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 Gammabutyrobetaine hydroxylase gene bbh from Pseudomonas sp.L-1

摘要:

摘要:【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46.5kDa，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。

Abstract:

Abstract:［Objective］Gamma-butyrobetaine hydroxylase is an enzyme that catalyzes the last step in the biosynthesis of Lcarnitine． 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.