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α烯烃是一种独特的微生物天然产物.由P450脂肪酸脱羧酶催化的中长链(C_(12)-C_(20))脂肪酸脱羧反应生成的α烯烃因其在生物燃料和生物材料领域的潜在应用前景备受关注.通过将脂肪酶TlL的甘油三酯水解活性与P450脱羧酶OleT_(JE)的脂肪酸脱羧活性偶联,可将油脂原料两步转化为α烯烃.为了提高油脂到α烯烃的转化效率,并降低两种酶的分步制备成本,本研究通过20个氨基酸的linker序列将双酶共价连接形成重组融合蛋白NHis6-OleT_(JE)-linker-TlL(FusC).在大肠杆菌中表达的FusC经镍柱层析纯化后成功实现双功能活性.经气相色谱分析,融合蛋白催化的甘油三酯顺序水解-脱羧偶联反应的产烃率达到31.7%,催化效率相对于双酶游离混合体系明显提升,展现出良好的底物通道效应,具有潜在的实际应用前景.
α-Olefin is a unique natural product of microorganism.The α-olefins produced by the decarboxylation of medium-long chain (C_ (12) -C_ (20)) fatty acids catalyzed by P450 fatty acid decarboxylase due to their potential in the field of biofuels and biomaterials The prospect of application is gaining attention.Dipopeptide can be converted into α-olefins in two steps by coupling the triglyceride hydrolysis activity of lipase TlL with the fatty acid decarboxylation activity of P450 decarboxylase OleT_ (JE) In this study, two enzymes were covalently linked to form recombinant fusion protein NHis6-OleT_ (JE) -linker-TlL (FusC) through a 20 amino acid linker sequence. In E. coli FusC was successfully purified by nickel column chromatography to achieve bifunctional activity.According to gas chromatography analysis, the yield of hydrocarbon-catalyzed decarboxylation of triglyceride catalyzed by fusion protein reached 31.7%, and the catalytic efficiency was higher than that of double enzyme The free-mixing system has been significantly improved, showing a good substrate channel effect, with potential practical applications.