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

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.