一株假单胞菌(Pseudomonas sp.)L-1菌株γ-丁基甜菜碱羟化酶基因bbh的克隆、表达及酶学性质

Cloning,expression and characterization of a Gamma-butyrobetaine hydroxylase gene bbh from Pseudomonas sp. L-1

【目的】γ-丁基甜菜碱羟化酶是生物体内合成L-肉碱的关键酶。从假单胞菌(Pseudomonas sp.)L-1中克隆γ-丁基甜菜碱羟化酶基因,实现其在大肠杆菌(Escherichia coli)中的高效表达,并对表达产物进行酶学性质分析,为生物转化生产L-肉碱奠定基础。【方法】通过PCR克隆γ-丁基甜菜碱羟化酶基因,并将其开放阅读框(ORF)克隆至融合表达载体pET-15b;表达产物经His.Bind Resin纯化后对BBH进行酶学性质及三维空间结构分析;并以静止细胞进行L-肉碱的转化。【结果】成功地克隆了一个γ-丁基甜菜碱羟化酶基因bbh(GenBank:JQ250036),并实现了其在E.coli中的高效表达。融合蛋白以同源二聚体的形式存在,单个亚基的分子量约46.5 kDa,最适反应温度为30℃,最适反应pH为7.5。该酶在45℃以下稳定。在pH6.0时该酶有最高的pH稳定性。以表达bbh基因的重组大肠杆菌静止细胞转化L-肉碱,L-肉碱产量可达12.7mmol/L。【结论】Pseudomonas sp.L-1γ-丁基甜菜碱羟化酶与现有报道的bbh基因有较大的差异。由该基因表达的γ-丁基甜菜碱羟化酶能有效地转化γ-丁基甜菜碱生成L-肉碱。本研究不仅丰富了γ-丁基甜菜碱羟化酶基因资源,而且为L-肉碱的生物转化提供了一种新的转化方案。

[Objective] Gamma-butyrobetaine hydroxylase is an enzyme that catalyzes the last step in the biosynthesis of L-carnitine.We cloned,expressed and characterized a gamma-butyrobetaine hydroxylase gene bbh from Pseudomonas sp.L-1,to facilitate the production of L-carnitine using microorganisms.[Methods] We cloned bbh gene by PCR,and then cloned the open reading frame of bbh into pET-15b vector and expressed by Isopropyl β-D-1-thiogalactopyranoside（IPTG） induction.After His-Bind Resin purification,the characteristics of BBH were studied.The three-dimensional structure of BBH monomer was modeled by SWISS-MODEL Workspace and resting cells were used for L-carnitine transformation.[Results] We cloned a gamma-butyrobetaine hydroxylase gene bbh（GenBank： JQ250036） from Pseudomonas sp.L-1 and expressed the gene in Escherichia coli BL21（DE3）.BBH fusion protein was a homodimer,and the molecular weight of subunit was about 46.5kDa.The optimal temperature and pH was 30 ℃ and pH 7.5.The enzyme was stable below 45 ℃.The enzyme was most stable at pH 6.0.We used resting cells of recombinant E.coli for L-carnitine biotransformation,after incubated at 30 ℃ and pH 7.0 for 31 h,the concentration of L-carnitine reached 12.7 mmol/L.[Conclusion] The bbh gene from Pseudomonas sp.L-1 strain is remarkably different from that of reported one.The gamma-butyrobetaine hydroxylase expressed by this gene could effectively transform γ-butyrobetaine for L-carnitine production.Beside by reporting of a bbh gene from bacteria,this research also provided a new process for biotransformation of L-carnitine.