Isolation,Purification And Characterization Of Cellulase Produced From Aspergillus Niveus MA35 In The Gut Of The Megalobrama Amblycephala

Cellulase is a kind of enzyme that catalyzes the hydrolysis of cellulose chain?-1,4-glycosidic bond and converts cellulose into glucose,cellobiose,and fiber oligosaccharide.Traditionally,it is divided into three categories:endoglucanase,exoglucanase,and?-glucosidase.Cellulase can be widely used in food,feed,textile,fermentation and other fields.Cellulase comes from a wide range of sources,such as insects,animals,bacteria and fungi,etc.And cellulase is mostly from microorganisms,especially bacteria and fungi.In this study,the cellulase of produced by fermenting Aspergillus niveus MA35 to obtain an intracellular endocellulase,and enzymology properties of the enzymes to do the research,to better serve vegetarian fish feed proportioning provides technical support,and to build fungi strains of cellulose enzyme engineering provides excellent genetic source.The cellulose purified from Aspergillus niveus MA35 fermentation bacteria by ammonium sulfate fractional precipitation,Q-Sepharose gel Fast Flow anion exchange chromatography,Sephadex G-100 chromatography.The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific was determined to be 36.6U/mL,the purification ratio was 6.13,and the recovery was 34%.According to SDS-PAGE polypropylene gel electrophoresis,the molecular weight of the enzyme was about 45 kD.The enzymatic properties indicated that the optimal reaction temperature was45°C,the optimum pH was 4.5,the Michaelis constant Km was 5.92 mg/mL,and the maximum reaction rate Vmax is 1.72×10^-1 mg/?mL·min?.In the temperature between 30to 55?and pH 4.0 8.0 has a good stability.The effects of several metal ions on the enzyme activity had been studied.The results showed that:Cl^-,I^-,Br^-,Na⁺,Li⁺,K⁺had no significant effect on enzyme activity,Zn²⁺,Mn²⁺had significant activation on enzyme activity,and Ca²⁺had slight activation effect on enzyme activity at low concentration,and had no significant effect on enzyme activity at high concentration.Cu²⁺,Pb²⁺,Fe²⁺have a strong inhibitory effect on the enzyme and are irreversible inhibition.The organic reagents ethanol,ethylene glycol,acetone and phenol have strong inhibitory effects on the cellulase,and their IC50 are 18.3%,8.3%,0.8%,and0.5%,respectively.So it can be judged that phenol has the strongest inhibitory effect on the cellulase,and ethanol has a slight inhibitory effect on the cellulase,which is consistent with the general rule of enriching proteins?enzymes?by the alcohol precipitation method in industrial production.The enzyme was modified respectively by several chemical modification reagents,such as bromoacetic acid,dithiothreitol,acetic anhydride,phenylmethylsulfonyl fluoride,p-chloromercuric acid,hydrogen peroxide,acetylacetone),and explore the necessary groups of the enzyme activity center,the results show that histidine imidazole and lysine epsilon-amino groups is the necessary of the enzyme activity center,sulphur,arginine residues,serine residue and methionine residue is not the necessary groups on the enzyme,disulfide bond has nothing to do with the enzyme activity.The inhibitory effect of bromoacetic acid on cellulase was reversible mixed inhibition,and the inhibitory effect of acetic anhydride on cellulase was irreversible non-competitive inhibition.