Mutations in the substrate entrance region of β-glucosidase from Trichoderma reesei improve enzyme activity and thermostability.
β-Glucosidase (EC 3.2.1.21) plays an essential role in biofuel production since it can cleave β-1,4-glycosidic bond to convert cellobiose into fermentable glucose. Based on the structure of Trichoderma reesei β-glucosidase 2 (TrBgl2) we solved, the amino acids in the outer channel of active site were mutated in this study. Mutants P172L and P172L/F250A showed the most enhanced k(cat)/K(m) and k(cat) values by 5.3- and 6.9-fold, respectively, compared to the wild type (WT) toward 4-nitrophenyl-β-D-glucopyranoside (p-NPG) substrate at 40°C. L167W and P172L/F250A mutations resulted in shift of optimal temperature to 50°C, at which WT was almost inactive. However, thin-layer chromatography analysis revealed that mutant L167W had the best synergism with T. reesei cellulases on degrading cellulosic substrates into glucose. This study enhances our understanding on the roles of amino acids in the substrate entrance region away from the active site and provides engineered T. reesei β-glucosidases with better activity and/or thermostability to hydrolyze cellobiose.
Institute of Biological Chemistry,  Academia Sinica, Taipei 11529,  Taiwan.
Hsiao-Lin
HL
Lee
Chih-Kang
CK
Chang
Wen-Yih
WY
Jeng
Andrew H-J
AH
Wang
Po-Huang
PH
Liang
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002475:Cellobiose / Q000378:metabolism; D044602:Cellulases / Q000378:metabolism; D004795:Enzyme Stability; D005960:Glucosides / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D005960:Glucosides; D002475:Cellobiose; C025193:4-nitrophenyl beta-D-glucoside; D044602:Cellulases; D001617:beta-Glucosidase