Screening and Characterization of Thermostable Phytase Mutants

doi:10.13523/j.cb.2010033

China Biotechnology

2021, Vol. 41

Issue (2/3): 30-37 DOI: 10.13523/j.cb.2010033

Screening and Characterization of Thermostable Phytase Mutants

CHEN Zhong-wei,ZHENG Pu,CHEN Peng-cheng,WU Dan(

)

School of Biotechnology, Jiangnan University, Wuxi 214122, China

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Abstract

Objective: Directed evolution of Escherichia coli phytase gene to obtain phytase mutants with improved thermostability. Methods: The mutant gene was obtained by error-prone PCR and the high-throughput screening methods of 96-well plate. After heterologous expression and purification, the mutant enzymes were characterized. Results: Three phytase mutants APPA1, APPA2 and APPA3 with significantly improved thermal stability were obtained through screening. The results of enzymatic properties analysis showed that the molecular weight of the three mutants was about 55kDa, and the optimum pH was 4.5, which was not significantly different from the wild-type. The thermal stability of mutants was significantly improved compared with the wild-type. Among them, the suitable temperature of APPA3 is 65℃, which is 5℃ higher than wild enzyme, and 50% of the enzyme activity is retained after treatment at 90℃ for 10 minutes. The three-dimensional structure simulation of the enzyme showed that the five mutation sites introduced new hydrogen bonds in the overall structure of the phytase. Conclusion: Obtaining Escherichia coli phytase mutants with improved thermal stability through directed evolution is of great significance to the industrial application of phytase and the study of the relationship between phytase structure and function.

Key words： Phytase Error-prone PCR Thermostability Pichia pastoris

Received: 25 October 2020 Published: 08 April 2021

ZTFLH:

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Corresponding Authors: Dan WU E-mail: wudan@jiangnan.edu.cn

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	Zhong-wei CHEN
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	Dan WU

Cite this article:

CHEN Zhong-wei,ZHENG Pu,CHEN Peng-cheng,WU Dan. Screening and Characterization of Thermostable Phytase Mutants. China Biotechnology, 2021, 41(2/3): 30-37.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2010033 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I2/3/30

Table 1 Crude enzyme activity of phytase wild-type and mutants

Fig.1 SDS-PAGE analysis of wild-type and mutant purified products of phytase M:Marker;1:Purified product of wild enzyme APPA;2:Purified product of mutant APPA1;3:Purified product of mutant APPA2;4:Purified product of mutant APPA3

Fig.2 Optimal pH of phytase and its mutants

Fig.3 The pH stability of phytase and its mutants

Fig.4 Optimal temperature of phytase and its mutants

Fig.5 Thermal stability of phytase and its mutants (heat treatment for 10 minutes)

Table 2 Kinetic parameters of phytase and its mutants

Fig.6 Protein 3-D structure of phytase from Escherichia coli (a) Wild enzyme homology modeling three-dimensional structure (b) Active center of phytase

Fig.7 The changes of 3-D structure caused by mutation sites (a)Three-dimensional structure of wild enzyme A138 site (b)Three-dimensional structure of S138 mutation site (c)Three-dimensional structure of wild enzyme K180 site (d)Three-dimensional structure of N180 mutation site (e)Three-dimensional structure of wild enzyme D185 site (f)Three-dimensional structure of E185 mutation site (g)Three-dimensional structure of wild enzyme S189 site (h)Three-dimensional structure of D189 mutation site (i)Three-dimensional structure of wild enzyme K363 site (j)Three-dimensional structure of T363 mutation site


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