Study On Mutation Breeding Of Higy-yield Aminoacylase Producing Aspergillus Oryzae And Fermentation Conditions

Aminoacylase is a kind of specificity of extracellular enzyme hydrolysis of DL-amino acids.DL-amino acids can be broken up for D-amino acids and L-amino acids and thus has important applicationin food, medicine and other fields.This paper use laboratory preservation of aspergillus oryzae A0as strains, will take place afterthe preparation of protoplast uv-lithium chloride mutagenesis, eventually selected a strain A15ofhigh-yield aminoacylase and good genetic stability mutagenesis.The best condition of the strain of the preparation of protoplast: fungus age of30h, ratio ofcompound enzyme for1.5%snail enzyme,1%lysozyme，1.5%cellulose enzyme, enzymolysis reactiontemperature for28℃, enzymolysis reaction timeof3h,0.8mol/L NaCl as osmotic pressure stabilizer.Under this condition, the rate of protoplast formation and regeneration of Aspergillus oryzae strain A0isgood.The best condition of the uv-lithium chloride mutation: mutagenic time of80s, mutagenicstrength to10cm, lithium chloride concentration of0.9%. In the power of15W under the irradiation ofultraviolet lamp, strain A0is mutagenized according to the above conditions. After initial screening,selected15mutagenic strains of discoloration circle diameter and colony diameter ratio is greater than thestarting strain.After measuring enzyme activity,5mutagenic strains’s enzyme activity are better than theparent strain.The enzyme activity of the mutant A15increased by95.4%than the enzyme activity of thestarting strain A0, up to589.7U/g.According to study the carbon sources, nitrogen sources, the ratio of carbon and nitrogen, theratio of material and water, inoculation, activators, inorganic salt, initial pH and temperature on theinfluence of high yield aminoacylase, the experimental result shows that the best carbon source is wheatbran, the best nitrogen source is soybean meal, the best ratio of carbon and nitrogen is8:2, the best ratio ofmaterial and water is1:1, best inoculation amount is8%, the best activator is0.1%twain-80, the bestinorganic salt is MgSO4, the optimal initial pH is7.0, the optimal temperature is30℃.According to the results of single factor experiment,we do the PB and BBD experiments, and find that the high-yield aminoacylase of the three main factors are wheat bran, soybean meal, and the ratio ofmaterial and water. Sythesizing the single factors results, we optimize the culture medium composition andculture conditions of enzyme production, is as follows:250mL shake bottle sample amount is wheat bran8.12g, soybean meal2.06g and the ratio of material and water is1.05,0.2%(mass fraction) MgSO4,adding1%(mass fraction) twain-80, the initial pH7.0, culture temperature30℃, inoculation amount8%.