Improving the Activity and Thermal Stability of <i>Thermomyces lanuginosus</i> Lipase by Rational Design

doi:10.13523/j.cb.2011036

China Biotechnology

2021, Vol. 41

Issue (2/3): 63-69 DOI: 10.13523/j.cb.2011036

Improving the Activity and Thermal Stability of Thermomyces lanuginosus Lipase by Rational Design

WEI Zi-xiang,ZHANG Liu-qun,LEI Lei,HAN Zheng-gang,YANG Jiang-ke(

)

College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China

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Abstract

Objective:In order to obtain a lipase with high enzyme activity and high temperature resistance, the Thermomyces lanuginosus lipase was modified by rational design, which laid the foundation for the application of lipase in the feed industry, oil processing and biodiesel.Methods:A candidate site was found by phylogenetic analysis of the lid and loop regions of the typical domain of lipase. A lipase recombinant with significantly improved lipase activity and high temperature resistance was obtained through rational design and experimental verification, and a multi-copy vector was constructed to complete the evaluation of the enzyme production ability in a 50L fermentor.Results:The thermal stability of lipase was significantly improved after designing. The crude enzyme solution still retained 78.94% of the enzyme activity after being placed at 80℃ for 12h. After fermentation for 168h in a 50L fermentor, the enzyme activity of the supernatant reached 29 000U/mL.Conclusion:A novel high activity and high thermal stable lipase was successfully designed and obtained, which facilitates its large-scale production and industrial application.

Key words： Lipase Rational design High activity Thermal stability

Received: 19 November 2020 Published: 08 April 2021

ZTFLH:

Q814.9

Corresponding Authors: Jiang-ke YANG E-mail: jiangke.yang@gmail.com

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	Zi-xiang WEI
	Liu-qun ZHANG
	Lei LEI
	Zheng-gang HAN
	Jiang-ke YANG

Cite this article:

WEI Zi-xiang,ZHANG Liu-qun,LEI Lei,HAN Zheng-gang,YANG Jiang-ke. Improving the Activity and Thermal Stability of Thermomyces lanuginosus Lipase by Rational Design. China Biotechnology, 2021, 41(2/3): 63-69.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2011036 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I2/3/63

Fig.1 The results of protein comparison with HotSpot Wizard 3.0 of TLL

Fig.2 3D structure indicate the mutation site of lipase TLL (a) 3D structure indicate of Gly91 (c) 3D structure indicate of Leu227 (b),(d) Structure indicate of mutations

Fig.3 Construction of recombinants and expression of original and optimized TLL (a)Plasmid map of pPIC-tll’s recombinant (b)Results of pPIC-tll, pPIC-tll2a and pPIC-tll4a enzymatic digestion (c)The SDS-PAGE of recombinant expression (d) Analysis of TLL activity and protein content before and after mutation

Fig.4 The results of enzymatic properties of original and optimized lipase (a) Effects of pH on lipase activity (b)Effects of temperature on lipase activity (c) Effect of treatment at 80℃ for 0-60min on enzyme activity (d)Effect of treatment at 80℃ for 0-12h on enzyme activity

Fig.5 Optimal lipase induced expression in fermentation tank (a) SDS-PAGE results of fermentation in 50L fermentor (b) Analysis of enzyme activity and protein content in induction stage


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