

    Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery.

  8. Histone acetylation: a switch between repressive and permissive chromatin

    PubMed Central

    Eberharter, Anton; Becker, Peter B.

    2002-01-01

    The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation. PMID:11882541

  9. 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).

  10. Bioassay technique using nonspecific esterase activities of Tetrahymena pyriformis for screening and assessing cytotoxicity of xenobiotics

    SciTech Connect

    Bogaerts, P.; Senaud, J.; Bohatier, J. |

    1998-08-01

    A simple and rapid test for screening and assessing the cytotoxicity of xenobiotics was developed with Tetrahymena pyriformis. The method estimates the activities of nonspecific esterases of a cell by concentrating within it a specific amount of fluorescence associated with fluorescein dye. The 2-h median effective concentration (EC50) values of 10 inorganic and eight organic substances are presented and compared to those of three other bioassays: the conventional T. pyriformis proliferation rate 9-h median inhibitory concentrations, the Microtox 30-min EC50s, and the Daphnia magna 4-methylumbelliferyl {beta}-D galactoside 1-h EC50s. A highly significant correlation was found between the results obtained with the fluorescein diacetate test and those obtained with the growth inhibition and Microtox tests. This in vivo enzymatic test showed high sensitivity to all compounds tested except Cr{sup 6+} and sodium dodecyl sulfate.

  11. Functional-based screening methods for lipases, esterases, and phospholipases in metagenomic libraries.

    PubMed

    Reyes-Duarte, Dolores; Ferrer, Manuel; García-Arellano, Humberto

    2012-01-01

    The use of metagenomic techniques for enzyme discovery constitutes a powerful approach. Functional screens, in contrast to sequence homology search, enable us to select enzymes based on their activity. It is noteworthy that they additionally guarantee the identification of genes coding for enzymes that exhibited no sequence similarity to known counterparts from public databases and that even do not match any putative catalytic residues, involved in the selected catalytic function. Therefore, this strategy not only provides new enzymes for new biotechnological applications, but also allows functional assignment of many proteins, found in abundance in the databases, currently designated as "hypothetical" or "conserved hypothetical" proteins. In the past decade, there has been an exponential increase in the design of functional screening programmes, the majority of them established for hydrolases and oxidoreductases. Here, functional screening methods that guarantee the greatest enzyme diversity, for mining esterases and lipases, are described. PMID:22426714

  12. Addition of feruloyl esterase and xylanase produced on-site improves sugarcane bagasse hydrolysis.

    PubMed

    Braga, Cleiton Márcio Pinto; Delabona, Priscila da Silva; Lima, Deise Juliana da Silva; Paixão, Douglas Antônio Alvaredo; Pradella, José Geraldo da Cruz; Farinas, Cristiane Sanchez

    2014-10-01

    Accessory enzymes that assist biomass degradation could be used to improve the recovery of fermentable sugar for use in biorefineries. In this study, different fungal strains isolated from the Amazon rainforest were evaluated in terms of their ability to produce feruloyl esterase (FAE) and xylanase enzymes, and an assessment was made of the contributions of the enzymes in the hydrolysis of pretreated sugarcane bagasse. In the selection step, screening using plate assays was followed by shake flask submerged cultivations. After carbon source selection and cultivation in a stirred-tank bioreactor, Aspergillusoryzae P21C3 proved to be a promising strain for production of the enzymes. Supplementation of a commercial enzyme preparation with 30% (v/v) crude enzymatic complex from A. oryzae P21C3 increased the conversion of cellulose derived from pretreated sugarcane bagasse by 36%. Supplementation with FAE and xylanase enzymes produced on-site can therefore be used to improve the hydrolysis of sugarcane bagasse. PMID:25151076

  13. How well do the substrates KISS the enzyme? Molecular docking program selection for feruloyl esterases

    PubMed Central

    Udatha, D. B. R. K. Gupta; Sugaya, Nobuyoshi; Olsson, Lisbeth; Panagiotou, Gianni

    2012-01-01

    Molecular docking is the most commonly used technique in the modern drug discovery process where computational approaches involving docking algorithms are used to dock small molecules into macromolecular target structures. Over the recent years several evaluation studies have been reported by independent scientists comparing the performance of the docking programs by using default ‘black box’ protocols supplied by the software companies. Such studies have to be considered carefully as the docking programs can be tweaked towards optimum performance by selecting the parameters suitable for the target of interest. In this study we address the problem of selecting an appropriate docking and scoring function combination (88 docking algorithm-scoring functions) for substrate specificity predictions for feruloyl esterases, an industrially relevant enzyme family. We also propose the ‘Key Interaction Score System’ (KISS), a more biochemically meaningful measure for evaluation of docking programs based on pose prediction accuracy. PMID:22435086

  14. Potential of Ophiostoma piceae sterol esterase for biotechnologically relevant hydrolysis reactions

    PubMed Central

    Barba Cedillo, Víctor; Prieto, Alicia; Martínez, María Jesús

    2013-01-01

    The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity toward p-nitrophenol, glycerol, and sterol esters. Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris under the AOX1 methanol-inducible promoter (PAOX1) using sorbitol as co-susbtrate, and the hydrolytic activity of the recombinant protein (OPE*) turned out to be improved from a kinetic point of view. In this study, we analyze the effects of sorbitol during the expression of OPE*, at first added as an additional carbon source, and methanol as inducer. The O. piceae enzyme was successfully used for PVAc hydrolysis, suggesting its potential applicability in recycled paper production to decrease stickies problems. PMID:23138020

  15. Structure of EstA esterase from psychrotrophic Pseudoalteromonas sp. 643A covalently inhibited by monoethylphosphonate

    SciTech Connect

    Brzuszkiewicz, Anna; Nowak, Elzbieta; Dauter, Zbigniew; Dauter, Miroslawa; Cieslinski, Hubert; Dlugolecka, Anna; Kur, Józef

    2010-10-28

    The crystal structure of the esterase EstA from the cold-adapted bacterium Pseudoalteromonas sp. 643A was determined in a covalently inhibited form at a resolution of 1.35 {angstrom}. The enzyme has a typical SGNH hydrolase structure consisting of a single domain containing a five-stranded {beta}-sheet, with three helices at the convex side and two helices at the concave side of the sheet, and is ornamented with a couple of very short helices at the domain edges. The active site is located in a groove and contains the classic catalytic triad of Ser, His and Asp. In the structure of the crystal soaked in diethyl p-nitrophenyl phosphate (DNP), the catalytic serine is covalently connected to a phosphonate moiety that clearly has only one ethyl group. This is the only example in the Protein Data Bank of a DNP-inhibited enzyme with covalently bound monoethylphosphate.

  16. Disruption of lysosomal targeting is associated with insecticidal potency of juvenile hormone esterase

    PubMed Central

    Bonning, Bryony C.; Ward, Vernon K.; van Meer, Marnix M. M.; Booth, Tim F.; Hammock, Bruce D.

    1997-01-01

    Juvenile hormone esterase (JHE; EC 3.1.1.1), which is intrinsically involved in regulation of development of some insect larvae, is rapidly removed from the hemolymph by the pericardial cells. Lys-29 and Lys-524, which are implicated in the degradation of JHE, were mutated to Arg. Neither the half-life of the modified JHE in the hemolymph nor the catalytic parameters were changed significantly, but when combined, these mutations resulted in apparent failure of lysosomal targeting in the pericardial cell complex. A hypothesis for the mechanism of reduced efficiency of lysosomal targeting is presented. Infection of larvae with a recombinant baculovirus expressing the modified JHE resulted in a 50% reduction in feeding damage compared with larvae infected with the wild-type virus, thus demonstrating improved properties as a biological insecticide. These data demonstrate that alteration of specific residues of JHE that disrupted lysosomal targeting, dramatically increased the insecticidal activity of this protein. PMID:9177159

  17. Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

    PubMed

    Damásio, André R L; Braga, Cleiton Márcio Pinto; Brenelli, Lívia B; Citadini, Ana Paula; Mandelli, Fernanda; Cota, Junio; de Almeida, Rodrigo Ferreira; Salvador, Victor Hugo; Paixao, Douglas Antonio Alvaredo; Segato, Fernando; Mercadante, Adriana Zerlotti; de Oliveira Neto, Mario; do Santos, Wanderley Dantas; Squina, Fabio M

    2013-08-01

    The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production. PMID:23229566

  18. Detection of feruloyl- and cinnamoyl esterases from basidiomycetes in the presence of interfering laccase.

    PubMed

    Haase-Aschoff, Paul; Linke, Diana; Berger, Ralf G

    2013-02-01

    Little is known on basidiomycete sources of feruloyl esterases (FAEs), although many wood-rotting representatives of these fungi typically grow on feruloyl-rich substrates. A major reason is that the almost ubiquitous presence of laccases interferes with the detection of FAE activity. Laccases polymerize the liberated ferulic acid (FA) in situ, thus detracting the product of enzymatic hydrolysis from its detection. A rapid HPLC-UV method was developed to detect the loss of FA, but also to quantify the hydrolysis of FA esters. The method allows at the same time to evaluate the substrate specificity of a FAE. Forty one basidiomycetes were tested for their FAE activities, and 25 out of the set were positive. The basidiomycetes hydrolyzing cinnamates with the highest conversion rates were Auricularia auricula-judae and Marasmius scorodonius. Moreover, a new FAE inducer, the nonionic detergent Tween 80, was found. This is the first comprehensive study on basidiomycete sources of FAEs. PMID:23306132

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

    SciTech Connect

    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 is 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.

  20. Production of Feruloyl Esterase from Aspergillus niger by Solid-State Fermentation on Different Carbon Sources

    PubMed Central

    Ou, Shiyi; Zhang, Jing; Wang, Yong; Zhang, Ning

    2011-01-01

    A mixture of wheat bran with maize bran as a carbon source and addition of (NH4)SO4 as nitrogen source was found to significantly increase production of feruloyl esterase (FAE) enzyme compared with wheat bran as a sole carbon and nitrogen source. The optimal conditions in conical flasks were carbon source (30 g) to water 1 : 1, maize bran to wheat bran 1 : 2, (NH4)SO4 1.2 g and MgSO4 70 mg. Under these conditions, FAE activity was 7.68 mU/g. The FAE activity on the mixed carbon sources showed, high activity against the plant cell walls contained in the cultures. PMID:21603274