Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum.
Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4-5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.
Department of Environmental Biology, Shandong Agricultural University, Tai'an, Shandong 271018, People's Republic of China.
Xiu-Juan
XJ
Wang
Yan-Jie
YJ
Peng
Li-Qing
LQ
Zhang
An-Na
AN
Li
Duo-Chuan
DC
Li
D000595:Amino Acid Sequence; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002597:Chaetomium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation
D005656:Fungal Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase