Screening Of Chitosanase-Producing Mutant And Study On Its Fermentation Technology

Three strains which could produce a high endo-chitosanase activity were isolatedfrom nature, based on enriched culture, primary screening, secondary screening, viscosityrapid determination and TLC analysis. Througth identifying their morphological,physiological and biochemical characteristics and 16 S rDNA, all of strains wereattributed to Mitsuaria sp., named Mitsuaria sp. 013, 141 and 281, respectively, in whichthe Mitsuaria sp. 141 with the highest chitosanase activity.To take Mitsuaria sp. 141 as the original strain, conduced by UV and ⁶⁰Coγ-raymutagenesis, the mutant Mitsuaria sp. 141-2 whose chitosanase activity increased 30.01ï¼…was obtained, and the high chitosanase activity of the mutant could be inherited aftercontinuous passages culture.By the single factor test and orthogonal test, the optimal condition of Mitsuaria sp.141-2 for the production of chitosanase was determined: colloid chitosan 0.50ï¼…, mediumvolume 100 mL in 500 mL flask, initial pH7.0, inoculation amount 2ï¼…, temperature 30℃,shaker speed 180 r/min, culture time 48 h. Under these conditions, the chitosanaseactivity reached 3.631 U/mL. Expand culture in 10 L fermenter, with medium volume 7L, agitation intensity 200 r/min, aeration rate 1 vvm, culture time 36 h, the chitosanaseactivity of fermentation liquid reached the highest at 3.258 U/mL.TLC analysis showed disaccharide or other chitooligosaccharides exist in thehydrolysates of chitosan. To determine the number average molecular mass of chitosanwith the acetylacetone method, the result indicated that the ratio of volume of 1.5ï¼…colloidal chitosan to crude enzyme was 15:1, 37℃, reaction 30 min, the number averagemolecular weight of hydrolysates dropped to 10921. Change the volume ratio to 10:1, 5:1,2:1, 1:1 and 1:2, the molecular weight of hydrolysates became smaller and smaller,respectively as 5683, 3018, 1247, 779 and 747.The induced-chitosanase of Mitsuaria sp. 141-2 was purified by ammonium sulfateprecipitation, centrifugal ultrafiltration and Sephadex G-200 gel chromatography. Themolecular weight of chitosanase was estimated to be about 33.6 kD by SDS-PAGE. Theoptimal pH and temperature were 4.0 and 37℃-42℃, respectively. The enzyme activitywas relatively stable below 45℃in a range of pH4.0～pH8.0.