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

Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou 310058, China.
ACTA MICROBIOLOGICA SINICA 05/2012; 52(5):602-10.
Source: PubMed

ABSTRACT

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.
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.
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.
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.

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    • ") and Pseudomonas sp. L1 (Lu et al., 2012), and both enzymes are homologous to the animal c-butyrobetaine hydroxylase in their requirement for oxygen and the cofactors iron, ascorbate, and a-ketogluterate (Lindstedt & Lindstedt, 1970; Lindstedt et al., 1968). "
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    ABSTRACT: Carnitine is a quaternary amine compound found at high concentration in animal tissues, particularly muscle, and is most well-studied for its contribution to fatty acid transport into mitochondria. In bacteria, carnitine is an important osmoprotectant and can also enhance thermotolerance, cryotolerance, and barotolerance. Carnitine can be transported into the cell or acquired from metabolic precursors, where it can serve directly as a compatible solute for stress protection, or be metabolized through one of a few distinct pathways as a nutrient source. In this review, we summarize what is known about carnitine physiology and metabolism in bacteria. In particular, recent advances in the aerobic and anaerobic metabolic pathways as well as the use of carnitine as an electron acceptor have addressed some long-standing questions in the field.
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