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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2023, Vol. 43 Issue (5): 24-36    DOI: 10.13523/j.cb.2210029
    
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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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     
Received: 19 October 2022      Published: 01 June 2023
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ZHOU Li-ya
NIU Yu-jie
ZHENG Xiao-bing
JIANG Yan-jun
MA Li
BAI Jing
HE Ying
Cite this article:

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.

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

Fig.1 The reaction equation of synthesis of GABA (a) and geminal aldimine (b) E represents enzyme, R represents amino acid side chain
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
Table 1 Factors and levels of Box-Behnken experimental design
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]
Table 2 Enzyme activities of GADs derived from several origins
Fig.2 Active pockets volume prediction of GadBs (a)GadB-WT (b)M1 (c)M2 (d)M3
Fig.3 SDS-PAGE analysis of GadBs (a) M: Protein marker; Lanes1-2: GadB-WT, M1; (b) M: Protein marker; Lanes1-2: M2 and M3
Fig.4 Enzyme activities of GadBs at pH 4.5 and pH 6.0
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
Table 3 The experimental results of Box-Behnken Design
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
Table 4 The experimental variance analysis of Box-Behnken Design
Fig.5 The interactive effects of variables on Enzyme activity of M2 (a) The interactive effect of glycerol concentration and compound nitrogen source concentration (b) The interactive effect of glycerol concentration and ratio of compound nitrogen source (c) The interactive effect of glycerol concentration and phosphate concentration (d) The interactive effect of nitrogen source concentration and ratio of compound nitrogen source (e) The interactive effect of nitrogen source concentration and phosphate concentration (f) The interactive effect of ratio of compound nitrogen source and phosphate concentration
Fig.6 Effect of induction conditions on fermentation of M2 (a) IPTG concentration (b) Induction temperature (c) Induction time
Fig.7 Optimal pH (a) and temperature (b) of GadB-WT and M2
Fig.8 pH (a) and thermal (b) stabilities of GadB-WT and M2
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
Table 5 The kinetic parameters of GadB-WT and M2
Fig.9 3D demonstration of active sites of GadBs Aldamine formed between the PLP-Lys276 Schiff base and His465 of GadB-WT(a), M2(b), M3(c). Molecular docking of L-MSG with active site of GadB-WT(d), M2(e), M3(f). Surface represents active pocket; Green sticks represent L-MSG; Pink sticks represent residues which may from interaction with L-MSG or PLP; Yellow sticks represent mutant residues
Fig.10 Schematic diagrams of electrostatic potential and B-factor of GadB-WT and M2 (a) Electrostatic potential of GadB-WT (b) Electrostatic potential of M2 (c) B-factor of GadB-WT (d) B-factor of M2
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