CPC乙酰化酶底物结合区域Loop上脯氨酸对其催化特性的影响<sup>*</sup>

doi:10.13523/j.cb.20171205

中国生物工程杂志

2017, Vol. 37

Issue (12): 34-39 DOI: 10.13523/j.cb.20171205

研究报告

CPC乙酰化酶底物结合区域Loop上脯氨酸对其催化特性的影响^*

唐存多^1,^2*,史红玲^1*,焦铸锦¹,刘飞¹,许建和^2**(

),阚云超^1**(

),姚伦广^1**(

)

1 南阳师范学院昆虫生物反应器河南省工程实验室河南省南水北调中线水源区生态安全重点实验室南阳 473061
2 华东理工大学生物反应器工程国家重点实验室上海 200237

Effect of Prolines in the Loop of CPC Acylase Substrate Binding Region on Its Catalytic Properties

Cun-duo TANG^1,^2*,Hong-ling SHI^1*,Zhu-jin JIAO¹,Fei LIU¹,Jian-he XU^2**(

),Yun-chao KAN^1**(

),Lun-guang YAO^1**(

)

1 Henan Provincial Engineering Laboratory of Insect Bio-reactor, Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North, Nanyang Normal University, Nanyang 473061, China
2 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

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摘要：

低温CPC乙酰化酶在7-ACA的生物合成中具有重要作用和显著的优势,开发低温CPC乙酰化酶具有重大的经济价值。为了获得在低温下具有更高催化活性的CPC乙酰化酶,在前期的研究基础上,以先前获得的CA III^M为亲本,借助分子对接的手段确定了它的底物结合区域,并利用pyMOL软件找出了底物结合区域Loop上关键的脯氨酸残基,分析后将选定的脯氨酸用甘氨酸进行替换。借助pET32a质粒在E. coli BL21(DE3)中进行了可溶性表达研究,除P272G外,其它突变体均实现了可溶性表达。P238G、P582G和P679G在13℃对CPC的催化活性分别为1.25、1.04和1.38 U/mg,较亲本的0.85 U/mg有了显著的提高。此外,分别考察了亲本及突变体的温度稳定性,它们之间无明显的差异。然后,在13℃下进行了7-ACA低温生物合成的研究,结果表明反应24 h后CPC的转化率也能达到80%以上。由此可见在CPC乙酰化酶冷适应性改造方面取得了较为理想的结果,为进一步的改造及应用奠定了坚实的基础,也为其它低温酶的创制提供了可资借鉴的经验。

关键词： CPC乙酰化酶; 冷适应性; 分子改造; 理性设计; 活性检测

Abstract:

Cold-active CPC acylase plays an important role and has significant advantage in the biosynthesis of 7-ACA, thus developing cold-active CPC acylase has significant economic value. In order to obtain the CPC acylase with higher catalytic activity at low temperature, on the basis of previous research, the previously obtained CA III^M was taken as a parent, confirmed its substrate binding region by the method of molecular docking, and found the key proline residues in the loop of CPC acylase substrate binding region by pyMOL. After analyzing, the selected prolines were mutated into glycines, respectively. The study of soluble expression was carried out in E. coli BL21(DE3) with pET32a plasmid, and soluble expression was achieved in the other mutants except P272G. Catalytic activity of P238G, P582G and P679G to CPC was 1.25, 1.04 and 1.38 U/mg respectively at 13℃, and there was a significant improvement compared with parental 0.85 U/mg. Moreover, the stability of parent and mutants was investigated, and there was no obvious difference between them. Afterwards, the low temperature biosynthesis of 7-ACA was carried out at 13℃, and the results showed that the conversion rate of CPC can reach 80% and above after 24h . The ideal results in cold adaptation improvement of CPC acylase were gained. So a solid foundation for further transformation and application was established, it provided a useful experience for the creating of other low-temperature enzymes.

Key words: CPC acylase Cold adaptation Molecular modification Rational design Activity assay

收稿日期: 2017-05-25 出版日期: 2017-12-16

ZTFLH:

Q819

基金资助: 国家自然科学基金(31371381);河南省科技攻关项目(162102210116);河南省高等学校重点科研项目计划(15A416008);河南省南水北调中线水源区水生态安全创新型科技团队专项(17454)

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	唐存多
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	刘飞
	许建和
	阚云超
	姚伦广

引用本文:

唐存多,史红玲,焦铸锦,刘飞,许建和,阚云超,姚伦广. CPC乙酰化酶底物结合区域Loop上脯氨酸对其催化特性的影响^*[J]. 中国生物工程杂志, 2017, 37(12): 34-39.

Cun-duo TANG,Hong-ling SHI,Zhu-jin JIAO,Fei LIU,Jian-he XU,Yun-chao KAN,Lun-guang YAO. Effect of Prolines in the Loop of CPC Acylase Substrate Binding Region on Its Catalytic Properties. China Biotechnology, 2017, 37(12): 34-39.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171205 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I12/34

图1 CPC与CAⅢM的三维结构的分子对接模型

表1 CA IIIM编码基因定点突变所需的引物

图2 重组大肠杆菌表达产物的SDS-PAGE分析

图3 纯化后的重组CPC乙酰化酶的SDS-PAGE分析

表2 亲本及突变体的温度稳定性

图4 CPC在低温下的转化率

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