Optimizations Of The Fermentative Condition And Application Of Aminopeptidase By Bacillus Subtilis Zj016

The solubility, the thermo stability and the nutrition of protein are obviously improved after hydrolysised, but its hydrolysate generally appeared bitter and was limited in application of food. Aminopeptidases can be debittering exopeptidases that selectively release N-terminal hydrophobic amino acid residues from bitter polypeptides. In this paper, the fermentation conditions, the enzyme properties and the application in SPI were investigated.Optimizations of the fermentation condition was carried out as follows: Corn starch 40g/L, Soybean dregs 30g/L, K2HPO4 3.216g/L, KH2PO4 0.074g/L, MgSO4?7H2O 0.3 g/L, NaCl 1 g/L, CoCl2 1mL(0.1mol/L), temperature 40oC, rotate speed 200r/min, pH 7.5, inoculum size 8%, volume 20mL/250mL.The aminopeptidase activity was2685.6U/mL after optimization, which increased 1.71 times. The optimum conditions were investigated on aminopeptidase in 7L jar fermentor were analyzed, the aminopeptidase activity reached 1200 U/mL.As for aminopeptidase produced by Bacillus subtis Zj016 after optimization, the purification procedure of ethanol fractionation(20%～40%) was studied on. The activity was optimal at pH 8.5 and 55°C. The temperature stability was 20～70℃and the pH stability was 8.0～10.0. While inhibited by the gelatin and other chemicals, the enzyme activity was not significantly influenced by Glycerol and peptone. Inhibited by several divalent cations (Zn2+,Ni2+) and EDTA. Activated by Co2+. The activation region of the enzyme seemed to had two Zn2+, and each subunit had one. Km of the aminopeptidase was 0.53m mol/L and Vmax was 19.23μmol/L/min.The limited hydrolysis optimum conditions of SPI by aminopeptidase and basic protease was studied as follows: hydrolyzing temperature 50℃, pH8.5, ratios of both enzymes /substrate 10000U/g, concentration of substrate 60g/L, total hydrolysis time 4h, hydrolysis degree of protein 25.0% and its hydrolysis product is a few bitter. The comparisons of bitter value, distribution of molecular weight, hydrophobic value and free amino acid between the hydrolysis product by the synergetic hydrolysis and the hydrolysis product by basic protease: their types of elution and distribution and proportion of molecular weight were different. the fomer was less than the latter in the hydrophobic value. and as for free amnio acid ,the fomer was 1610mg/L, and the latter was 1250mg/L.