Breeding On Aminopeptidase-producing Strains And Optimization Of Its Fermentation Conditions

Aminopeptidase, a collection of enzymes which can remove amino acid from the N-terminus of peptides one by one, is very important in protein hydrolysate production and debittering. The purpose in the paper is to raise productivity of aminopeptidase by mutation breeding and optimization of fermentation nutrient medium. Two treatment methods, Co⁶⁰-rays and N⁺ implantation, were used in the mutation experiments. N⁺ implantation is more effective than Co⁶⁰-rays treatment, and raise productivity from 231.5 u/ml to 323.1 u/ml. After preliminary optimization on fermentation condition the yield reach 547.1u/ml. Study on fermentation condition shows that supplying monosaccharide and surfactant, lengthening the fermentation time can raise fermentation productivity.Nutrient medium formula is optimized using statistic method. Plackett-Burman experiment shows that wheat bran, syrup and KH2PO4 have noticeable effect on the aminopeptidase productivity. Set the value of the three factors near the right zone in the rapid-uphill experiment and conduct RSA experiment. Result is analyzed in Minitab, and the optimum formula is wheat bran 81.0g/L, syrup 36.0g/L, KH2PO410.3g/L,corn steep 12.0g/L,MgSO4·7H2O0.4g/L,CaCl21.0g/L,(NH4)2SO41.0g/L,PEG1.0g/L. After optimization the productivity reach 643.9u/ml. On 5.0L fermentation tank, keep constant temperature at 36℃and regulate pH to 6.8 after fermentation 20 hours, the yield go up to 723.8u/ml.The aminopeptidase reach the maximal activity at 60℃and pH8.0 respectively, and show relative stability at pH 6.0～9.0. The optimal technique parameters for bone power degradation with aminopeptidase and protease are as follows: 45℃, pH7.0, 2% for enzyme quantity, 1: 3 for protein and aminopeptidase ratio.