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Enhancing Catalytic Efficiency of an Actinoplanes utahensis Echinocandin B Deacylase through Random Mutagenesis and Site-Directed Mutagenesis

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Abstract

Echinocandin B deacylase (EBDA), from Actinoplanes utahensis ZJB-08196, is capable of cleaving the linoleoyl group from echinocandin B (ECB), forming the echinocandin B nucleus (ECBN), which is a key precursor of semisynthetic antifungal antibiotics. In the present study, molecular evolution of AuEBDA by random mutagenesis combined with site-directed mutagenesis (SDM) and screening was performed. Random mutagenesis on the wild-type (WT) AuEBDA generated two beneficial substitutions of G287Q, R527V. The “best” variant AuEBDA-G287Q/R527V was obtained by combining G287Q with R527V through SDM, which was most active at 35 °C, pH 7.5, with Km and vmax values of 0.68 mM and 395.26 U/mg, respectively. Mutation of G287Q/R527V markedly increased the catalytic efficiency kcat/Km by 290% compared with the WT-AuEBDA.

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Funding

This work was financially supported by National Natural Science Foundation of China (21878274).

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, People’s Republic of China

    Ying-Nan Cheng, Shuai Qiu, Feng Cheng, Chun-Yue Weng, Ya-Jun Wang & Yu-Guo Zheng

  2. Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, People’s Republic of China

    Ying-Nan Cheng, Shuai Qiu, Feng Cheng, Chun-Yue Weng, Ya-Jun Wang & Yu-Guo Zheng

  3. The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Ying-Nan Cheng, Shuai Qiu, Feng Cheng, Chun-Yue Weng, Ya-Jun Wang & Yu-Guo Zheng

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  1. Ying-Nan Cheng
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  2. Shuai Qiu
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Correspondence to Ya-Jun Wang.

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Cheng, YN., Qiu, S., Cheng, F. et al. Enhancing Catalytic Efficiency of an Actinoplanes utahensis Echinocandin B Deacylase through Random Mutagenesis and Site-Directed Mutagenesis. Appl Biochem Biotechnol 190, 1257–1270 (2020). https://doi.org/10.1007/s12010-019-03170-3

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