Studies On The Optimization Of Fermentation And Preliminary Application About Aeromonas Sp.Ch2 Producing Chitinase

Chitinase(Ec.3.2.14),a group of enzymes capable of degrading chitin directly to chitin oligosaccharides or N-acetylglucosamine,is produced by a wide variety of organisms including plants,microorganism and animals.Chitinase has received increased attention because of its potential application in biocontrol of plant-pathogenic fungi and pests,as well as in bioconversion of shellfish chitin wastes.Ch2 was one of chitinase-producing strains conserved in our lab. Base on the results of physiological and biochemical characteristics determined by standard methodology and and Biolog,it was identified belonging to the genus Aeromonas sp.Ch2 and named as Aeromonas sp.Ch2.The growth property and growth conditions were also identifiedThe conditions of chitinase production by Aeromonas sp.Ch2 were then evaluated.The results showed that the optimal conditions of fermentation of Ch2 were as follows:using 0.5%colloid chitin as the carbon source,1.2%peptone and 0.2%urea as the nitrogen source,0.05% MgSO₄,0.03%K₂HPO₄,0.5%NaCl,50ml media in 250ml flask. Inoculum was 6%,inoculum age 16 hours,initial pH value 6.5-7.0, culturing temperature 28℃,rotated speed 180 r/min,and incubated it for 3 days.The chitinase activity of fermented culture medium could reach 1.69 U/ml under the optimal condition,which was 7.5 times higher than pre-optimization.The batch and feed-batch process of chitinase production by Aeromonas sp.Ch2 in a 3 L stirred fermenter was then evaluted.It was found that the feed-batch process was helpful to increase the chtinase production with the highest chtinase yield of 1.802 U/ml and that the peak of activity appeared in 65 hours,7 hours prior to that by batch process.After precipitation with ammonium sulfate(60%saturation,4℃)and affinity chromatography on regenerated chitosan,the chitinase extract was purified to be as one band after SDS-PAGE.The potential of purified chitinase from Aeromonas sp.Ch2 as antifugi agent and depolymerization of chitosan was evaluated.It was found that the crude chitinase showed better antifungi effect than that of whole cell of Aeromonas sp.Ch2.The results of chitosan depolymerization showed that the crude chitinase could easyly degrade chitosan with lower degree of deacetylation.