scholarly journals Xylanase and Acetyl Xylan Esterase Activities of XynA, a Key Subunit of the Clostridium cellulovorans Cellulosome for Xylan Degradation

2002 ◽  
Vol 68 (12) ◽  
pp. 6399-6402 ◽  
Author(s):  
Akihiko Kosugi ◽  
Koichiro Murashima ◽  
Roy H. Doi
Keyword(s):  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Acetyl Xylan Esterase ◽  
Clostridium Cellulovorans ◽  
Broad Substrate Specificity ◽  
Xylan Degradation ◽  
Hydrolysis Of ◽  
Esterase Activities

ABSTRACT The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-β-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans.

scholarly journals Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Svini Dileepa Marasinghe ◽  
Eunyoung Jo ◽  
Sachithra Amarin Hettiarachchi ◽  
Youngdeuk Lee ◽  
Tae-Yang Eom ◽  
...  
Keyword(s):  
Lignocellulosic Biomass ◽  
Glycoside Hydrolase ◽  
Synergetic Effect ◽  
Value Added ◽  
Industrial Applications ◽  
Affinity Column ◽  
Glycoside Hydrolase Family ◽  
Acetyl Xylan Esterase ◽  
Xylan Degradation ◽  
Hydrolysis Of

Abstract Background Xylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the β-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis. Results xynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50–55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold. Conclusion The supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.

scholarly journals Purification and Cloning of a Broad Substrate Specificity Human Liver Carboxylesterase That Catalyzes the Hydrolysis of Cocaine and Heroin

1997 ◽  
Vol 272 (23) ◽  
pp. 14769-14775 ◽  
Author(s):  
Evgenia V. Pindel ◽  
Natalia Y. Kedishvili ◽  
Trent L. Abraham ◽  
Monica R. Brzezinski ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Human Liver ◽  
Broad Substrate Specificity ◽  
Hydrolysis Of

scholarly journals The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains

2016 ◽  
Vol 72 (10) ◽  
pp. 750-761 ◽  
Author(s):  
Emma L. Summers ◽  
Christina D. Moon ◽  
Renee Atua ◽  
Vickery L. Arcus
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Binding Domain ◽  
Carbohydrate Binding ◽  
Catalytic Core ◽  
Binding Domains ◽  
Carbohydrate Binding Modules ◽  
Tim Barrel ◽  
Domain Arrangement ◽  
Hydrolysis Of

Glycoside hydrolase (GH) family 29 consists solely of α-L-fucosidases. These enzymes catalyse the hydrolysis of glycosidic bonds. Here, the structure of GH29_0940, a protein cloned from metagenomic DNA from the rumen of a cow, has been solved, which reveals a multi-domain arrangement that has only recently been identified in bacterial GH29 enzymes. The microbial species that provided the source of this enzyme is unknown. This enzyme contains a second carbohydrate-binding domain at its C-terminal end in addition to the typical N-terminal catalytic domain and carbohydrate-binding domain arrangement of GH29-family proteins. GH29_0940 is a monomer and its overall structure consists of an N-terminal TIM-barrel-like domain, a central β-sandwich domain and a C-terminal β-sandwich domain. The TIM-barrel-like catalytic domain exhibits a (β/α)8/7arrangement in the core instead of the typical (β/α)8topology, with the `missing' α-helix replaced by a long meandering loop that `closes' the barrel structure and suggests a high degree of structural flexibility in the catalytic core. This feature was also noted in all six other structures of GH29 enzymes that have been deposited in the PDB. Based on sequence and structural similarity, the residues Asp162 and Glu220 are proposed to serve as the catalytic nucleophile and the proton donor, respectively. Like other GH29 enzymes, the GH29_0940 structure shows five strictly conserved residues in the catalytic pocket. The structure shows two glycerol molecules in the active site, which have also been observed in other GH29 structures, suggesting that the enzyme catalyses the hydrolysis of small carbohydrates. The two binding domains are classed as family 32 carbohydrate-binding modules (CBM32). These domains have residues involved in ligand binding in the loop regions at the edge of the β-sandwich. The predicted substrate-binding residues differ between the modules, suggesting that different modules bind to different groups on the substrate(s). Enzymes that possess multiple copies of CBMs are thought to have a complex mechanism of ligand recognition. Defined electron density identifying a long 20-amino-acid hydrophilic loop separating the two CBMs was observed. This suggests that the additional C-terminal domain may have a dynamic range of movement enabled by the loop, allowing a unique mode of action for a GH29 enzyme that has not been identified previously.

scholarly journals Nudix proteins affecting microbial pathogenesis

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1110-1114 ◽  
Author(s):  
Elzbieta Kraszewska ◽  
Joanna Drabinska
Keyword(s):  
Substrate Specificity ◽  
The State ◽  
Microbial Pathogenesis ◽  
Cellular Functions ◽  
Broad Substrate Specificity ◽  
Domains Of Life ◽  
Hydrolysis Of

Nudix proteins catalyse hydrolysis of pyrophosphate bonds in a variety of substrates and are ubiquitous in all domains of life. Their widespread presence and broad substrate specificity suggest that they have important cellular functions. In this review, we summarize the state of knowledge on microbial Nudix proteins involved in pathogenesis.

scholarly journals GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Yusuke Nakamichi ◽  
Tatsuya Fujii ◽  
Thierry Fouquet ◽  
Akinori Matsushika ◽  
Hiroyuki Inoue
Keyword(s):  
Glycoside Hydrolase ◽  
Glucuronic Acid ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Promising Tool ◽  
Talaromyces Cellulolyticus ◽  
Food And Feed ◽  
Cellulose Binding ◽  
Hydrolysis Of

ABSTRACT Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (XnU4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing XnU4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system. IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.

scholarly journals A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

2014 ◽  
Vol 70 (11) ◽  
pp. 2950-2958 ◽  
Author(s):  
Franz J. St John ◽  
Diane Dietrich ◽  
Casey Crooks ◽  
Edwin Pozharski ◽  
Javier M. González ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Amino Acid Identity ◽  
Glycoside Hydrolase Family ◽  
High Sequence Identity ◽  
Loop Region ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 30

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A fromClostridium papyrosolvens(CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8–α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition.CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme fromBacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes,CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8–α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function.

Structural and biochemical basis of a marine bacterial glycoside hydrolase family 2 β-glycosidase with broad substrate specificity

2021 ◽  
Author(s):  
Jian Yang ◽  
Shubo Li ◽  
Yu Liu ◽  
Ru Li ◽  
Lijuan Long
Keyword(s):  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Structural Complexity ◽  
Glycoside Hydrolase Family ◽  
Biochemical Basis ◽  
Broad Substrate Specificity ◽  
Biochemical Analyses ◽  
Transformation Of Organic Matter ◽  
Enzymatic Machinery ◽  
Hydrolase Family

Uronic acids are commonly found in marine polysaccharides and increase structural complexity sanand intrinsic recalcitrance to enzymatic attack. The glycoside hydrolase family 2 (GH2) include proteins that target sugar conjugates with hexuronates and are involved in the catabolism and cycling of marine polysaccharides. Here, we reported a novel GH2, Aq GalA from a marine algae-associated Bacteroidetes with broad-substrate specificity. Biochemical analyses revealed that Aq GalA exhibits hydrolyzing activities against β-galacturonide, β-glucuronide, and β-galactopyranoside via retaining mechanisms. We solved the Aq GalA crystal structure in complex with galacturonic acid (GalA) and showed (via mutagenesis) that charge characteristics at uronate-binding subsites controlled substrate selectivity for uronide hydrolysis. Additionally, conformational flexibility of the Aq GalA active site pocket was proposed as a key component for broad substrate enzyme selectivity. Our Aq GalA structural and functional data augments the current understanding of substrate recognition of GH2 enzymes and provided key insights into the bacterial use of uronic acid containing polysaccharides. IMPORTANCE The decomposition of algal glycans driven by marine bacterial communities represents one of the largest heterotrophic transformation of organic matter fueling marine food webs and global carbon cycling. However, our knowledge of the carbohydrate cycling is limited due to structural complexity of marine polysaccharides and the complicated enzymatic machinery of marine microbes. To degrade algal glycan, marine bacteria such as members of Bacteroidetes produce a complex repertoire of carbohydrate-active enzymes (CAZymes) matching the structural specificity of the different carbohydrates. In this study, we investigated an extracellular GH2 β-glycosidase, Aq GalA from a marine Bacteroidetes to identify the key components responsible for glycuronides recognition and hydrolysis. The broad substrate specificity of Aq GalA against glycosides with diverse stereochemical substitutions indicates its potential in processing complex marine polysaccharides. Our findings promote a better understanding of microbially-driven mechanisms of marine carbohydrate cycling.

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