Chitinase From Brevibacillus Brevis FM4B: Fermentation, Cloning And Enzymatic Properties

Chitinase can hydrolysis the beta-1,4 glycosidic bond of chitin which is the second-d sugar except for cellulose to N-acetyl glucosamine and chitosan oligosaccharide. E-zymatic degradation of chitin is the most environmentant friendly and efficient method up to now. At the same time chitinase can kill pathogenic fungi.Therefore it have hug-e application prospect in biological control of plant diseases and pests and diseases dur-ing storage of fruit and vegetable.In this paper, we studied the purification and characterization of the chitinase from B revibacillus brevis FM4B firstly. Chitinase was purified by centrifugation, ethanol prec ipitation and Sephadex G-100 chromatography. Purified chitinase with a purification fa ctor of 7.19 and a recovery of 30.6% was obtained. SDS-PAGE determinated the mol-ecular weight. The enzyme had an optimum temperature of 50 ℃ and an optimum pH of 6. O,molecular weight of 66kDa. It also showed good stability in the pH range from 5.0-9.0. The enzyme was inhibited intensively by Cu2+, Hg2+, Pb2+, Co2+and Zn2+ wi-th 0.0 01mol/L, while was slightly activated by Mg2+ and Ca2+ with 0.001mol/L. FM4B chitinase can obviously inhibit different kinds of fungi. The Brevibacillus brevis FM4B chitinase with high thermal stability,good pH adaptability and significant antifu-ngal effect.On the basis of the above research,this paper then study the fermentation process of the chitinase.The optimal fermentation conditions:colloidal chitin is the most suitable carbonsource,peptone was the best nitrogen source,the most suitable to their contents were 1%.4% inoculation quantity, pH6.0 of initial fermentation loading volume and 24h,210r/min at 28 ℃, is the most suitable speed fermentation,the enzym eactivity of 312U/mL.In order to obtain the chitinase, control of plant pathogenic fungi more effectively. The third part of this paper researched the molecular biology of the chitinase and const ructed efficient inducible expression engineering strain for chitinase.Characterization and optimization of fermentation conditions and study of recombinant enzyme. Throu-gh the genebank,the primers were designed from the strain FM4B,success in obtaining the enzyme gene,by PET-22b plasmid of the gene into E.coliBL21 (DE3), in IPTG ind-uced conditions,the enzyme was highly expressed,the optimal reaction temperature-e and pH of the recombi inant enzyme were 50 ℃ and 6. At 20-50 ℃,pH5.0-7.0 has better temperature and pH stability; promoting and strongest inhibitory action on 0.001mol/L CaCl2 and MnCl2.4H2O using colloidal chitin as substrate;the Km value is 2.830Mg/mL and Vmax value was 5.35mg/mL·min-1, the target protein molecular weight is about 60KD, the phase i s almost with the wild enzyme molecular weight. When the 50mL/(250mL) triangle bottle inoculation of 1%, IPTG induced time of 6 hours,IPTG induced concentration of 67.2mol/L,induced bacteria concentration reached OD6002.403, induced by the temperature of 25 ℃, the speed of 200r/min indu ction, the recombinant enzyme activity is highest,the enzyme activity of more than 20 times than wild enzyme. The construction of engineering strain and expressing effect-ively lay a foundation for the industrial production of chitinase.