摘要
PAPS是生物体内磺化反应的惟一活性磺酸基团供体。腺苷磷酰硫酸激酶是催化APS和ATP生成PAPS和ADP的一类酶。为实现PAPS的酶法合成,作者通过密码子优化来自结核分枝杆菌(Mycobacterium tuberculosis AUSMDU00018547)的腺苷磷酰硫酸激酶编码基因与共表达硫氧还蛋白TrxB,实现了腺苷磷酰硫酸激酶在大肠杆菌Escherichia coli BL21(DE3)中的异源高活性表达。采用His-Tag标签实现了对腺苷磷酰硫酸激酶的纯化,进一步酶学性质分析结果表明,重组腺苷磷酰硫酸激酶最适温度、最适pH以及比酶活分别为35℃、7.5和(1.26±0.08)U/mg。腺苷磷酰硫酸激酶的异源活性表达与酶学性质分析为未来实现酶法合成PAPS奠定了基础。
PAPS is the only active sulfonic acid group donor for sulfation in living organisms.Adenylyl-sulfate kinase catalyzes the formation of PAPS and ADP from APS and ATP.In order to achieve the enzymatic synthesis of PAPS,the adenylyl-sulfate kinase encoding gene from Mycobacterium tuberculosis AUSMDU00018547 was codon-optimized and heterologously co-expressed with thioredoxin TrxB to achieve the active and high-level heterologous expression of adenosine phosphorylsulfate kinase(APK)in Escherichia coli BL21(DE3).His-tagged recombinant adenylyl-sulfate kinase was successfully purified.Further enzymatic property analysis results showed that the optimal temperature,pH and the specific enzyme activity of recombinant adenylyl-sulfate kinase were 35℃,pH 7.5 and(1.26±0.08)U/mg,respectively.Heterologous expression of adenylyl-sulfate kinase and enzymatic property analysis lay a foundation for the enzymatic synthesis of PAPS in the future.
作者
胥睿睿
王阳
史仲平
康振
XU Ruirui;WANG Yang;SHI Zhongping;KANG Zhen(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2021年第2期49-56,共8页
Journal of Food Science and Biotechnology
基金
国家“十三五”重点研发计划项目(2018YFA0901401)
国家轻工技术与工程一流学科自主课题(LITE2018-16)。
