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  

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

作　　者：刘浩 马世杰 周哲敏[1] 崔文璟[1] LIU Hao;MA Shi-jie;ZHOU Zhe-min;CUI Wen-jing(Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122)

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,无锡214122

出　　处：《生物技术通报》2023年第9期281-290,共10页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32171420)。

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(Pan D)催化L-天冬氨酸脱羧来合成,但普遍在用的Pan D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)Pan D在Escherichia coli中异源表达。对杰氏棒杆菌来源Pan D进行Alaph Fold2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p H均为6.5,且在p H 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OD_(600)=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。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 ofβ-alanine.Therefore,in this study,using enzyme mining,we obtained L-aspartic acidα-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 wildtype 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 wildtype 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 trans

关 键 词：Β-丙氨酸 虚拟突变 全细胞催化 

分 类 号：Q789[生物学—分子生物学]