Directed Evolution of Pseudomonas fluorescens Lipase Variants With Improved Thermostability Using Error-Prone PCR

Lijun Guan,Song Yan,Yang Gao,Zhihong Zhang,Nina Ji,Shuwen Lu,Ye Zhou,Kunlun Wang,Jialei Li

doi:10.3389/fbioe.2020.01034

Abstract

Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with Tm values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a Tm value of 68.0°C. Although the increase of Tm was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes.

Highlights

IntroductionLipases (triacylglycerol-ester-hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the synthesis of esters and the release of fatty acids via the hydrolysis of fats and oils
Lipases are ubiquitous enzymes that catalyze the synthesis of esters and the release of fatty acids via the hydrolysis of fats and oils
The phylogenetic analysis showed that Pseudomonas fluorescens lipase (PFL) belongs to family I.1, and shares higher sequence identities with family I.2, which had a slightly smaller size (30–32 kDa) than family I.1 (33 kDa) due to the deletion of several amino acids forming an anti-parallel β-strand on the surface of the lipase

Summary

Introduction

Lipases (triacylglycerol-ester-hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the synthesis of esters and the release of fatty acids via the hydrolysis of fats and oils. Lipases are ubiquitously produced by microorganisms, animals, and plants. Most lipases widely used in industry are derived from microbial sources, because the respective genes are relatively easy to access, can be efficiently expressed, and inexpensively produced by large-scale fermentation (Sharma et al, 2002). Many lipases from these microorganisms showed low thermostability and unsatisfactory stability in industrial processes, which was a limiting factor for their industrial application. Thermostability of the enzyme is a prerequisite for industrial processes that rely on high enzyme activity, mass transfer, reactant solubility, fine chemical synthesis and so on (Sharma et al, 2002). Lipase with higher thermostability play a vital role in industrial applications, and it is urgent to improve the thermostability of unstable lipases (Wen et al, 2012; Miyakawa et al, 2015)

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Results

Conclusion