The Study On Chitosanase Production By Aspergillus Fumigatus ZX2019

Chitooligosaccharide,as the product of chitosan degradation,is more widely used in many fields such as health care products,medicine,food additives,feed and fertilizer than chitin and chitosan due to its good water solubility,high biological activity and easy absorption by organisms.Compared with acid method and oxidation method,enzymatic preparation of chitooligosaccharides is more ideal,with mild reaction conditions,controllable production and environmentally friendly.Therefore,screening strains with strong enzyme production ability and optimizing their enzyme production conditions become an important work,which has great practical application value.Firstly,the strains screened in the previous laboratory were identified,and then the fermentation conditions of shake flask and fermentation tank were optimized.The chitosanase was isolated and purified,and its enzymatic properties were preliminarily studied to provide theoretical basis for future industrial applications.The results are as follows:（1）In this study,a Aspergillus fumigatus strain with high chitosanase production was screened by the microbiology laboratory of Yantai University.ITS sequence was identified as Aspergillus fumigatus,and it was named Aspergillus fumigatus ZX2019.Firstly,single-factor experiments were conducted for metal ions,complex metal ions and complex nitrogen source concentration（w/v）,and based on this,further optimization of fermentation conditions using response surface methodology for three factors,temperature,p H and carbon source.The results showed that the chitosanase activity reached 10.18U/m L when the fermentation conditions were 0.94%chitosan（w/v）and 1.06%glucose（w/v）as complex carbon sources,0.5%（w/v）（NH₄）₂SO₄as nitrogen source,p H 5.4,temperature 26.6℃and 0.050%（w/v）Mn²⁺+0.025%（w/v）Fe²⁺as metal ion concentration.（2）Based on the optimum shake flask fermentation conditions,the fermentation process of this Aspergillus fumigatus was optimized in a 10 L fermenter.The results showed that:the optimum stirring speed of the fermenter was 150 r/min;the enzyme activity was higher and more stable when the fermentation was carried out at a constant p H 5.4;the suitable supplementation method for chitosan was supplementation method 1,and the maximum enzyme activity reached 19.75 U/m L.（3）The chitosanase produced by Aspergillus fumigatus was isolated and purified,and the crude enzyme solution was first purified by 70%saturation ammonium sulfate salting,dialysis bag desalting and DEAE-cellulose anion exchange column,and then the molecular weight of the enzyme was determined by SDS-PAGE electrophoresis,and the molecular weight was estimated to be about 29.1 k D.The enzymatic properties of the purified chitosanase were investigated,and the results showed that the enzymatic reaction was promoted by the metal ions Ca²⁺,Mg²⁺and Mn²⁺at an appropriate amount.The metal ions Ca²⁺,Mg²⁺and Mn²⁺,in moderate amounts,promoted the enzymatic reaction,with Mn²⁺having a stronger promotion effect,while Na⁺and Zn²⁺had almost no effect on the enzyme activity,while Al³⁺,Cu²⁺and Fe²⁺inhibited the enzymatic reaction at set concentrations,with Cu²⁺had the strongest inhibition effect.The optimum chitosan concentration during the enzymatic reaction was 1%（w/v）.