Improvement of low-temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site-directed mutagenesis.
By directed evolution and subsequent site-directed mutagenesis, cold-adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four-amino acid substitution variant RTN29 displayed a sixfold increase in caseinolytic activity in the temperature range of 15-25 degrees C, a down-shift of optimum temperature by approximately 15 degrees C, as well as a decrease in thermostability, indicating it follows the general principle of trade-off between activity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity was observed after heat-treatment at 60 degrees C for 2 h. Notably, RTN29 exhibited a lower hydrolytic activity toward suc-AAPF-pNA, due to an increase in K(m) and a decrease in k(cat), in contrast to other artificially cold-adapted subtilases with increased low-temperature activity toward small synthetic substrates. All mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the cold-adapted variants occurred within or near the substrate-binding region. None of these mutations, however, match the corresponding sites in naturally psychrophilic and other artificially cold-adapted subtilases, implying there are multiple routes to cold adaptation. Homology modeling and structural analysis demonstrated that these mutations led to an increase in mobility of substrate-binding region and a modulation of substrate specificity, which seemed to account for the improvement of the enzyme's catalytic activity toward macromolecular substrates at lower temperatures. Our study may provide valuable information needed to develop enzymes coupling high stability and high low-temperature activity, which are highly desired for industrial use.
Zhong
Chuan-Qi
CQ
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072.
Song
Shengli
S
Fang
Nan
N
Liang
Xiaoliang
X
Zhu
Hui
H
Tang
Xiao-Feng
XF
Tang
Bing
B
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism*; D003080:Cold Temperature; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D002364:Caseins; D020860:Subtilisin