Extracellular Expression And Fermentation Optimization Of Chitosanase From Aspergillus Fumigatus In Escherichia Coli

Chitosanase can specifically hydrolysis ?-1,4 glucosidic bond of chitosan andproduce the value chito-oligosaccharides(COS).Production of chito-oligosaccharides is the improtant industrial research on the chitin.Moreover,it has considerable application prospect in the food,medicine,agriculture and other industries.Therefore,it has been attached great importance by the researchers both at home and abroad.The bioactive and physiological effects of COS need specific degree of polymerization.A potential industrial endo-chitosanase from Aspergillus Fumigatus,further identified as a member of glycosyl hydrolase family 75,has show stronge endochitosanolytic activity toward chitosan with broad range of acetylation and can produce oligosaccharides with degrees of polymerization from 3-6.However,the enzyme is the same as that of the other G75 family of enzymes.Structure has not been resolved,The analysis of the structure and function of CSN is not only important for understanding the structure and function of the enzyme,but also can be used as a template for the structure prediction and molecular simulation of G75 family.at present,the chitosan enzyme is expressed in the form of inclusion body in E.coli.The study object is derived from the Chitosanase from Aspergillus Fumigatus,signal peptide pelB was used to the secretory expression.At first,The effect of pelB-CSN and histidine tag(6xHis-tag)fusion on its expression was investigated.To facilitate its later purification,N-terminus or C-termianl 6xHis epitope tag was added to the pelB-CSN protein fusion.Our results indicated that pelB-CSN without 6xHis-tag(pelB-CSN)or with N-terminus 6xHis-tag(pelB-CSN-N)can both be effectively secreted into the medium,while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies.Process optimization strategies were further developed to improve the secretion efficiency of recombinant pelB-CSN-Nin E.coli.Under the induction of 10 g/L lactose in shake-flask culture,the extracellular activity of CSN reached 6,015 U/mL at 25? in TB medium containing 1%glycine.Moreover,a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter,increasing the extracellular CSN to 1.65 mg/mLin 44 hours fermentationwith the optimal addition of lactose and glycine.This provided the conditions for the further study of the structure of the enzyme.