[Cloning, expression and characterization of a gamma-butyrobetaine hydroxylase gene bbh from Pseudomonas sp. L-1].
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 beta-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 degrees C and pH 7.5. The enzyme was stable below 45 degrees C. 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 degrees C and pH 7.0 for 31 h, the concentration of L-carnitine reached 12.7 mmol/L.


CONCLUSIONS
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 gamma-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.
Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou 310058, China. 05swkxlxf@sina.com
Xiangfeng
X
Lu
Pengcheng
P
Zhang
Qianyan
Q
Li
Hui
H
Liu
Xiaoqing
X
Lin
Xiaohang
X
Ma
D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D050563:gamma-Butyrobetaine Dioxygenase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D050563:gamma-Butyrobetaine Dioxygenase