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

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.