scholarly journals Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kanyisa Ndata ◽  
Walter Nevondo ◽  
Bongi Cekuse ◽  
Leonardo Joaquim van Zyl ◽  
Marla Trindade
Keyword(s):  
Glycosyl Hydrolase ◽  
Metagenomic Library ◽  
Bioinformatic Analysis ◽  
Glycoside Hydrolase Family ◽  
Kinetic Properties ◽  
Glycosyl Hydrolases ◽  
Remarkable Feature ◽  
Manure Compost ◽  
Horse Manure ◽  
Hydrolase Family

Abstract Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications. Conclusions Here we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.

scholarly journals Growth of Azospirillum irakense KBC1 on the Aryl β-Glucoside Salicin Requires either salA or salB

1999 ◽  
Vol 181 (10) ◽  
pp. 3003-3009 ◽  
Author(s):  
Denis Faure ◽  
Jos Desair ◽  
Veerle Keijers ◽  
My Ali Bekri ◽  
Paul Proost ◽  
...  
Keyword(s):  
Microbial Growth ◽  
Glycosyl Hydrolase ◽  
Cosmid Library ◽  
Glycosyl Hydrolases ◽  
Amino Acid Similarity ◽  
Functional Homology ◽  
Low Degree ◽  
Genetic Implication ◽  
Glycosyl Hydrolase Family 3 ◽  
Hydrolase Family

ABSTRACT The rhizosphere nitrogen-fixing bacteriumAzospirillum irakense KBC1 is able to grow on pectin and β-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring β-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl β-glucosides. A Δ(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB′ subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this β-glucosidase family in the utilization of β-glucosides for microbial growth.

scholarly journals Transcript Analysis of Genes Encoding a Family 61 Endoglucanase and a Putative Membrane-Anchored Family 9 Glycosyl Hydrolase from Phanerochaete chrysosporium

2002 ◽  
Vol 68 (11) ◽  
pp. 5765-5768 ◽  
Author(s):  
Amber Vanden Wymelenberg ◽  
Stuart Denman ◽  
Diane Dietrich ◽  
Jennifer Bassett ◽  
Xiaochun Yu ◽  
...  
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Glycoside Hydrolase ◽  
Glycosyl Hydrolase ◽  
Thermobifida Fusca ◽  
Glycoside Hydrolase Family ◽  
Transcript Analysis ◽  
Transcript Levels ◽  
Genes Encoding ◽  
Membrane Bound ◽  
Hydrolase Family

ABSTRACT Phanerochaete chrysosporium cellulase genes were cloned and characterized. The cel61A product was structurally similar to fungal endoglucanases of glycoside hydrolase family 61, whereas the cel9A product revealed similarities to Thermobifida fusca Cel9A (E4), an enzyme with both endo- and exocellulase characteristics. The fungal Cel9A is apparently a membrane-bound protein, which is very unusual for microbial cellulases. Transcript levels of both genes were substantially higher in cellulose-grown cultures than in glucose-grown cultures. These results show that P. chrysosporium possesses a wide array of conventional and unconventional cellulase genes.

Plant vascular system-feeding Psyllidae (Hemiptera) and Nematoda genomes encode family 12 glycosyl hydrolases

2019 ◽  
Vol 151 (3) ◽  
pp. 291-297
Author(s):  
Richard W. Jones
Keyword(s):  
Plant Cell Wall ◽  
Vascular Tissue ◽  
Vascular System ◽  
Glycosyl Hydrolase ◽  
Expression System ◽  
Root Tip ◽  
Glycosyl Hydrolases ◽  
Endoglucanase Activity ◽  
Intron Structure ◽  
Hydrolase Family

AbstractInsect-encoded cellulolytic plant cell wall hydrolases have thus far been found mostly from glycosyl hydrolase family 5, 9, 10, and 45. We now report the first evidence for genomic encoding of family 12 glycosyl hydrolases in vascular feeding Psyllidae (Hemiptera) and Nematoda. The genes were identified in three psyllids (Acanthocasuarina muellerianae Taylor, Pachypsylla venusta (Osten-Sacken), and Diaphorina citri Kuwayama) and a root tip feeding dagger nematode (Xiphinema index Thorne and Allen; Dorylaimida: Longidoridae). While the final gene products were highly similar, the genomic intron structure varied, having a 2 kB intron in P. venusta, a 283 base-pair intron in D. citri, and no intron in X. index. Endoglucanase activity was demonstrated using the D. citri genes in an Agrobacterium Conn (Rhizobiaceae) infiltration-based plant expression system. The presence of family 12 endoglucanases in this set of insects suggests a specific role in facilitating feeding on vascular tissue.

scholarly journals A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases

2014 ◽  
Vol 80 (11) ◽  
pp. 3426-3432 ◽  
Author(s):  
Zhongyuan Li ◽  
Xianli Xue ◽  
Heng Zhao ◽  
Peilong Yang ◽  
Huiying Luo ◽  
...  
Keyword(s):  
Specific Activity ◽  
Glycosyl Hydrolase ◽  
Beech Wood ◽  
Catalytic Efficiency ◽  
Cd Spectroscopy ◽  
Titration Calorimetry ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Sheep Rumen ◽  
Hydrolase Family

ABSTRACTEfficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed inEscherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

scholarly journals Functional metagenomics reveals novel β-galactosidases not predictable from gene sequences

2016 ◽  
Author(s):  
Jiujun Cheng ◽  
Tatyana Romantsov ◽  
Katja Engel ◽  
Andrew C. Doxey ◽  
David R. Rose ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Sinorhizobium Meliloti ◽  
Biochemical Analysis ◽  
Sequence Similarity ◽  
Metagenomic Library ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Galactosidase Activity ◽  
Functional Metagenomics ◽  
Hydrolase Family

AbstractA soil metagenomic library carried in pJC8 (an IncP cosmid) was used for functional complementation for β-galactosidase activity in bothα-Proteobacteria (Sinorhizobium meliloti)andγ-Proteobacteria (Escherichia coli).Oneβ-galactosidase, encoded by overlapping clones selected in both hosts, was identified as a member of glycoside hydrolase family 2. ORFs obviously encoding possible β-galactosidases were not identified in 19 other clones that were only able to complementS. meliloti.Based on low sequence similarity to known glycoside hydrolases but not β-galactosidases, three ORFs were examined further. Biochemical analysis confirmed that all encodedβ-galactosidase activity. Bioinformatic and structural modeling implied that Lac161_ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain.

scholarly journals Genome-Wide Identification and Characterization of Xyloglucan Endotransglycosylase/Hydrolase in Ananas comosus during Development

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 537 ◽  
Author(s):  
Qingyun Li ◽  
Huayang Li ◽  
Chongyang Yin ◽  
Xiaotong Wang ◽  
Qing Jiang ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
Ananas Comosus ◽  
Pcr Analysis ◽  
Glycoside Hydrolase Family ◽  
Xyloglucan Endotransglycosylase ◽  
Group Iii ◽  
C Terminus ◽  
Group I ◽  
A Cell ◽  
Hydrolase Family

Xyloglucan endotransglycosylase/hydrolase (XTH) is a cell-wall-modifying enzyme participating in diverse cell morphogenetic processes and adaptation to stress. In this study, 48 XTH genes were identified from two pineapple (Ananas comosus) cultivars (‘F153’ and ‘MD2’) and designated Ac(F153)XTH1 to -24 and Ac(MD2)XTH1 to -24 based on their orthology with Arabidopsis thaliana genes. Endoglucanase family 16 members were identified in addition to XTHs of glycoside hydrolase family 16. Phylogenetic analysis clustered the XTHs into three major groups (Group I/II, III and Ancestral Group) and Group III was subdivided into Group IIIA and Group IIIB. Similar gene structure and motif number were observed within a group. Two highly conserved domains, glycosyl hydrolase family 16 (GH16-XET) and xyloglucan endotransglycosylase C-terminus (C-XET), were detected by multiple sequences alignment of all XTHs. Segmental replication were detected in the two cultivars, with only the paralogous pair Ac(F153)XTH7-Ac(F153)XTH18 presented in ‘F153’ prior to genomic expansion. Transcriptomic analysis indicated that XTHs were involved in the regulation of fruit ripening and crassulacean acid metabolism with tissue specificity and quantitative real-time PCR analysis suggested that Ac(MD2)XTH18 was involved in root growth. The results enhance our understanding of XTHs in the plant kingdom and provide a basis for further studies of functional diversity in A. comosus.

scholarly journals Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

2017 ◽  
Author(s):  
Luana de Fátima Alves ◽  
Tiago Cabral Borelli ◽  
Cauã Antunes Westmann ◽  
Rafael Silva-Rocha ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Microbial Communities ◽  
Glycosyl Hydrolase ◽  
Metagenomic Library ◽  
Current Method ◽  
Hydrolytic Activity ◽  
Industrial Applications ◽  
Glycosyl Hydrolases ◽  
Coding Region ◽  
Ph Indicator ◽  
Wide Range

AbstractThe vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from microbial communities inhabiting a soil sample by using thelacZα-based plasmid pSEVA232 in the generation of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragmentsin framewithin the coding region of thelacZαalpha gene present in the original vector. We concluded that the current method has a higher tendency for false positive recovery of clones, when used in combination with alacZα-based vector. Finally, we discuss the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in metagenomic screenings methodologies.

Molecular determinants of substrate specificity revealed by the structure ofClostridium thermocellumarabinofuranosidase 43A from glycosyl hydrolase family 43 subfamily 16

2016 ◽  
Vol 72 (12) ◽  
pp. 1281-1289 ◽  
Author(s):  
Arun Goyal ◽  
Shadab Ahmed ◽  
Kedar Sharma ◽  
Vikas Gupta ◽  
Pedro Bule ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Glycosyl Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Side Chains ◽  
Glycosyl Hydrolase Family ◽  
Molecular Determinants ◽  
Glycoside Hydrolase Family 43 ◽  
Binding Cleft ◽  
Hydrolase Family

The recent division of the large glycoside hydrolase family 43 (GH43) into subfamilies offers a renewed opportunity to develop structure–function studies aimed at clarifying the molecular determinants of substrate specificity in carbohydrate-degrading enzymes. α-L-Arabinofuranosidases (EC 3.2.1.55) remove arabinose side chains from heteropolysaccharides such as xylan and arabinan. However, there is some evidence suggesting that arabinofuranosidases are substrate-specific, being unable to display a debranching activity on different polysaccharides. Here, the structure ofClostridium thermocellumarabinofuranosidase 43A (CtAbf43A), which has been shown to act in the removal of arabinose side chains from arabinoxylan but not from pectic arabinan, is reported.CtAbf43A belongs to GH43 subfamily 16, the members of which have a restricted capacity to attack xylans. The crystal structure ofCtAbf43A comprises a five-bladed β-propeller fold typical of GH43 enzymes.CtAbf43A displays a highly compact architecture compatible with its high thermostability. Analysis ofCtAbf43A along with the other member of GH43 subfamily 16 with known structure, theBacillus subtilisarabinofuranosidase BsAXH-m2,3, suggests that the specificity of subfamily 16 for arabinoxylan is conferred by a long surface substrate-binding cleft that is complementary to the xylan backbone. The lack of a curved-shaped carbohydrate-interacting platform precludes GH43 subfamily 16 enzymes from interacting with the nonlinear arabinan scaffold and therefore from deconstructing this polysaccharide.

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