Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis

PloS One
Boyang GuoKouichi Nozaki

Abstract

Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5-9.0 and possessed high thermal stability up to 50 °C with 80% of the residual activities compared with the stability seen at the pH range of 6.5-7.0 and temperatures of up to 40 °C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass to...Continue Reading

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Citations

Jan 6, 2017·Biotechnology for Biofuels·Silja Kuusk, Priit Väljamäe
Jan 18, 2019·International Journal of Molecular Sciences·Diego César Batista MarianoRaquel Cardoso de Melo-Minardi
Jul 3, 2020·BMC Molecular and Cell Biology·Diego MarianoRaquel Cardoso de Melo-Minardi
Aug 28, 2020·Frontiers in Bioengineering and Biotechnology·Shohreh AriaeenejadGhasem Hosseini Salekdeh
Sep 7, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Leon Sulfierry Corrêa CostaLeonardo Henrique Franca de Lima
Mar 12, 2020·Biotechnology and Bioengineering·Misha AliQayyum Husain
Aug 11, 2021·Microbiological Research·Zedong JiangYanbing Zhu
Dec 11, 2021·Applied Microbiology and Biotechnology·Ruiqin ZhangYanjun Li

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Methods Mentioned

BETA
transglycosylation
PCR
gel filtration
electrophoresis
Protein Thermal Shift

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