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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  2023, Vol. 43 Issue (5): 24-36    DOI: 10.13523/j.cb.2210029
研究报告     
谷氨酸脱羧酶的分子改造及酶学性质研究*
周丽亚1,牛玉杰1,郑晓冰1,姜艳军1,**(),马丽1,白敬2,贺莹1,**()
1 河北工业大学化工学院 天津 300130
2 河北科技大学食品与生物学院 石家庄 050018
Molecular Modification and Enzymatic Properties of Glutamate Decarboxylase
ZHOU Li-ya1,NIU Yu-jie1,ZHENG Xiao-bing1,JIANG Yan-jun1,**(),MA Li1,BAI Jing2,HE Ying1,**()
1 School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China
2 College of Food Science and Biology, Hebei University of Science & Technology, Shijiazhuang 050018, China
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摘要：

以大肠杆菌(Escherichia coli)来源的谷氨酸脱羧酶(GadB)为研究对象,通过组合突变,获得了pH适用范围拓宽、催化活力提高和稳定性增强的组合突变体M2。与野生型GadB-WT相比,组合突变体M2的pH适用范围有效拓宽,在pH6.0时催化活力比GadB-WT提高113.43%。之后对含有M2突变体基因重组菌的发酵培养基和诱导条件进行优化,优化后单位培养基酶活力比未优化时提高了104.13%。在此基础上对M2的酶学性质进行测定,测得其最适pH为5.0,最适温度为37℃。通过稳定性测定M2的pH稳定性和热稳定性与野生型GadB-WT相比都有一定程度的增强。M2的动力学参数Km值为7.316 μmol/L,kcat为13.387 s-1,kcat/Km为1.830 L/(s·μmol)。研究获得的组合突变体M2进一步丰富了催化合成γ-氨基丁酸的GadB突变体酶库,具有良好应用前景。
关键词： 谷氨酸脱羧酶;  γ-氨基丁酸;  定点突变;  发酵优化    
Abstract:

By combining and screening several mutations of glutamate decarboxylase (GadB) derived from E. coli, a combined mutant M2 with a wider pH range, higher catalytic activity and higher pH and thermal stabilities was obtained. Compared with the wild-type GadB-WT, the pH range of the combined mutant M2 was effectively broadened, and it produced a higher catalytic activity of 113.43% over the wild-type GadB-WT under pH6.0. After the optimization for fermentation medium and induction conditions of the recombinant bacteria, an overall 104.13% increase of enzyme activity was gained over the unoptimized medium. On this basis, the enzymatic properties of M2 were determined. The optimum pH was 5.0 and the optimum temperature was 37℃. Compared with wild-type Gad-WT, the pH stability and thermal stability of M2 were enhanced to some extent. The kinetic parameters of M2 were determined as follows:Km was 7.316 μmol/L, kcat was 13.387 s-1, and kcat/Km was 1.830 L/(s·μmol). The combined mutant M2 obtained in this study further enriches the GAD mutant enzyme library for the synthesis of γ-aminobutyric acid and has a promising application prospect.
Key words: Glutamate decarboxylase    γ-Aminobutyric acid    Site-directed mutagenesis    Fermentation optimization
收稿日期: 2022-10-19 出版日期: 2023-06-01
ZTFLH:  Q814  
基金资助: *国家自然科学基金(21878068);国家自然科学基金(2217083);河北省省级科技计划(20372802D);河北省自然科学基金(B2020202036)
通讯作者: **电子信箱: yanjunjiang@hebut.edu.cn; heying1980@hebut.edu.cn   
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周丽亚
牛玉杰
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姜艳军
马丽
白敬
贺莹

引用本文:

周丽亚, 牛玉杰, 郑晓冰, 姜艳军, 马丽, 白敬, 贺莹. 谷氨酸脱羧酶的分子改造及酶学性质研究*[J]. 中国生物工程杂志, 2023, 43(5): 24-36.

ZHOU Li-ya, NIU Yu-jie, ZHENG Xiao-bing, JIANG Yan-jun, MA Li, BAI Jing, HE Ying. Molecular Modification and Enzymatic Properties of Glutamate Decarboxylase. China Biotechnology, 2023, 43(5): 24-36.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2210029    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I5/24

图1  GABA(a)和偕二胺(b)合成反应方程式
编号 因素 水平
-1 0 1
A 甘油浓度/(g/L) 4 8 12
B 复合氮源浓度/(g/L) 20 25 30
C 复合氮源比例/(g:g) 0.5 1 1.5
D 磷酸盐浓度/(mmol/L) 100 120 140
表1  Box-Behnken实验设计的因素与水平
来源 名称 pH 温度/℃ 比酶活/(U/mg) 参考文献
E.coli GadB-WT 4.5 40 86 本文
M1 4.0 37 83 本文
M2 5.0 37 137 本文
M3 4.0 37 110 本文
Glu89Gln/Δ452-466 4.0 37 32~35 [18]
Glu89Gln/His465Ala 4.0 37 100~105 [18]
L.brevis CGMCC 1306 Wild-type GAD 4.8 48 7.2~7.5 [19]
GADΔC 4.8 48 7.5~8.0 [19]
L.brevis Lb85 GadB1 4.6 40 24~26 [20]
GadB1T17I/D294G/E312S/Q346H 4.0 40 62~65 [20]
L.plantarum ATCC 14917 GAD-WT 5.0 40 8.9 [21]
GAD Δ11 5.0 37 9.8 [21]
E.raffinosus GAD 4.6 45 21.7 [27]
N.crassa GAD 5.0 37 2.76 [28]
A. oryzae GAD 5.5 60 48.2 [29]
T.kodakaraensis GAD 8.0~9.0 90 NR [30]
P.horikoshii PhGAD 8.0 >97 0.26 [31]
表2  几种来源的GAD的活性比较
图2  GadBs活性口袋体积预测
图3  GadBs的SDS-PAGE分析
图4  GadBs在pH4.5和pH6.0的酶活力
实验编号 A B C D 比酶活/(U/mL)
1 0 0 0 0 4.040
2 1 0 -1 0 3.415
3 0 -1 1 0 3.667
4 0 0 -1 -1 2.680
5 0 1 0 1 3.234
6 0 -1 -1 0 3.218
7 0 1 -1 0 3.623
8 0 0 0 0 3.866
9 -1 0 -1 0 3.015
10 1 1 0 0 3.083
11 0 -1 0 1 3.605
12 -1 0 0 1 3.280
13 -1 0 0 -1 3.080
14 1 0 1 0 2.677
15 0 1 1 0 2.404
16 -1 1 0 0 3.171
17 0 -1 0 -1 3.484
18 0 0 1 -1 3.605
19 0 0 0 0 3.898
20 0 0 1 1 2.448
21 0 1 0 -1 3.230
22 -1 -1 0 0 3.450
23 0 0 -1 1 3.814
24 -1 0 1 0 3.152
25 1 0 0 1 3.251
26 1 0 0 -1 3.410
27 1 -1 0 0 3.565
表3  Box-Behnken Design实验结果
来源 平方和 自由度 均方 F 值 P值
Model 4.539 4 14 0.324 2 64.393 4 < 0.000 1 显著
A 0.005 3 1 0.005 3 1.055 1 0.324 6
B 0.420 0 1 0.420 0 83.410 6 < 0.000 1
C 0.273 3 1 0.273 3 54.278 2 < 0.000 1
D 0.001 7 1 0.001 7 0.336 1 0.572 8
AB 0.010 2 1 0.010 2 2.025 9 0.180 1
AC 0.191 6 1 0.191 6 38.055 9 < 0.000 1
AD 0.032 2 1 0.032 2 6.398 8 0.026 4
BC 0.695 1 1 0.695 1 138.051 6 < 0.000 1
BD 0.003 5 1 0.003 5 0.685 5 0.423 9
CD 1.312 2 1 1.312 2 260.591 4 < 0.000 1
A2 0.771 8 1 0.771 8 153.280 8 < 0.000 1
B2 0.287 9 1 0.287 9 57.183 3 < 0.000 1
C2 1.251 8 1 1.251 8 248.610 1 < 0.000 1
D2 0.508 5 1 0.508 5 100.995 0 < 0.000 1
残差 0.060 4 12 0.005 0
失拟值 0.043 4 10 0.004 3 0.508 8 0.809 4 不显著
纯误差 0.017 0 2 0.008 5
总离差 4.599 8 26
表4  Box-Behnken Design实验方差分析
图5  各变量对M2酶活力的交互影响
图6  诱导条件对M2发酵的影响
图7  GadB-WT和M2的最适pH(a)和最适温度(b)
图8  GadB-WT和M2的pH稳定性(a)和热稳定性(b)
Km/(μmol/L) kcat/(s-1) kcat/Km/
[L/(s·μmol)]
GadB-WT 8.155 3.446 0.423
M2 7.316 13.387 1.830
表5  GadB-WT和M2的动力学参数
图9  GadBs活性位点的3D结构
图10  GadB-WT和M2的表面静电势和B-factor值分布示意图
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