Purification And Characterization Of Two Proteases Related To The Flavor Of Wine-lees Fish

Wine-lees fish, a kind of important traditional food, is popular for its special flavor and longer shelf life. During the processing and fermentation of wine-lees fish, various endogenous and exogenous proteases hydrolyze proteins into peptides and amino acids, which are converted into small molecular compounds by further reaction with respect to the flavor and quality of product. However, the effects of proteases on the formation mechanism of unique flavor, is still poorly understood.In order to investigate the effects of muscle endogenous enzyme on wine-lees fish flavor, an aminopeptidase was purified and characterized from black carp muscle. The peptide mass fingerprinting of this enzyme suggested that it was a puromycin-sensitive aminopeptidase which was purified to homogeneity by ammonium sulfate fractionation and three chromatographies. Puromycin was further confirmed as a competitive inhibitor with Ki value of 0.25 μmol/L. The enzyme was named bcPSA. The molecular masse of bcPSA was approximately 100 k Da, its optimum temperature and pH were 40 ℃ and 7.5, thermal stability of it was poor. Among many fluorescent substrates for aminopeptidase, the bcPSA preferentially hydrolyzed substrate Lys-MCA and Leu-MCA. Using LysMCA、Arg-MCA and Val-MCA as substrates, the Km of bcPSA were 4.8 μmol/L, 2.9 μmol/L, 3.6 μmol/L; the Vmax of bc PSA were 200 nmol·L^-1·min^-1, 210 nmol·L^-1·min^-1, 56 nmol·L^-1·min^-1, respectively. Salt and alcohol had a certain inhibitory effect on the activity of bcPSA. At the concentration of 3.2% NaCl and 5% ethanol, the enzyme remained 58.5% and 87.4% of its initial activity, respectively. The aminopeptidase（s） activity decreased slowly and remained 41.1% of its initial activity even after twelve days salting of black carp muscle. These results suggested the possible contribution of fish aminopeptidases to free amino acid formation and flavor generation in wine-lees fish.One bacterial strain, producing high yield protease, was isolated from unsterilized wine-lees fish by the way of casein plate method and UV spectrophotometry. It was suggested that strain Bs. Lee was the closest relative of Bacillus subtilis based on physiobiochemical characteristics and phylogenetic analysis of 16 S rDNA of the strain. The logarithmic phase growth of the strain was 2⁸ h and it produced a lot spores at stable stage, the rate of spores was more than 90% after culturing of 20 h. Na Cl could restrain the growth of strain Bs. Lee. The value of OD600 was 20% of the control group at the concentration of 9% Na Cl, suggesting the strain was salinity tolerant. An extracellular protease, molecular weight of about 60 kDa, was purified to homogeneity through the chromatographies procedure as like that of bcPSA. Its properties were studied by casein zymography. The optimum temperature and pH of the protease were 40℃ and 8.0. The protease activity was almost completely inhibited by 10 mmol/L EDTA, 10 mmol/L EGTA, and it was slightly activated by 5 mmol/L benzamidine. Ca²⁺, Ba²⁺ could obviously enhance the enzyme activity. In the range of 0.5³ mol/L, the enzyme activity enhanced with the increase of concentrations of NaCl and the protease behaved high salt tolerance in 18% NaCl. These results showed the enzyme was a neutral high salt tolerant metalloprotease. It was suggested that Bacillus subtilis could grow well and secreted a lot protease under the conditions of wine-lees fish processing and the protease would contribute to the degradation of protein so as to influence the quality of the products.