Improving the Thermostability of Rhizopus chinensis Lipase Through Site-Directed Mutagenesis Based on B-Factor Analysis

Zhanbao Jiang; Chengbo Zhang; Minyuan Tang; Bo Xu; Lili Wang; Wen Qian; Jiandong He; Zhihong Zhao; Qian Wu; Yuelin Mu; Junmei Ding; Rui Zhang; Zunxi Huang; Nanyu Han

doi:10.3389/fmicb.2020.00346

Improving the Thermostability of Rhizopus chinensis Lipase Through Site-Directed Mutagenesis Based on B-Factor Analysis

Front Microbiol. 2020 Mar 3:11:346. doi: 10.3389/fmicb.2020.00346. eCollection 2020.

Authors

Zhanbao Jiang¹, Chengbo Zhang², Minyuan Tang¹, Bo Xu^{1

2

3

4}, Lili Wang⁵, Wen Qian⁵, Jiandong He⁵, Zhihong Zhao⁵, Qian Wu^{1

2

3

4}, Yuelin Mu^{1

2

3

4}, Junmei Ding^{1

2

3

4}, Rui Zhang^{1

2

3

4}, Zunxi Huang^{1

2

3

4}, Nanyu Han^{1

2

3

4}

Affiliations

¹ School of Life Sciences, Yunnan Normal University, Kunming, China.
² Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, China.
³ Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan Normal University, Kunming, China.
⁴ Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, China.
⁵ Yunnan Walvax Biotechnology Co., Ltd., Kunming, China.

Abstract

In order to improve the thermostability of lipases derived from Rhizopus chinensis, we identified lipase (Lipr27RCL) mutagenesis sites that were associated with enhanced flexibility based upon B-factor analysis and multiple sequence alignment. We found that two mutated isoforms (Lipr27RCL-K64N and Lipr27RCL-K68T) exhibited enhanced thermostability and improved residual activity, with respective thermal activity retention values of 37.88% and 48.20% following a 2 h treatment at 50°C relative to wild type Lipr27RCL. In addition, these Lipr27RCL-K64N and Lipr27RCL-K68T isoforms exhibited 2.4- and 3.0-fold increases in enzymatic half-life following a 90 min incubation at 60°C. Together these results indicate that novel mutant lipases with enhanced thermostability useful for industrial applications can be predicted based upon B-factor analysis and constructed via site-directed mutagenesis.

Keywords: B-factor analysis; lipase; multiple sequence alignment; site-directed mutagenesis; thermostability.