β-glucosidase Study From Mutation And Selection Of Aspergillus Niger

β-glucosidas is an enzyme which can hydrolyze cello-oligosaccharide and cellobiose to glucose.It is widely existent in various living things.It plays an important role in the hydrolysis of cellulose by converting cellobiose to glucose,and in food-making and pharmacy and so on. Breeding of good strains and optimization of enzyme production medium condition is the focus of the study.The study is to obtain mutated strains of high-activityβ-glucosidase and optimization of enzyme production medium condition of good mutated strains which treated by UV light.The main results are as follows:(1)Optimization of enzyme production medium condition of Aspergillus niger 30786.The effect of carbon sources, nitrogen source,initial pH on producedβ-glucosidase was studied,based on which the conditions for producingβ-glucosidase by Aspergillus niger were optimized by response surface methodolog.Results show that the three factors all have effect onβ-glucosidase activity,and the optimal conditions are as follows:corn-core 21.5g/L,(NH4)2SO46.4g/L and initial pH5.2,and theβ-glucosidase activity can be up to 5.34U/ml.(2)Breeding of mutated strains C112.Aspergillus niger 30786 treated by UV light for certain time,the mutated strains were selected by shake flasks fermentation,and obtained a high-yield strains Aspergillus niger C112,and the enzymatic ability was good stability.(3)Optimization of enzyme production medium condition of mutated strains C112.Plackett-Burman design (PBD) was used to evaluate the effects of eight medium components on the production ofβ-glucosidase by Aspergillus niger C112, which showed that corn-core,(NH4)2SO4 and KH2(PO4) were the most significant ingredients (P<0.05).The steepest ascent experiment was adopted to determine the optimal region of the medium composition.The optimum composition of the fermentation medium for maximumβ-glucosidase yield,as determined on the basis of central composite design (CCD),was obtained by response surface methodology.The high correlation between the predicted and observed values indicated the validity of the model. A maximumβ-glucosidase yield of 7.22U/mlwas obtained at 30.7g/L corn-core,7g/L(NH4)2SO4 and 6g/L KH2(PO4),and the production was increased by 35% compared with Aspergillus niger 30786.(4)genetic and variation of reference strain and mutated strains.The randomly amplified polymorphic DNA(RAPD) patterns of whole-cell lysates from five Aspergillus niger strains,including one reference strain,four mutated strains C112,C206,C12 and C89,were investigated.PCR-RAPD analysis of genomic DNA was performed using twenty primers (S65,S66,S2160,S2010,S10,S1404, S13,S1211, S1301,S1209,S212,S25,S177,S1103,S1121,S1125,S451,S1037,S1016 and S1214).The RAPD assay discriminated between five strains.The data indicates that the the similarity coefficient varied from 0.87 to 0.89 indicating variability among the four mutated strains and reference strain.