Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (9): 281-290.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0085
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LIU Hao(), MA Shi-jie, ZHOU Zhe-min, CUI Wen-jing()
Received:
2023-02-07
Online:
2023-09-26
Published:
2023-10-24
Contact:
CUI Wen-jing
E-mail:6200208115@stu.jiangnan.edu.cn;wjcui@jiangnan.edu.cn
LIU Hao, MA Shi-jie, ZHOU Zhe-min, CUI Wen-jing. Improving the Activity of L-aspartate-a-decarboxylase from Corynebacterium jeikeium Through Semi-rational Design and Whole-cell Catalytic Synthesis of β-alanine[J]. Biotechnology Bulletin, 2023, 39(9): 281-290.
Fig. 1 SDS-PAGE analysis of recombinant enzyme CJpanD and mutants M: Protein marker; 1: E. coli BL21/pET28a cell breaking supernatant; 2: E. coli BL21/pET28a-CJPanD cell breaking supernatant; 3: E. coli BL21/pET28a-CJPanD-L39A cell breaking supernatant; 4: purified pET28a-CJPanD; 5: purified pET28a-CJPanD-L39A
Mutant | ddG/(Kal·mol-1) |
---|---|
H11 | -3.572 |
C26 | -4.946 |
L39 | -3.137 |
I49 | -1.518 |
N72 | -3.458 |
L55 | -2.940 |
T56 | -2.283 |
Table 1 Selection of pseudo-mutant amino acids
Mutant | ddG/(Kal·mol-1) |
---|---|
H11 | -3.572 |
C26 | -4.946 |
L39 | -3.137 |
I49 | -1.518 |
N72 | -3.458 |
L55 | -2.940 |
T56 | -2.283 |
Fig. 4 Optimal temperature and temperature stability, optimal pH and pH stability of recombinant enzymes A: Optimal temperature for recombinant enzymes. B: Effect of temperature on the stability of recombinant enzymes. C: Optimal pH for recombinant enzymes. D: Effect of pH on the stability of recombinant enzymes
Mutant | Km/ (mmol·L-1) | Kcat/ (s-1) | Kcat/Km / (mmol·L-1·s-1) |
---|---|---|---|
WT | 3.6±0.1 | 102.1±0.3 | 28.1 |
L39A | 3.3±0.1 | 132.5±0.3 | 39.3 |
Table 2 Kinetic parameters of WT and L39A
Mutant | Km/ (mmol·L-1) | Kcat/ (s-1) | Kcat/Km / (mmol·L-1·s-1) |
---|---|---|---|
WT | 3.6±0.1 | 102.1±0.3 | 28.1 |
L39A | 3.3±0.1 | 132.5±0.3 | 39.3 |
Fig. 7 Whole cell catalytic synthesis of β-alanine A: 1 mol/L substrate, change in substrate and product. B: 1 mol/L substrate, change of conversion and yield. C: 1.5 mol/L substrate, change of substrate and product. D: 1.5 mol/L substrate, change of conversion and yield
26 h | Conversion/% | β-alanine yield/(g·L-1) |
---|---|---|
WT | 73.1 | 37.55 |
L39A | 80.2 | 71.22 |
Table 3 Conversion and yield at 26 h per batch
26 h | Conversion/% | β-alanine yield/(g·L-1) |
---|---|---|
WT | 73.1 | 37.55 |
L39A | 80.2 | 71.22 |
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