Studies On L-carnitine Metabolising Enzymes And Transformation Of A Strain Pseudomonas Sp.L-1

L-carnitine, namely L-β-hydroxy-y-trimethylaminobutyrate, vitamin BT, is an ubiquitous substance. L-carnitine is a basic component in cell and functions as a vector in the transport of long-chain fatty acid from cytoplasm to the mitochondrial matrix, where β-oxidation occurs. So far, L-carnitine has been widely used in the fields of medicine, health, food and animal feed. The continuous growth of market demand urge people to study and improve the production process of L-carnitine. Because of low cost, less pollution and lower energy consumption, microbial enzymatic conversion method is more popular than chemical synthesis in industry.In this study, a bacterium named L-1which can convert y-butyrobetaine to L-Carnitine was isolated from soil and it was identified subsequently as a strain of the genus Pseudomonas. To this train, the conversion of L-carnitine and enzymes involved in the metabolism of L-carnitine have been studied. The main results are as follows:1. The isolation and identification of L-carnitine producing strain:A bacterium named L-1which can convert y-butyrobetaine to L-Carnitine was isolated from soil after enrichment cultivation and streaking plate purification. Based on its morphological studies, physiochemical identified,16S rDNA sequencing and phylogenetic analysis, strain L-1was identified to be Pseudomonas sp..2. Cloning and expression of the gene of y-butyrobetaine hydroxylase (BBH): The y-butyrobetaine hydroxylase gene bbh (GenBank:JQ250036) was amplified by PCR. Results of multiple alignment and phylogenetic analysis to the amino acid sequence of the gene showed that the enzyme was closely related with members of BBH from Pseudomonas and Burkholderia. But the highest similarity was only76%, indicated that BBH of L-1was quite different with those from other reports. The predicted three-dimensional structure of BBH monomer showed that it contains two domains:a double-strand β-helix catalytic domain and a smaller N-terminal domain of unknow function. The ORF of bbh was inserted into pET-15b expression vector and expressed in E.coli BL21(DE3) by IPTG induction.3. Purification and characterization of recombinant BBH:The recombinant E.coli Cells were harvested by centrifugation after IPTG induction and the cells were disrupted with ultrasonic to obtain crude enzyme. After His·Bind Resin chromatography and precipitation of ammonium sulfate, the specific activity of the enzyme reached0.115U/mg. SDS-PAGE and gel filtration analysis showed that the BBH was a homodimer, and the molecular weight of subunit was about46.5kDa. The optimal temperature and pH was30℃and pH7.5. The enzyme was stable below45℃. The enzyme was most stable at pH6.0. We used resting cells of recombinant E.coli for L-carnitine biotransformation, after incubated at30℃and pH7.0for31h, the concentration of L-carnitine reached12.7mmol/L.4. Knocking out of the L-carnitine dehydrogenase gene cdh and the study of y-butyrobetaine metabolic pathways of strain L-1:The L-carnitine dehydrogenase gene was amplified by PCR. The ORF of the gene was inserted into pET-15b expression vector and expressed in E.coli BL21(DE3) by IPTG induction. Confirmed that the gene bbh encoding an enzyme with L-carnitine dehydrogenase activity. After knocking out of the gene cdh, L-carnitine productivity of the strain L-1-△cdh was3.2fold higher. HPLC-MS analysis indicated that there was another γ-butyrobetaine metabolic pathway, which degrade y-butyrobetaine by demethylation of trimethylamine of γ-butyrobetaine.