scholarly journals Thermostable mutants of glycoside hydrolase family 6 cellobiohydrolase from the basidiomycete Phanerochaete chrysosporium

2020 ◽  
Author(s):  
Sora Yamaguchi ◽  
Naoki Sunagawa ◽  
Mikako Tachioka ◽  
Kiyohiko Igarashi ◽  
Masahiro Samejima
Keyword(s):  
Thermal Stability ◽  
Phanerochaete Chrysosporium ◽  
Glycoside Hydrolase ◽  
Thermal Inactivation ◽  
Renewable Resource ◽  
Cellulosic Biomass ◽  
Limiting Factor ◽  
Glycoside Hydrolase Family ◽  
Hydrolytic Activities ◽  
Hydrolase Family

AbstractThermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) is a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass. However, while the catalytic rate of enzymes generally becomes faster at higher temperatures, Cel6 CBHs are inactivated at lower temperatures than Cel7 CBHs, and this represents a limiting factor for industrial utilization. In this study, we produced a series of mutants of the glycoside hydrolase family 6 cellobiohydrolase PcCel6A from the fungus Phanerochaete chrysosporium, and compared their thermal stability. Eight mutants from a random mutagenesis library and one rationally designed mutant were selected as candidate thermostable mutants and produced by heterologous expression in the yeast Pichia pastoris. Comparison of the hydrolytic activities at 50 and 60 °C indicated that the thermal stability of PcCel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.

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.

scholarly journals Thermostable Mutants of Glycoside Hydrolase Family 6 Cellobiohydrolase from the Basidiomycete Phanerochaete chrysosporium

2020 ◽  
Vol 67 (3) ◽  
pp. 79-86
Author(s):  
Sora Yamaguchi ◽  
Naoki Sunagawa ◽  
Mikako Tachioka ◽  
Kiyohiko Igarashi ◽  
Masahiro Samejima
Keyword(s):  
Phanerochaete Chrysosporium ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

scholarly journals Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungusRasamsonia emersonii

2016 ◽  
Vol 72 (7) ◽  
pp. 860-870 ◽  
Author(s):  
Mikael Gudmundsson ◽  
Henrik Hansson ◽  
Saeid Karkehabadi ◽  
Anna Larsson ◽  
Ingeborg Stals ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Cellulosic Biomass ◽  
Glycoside Hydrolase Family ◽  
Hypocrea Jecorina ◽  
Moderately Thermophilic ◽  
Functional Studies ◽  
Excellent Candidate ◽  
Hydrolase Family

The filamentous fungusHypocrea jecorinaproduces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase fromH. jecorina(HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungusRasamsonia emersonii(ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization ofReCel3A, heterologously produced inH. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies ofReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics ofReCel3A compared withHjCel3A and mixtures containingHjCel3A makeReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Crystal structures of glycoside hydrolase family 3 β-glucosidase 1 from Aspergillus aculeatus

2013 ◽  
Vol 452 (2) ◽  
pp. 211-221 ◽  
Author(s):  
Kentaro Suzuki ◽  
Jun-Ichi Sumitani ◽  
Young-Woo Nam ◽  
Toru Nishimaki ◽  
Shuji Tani ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Glycoside Hydrolase ◽  
Degree Of Polymerization ◽  
Cellulosic Biomass ◽  
Protein Crystal ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Aspergillus Aculeatus ◽  
Technical Improvement ◽  
Hydrolase Family

GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.

scholarly journals Characterization of an Endoglucanase Belonging to a New Subfamily of Glycoside Hydrolase Family 45 of the Basidiomycete Phanerochaete chrysosporium

2008 ◽  
Vol 74 (18) ◽  
pp. 5628-5634 ◽  
Author(s):  
Kiyohiko Igarashi ◽  
Takuya Ishida ◽  
Chiaki Hori ◽  
Masahiro Samejima
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Phanerochaete Chrysosporium ◽  
Glycoside Hydrolase ◽  
Methylotrophic Yeast ◽  
Hydrolytic Activity ◽  
Cellulosic Biomass ◽  
Cellulolytic Enzymes ◽  
Glycoside Hydrolase Family ◽  
Amorphous Cellulose

ABSTRACT The wood decay fungus Phanerochaete chrysosporium has served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast Pichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (<22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley β-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.

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