Cloning, Overexpression And Properties Of Ldh Gene From Lactobacillus Paracasei

Phenyllactic acid(PLA) is a novel antimicrobial compound. Lactatedehydrogenase(LDH) is the main enzyme for converting phenylpyruvate(PPA) to PLAin lactic acid bacteria(LAB).By bioinformatics tools provided by websites of NCBI and ExPasy and combiningelse complicated bioinformatics software package such as ClustalX,VMD, to identifysequence encoding LDH protein from database. The coding sequence was analyzed forthe characteristics of the deduced protein, including the physical-chemicalcharacteristics, post-translational modification sites, topological structure, secondarystructures,3D and domains. And compared with sequence encoding LDH from othermicroorganisms to find the conserved sequence. The gene codes for335amino acids.The theoretical molecular weight of the deduced protein is36608.8. The coding proteinis demonstrated to have3trans-membrane regions. And the comparison of amino acidsequence shows that LDH from Lactobacillus paracasei has high homology with LDHfrom Lactobacillus casei. The structure of ldh gene and LDH protein fromLactobacillus paracasei were analyzed and predicted with bioinformatics. Thisprovided theory evidence for L.paracaseiW2research of our lab, and it was useful forexpressing activated LDH.The gene ldh encoding LDH was amplified from genome DNA of L.paracaseiW2using PCR technique. The PCR product was cloned into pMD19-T vector. Thefollowing sequencing step showed that in L.paracaseiW2ldh is1008bp. Compared withldh from L.paracasei subsp.Paracasei, the PCR sequence homologous rates of nucleotide and amino acids were99.6%and100%.This was accompanied by highlyhomologous amino acid sequences of LDH from L.paracasei.. The DNA fragment of ldh double digested with restriction endonucleases, andinsert into expression vector pET-22b(+). Then transformed recombinants expressionplasmid pET22b-ldh into E. coliBL21and the specific fusion protein was expressedwith IPTG. The molecular weight of LDH were37kD respectively shown bySDS-PAGE. The levels of LDH activity were up to74.5U/mL. The result show that theexpression with biological activity. The activity of LDH were determined at50℃,pH6.0, and thermal stability was well. The Kmvalue for LDH were2.512mM usingPPA.