Directed evolution and immobilization of new lipase Lip 906

Shuang Dai,He Li,Xiong Xiao,Zujun Deng,Yun Jeonyun,Shan Liu

doi:10.1051/e3sconf/202123302030

Abstract

In this experimental study, a new lipase named Lip 906 was screened out from a metagenomic library in the laboratory. To improve the stability of the enzyme and develop and apply it as soon as possible, we adopted directed evolution and immobilization methods. A random mutation library was constructed by error-prone PCR and finally, a mutant lipase Lip 5-D with increased enzyme activity was screened out and immobilized. The activity of the mutant enzyme Lip 5-D was improved by 4 times compared with the wild-type lipase Lip 906. The optimal reaction temperature rose by 4 °C, and by 3 °C after immobilization. The optimal reaction pH increased from 7.8 to 7.5. Both temperature stability and pH stability were improved. The mutant enzyme Lip 5-D can maintain about 70% of the relative activity after incubation at 65 °C for 2 h, and it can keep 60% at pH 3-10. Error-prone PCR and immobilization improve the catalytic activity and stability of the enzyme, and promote its development and application in many industries.

Highlights

IntroductionLipase (E.C. 3.1.1.3), known as acylglycerol hydrolases, is widely present in prokaryotes (e.g. bacteria[1,2]), eukaryotes (e.g. molds[3]), mammals, and plants[4,5]
Lipase (E.C. 3.1.1.3), known as acylglycerol hydrolases, is widely present in prokaryotes, eukaryotes, mammals, and plants[4,5]
The mutation rate in this study meets the ideal low mutation rate (0~4 bases are mutated per 1 kb gene fragment)[34,35], which ensures that the subsequent screening work can be proceeded smoothly

Summary

Introduction

Lipase (E.C. 3.1.1.3), known as acylglycerol hydrolases, is widely present in prokaryotes (e.g. bacteria[1,2]), eukaryotes (e.g. molds[3]), mammals, and plants[4,5]. Lipase can hydrolyze esters to release monoglycerides, diglycerides, glycerol and free fatty acids[6,7,8]. In addition to catalyzing the hydrolysis[9] and synthesis of glycerides[10,11,12], lipase can catalyze the transesterification[6]and synthesis of biosurfactants[13], peptides[8], polymers[9] and drugs[14]. The stereospecificity of certain lipases can be adopted to catalyze the resolution of optical isomers and the synthesis of chiral drugs. Lipase and its modified preparations are used in many fields, such as food and nutrition, daily chemical, oleochemical and agrochemical industries, paper industry, detergent and biosurfactant synthesis, and drug synthesis. Since the current production cost of lipases is still higher than traditional chemical catalysts and to meet the needs of industrial production and mining, developing new types of microbial lipases with high catalytic activity and stability is an urgent need for industrialization

Methods

Results

Conclusion