摘要：β-丙氨酸是多个药物合成的重要砌块，可通过天冬氨酸α脱羧酶（PanD）催化L-天冬氨酸脱羧合成，但PanD酶活性不高是全细胞催化合成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PanD在Escherichia coli中异源表达。对其进行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。酶学性质数据表明，野生型酶和突变体最适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。对突变体结构解析发现：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，使活性中心周围区域稳定性提高，从而提高了催化活性。全细胞催化结果表明，在OD600=40的菌体浓度下，L39A 4 h够转化70%的L-天冬氨酸，而野生型4 h仅能转化50%，L39A在12 h能够完全转化1 mol/L的L-天冬氨酸，突变体L39A在转化效率上略优于野生型。
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Improving the activity of L-aspartate-α-decarboxylase from Corynebacteriumjeikeium through semi-rational design and whole-cell catalytic synthesis of β-alanine
Abstract：β-alanine is an important building block in the synthesis of many drugs. At present, β-alanine is synthesized mainly by decarboxylation of aspartic L-aspartate-α -decarboxylase (PanD)，however, the low activity of PanD enzyme is the bottleneck of whole cell catalytic synthesis of β-alanine. Therefore, in this study, through enzyme mining, PanD from Corynebacterium jeikeium was heterologous expressed in Escherichia coli. AlaphFold2 modeling and molecular docking were conducted by Corynebacterium gerii derived PanD. The mutation hotspot was determined by Rosetta virtual mutation, and the mutant L39A was finally screened by using thin layer chromatography (TLC) and re-screening after purification. Its specific enzyme activity was 13.45 U/mg, which was 1.4 times higher than that of wild type (9.6 U/mg). The results showed that the optimum pH of wild-type enzyme and L39A mutant was 6.5, and the enzyme activity was stable between pH 6.0 and 7.0. The optimum temperature of L39A and wild type was 55 ℃, but the thermal stability of L39A was higher than that of the wild type. The catalytic efficiency of the mutant was 1.4 times higher than that of the wild type. The structural analysis of the mutant showed that the 39th position was replaced by alanine with smaller side chain groups, which enhanced the hydrophilicity and increased the interaction between the key catalytic amino acid tyrosine at 58th position and other amino acids, which improved the stability of the region around the active center, thus improving the catalytic activity. Whole-cell catalytic data showed that L39A could convert 70% L-aspartic acid at 4 h, while the wild type could only convert about 50% L-aspartic acid at 4 h, and L39A could convert 90% L-aspartic acid at 10 h, and completely convert 1 mol/L L-aspartic acid at 12 h. The conversion efficiency of the mutant L39A was slightly better than that of the wild type 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.
Keywords： Bioengineering β-alanine Virtual mutation Whole cell catalysis
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