High-effective Catalytic Characteristics Of Glycerol Dehydratase From K.pneumoniae Xjpd-li

Compared with chemical synthesis, biosynthesis of 1,3-PD have many obvious advantages, such as the environmental friendship and low cost, which makes it more promising. In the former research,we had reported that one new strain identified and named as K. pneumoniae XJPD-Li which exhibits good properties for the production of 1,3-propendiol from glycerol, such as higher fermentation temperature(40℃) and optimal pH(8.0), and faster glycerol consuming with higher molar yield of 1,3-PD than that of other reported.Good properties of glycerol dehydratase from K. pneumoniae XJPD-Li draw our attention to the catalysis system in this strain. The gene of glycerol dehydratase (dhaBCE) was amplified from the genomic DNA of K. pneumoniae XJPD-Li by PCR. The over-expression system E.coli BL21(DE3) PET-28a(+) plasmid was then successfully constructed. The active protein was induced for 5 hours by 1.0mM IPTG at 30℃while OD600 reached 0.6. The optimal reaction temperature and pH were 45℃and 8.0, respectively. The special activity of recombinant glycerol dehydratase was 40.2 U·mg^-1 which was about 80 times compared with that of wild strain. Sequence analysis showed that the gene of dhaBCE is 2693bp, The gene and amino acid sequence of glycerol dehydratase (dhaBCE) from K. pneumoniae XJPD-Li have a significant level of identity with GDHt from K. pneumoniae(U60992) (99.48% and 99.55% identity, respectively).Among three subunits of glycerol dehydratase, amino acid residues S193, E407 ofαsubunit are different with what had been reported. M193α(S193C) and M407α(E407A) mutants were designed and constructed by megaprimer PCR and one step inverse PCR. The recombinant plasmid M193αand M407αwere expressed in E. coli BL21(DE3)strain. The result showed that the special activity of the mutation M193αwas 37.8 U·mg^-1 ,the optimal reaction temperature and pH are 45℃and 8.0.The special activity of the mutation M407αwas 14.7 U·mg^-1,only 36.6 % of dhaBCE,the optimal reaction temperature and pH were 40℃and 8.0,5℃lower than that of the pET-28a-dhaBCE. According to the sequence analysis ofβsubunits of glycerol dehydratase, M47β(N47I) and M189β(V189I) mutants were designed and constructed by one step inverse PCR. The recombinant plasmid M47βand M189βwere expressed in E. coli BL21(DE3)strain. The result show that the special activity of the mutation M47βis 38.7 U·mg^-1 ,the optimal reaction temperature and pH were 40℃and 8.0, lower than that of the pET-28a-dhaBCE;the special activity of the mutation M189βwas 39.2 U·mg^-1, the optimal reaction temperature and pH were 45℃and 8.0.The gene of yqhD was amplified from the genomic DNA of E.coli K-12 by PCR and inserted into PET-28a(+)–dhaBCE plasmid. SDS-PAGE shows four protein bands with molecular weight of 66,43,24 and 16KD。Site-directed mutagenesis results in this research will provide the guidance for the construction of the newly catalysis sysetem and directed evolution of glycerol dehydratase.