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

Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH...

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Published inJournal of Applied Glycoscience Vol. 67; no. 3; pp. 79 - 86
Main Authors Yamaguchi, Sora, Sunagawa, Naoki, Tachioka, Mikako, Igarashi, Kiyohiko, Samejima, Masahiro
Format Journal Article
LanguageEnglish
Published Tsukuba The Japanese Society of Applied Glycoscience 03.09.2020
Japan Science and Technology Agency
Subjects
Biodegradation
Biomass
Carbohydrates
Cellobiohydrolase
Cellulase
Deactivation
Disulfide bonds
enzymatic saccharification
Enzymes
Fungi
Glycosidases
Glycoside hydrolase
Glycosides
Hydrolase
Inactivation
Mutants
Phanerochaete chrysosporium
protein engineering
Random mutagenesis
Regular Paper
Renewable resources
Thermal stability
Yeasts
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Abstract Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are 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.
AbstractList Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are 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.
Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are 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 Pc Cel6A 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 Pc Cel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.
Author Samejima, Masahiro
Tachioka, Mikako
Sunagawa, Naoki
Igarashi, Kiyohiko
Yamaguchi, Sora
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  organization: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo
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SubjectTerms Biodegradation
Biomass
Carbohydrates
Cellobiohydrolase
Cellulase
Deactivation
Disulfide bonds
enzymatic saccharification
Enzymes
Fungi
Glycosidases
Glycoside hydrolase
Glycosides
Hydrolase
Inactivation
Mutants
Phanerochaete chrysosporium
protein engineering
Random mutagenesis
Regular Paper
Renewable resources
Thermal stability
Yeasts
Title Thermostable Mutants of Glycoside Hydrolase Family 6 Cellobiohydrolase from the Basidiomycete Phanerochaete chrysosporium
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