Identification And Condition Optimization Of Chitosanase-producing Strain Bacillus Subtilis GZ1 From Guangxi Subtropical Marine Mangrove Soil Sediments

Chitosan is the unique natural cationic polysaccharide with a great reserve in nature,and it is widely distributed in the environment.As the hydrolytic product of chitosan,it has the same properties with chitosan just like inhibition,anti-tumor and antioxidant.Furthermore,it can be used in industrial production more appropriately because of its low molecular weight,high water solubility and absorbable.Mangrove ecosystems is of complex structure,and there is a lot of shellfish living in it,it is an important source of natural chitosan.Thus,we isolated four chitosanase producing strains which could produce hydrolysis circles on chitosan agarose plates in the marine sample by pure culturing method,then we cultured these four strain in liquid secondary screen medium to determine their chitosanase activity,and the GZ1 was selected to be identificated because of its highest activity in the four strains.Identification results indicated that the GZ1 was a gram-positive strain with spore and capsule,and its bacterial colony was yellow and round,its edge of colony was irregular wave.We constructed the 16S rDNA phylogenetic tree of GZ1,combined the phylogenetic tree with the result of physiological and biochemical properties identification,we identified the GZ1 to be Bacillus subtilis,and it is named after Bacillus subtilis GZ1.DNS colorimetry method was applied to determine the chitosanase activity of fermentation broth.The optimum reaction temperature of crude enzyme was determined,and the optimum reaction temperature and pH was 55 ? and 5.5 respectively,besides,there was 45%Relative activity remained when the reaction temperature came to 85 ?.The single factor experiment contained culture time,nitrogen source,carbon source,pH value,temperature,rotate speed,liquid volume and chitosan concentration was performed,and the four-factor and three-level orthogonal experiment was designed based on the result of single factor experiment.The result of orthogonal experiment indicated that the optimum culture temperature,pH value,liquid volume,nitrogen concentration,carbon concentration,rotate speed and chitosan concentrati-on was 20 ?,pH 7.5,80 mL/250 mL,1.0%typtone,1%soluble starch,180 rpm and 0.75%respectively.In the end,the highest chitosanase activity of fermentation broth is 1.886 U/mL.In our studies,the results of orthogonal experiment were different from single factor experiment,which indicated that the interaction of various factors had a great influence,thus,optimuming the fermentation condition of Bacillus subtilis GZ1 with response surface method could be better.Compared with the recombinant chitosan enzyme Bacillus subtilis reported,the enzyme-producing ability of Bacillus subtilis GZ1 was lower due to its unmodified,so we could improve the enzyme-producing ability of Bacillus subtilis GZ1 by the method of reforming the chitosan gene's promoter and copy number.Bacillus subtilis is a common addition in animal feed,The chitosan of Bacllus subtilis GZ1 had a high reaction temperature of 55 ?,which was beneficial the production of animal feed industry.