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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (2/3): 30-37    DOI: 10.13523/j.cb.2010033
技术与方法     
耐热植酸酶突变体的筛选及性质研究 *
陈中伟,郑璞,陈鹏程,吴丹()
江南大学生物工程学院 无锡 214112
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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摘要：

目的: 对大肠杆菌Escherichia coli植酸酶基因进行定向进化,获得热稳定性提高的植酸酶突变体。方法: 利用易错PCR技术和96微孔板高通量筛选方法获得突变体基因,并对突变酶进行异源表达、纯化及性质研究。结果: 通过筛选获得3株热稳定性明显提高的植酸酶突变体APPA1、APPA2、APPA3。酶学性质分析结果表明,3个突变体分子量均约为55kDa,最适pH均为4.5,与野生型无明显差别,热稳定性较野生型均有显著提高,其中突变体APPA3的最适温度为65℃,较野生酶提高5℃,在90℃处理10min后保留50%的酶活。酶的三维结构模拟显示,5个突变位点在植酸酶整体结构上均引入新氢键。结论: 通过定向进化获得热稳定性提高的大肠杆菌Escherichia coli植酸酶突变体,对植酸酶的工业应用和研究植酸酶结构与功能关系具有重要意义。
关键词： 植酸酶;  易错PCR;  热稳定性;  毕赤酵母    
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
收稿日期: 2020-10-25 出版日期: 2021-04-08
ZTFLH:  Q814  
基金资助: * 江苏省自然科学基金资助项目(BK20171261)
通讯作者: 吴丹     E-mail: wudan@jiangnan.edu.cn
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引用本文:

陈中伟,郑璞,陈鹏程,吴丹. 耐热植酸酶突变体的筛选及性质研究 *[J]. 中国生物工程杂志, 2021, 41(2/3): 30-37.

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.

链接本文:

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

名称 粗酶液酶活
(U/mL)		 蛋白质浓度
(mg/mL)		 比酶活
(U/mg)
野生酶APPA 17.076 0.586 29.140
突变体APPA1 23.265 0.695 33.475
突变体APPA2 16.843 0.574 29.343
突变体APPA3 20.837 0.601 34.671
表1  植酸酶野生型和突变体粗酶液的活性
图1  植酸酶野生型和突变体纯化产物SDS-PAGE分析
图2  植酸酶野生型和突变体的最适pH
图3  植酸酶野生型和突变体的pH稳定性
图4  植酸酶野生型和突变体的最适温度
图5  植酸酶野生型和突变体的热稳定性(热处理10min)
名称 Km
(mmol/L)		 kcat
(s-1)		 kcat/Km
[L/(mmol·s)]
野生酶APPA 8.85 957.34 108.17
突变体APPA1 8.43 903.71 107.20
突变体APPA2 8.82 972.93 110.31
突变体APPA3 8.96 983.38 109.75
表2  植酸酶野生型和突变体的动力学参数
图6  大肠杆菌植酸酶三维建模结构
图7  突变位点引起的三维结构变化
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