杰氏棒杆菌L-天冬氨酸α脱羧酶半理性改造及全细胞催化合成β-丙氨酸

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

摘要/Abstract

摘要：

β-丙氨酸是多个药物合成的重要砌块，可以通过天冬氨酸α脱羧酶（PanD）催化L-天冬氨酸脱羧来合成，但普遍在用的PanD酶活性不高是制约全细胞催化合成β-丙氨酸的瓶颈。因此，本研究通过酶的挖掘，选择将杰氏棒杆菌来源（Corynebacterium jeikeium）PanD在Escherichia coli中异源表达。对杰氏棒杆菌来源PanD进行AlaphFold2建模和分子对接，采用Rosetta虚拟突变确定突变热点，结合薄层层析初筛和纯化后复筛，最终筛选到突变体L39A，其比酶活为13.45 U/mg，相比野生型酶的比酶活（9.6 U/mg）提升了1.4倍。酶学性质表征数据表明，野生型酶和L39A突变体最适pH均为6.5，且在pH 6.0-7.0 之间酶活性稳定；两者最适温度为55℃，但L39A热稳定性较野生型提高；突变体酶的催化效率比野生型提升了1.4倍。对突变体进行结构解析发现，39位取代为侧链基团更小的丙氨酸，亲水性增强，增加了关键催化氨基酸58位酪氨酸与其他氨基酸的相互作用，使活性中心周围的区域稳定性提高，从而提高了催化活性。全细胞催化数据表明，在OD₆₀₀=40的菌体浓度下，L39A在4 h能够转化70%的L-天冬氨酸，而野生型4 h仅能转化约50%，L39A在10 h能够转化90% L-天冬氨酸，在12 h能够完全转化1 mol/L的L-天冬氨酸，这种全细胞转化效率的提升在1.5 mol/L的底物条件下更加明显。本研究筛选到的突变体具有工业化应用潜力，建立了绿色、高效的β-丙氨酸生物合成法，为生物法大规模合成β-丙氨酸提供了重要的技术基础。

关键词: β-丙氨酸, 虚拟突变, 全细胞催化

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

刘浩, 马世杰, 周哲敏, 崔文璟. 杰氏棒杆菌L-天冬氨酸α脱羧酶半理性改造及全细胞催化合成β-丙氨酸[J]. 生物技术通报, 2023, 39(9): 281-290.

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.

图/表 10

图1 重组酶CJpanD及突变体蛋白电泳 M：标准分子量蛋白Marker；1：pET28a空载破碎上清；2；pET28a-CJPanD破碎上清；3：pET28a-CJPanD-L39A破碎上清；4：pET28a-CJPanD纯酶；5：pET28a-CJPanD-L39A纯酶

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

表1 拟突变氨基酸的选取

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

图2 薄层层析结果 WT：野生型；M1-15：不同的突变体

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

图3 不同突变体比酶活

Fig. 3 Specific enzyme activity in different mutants

图4 最适温度及温度稳定性、最适pH及pH稳定性 A：重组酶的最适温度；B：温度对重组酶稳定性的影响；C：重组酶的最适pH；D：pH对重组酶稳定性的影响

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

图5 动力学常数拟合

Fig. 5 Kinetic constant fitting

表2 动力学常数测定

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

图6 突变体L39A和野生型结构信息 A：突变体L39a结构信息；B：野生型结构信息

Fig. 6 Structural information of L39A and WT A: Structural information of mutant l39A. B: Wild type structure information

图7 全细胞催化合成β-丙氨酸 A：1 mol/L底物，底物与产物的变化；B：1 mol/L底物，转化率与产量的变化；C：1.5 mol/L底物，底物与产物的变化；D：1.5 mol/L底物，转化率与产量的变化

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

表3 一批次26 h转化率与产量

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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