Enhancing thermostability of Yarrowia lipolytica lipase 2 through engineering multiple disulfide bonds and mitigating reduced lipase production associated with disulfide bonds

Lilang Li; Shihai Zhang; Weikun Wu; Wutai Guan; Zixiao Deng; Hanzhen Qiao

doi:10.1016/j.enzmictec.2019.03.008

Enhancing thermostability of Yarrowia lipolytica lipase 2 through engineering multiple disulfide bonds and mitigating reduced lipase production associated with disulfide bonds

Enzyme Microb Technol. 2019 Jul:126:41-49. doi: 10.1016/j.enzmictec.2019.03.008. Epub 2019 Mar 30.

Authors

Lilang Li¹, Shihai Zhang², Weikun Wu³, Wutai Guan⁴, Zixiao Deng³, Hanzhen Qiao³

Affiliations

¹ College of Animal Science, South China Agricultural University, Guangzhou 510640, China; Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. Electronic address: lililang@stu.scau.edu.cn.
² College of Animal Science, South China Agricultural University, Guangzhou 510640, China; Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China. Electronic address: zhangshihai@scau.edu.cn.
³ College of Animal Science, South China Agricultural University, Guangzhou 510640, China; Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
⁴ College of Animal Science, South China Agricultural University, Guangzhou 510640, China; Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of Animal Science, South China Agricultural University, Guangzhou 510642, China; College of Animal Science and National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China. Electronic address: wtguan@scau.edu.cn.

PMID: 31000163
DOI: 10.1016/j.enzmictec.2019.03.008

Abstract

The limited thermostability of Yarrowia lipolytica lipase 2 (Lip2) hampers its industrial application. To improve its thermostability, we combined single disulfide bonds which our group identified previously. In this study, combining different regional disulfide bonds had greater effect than combining same regional disulfide bonds. Furthermore, mutants with 4, 5, and 6 disulfide bonds exhibited dramatically enhanced thermostability. Compared with the wild-type, sextuple mutant 6s displayed a 22.53 and 31.23 ℃ increase in the melting temperature (T_m) and the half loss temperature at 15 min (T15 50), respectively, with greater pH stability and a wider reaction pH range. Molecular dynamics simulation revealed that multiple disulfide bonds resulted in more rigid structures of mutants 4s, 5s and 6s, and prolonged enzyme unfolding times. Moreover, secretions of mutants 5s and 6s were significantly increased by 60% and 80% by co-expressing with the chaperone protein disulfide isomerase (PDI), which mitigated the reduced production issue caused by multiple disulfide bonds. Results of this study indicated that enhanced heat endurance giving more potential for industrial application.

Keywords: Disulfide bonds; Molecular dynamics simulation; Secretion; Thermostability; Yarrowia lipolytica lipase 2.

MeSH terms

Carboxylic Ester Hydrolases / chemistry*
Carboxylic Ester Hydrolases / genetics
Carboxylic Ester Hydrolases / metabolism*
Disulfides / chemistry*
Enzyme Stability
Fungal Proteins / chemistry*
Fungal Proteins / genetics
Fungal Proteins / metabolism*
Molecular Dynamics Simulation
Mutagenesis, Site-Directed
Mutation
Protein Conformation
Protein Disulfide-Isomerases / metabolism
Temperature*
Transcription Factors / metabolism
Yarrowia / enzymology*

Substances

Disulfides
Fungal Proteins
Transcription Factors
Carboxylic Ester Hydrolases
YLLIP2 protein, Yarrowia lipolytica
Protein Disulfide-Isomerases