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 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 Evolutionary variance analysis of the Glycoside Hydrolase Family 13: Structural evidence in classification and evolution

2017 ◽  
Author(s):  
Jose Sergio Hleap ◽  
Christian Blouin
Keyword(s):  
Glycoside Hydrolase ◽  
Structural Information ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Sequence Information ◽  
Novel Method ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13 ◽  
Structural Shifts

AbstractGlycoside Hydrolase Family 13 (GH13) structures are responsible for the hydrolysis of starch into smaller carbohydrates. They important in industrial applications and evolutionary studies. This family has been thoroughly documented in the the Carbohydrate-Active enZYmes Database (CAZY), and divided into subfamilies based mainly in sequence information. Here we give structural evidence into GH13 classification and evolution using structural information. Here we proposed a novel method that is sensitive enough to identify miss-classifications, or to provide evidence for further partition that can be of interests to bio-engineers and evolutionary biologists. We also introduced a method to explore the relative importance of residues with respect to the overall deformation that it causes to the overall structure in an evolutionary time scale. We found that the GH13 family can be classified into three main structural groups. There is a hierarchical structure within these clusters that can be use to inform other classification schemes. We also found that by using structural information, subtle structural shifts can be identified and that can be missed in sequence/phylogeny-only based classifications. When each structural group is explored, we found that identifying the most structurally variable sites can lead to identification of functionally (both catalytically and structurally) important residues.

scholarly journals Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuya Kuritani ◽  
Kohei Sato ◽  
Hideo Dohra ◽  
Seiichiro Umemura ◽  
Motomitsu Kitaoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metabolic Pathway ◽  
Glycoside Hydrolase ◽  
Gene Locus ◽  
Glycoside Hydrolase Family ◽  
Sequential Reaction ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Hydrolase Family

AbstractLevoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-d-glucose by LgdB2; next, LgdC reduces 3-keto-d-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

scholarly journals The response to selection in Glycoside Hydrolase Family 13 structures: A comparative quantitative genetics approach

2017 ◽  
Author(s):  
Jose Sergio Hleap ◽  
Christian Blouin
Keyword(s):  
Glycoside Hydrolase ◽  
Fitness Landscape ◽  
Protein Structures ◽  
Purifying Selection ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Response To Selection ◽  
Effect Change ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

AbstractThe Glycoside Hydrolase Family 13 (GH13) is both evolutionary diverse and relevant to many industrial applications. Its members perform the hydrolysis of starch into smaller carbohydrates. Members of the family have been bioengineered to improve catalytic function under industrial environments. We introduce a framework to analyze the response to selection of GH13 protein structures given some phylogenetic and simulated dynamic information. We found that the TIM-barrel is not selectable since it is under purifying selection. We also show a method to rank important residues with higher inferred response to selection. These residues can be altered to effect change in properties. In this work, we define fitness as inferred thermodynamic stability. We show that under the developed framework, residues 112Y, 122K, 124D, 125W, and 126P are good candidates to increase the stability of the truncated protein 4E2O. Overall, this paper demonstrate the feasibility of a framework for the analysis of protein structures for any other fitness landscape.

scholarly journals Unravelling the diversity of glycoside hydrolase family 13 α-amylases from Lactobacillus plantarum WCFS1

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Laura Plaza-Vinuesa ◽  
Oswaldo Hernandez-Hernandez ◽  
F. Javier Moreno ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Glycoside Hydrolase ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Structural Features ◽  
Biochemical Properties ◽  
Glycoside Hydrolase Family ◽  
Reaction Conditions ◽  
Genes Encoding ◽  
Hydrolysis Of

Abstract Background α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included. Results In this study, Lp_0179 and Lp_2757 L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases. Conclusions The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions.

scholarly journals Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation

2015 ◽  
Vol 81 (18) ◽  
pp. 6380-6392 ◽  
Author(s):  
Manuel Nieto-Domínguez ◽  
Laura I. de Eugenio ◽  
Jorge Barriuso ◽  
Alicia Prieto ◽  
Beatriz Fernández de Toro ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Intronless Gene ◽  
Content Type ◽  
Alkyl Glycosides ◽  
Beechwood Xylan ◽  
Dimeric Form ◽  
Culture Supernatants ◽  
Hydrolysis Of

ABSTRACTThis paper reports on a novel β-xylosidase from the hemicellulolytic fungusTalaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, andKmandVmaxvalues of 0.17 mM and 52.0 U/mg, respectively, using commercialp-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.

scholarly journals Polymer Extraction from Lignocellulosic Biomass (Water Hyacinth)

2020 ◽  
Vol 2 (1) ◽  
pp. 77
Keyword(s):  
Lignocellulosic Biomass ◽  
Water Hyacinth ◽  
Plant Biomass ◽  
Alkali Treatment ◽  
Specific Effect ◽  
Value Added ◽  
Grape Juice ◽  
Lignocellulosic Wastes ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

Bioconversion of renewable lignocellulosic biomass to biofuel and value-added products is globally gaining significant importance. Lignocellulosic wastes are the most promising feedstock considering its great availability and low cost. The biomass conversion process involves mainly two steps: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars and fermentation of the sugars to ethanol and other bioproducts. However, sugars necessary for fermentation are trapped inside the recalcitrant structure of the lignocellulose. Hence, pretreatment of lignocellulosic wastes is always necessary to alter and/or remove the surrounding matrix of lignin and hemicelluloses in order to improve the hydrolysis of cellulose. These pretreatments cause physical and/or chemical changes in the plant biomass in order to achieve this result. Each pretreatment has a specific effect on the cellulose, hemicellulose, and lignin fraction. Thus, the pretreatment methods and conditions should be chosen according to the process configuration selected for the subsequent hydrolysis steps. In general, pretreatment methods can be classified into four categories, including physical, physicochemical, chemical, and biological pretreatment. Bioresource utilization of biopolymeric materials has now gained recent attention. Cellulose was extracted from water hyacinth by acid, alkali treatment & extracted cellulose was grafted with curcumin, pesticide, grape juice, magnetorheological fluid, and the grafted composite material was evaluated for release of respective grafted materials. In the present study, a polymer extracted from water hyacinth was evaluated for various applications. The present study would suggest the possible utilization of water hyacinth composite as the biomaterial for diverse applications.

scholarly journals Novel Members of Glycoside Hydrolase Family 13 Derived from Environmental DNA

2008 ◽  
Vol 74 (6) ◽  
pp. 1914-1921 ◽  
Author(s):  
Antje Labes ◽  
Eva Nordberg Karlsson ◽  
Olafur H. Fridjonsson ◽  
Pernilla Turner ◽  
Gudmundur O. Hreggvidson ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Environmental Dna ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Biotechnological Applications ◽  
Consensus Primer ◽  
Thermophilic Conditions ◽  
Maltogenic Amylase ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

ABSTRACT Starch and pullulan-modifying enzymes of the α-amylase family (glycoside hydrolase family 13) have several industrial applications. To date, most of these enzymes have been derived from isolated organisms. To increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using DNA from enrichments from geothermal habitats. With this approach, we succeeded in isolating three new enzymes: a neopullulanase and two cyclodextrinases. Both cyclodextrinases displayed significant maltogenic amylase side activity, while one showed significant neopullulanase side activity. Specific motifs and domains that correlated with enzymatic activities were identified; e.g., the presence of the N domain was correlated with cyclodextrinase activity. The enzymes exhibited stability under thermophilic conditions and showed features appropriate for biotechnological applications.

Bacillus licheniformistrehalose-6-phosphate hydrolase structures suggest keys to substrate specificity

2016 ◽  
Vol 72 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Min-Guan Lin ◽  
Meng-Chun Chi ◽  
Vankadari Naveen ◽  
Yi-Ching Li ◽  
Long-Liu Lin ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Bacillus Licheniformis ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Positive Charge ◽  
Glycoside Hydrolase Family ◽  
Erwinia Rhapontici ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures ofBacillus licheniformisTreA (BlTreA) and its R201Q mutant complexed withp-nitrophenyl-α-D-glucopyranoside (R201Q–pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure ofBlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site ofBlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions ofBacillus cereusoligo-1,6-glucosidase (BcOgl) andErwinia rhaponticiisomaltulose synthase (NX-5), the surface potential of theBlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity ofBlTreA. Strikingly, the281HHLK284motif and Lys292 play critical roles in substrate discrimination byBlTreA.

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