Improving the Activity of L-aspartate-a-decarboxylase from Corynebacterium jeikeium Through Semi-rational Design and Whole-cell Catalytic Synthesis of β-alanine

doi:10.13560/j.cnki.biotech.bull.1985.2023-0085

Abstract

Abstract:

β-alanine is an important block in the synthesis of many drugs, which can be obtained by catalyzing the decarboxylation of L-aspartic acid. At present, the low activity of PanD enzyme is the bottleneck of whole cell catalytic synthesis of β-alanine. Therefore, in this study, using enzyme mining, we obtained L-aspartic acid α-decarboxylase from Corynebacterium jeikeium, and successfully expressed it in Escherichia coli. We leveraged AlaphFold2 to model the structure of the enzyme and docked L-aspartate to it. The hotspot residues to be mutated was determined by Rosetta virtual mutation, The mutant L39A was finally screened out by using thin layer chromatography（TLC）and was purified for characterization. The enzymatic characterization data showed that the specific enzyme activity was 13.45 U/mg, which was 1.4-fold higher than that of wild type（9.6 U/mg）. The optimal pH of both the wild-type enzyme and L39A mutant were 6.5. Moreover, the wild-type enzyme and the L39A mutant were both stable between pH 6.0 and 7.0. The optimal temperature of both the L39A mutant and the wild-type enzyme were 55℃, and the thermal stability of L39A was higher than that of the wild-type enzyme. Besides, the catalytic efficiency of the mutant was 1.4-fold higher than that of the wild-type enzyme. Structural analysis of the mutant revealed that the hydrophilicity was enhanced when the position 39 was replaced by alanine with smaller side chain group, and accordingly the interaction between the key catalytic amino acid tyrosine 58 and other surrounding residues was reinforced, which improved the stability of the region around the active center. Eventually, the catalytic activity of L39A mutant was augmented. The whole-cell catalytic results showed that L39A converted 70% L-aspartate after 4 h biotransformation, while the wild-type enzyme only converted approximately 50% substrate in the same time. Along with the process of transformation, L39A converted 90% substrate at 10 h, and eventually completely converted 1 mol/L substate at 12 h. The improvement of whole cell transformation efficiency was more obvious under the substrate condition of 1.5 mol/L. The mutant screened in this study has the potential for industrial application, and a green and efficient β-alanine biosynthesis method has been established, which lays an important foundation for the industrialization of β-alanine biosynthesis.

Key words: β-alanine, virtual mutation, whole cell catalysis

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.

Figures/Tables 10

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

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. 2 Results by thin layer chromatography WT: Wild type. M1-15: Different mutants

Fig. 3 Specific enzyme activity in different mutants

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

Fig. 5 Kinetic constant fitting

Table 2 Kinetic parameters of WT and L39A

Mutant	K_m/ （mmol·L-¹）	K_cat/ （s^-1）	K_cat/K_m / （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. 6 Structural information of L39A and WT A: Structural information of mutant l39A. B: Wild type structure information

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

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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