Affiliation:
1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou P. R. China
2. Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education Zhejiang University of Technology Hangzhou Zhejiang P. R. China
3. The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals Zhejiang University of Technology Hangzhou P. R. China
Abstract
AbstractThe aldo‐keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) to t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer‐aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6. Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant KdAKRM10 (KdAKRM6‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T5015 and Tm values were increased by 10.2 and 10.0°C, respectively, and half‐life (t1/2) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6. Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10, thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs.
Funder
National Key Research and Development Program of China
National Natural Science Foundation of China