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Martin Schulein - One of the best experts on this subject based on the ideXlab platform.
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anatomy of glycosynthesis structure and kinetics of the Humicola insolens cel7b e197a and e197s glycosynthase mutants
Chemistry & Biology, 2004Co-Authors: Valerie M A Ducros, Martin Schulein, Torben Peter Frandsen, Ingemar Von Ossowski, Chris A Tarling, David L Zechel, Marek A Brzozowski, Stephen G Withers, Gideon J. DaviesAbstract:The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).
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structural basis for ligand binding and processivity in cellobiohydrolase cel6a from Humicola insolens
Structure, 2003Co-Authors: Annabelle Varrot, Martin Schulein, Hugues Driguez, Torben Peter Frandsen, Ingemar Von Ossowski, Viviane Boyer, Sylvain Cottaz, Gideon J. DaviesAbstract:Abstract The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7–1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.
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fluorescent cellulose microfibrils as substrate for the detection of cellulase activity
Biomacromolecules, 2003Co-Authors: William Helbert, Martin Schulein, Henri Chanzy, Tommy Lykke Husum, Steffen ErnstAbstract:To devise a sensitive cellulase assay based on substrates having most of the physical characteristics of native cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to prepare suspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a series of commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well as crude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as reducing sugars. After adequate centrifugation, these soluble products were analyzed as a function of grafting content, digestion time, and cellulase characteristics. The resulting data allowed the grafting conditions to be optimized in order to maximize the quantity of soluble products and therefore to increase the sensitivity of the detection. A comparison between the amount of released fluorescence and that of released reducing sugar allowed the differentiation between processive exo a...
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optimized mixtures of recombinant Humicola insolens cellulases for the biodegradation of crystalline cellulose
Biotechnology and Bioengineering, 2001Co-Authors: Claire Boisset, Henri Chanzy, Bernard Henrissat, Cecile Petrequin, Martin SchuleinAbstract:The digestion of bacterial cellulose ribbons by ternary mixtures of enzymes consisting of recombinant cellulases (two cellobiohydrolases, Cel6A and Cel7A, and the endoglucanase Cel45A) from Humicola insolens was investigated over a wide range of mixture composition. The extent of digestion was followed by soluble sugar release (saccharification) analysis together with transmission electron microscopy (TEM) observations. It was found that the addition of minute quantities of Cel45A induced a spectacular increase in saccharification of the substrate with either Cel7A or the mixture of Cel6A and Cel7A. Conversely, only a moderate saccharification resulted from the mixing of Cel45A and Cel6A. This difference is believed to originate from (1) the occasional endo character of Cel6A and (2) the competition of Cel6A and Cel45A for the substrate sites that are sensitive to endo activity. Interestingly, the mixture of enzymes giving rise to the highest saccharification rate did not always correspond to mixtures of enzymes generating the highest synergy. TEM images revealed that the bacterial cellulose ribbons became at the same time cut and narrowed down under the action of an optimized mixture of the three enzymes.
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direct electron transfer of cellobiose dehydrogenase from various biological origins at gold and graphite electrodes
Journal of Electroanalytical Chemistry, 2001Co-Authors: Annika Lindgren, Tautgirdas Ruzgas, Lo Gorton, Dietmar Haltrich, Ursula Baminger, Martin SchuleinAbstract:Direct electron transfer was observed for cellobiose dehydrogenases (CDH) from three different fungi, viz. CDH from Phanerochaete chrysosporium, Sclerotium rolfsii and Humicola insolens, in the presence of cellobiose when the enzymes were adsorbed on graphite electrodes. The redox wave of the heme cofactor of CDH could be demonstrated on thiol modified gold electrodes using cyclic voltammetry for Phanerochaete CDH and Humicola CDH; however, the electrocatalytic current for cellobiose oxidation could only be registered for Phanerochaete CDH.
Gideon J. Davies - One of the best experts on this subject based on the ideXlab platform.
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anatomy of glycosynthesis structure and kinetics of the Humicola insolens cel7b e197a and e197s glycosynthase mutants
Chemistry & Biology, 2004Co-Authors: Valerie M A Ducros, Martin Schulein, Torben Peter Frandsen, Ingemar Von Ossowski, Chris A Tarling, David L Zechel, Marek A Brzozowski, Stephen G Withers, Gideon J. DaviesAbstract:The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).
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structural basis for ligand binding and processivity in cellobiohydrolase cel6a from Humicola insolens
Structure, 2003Co-Authors: Annabelle Varrot, Martin Schulein, Hugues Driguez, Torben Peter Frandsen, Ingemar Von Ossowski, Viviane Boyer, Sylvain Cottaz, Gideon J. DaviesAbstract:Abstract The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7–1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.
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distortion of a cellobio derived isofagomine highlights the potential conformational itinerary of inverting β glucosidases
Chemical Communications, 2003Co-Authors: Annabelle Varrot, James Macdonald, Robert V Stick, Gavin Pell, Harry J Gilbert, Gideon J. DaviesAbstract:A cellobio-derived isofagomine glycosidase inhibitor (Ki ∼ 400 nM) displays an unusual distorted 2,5B (boat) conformation upon binding to cellobiohydrolase Cel6A from Humicola insolens, highlighting the different conformational itineraries used by various glycosidases, with consequences for the design of therapeutic agents.
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structure of the Humicola insolens cellobiohydrolase cel6a d416a mutant in complex with a non hydrolysable substrate analogue methyl cellobiosyl 4 thio β cellobioside at 1 9 a
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Annabelle Varrot, Hugues Driguez, Torben Peter Frandsen, Gideon J. DaviesAbstract:The enzymatic degradation of cellulose continues to be one of the most important enzyme-catalysed reactions. Glycoside hydrolases from family GH-6 hydrolyse cellulose with inversion of the configuration of the anomeric carbon. Whilst the catalytic proton donor has been clearly identified (Asp226 in Humicola insolens Cel6A), the identification and even the existence of a potential Bronsted base remains unclear. Equally controversial is the role of surface-loop flexibility. Here, the structure of the D416A mutant of the H. insolens cellobiohydrolase Cel6A in complex with a non-hydrolysable thiooligosaccharide methyl cellobiosyl-4-thio-β-cellobioside at 1.9 A resolution is presented. Substrate distortion in the −1 subsite, to a 2S0 skew-boat conformation, is observed, similar to that seen in the analogous Trichoderma reesei Cel6A structure [Zou et al. (1999), Structure, 7, 1035–1045], but the active-centre N-terminal loop of the H. insolens enzyme is found in a more open conformation than described for previous structures.
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highly efficient synthesis of β 1 4 oligo and polysaccharides using a mutant cellulase
Journal of the American Chemical Society, 2000Co-Authors: Sebastien Fort, Martin Schulein, Gideon J. Davies, Viviane Boyer, Lars Christiansen, Lionel Greffe, Olga V Moroz, And Sylvain Cottaz, Hugues DriguezAbstract:This report describes an efficient chemoenzymatic synthesis of a variety of regioselectively modified β(1→4)-oligo- and -polysaccharides. This successful approach was based on: (i) the use of a “glycosynthase” which is a Glu-197-Ala nucleophile mutant of the retaining cellulase endoglucanase I (Cel7B) from Humicola insolens and (ii) the rational design of modified acceptor and donor molecules through a careful examination of information given by the X-ray structures of wild type and mutated enzymes. The mutant was able to catalyze, in high yield, the regio- and stereoselective glycosylation of α-glycobiosyl fluorides both unsubstituted and modified with various mono- and disaccharide acceptors, as well as the polymerization of these donors through a single-step inverting mechanism.
Wei Zhang - One of the best experts on this subject based on the ideXlab platform.
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CRISPR/Cas9-mediated genome editing directed by a 5S rRNA–tRNAGly hybrid promoter in the thermophilic filamentous fungus Humicola insolens
'Springer Science and Business Media LLC', 2021Co-Authors: Chao Fan, Huiying Luo, Bin Yao, Wei Zhang, Bo Liu, Yuhong Zhang, Huoqing HuangAbstract:Abstract Background Humicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism. Results Herein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA–tRNAGly promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation. Conclusions The established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production
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overexpression and characterization of a novel endo β 1 3 4 glucanase from thermophilic fungus Humicola insolens y1
Protein Expression and Purification, 2017Co-Authors: Bo Liu, Yuhong Zhang, Wei ZhangAbstract:A novel endo-β-1,3(4)-glucanase gene, cel16A, was cloned from the fungus Humicola insolens Y1. The 988-bp full-length gene encoded a 286-residue polypeptide consisting of a putative signal peptide of 20 residues and a catalytic domain belonging to glycosyl hydrolase family 16. It was successfully overexpressed in Pichia pastoris GS115. The purified recombinant Cel16A exhibited highest specific activity toward barley β-glucan, followed by lichenan and laminarin, but not toward CMC-Na, birchwood xylan, Avicel and filter paper, indicating that Cel16A is an endo-β-1,3(4)-glucanases. Recombinant Cel16A had a pH optimum at 5.5 and a temperature optimum at 55 °C with a specific activity of 693 U/mg toward barley β-glucan. It exhibited good stability over pH 5.0-9.0 and at temperatures up to 50 °C, retaining over 80% maximum activity. The Km and Vmax values of Cel16A for barley β-glucan were 0.91 mg ml-1 and 1530 μmol min-1·mg-1, respectively. All these favorable enzymatic properties of Cel16A make it a good candidate for applications in various industries.
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the use of t dna insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens y1
Scientific Reports, 2016Co-Authors: Bo Liu, Yuhong Zhang, Yunliu Fan, Wei ZhangAbstract:Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41% and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.
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functional diversity of family 3 β glucosidases from thermophilic cellulolytic fungus Humicola insolens y1
Scientific Reports, 2016Co-Authors: Wei Xia, Yingguo Bai, Ying Cui, Lichun Qian, Wei Zhang, Xiuan ZhanAbstract:The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50–60 °C and pH 5.5–6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.
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a neutral thermostable β 1 4 glucanase from Humicola insolens y1 with potential for applications in various industries
PLOS ONE, 2015Co-Authors: Wei Zhang, Bo Liu, Yuhong Zhang, Zhifang Zhang, Yunliu FanAbstract:We cloned a new glycoside hydrolase family 6 gene, Hicel6C, from the thermophilic fungus Humicola insolens Y1 and expressed it in Pichia pastoris. Using barley β-glucan as a substrate, recombinant HiCel6C protein exhibited neutral pH (6.5) and high temperature (70°C) optima. Distinct from most reported acidic fungal endo-β-1,4-glucanases, HiCel6C was alkali-tolerant, retaining greater than 98.0, 61.2, and 27.6% of peak activity at pH 8.0, 9.0, and 10.0, respectively, and exhibited good stability over a wide pH range (pH 5.0−11.0) and at temperatures up to 60°C. The Km and Vmax values of HiCel6C for barley β-glucan were 1.29 mg/mL and 752 μmol/min·mg, respectively. HiCel6C was strictly specific for the β-1,4-glucoside linkage exhibiting activity toward barley β-glucan, lichenan, and carboxy methylcellulose sodium salt (CMC-Na), but not toward laminarin (1,3-β-glucan). HiCel6C cleaved the internal glycosidic linkages of cellooligosaccharides randomly and thus represents an endo-cleaving enzyme. The predominant product of polysaccharide hydrolysis by HiCel6C was cellobiose, suggesting that it functions by an endo-processive mechanism. The favorable properties of HiCel6C make it a good candidate for basic research and for applications in the textile and brewing industries.
Bernard Henrissat - One of the best experts on this subject based on the ideXlab platform.
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Production of α-1,3-L-arabinofuranosidase active on substituted xylan does not improve compost degradation by Agaricus bisporus
PLoS ONE, 2018Co-Authors: Aurin Vos, Bernard Henrissat, Edita Jurak, Peter De Gijsel, Robin Ohm, Luis Lugones, Mirjam Kabel, Han WöstenAbstract:Agaricus bisporus consumes carbohydrates contained in wheat straw based compost used for commercial mushroom production. Double substituted arabinoxylan is part of the ~40% of the compost polysaccharides that are not degraded by A. bisporus during its growth and development. Genes encoding α-1,3-l-arabinofuranosidase (AXHd3) enzymes that act on xylosyl residues doubly substituted with arabinosyl residues are absent in this mushroom forming fungus. Here, the AXHd3 encoding hgh43 gene of Humicola insolens was expressed in A. bisporus with the aim to improve its substrate utilization and mushroom yield. Transformants secreted active AXHd3 in compost as shown by the degradation of double substituted arabinoxylan oligomers in an in vitro assay. However, carbohydrate composition and degree of arabinosyl substitution of arabinoxylans were not affected in compost possibly due to inaccessibility of the doubly substituted xylosyl residues.
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optimized mixtures of recombinant Humicola insolens cellulases for the biodegradation of crystalline cellulose
Biotechnology and Bioengineering, 2001Co-Authors: Claire Boisset, Henri Chanzy, Bernard Henrissat, Cecile Petrequin, Martin SchuleinAbstract:The digestion of bacterial cellulose ribbons by ternary mixtures of enzymes consisting of recombinant cellulases (two cellobiohydrolases, Cel6A and Cel7A, and the endoglucanase Cel45A) from Humicola insolens was investigated over a wide range of mixture composition. The extent of digestion was followed by soluble sugar release (saccharification) analysis together with transmission electron microscopy (TEM) observations. It was found that the addition of minute quantities of Cel45A induced a spectacular increase in saccharification of the substrate with either Cel7A or the mixture of Cel6A and Cel7A. Conversely, only a moderate saccharification resulted from the mixing of Cel45A and Cel6A. This difference is believed to originate from (1) the occasional endo character of Cel6A and (2) the competition of Cel6A and Cel45A for the substrate sites that are sensitive to endo activity. Interestingly, the mixture of enzymes giving rise to the highest saccharification rate did not always correspond to mixtures of enzymes generating the highest synergy. TEM images revealed that the bacterial cellulose ribbons became at the same time cut and narrowed down under the action of an optimized mixture of the three enzymes.
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imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase cel6a from Humicola insolens and its mode of synergy with cellobiohydrolase cel7a
Applied and Environmental Microbiology, 2000Co-Authors: Claire Boisset, Martin Schulein, Bernard Henrissat, Carole Fraschini, Henri ChanzyAbstract:Dispersed cellulose ribbons from bacterial cellulose were subjected to digestion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an excess of beta-glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than Cel6A. In combination, these enzymes showed substantial synergy culminating with a molar ratio of approximately two-thirds Cel6A and one-third Cel7A. Ultrastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations, together with the examination of the digestion kinetics, indicate that Cel6A can be classified as an endo-processive enzyme, whereas Cel7A is essentially a processive enzyme. Thus, the synergy resulting from the mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an appropriate substrate, superior to Valonia or bacterial cellulose microcrystals, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A.
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structures of oligosaccharide bound forms of the endoglucanase v from Humicola insolens at 1 9 a resolution
Biochemistry, 1995Co-Authors: Gideon J. Davies, Bernard Henrissat, S P Tolley, Carsten M Hjort, Martin SchuleinAbstract:Cellulose, a polymer of beta-1,4-linked glucose residues, is the major polysaccharide component of plant cell walls and the most abundant biopolymer. The underlying mechanisms of the enzymatic degradation of cellulose are of increasing commercial and ecological significance. Endoglucanase V, from the cellulolytic soil hyphomycete Humicola insolens, is an endocellulase, the catalytic core of which consists of 210 amino acids and is known to hydrolyze the beta-1,4 links with inversion of configuration at the anomeric carbon. The major products of cellulose hydrolysis are cellobiose and cellotriose. The crystal structures of the endoglucanase V (EGV) from H. insolens, in native, product (cellobiose), inactive mutant (D10N), and oligosaccharide-bound [(D10N)-cellohexaose] forms, have been determined at resolutions of 1.9 A or better. EGV consists of a six-stranded beta-barrel domain with long interconnecting loops. A 40 A groove exists along the surface of the enzyme, and this contains the catalytic residues, Asp 10 and Asp 121. The two catalytic aspartates sit to either side of the substrate binding groove in an ideal conformation for facilitating cleavage by inversion, their carboxyl groups being separated by approximately 8.5 A. The complex between substrate and inactive mutant reveals excellent density for an oligosaccharide in six of the enzyme's seven substrate binding subsites. No sugar moiety, however, is seen bound to the -1 subsite at the point of cleavage. The geometry of the cleavage site suggests that the enzyme would favor the binding of sugars with an elongated glycosidic bond, as found in the transition state, as opposed to the binding of substrate. The oligosaccharide complexes reveal solvent water suitably placed for participation in a single displacement reaction as first suggested by Koshland in 1953 [Koshland, D. E. (1953) Biol. Rev. 28, 416-436]. A large conformational change takes place upon substrate binding. This "lid flipping" has the effect of increasing the hydrophobic environment of the catalytic proton donor, enclosing the active site at the point of cleavage, and bringing a third aspartate (Asp 114) in close proximity to the substrate. Site-directed mutagenesis of the catalytic residues has been used to confirm their significance in catalysis.
Wei Xia - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE High Level Expression of a Novel Family 3 Neutral β-Xylosidase from Humicola insolens Y1 with High Tolerance to D-Xylose
2016Co-Authors: Wei Xia, Pengjun Shi, Ying Cui, Lichun Qian, Mengjuan Xia, Bin YaoAbstract:A novel β-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pas-torisGS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimi-zation. Recombinant Xyl3A showed both β-xylosidase and α-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0–9.0 (in-cubated at 37°C for 1 h) and at temperatures of 60°C and below, retaining over 80%maxi-mum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 β-xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xy-lose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the samemicrobial source showed significant synergistic effects on the degrada-tion of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good po-tential applications in the bioconversion of hemicelluloses to biofuels
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functional diversity of family 3 β glucosidases from thermophilic cellulolytic fungus Humicola insolens y1
Scientific Reports, 2016Co-Authors: Wei Xia, Yingguo Bai, Ying Cui, Lichun Qian, Wei Zhang, Xiuan ZhanAbstract:The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50–60 °C and pH 5.5–6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.
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high level expression of a novel family 3 neutral β xylosidase from Humicola insolens y1 with high tolerance to d xylose
PLOS ONE, 2015Co-Authors: Wei Xia, Ying Cui, Lichun Qian, Mengjuan XiaAbstract:A novel β-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pastoris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimization. Recombinant Xyl3A showed both β-xylosidase and α-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0–9.0 (incubated at 37°C for 1 h) and at temperatures of 60°C and below, retaining over 80% maximum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 β-xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xylose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the same microbial source showed significant synergistic effects on the degradation of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good potential applications in the bioconversion of hemicelluloses to biofuels.