Kex2蛋白酶K291突变体性质和动力学研究

杨帆; 刘晓; 王之可; 李素霞

doi:10.14135/j.cnki.1006-3080.20201009001

Kex2蛋白酶K291突变体性质和动力学研究

doi: 10.14135/j.cnki.1006-3080.20201009001

杨帆^1,,
刘晓²,
王之可²,
李素霞^1, ,

1.
华东理工大学生物反应器国家重点实验室，上海 200237
2.
上海雅心生物技术有限公司，上海 200231

详细信息

作者简介:
杨帆：杨　帆(1995-)，女，硕士生，主要研究方向为重组蛋白酶。E-mail：2258361193@qq.com

通讯作者:
李素霞，E-mail：lisuxia@ecust.edu.cn

中图分类号: Q78
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出版历程
- 收稿日期: 2020-10-09
- 网络出版日期: 2021-01-07

Properties and Kinetics of Kex2 K291 Mutants

YANG Fan^1
,,
LIU Xiao²,
WANG Zhike²,
LI Suxia^{1
, ,}

1.
State Key Laboratory of Bioreactors, East China University of Science and Technology, Shanghai 200237, China
2.
Shanghai Yaxin Biotechnology Co. Ltd, Shanghai 200231, China

摘要

摘要: 利用毕赤酵母GS115表达系统，成功表达并纯化获得了两种Kex2蛋白酶突变体Kex2-K291L和Kex2-K291H。对比研究了天然Kex2和这两种突变体的酶学性质和稳定性。实验结果表明，与天然Kex2相比，Kex2-K291H和Kex2-K291L两种突变体的稳定性得到了明显的改善。两种突变体的最适pH和最适温度与天然Kex2保持一致，均为pH 9.0和37 ℃，两种突变体的温度稳定性也与天然Kex2基本一致。与天然Kex2蛋白酶相比，突变体Kex2-K291H的pH稳定性范围扩大，从5.0~6.0扩大至5.0~7.0。酶促反应动力学研究表明，突变体Kex2-K291H和Kex2-K291L的K_cat/K_m值分别为天然Kex2的1.8倍和2.0倍。突变体Kex2-K291H和Kex2-K291L在改善天然Kex2自降解问题的同时，提高了pH稳定性和催化效率。
- Kex2蛋白酶 /
- 突变体 /
- 毕赤酵母表达系统 /
- 稳定性 /
- 动力学
Abstract: Two kinds of Kex2 protease mutants Kex2-K291L and Kex2-K291H were successfully expressed in Pichia. pastoris, which were induced by methanol, and purified with anion exchange chromatography (Q-FF). Finally, the enzymatic characteristics of these Kex2 proteases were characterized. It was showed that compared with the wild-type Kex2, the degradation of the two mutants Kex2-K291H and Kex2-K291L has been significantly improved. The wild-type Kex2 protease was degraded during the purification, and a non-single band appeared, while the mutants were not degraded during the purification, and it was still a single band. The optimum pH and optimum temperature of these two mutants were the same as those of the wild-type Kex2. Their optimal pH and temperature were at pH 9.0 and 37 ℃. Compared with the wild-type Kex2 protease, the pH stability range of the mutant Kex2-K291H was expanded, from the range of pH 5.0 to 6.0 to the range of pH 5.0 to 7.0; compared with the wild-type Kex2, Kex2-K291H was more stable at 4 ℃ to 37 ℃. Enzymatic reaction kinetics studies showed that the Kcat/Km values of mutant Kex2-K291H and Kex2-K291L were 1.8 fold and 2.0 fold higher than that of the wild-type Kex2 protease, respectively.
- Kex2 protease /
- mutant /
- Pichia pastoris /
- stability /
- kinetics

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图 1 Kex2蛋白酶结构示意图

Figure 1. Three-dimensional structure of Kex2 protease

blue-calcium ion; red-the catalytic triad Ser385-His213-Asp175; orange-K291 residue; yellow-K290 residue; Green-Whole protein structure

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图 2 （a）天然Kex2蛋白酶和突变体的诱导表达结果（b）Q-FF纯化天然Kex2蛋白酶和突变体

Figure 2. (a) The expression of the wild type Kex2 protease and its mutants (b) Purification of the wild type Kex2 protease and its mutants with Q-FF

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图 3 天然Kex2蛋白酶和突变体的最适温度

Figure 3. Optimal temperature of wild-type Kex2 protease and mutants.

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图 4 天然Kex2蛋白酶与突变体的温度稳定性比较

Figure 4. Comparison of temperature stability between wild-type Kex2 protease and mutants.

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图 5 天然Kex2蛋白酶与突变体的最适pH

Figure 5. Optimal pH of wild-type Kex2 protease and its mutants.

The substrates were dissolved in buffers of different pH and kept at 25 ℃. Then add protease and react.

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图 6 天然Kex2蛋白酶与突变体pH稳定性

Figure 6. pH stability of wild-type Kex2 protease and its mutants.

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图 7 Lineweaver-Burk双倒数图分析蛋白酶的动力学参数

Figure 7. Lineweaver-Burk plots analysis of the kinetics

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表 1 突变引物

Table 1. Primers of mutants

Primers	Sequences for primers (5’ to 3’)
F	CCGGAATTCTTGGTCTCCTCTCAACAAATC
R	ATAGTTTAGCGGCCGCTTATGGGGTATTTGGATC
F_291H	CAGATTTGGTGAAGCATGCTTTGGTTAAGG
R_291H	CCTTAACCAAAGCATGCTTCACCAAATCTG
F_291L	CAGATTTGGTGAAGTTGGCTTTGGTTAAGG
R_291L	CCTTAACCAAAGCCAACTTCACCAAATCTG

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表 2 Kex2蛋白酶动力学参数

Table 2. Kinetic parameters of Kex2 proteases

Proteases	K_m/(mmol·L⁻¹)	K_cat/s⁻¹	K_cat/K_m/(mol⁻¹·L·s⁻¹)
Wild Kex2 protease	0.037	151	4.081$ \times {10}^{7} $
Kex2-K291H mutant	0.018	136	7.556$ \times {10}^{7} $
Kex2-K291L mutant	0.019	159	8.368$ \times {10}^{7} $

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参考文献(15)

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