The Study Of Chitosanase-producing Strain On Mitsuaria Sp.C4

Chitosanase(EC 3.2.1.132)is an enzyme with systematic name chitosan N-acetylglucosaminohydrolase,which specifically hydrolyzed linear chitosan into oligosaccharide.The oligosaccharide with low molecular weight is soluble in water and can be absorbed by the human body easily.At present,the known chitosanase producing strains generally do not meet the requirements of industrial production.Then screening for new chitosanase strains and improving its enzyme production are waluable.We employed a chitosan plate assay to screen for chitosanase producing strains.We found one strain with high chitosanase-producing ability from the coastal soil of Fujian,Quanzhou,which belongs to Mitsuaria sp.and was referred to as C4.Subsequently,we obtained a high yield strain Mitsuaria sp.ZY3 through ultraviolet mutagenesis breeding.The Mitsuaria sp.ZY3 strain possesses both high genetic stability and enzyme activity.The enzyme activity of the chitosanase purified from the Mitsuaria sp.ZY3 strains was increased by 2.77-fold compared with that of the C4 strain.A series of single factor and Response Surface were studied on the fermentation conditions of the C4 strain,the best fermentation condition is(w/v):1.6%colloidal chitosan,0.14%K2HPO4.3H2O,0.06%KH2PO4,0.1%MgS04.7H20,0.6%peptone and 0.06%yeast were used as the fermentation medium.Fermentation were performed in Erlenmeyer flasks containing 35 mL of media at 30? with an intial pH 6.5;Flasks were maintained in an orbital shaker at 150 rpm for 72 hours.Under the best condition,the enzyme activity can reach 7.892 U/mL.The chitosanase was then purified by 70%-80%ammonium sulfate precipitation,dialysis and Sephadex G-75 gel filtration.SDS-PAGE analysis shows a clear protein band,suggesting the molecular weight of the chitosananse purified from the C4 strain is 34 kDa.The optimum hydrolysis conditions for the chitosanase is under pH7.2 and 40?,the conceltration of Ietal ions in the solution of 20 mol/L,Mg2+ can promote enzyme activity at 20 mmol/L,while Zn2+,Ca+,K+,Mn2+have different degrees of inhibition on the enzyme activity.When powder chitosan as the substrate,the enzymatic reaction apparent Km value is 2.449 g/L and Vmin is 4 × 102 g.L-1.min-1.The genome of Mitsuaria sp.C4 is extracted,we combine the two and three generations of sequencing.According to the sequencing results,we assemble the annotation and analysis,and obtain a complete circular genome of about 6 Mb.