Screening Of Polyhydroxyalkanoate (PHA) Synthesis Ocean Strain And Study Of Related Enzymes

The ocean is rich in material and microbial resources.Among them,the polyhydroxyalkanoate(PHA)is a kind of biological material presented in the ocean microorganism and widely used as the environmental protection and bio-medical materials because of its biocompatibility and biodegradability.As early as 1926,French scientist Lemoigne first discovered the most common monomer of PHA: polyhydroxybutyrate(PHB)in Bacillus megacus,which opened the PHA study tour.In this study,a strain produced PHB from methanol was isolated from the seaside soil sample.The strain was identified and the methanol concentration was optimized.At the same time,the three genes in the PHB synthesis pathway were cloned,expressed and purified.The enzyme activity and site-directed mutagenesis of key enzyme were studied.A strain that producing PHB used methanol as sole energy and carbon source was isolated from the marine soil sample.The genome of the strain was extracted,the 16 S rRNA gene sequence was amplified and the phylogenetic tree was constructed.The result showed that the isolated strain belonged to the Methylobacterium genus and was named Methylobacterium sp.1805.The suitable methanol initial concentration,the added methanol concentration for strain grow was studied.The degradation and tolerance ability of the strain for methanol was studied.The methanol initial concentration for PHB accumulated was optimum too.The results showed the bacteria in the initial culture medium of 0.4 % methanol concentration,added 0.5 % methanol per day were most conducive to the strain growth.The strain OD600 value could reach 7 at 72 hours under the optimum conditions,went into a stable growth state.The bacteria could tolerate high methanol and could grow well in the culture medium of the 4 % methanol concentration.The degradation ability of strain for methanol was also very strong too.It was appropriate for PHB accumulation that the strain was cultured in product accumulation culture medium of 0.5 % initial methanol concentration and 0.5 % methanol was added daily.Under the optimized conditions,the strain could accumulate 51.8 % PHB of cell dry weight.The strain was potential for industrial applications.The primers were designed by homologous multiple comparison with the sequences of phaA,phaB and phaC genes in Methylobacterium.The three genes were amplified from Methylobacterium sp.1805,sequenced and the three gene fragments were ligated into pET-28a(+)expression vector.The three enzyme proteins were expressed and the enzyme activity and kinetic were measured,and the three enzymes were analyzed by bioinformatics two.The potential key amino acids of PhaC(Cys at position 341,Asp at position 496,His at position 524)were mutated by RF(restriction free)cloning and activity of the mutational PhaC were measured.Using the primers designed by homologous comparison,the sequence of the three genes were successfully amplified and submitted to the NCBI database.The accession numbers were KY229163,KY229164 and KY229165,respectively.The enzyme activity of PhaA recombinase reached 0.41 U/mg and the value of Vmax and Km of Pha A were 0.15 ?M min-1 mg-1 protein and 0.25 mM,respectively.The enzyme belonged to cond-enzymes superfamily and catalyzed two molecule acetyl-CoAs to a molecule acetylacetyl-Co A by "ping pong" catalytic mechanism.The catalytic process was mainly performed by the "Cys89-His348-Cys378" catalytic domain.The enzyme activity of PhaB was 1.64 U and the values of Vmax and Km were 0.48 ?M min-1 mg-1 protein and 1.21 mM,respectively.The enzyme belonged to short-chain alcohol dehydrogenase in the NADB-Rossmann superfamily.The enzyme activity of PhaC was 33.51 U and the values of Vmax and Km were 0.1 ?M min-1 mg-1 protein and 0.14 mM,respectively.The enzyme belonged to class I PhaC enzyme of PhaC-N superfamily.The PhaC structural analysis showed PhaC had only 11.91 % similarity with human gastric lipase,and it was not similar to that of typical PhaC through SWISS-MODEL.The enzymatic activity was improved after site-directed mutagenesis of three potential key amino acids,indicated that the newly cloned PhaC might had a different catalytic mechanism than the typical I PhaC.