Screening Of Chitosanase-producing Strains And Optimization Of Fermentation Conditions

Chitooligosaccharide is a polysaccharide with wide application value.The traditional industrial production of chitooligosaccharides mostly uses concentrated acid and alkali,which pollutes the environment.The preparation of chitosan oligosaccharides by enzymatic degradation of chitosan has the characteristics of mild reaction,uniform products and no pollution,which is a chitosan oligosaccharide production method with research and application values.In this study,a chitosanase producing fungus was screened and the fermentation conditions of chitosanase production were optimized,and the enzymatic properties of the chitosanase were studied.This study lays a foundation for the production of chitosanase by fermentation and the chitosan oligosaccharides.Below are key research findings:?1?In this study,the rich resources of shrimp and crab in Yantai beach and the humus soil in Yantai university were selected as the isolated source of the strain.The colloid chitosan was used as the only carbon source in the primary screening plate.By comparing the growth period,the size of the bacterial colony and the transparent circle,and the ratio of the diameter of the transparent circle to the diameter of the bacterial colony,7 strains were screened out as the re-screening strain.Enzyme activity was determined by DNS method to further screen the strains with strong enzyme producing ability.By comparing the initial enzyme activity of these 7 strains,the strains with high enzyme producing activity were selected and identified as aspergillus by morphology.?2?Through optimization of single factor and orthogonal experiments,the optimal conditions for fermentation enzyme production of this strain were obtained:fermentation temperature 27?,initial pH 5.5,loading volume 30%?v/v?,inoculation volume 9%?v/v?,rotation speed 150r/min,fermentation time 120h.The optimum carbon source was 1.0%?w/v?chitosan+1%?w/v?glucose,carbon source was 0.5%?w/v??NH₄?₂SO₄,the optimal metal ions of Mn²⁺.Fermentation was carried out under the suitable conditions,and the enzyme activity of the fermentation broth was measured to reach 18.68 U/mL,which was80%higher than the initial enzyme activity.?3?The enzymatic properties of the chitosanase produced by the screened strain SM2019-6 in this experiment:the enzyme activity was relatively stable in the range of pH2.0⁶.0,and when the pH value was greater than 6,the enzyme activity gradually decreased with the increase of pH.When pH=9,enzyme activity is basically lost.In terms of temperature,when T?55?,the enzyme activity decreased rapidly,and when the temperature reached about 80?,the chitosan enzyme in the solution was basically destroyed.Moreover,5 mmol/L Mg²⁺had a significant activation effect on chitosanase.?4?Through the study of the enzymatic hydrolysis conditions of chitosan enzyme,the optimal conditions for the enzymatic reaction were obtained:the optimal reaction time between chitosan enzyme and substrate was 10 min,the optimal enzymatic hydrolysis temperature was 50?,and the optimal pH was 6.0.