Screening,Identification,and Research Of Fermentation Producing Chitinase Of A Strain Of Serratia Marcescens

As the most biomacromolecule reserves in nature,only second to cellulose,chitin is the important part of peritrophic membrane of insect,egg-shell of plant-pathogenic nematode,and cell-wall of plant-pathogenic fungus.It mainly acts as skeleton to support body structure and protect the body.Chitinase(EC 3.2.1.14)is a kind of glycosyl hydrolase which can specific degrade chitin.Therefore,chitinase can as a kind of biological control agent for improvement of plant disease-resistance for plant-pathogenic fungus and pests.In this study,by enrichment cultivation,primary screening and secondary screening,4 bacterial strains(MEW05,MEW06,MET14,and MET32)were obtained from water samples which had been collected from Wuhan Sand Lake,Hubei Province.Those isolated strains could grow on 1%colloidal chitin plates and form obviously hydrolysis circle.After shake-flask culture,the chitinase activity of MEW06 fermentation supernatant has reached to 71.74 U/mL,thus,MEW06 has been consider as the high-yielding chitinase strain for further study.After 24 h cultivate at 28 ?,MEW06 colony presented rounded,bright red,and the edge is smooth.Microscopic observation showed MEW06 thallus is rod-shaped and approximately spherical,peritrichous,no spore,no capsule,and Gram-negative.16S rDNA sequencing finally indicated MEW06 is a strain of Serratia marescens.MEW06 fermentation supernatant was separated by ammonium sulfate precipitation,affinity chromatography,and DEAE anion exchange column.The concentrations and chitinase activities of each sample from separation have been measured.The results show that the chitinase activity,purification fold,and recovery rate of the sample after DEAE anion exchange column was 14.67 U/mg,9.17,and 28.49%,respectively.Finally,in the SDS-PAGE,a single 50 kDa band was found in the lane of DEAE anion exchange column,thus,MEW06 fermented supernatant has a 50 kDa chitinase.The enzymatic properties show that the optimal reaction temperature and pH was 45 ?and 6 and enzymatic activity was relatively stable at 25 to 65 ? and pH 5 to 11.Zn2+ and K+can significantly promote chitinase activity.Mg2+,Fe2+,Mn2+,and Ag+ can significantly inhibit the chitinase activity.The order of inhibition ability of inhibitor against chitinase activity was:SDS>NaN3>EDTA.MEW06 genomic sequencing show that,the size of genome is 5.34 Mbp,the GC%amount is 60.99%,the predict gene number is 5058,gene average length is 914 bp,and the scaffold number is 48.MEW06 genome contain five kind of chitinase,GM000046,GM002360,GM002904,GM003301,and GM004442.Thereinto,GM002904 and GM004442 have signal peptide,and the molecular weight is 58.55 and 44.45 kDa,respectively.The different shaking flask fermentation conditions optimization show that the optimal fermentation conditions for chitinase production are pH 7.0,31 ?,3%inoculation,35%loaded-liquid,150 rpm of shaker speed and 48 h of incubation time.The response surface optimization of fermentation conditions optimization show that the optimized formula is:17.5 g/L of starch,8/1000(wt/vol)of colloidal chitin,20 g/L of peptone,0.56 g/L of K2HPO4,0.375 g/L of KH2PO4,0.5 g/L of MgSO4·7H2O,0.01 g/L of FeSO4·7H2O,and 0.01 g/L of ZnSO4.After the finally verification,the chitinase activity of fermentation supernatant using optimized medium has reached 197.32 U/mL,which is 2.58 times than initial medium.Both MEW06 fermentation liquor and fermented supernatant can significantly inhibit the growth of Bacillus thuringiensis 171.MEW06 fermentation liquor can significantly inhibit the growth of hypha of the plant pathogenic fungi,Botrytis cinerea and Physalospora piricola.The lethality of 1/4 of MEW06 fermentation liquor against Caenorhabditis elegans and Meloidogyn incognita was 100%and 58.74%,respectively.