Cloning of two cellobiohydrolase genes from Trichoderma viride and heterogeneous expression in yeast Saccharomyces cerevisiae.
Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml(-1) under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70 degrees C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.
Song
Jinzhu
J
Department of Life Science and Engineering, Harbin Institute of Technology, P.O. Box 437, No. 92, West Da-Zhi Street, Harbin, Heilongjiang, 150001, China. sjz@hit.edu.cn
Liu
Beidong
B
Liu
Zhihua
Z
Yang
Qian
Q
D015152:Blotting, Northern; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013696:Temperature; D014170:Transformation, Genetic; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics*
D012331:RNA, Fungal; D011994:Recombinant Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase