| The Branched-chain amino acid aminotransferas(BCAT) activity, enzyme-producing and transamination conditions of 5 Staphylococcus strains selected from ham were studied in this paper. Branched-chain keto acid decarboxylase, alcohol dehydrogenase and esterase activities and the production of flavor compounds in the branched-chain amino acid metabolic pathway leaded by BCAT were also detected. All studies were designed to provide basis of basic research for BCAT enzymatic property and branched-chain amino acid metabolism. Results as follows:Firstly,5 Staphylococcus strains were selected from ham with the ability of BCAT enzyme-producing, and Staphylococcus saprophyticus RG-2 was chosen to be the enzyme-producing conditions optimizing strain though growth curve and BCAT activity tests (using leucine as substrate, and the same below). With the method of single factor test and response surface methodology, the optimized enzyme-producing conditions were:pH7.09, temperature 34.87℃,2.21% NaCl (22.1g/L). And the BCAT activity of Staphylococcus saprophyticus RG-2 under this condition was 38.59±1.58U/mL.Both β-mercapto-ethanol and glutathione could promote BCAT activity, hydroxylamine hydrochloride, EDTA and some metallic ion like Zn2+, Cu2+ may cause activity inhibition(1mM). With the method of single factor test and orthogonal test, the optimized transamination conditions of Staphylococcus saprophyticus RG-2 were:temperature 37℃, pH 8.5,4mM CTAB and 4mM PLP. And the BCAT activity of Staphylococcus saprophyticus RG-2 under this condition was 42.69±1.34U/mL.Next the main enzymes in the branched-chain amino acid metabolic pathway:branched-chain keto acid decarboxylase, alcohol dehydrogenase and esterase activities of 5 Staphylococcus strains were studied. All 5 Staphylococcus strains showed branched-chain keto acid decarboxylase activity, and Staphylococcus saprophyticus RG-2 had the highest enzymic activity, which reached 36.05±3.42U/mL. Staphylococcus xylosus X-6 showed the highest alcohol dehydrogenase activity (P＜0.05), which reached 88.97±2.91U/mL. And esterase activity of Staphylococcus saprophyticus RG-2 was also significantly higher than other 4 strains(P<0.05), which reached 126.67±2.60U/mg Pro.Only 3-methyl-1-butanol could be detected in the bacterium solution reaction system of 5 Staphylococcus strains using leucine as substrate. And the 3-methyl-1-butanol production reached 0.088±0.035mM from Staphylococcus saprophyticus RG-2, which was significantly higher than other 4 strains(P<0.05). It was also found that 3-methyl butanal adding could increase 3-methyl-1-butanol production. Only 3-methyl-1-butanol metabolized from leucine could be detected in the BCAT reaction system using three branched-chain amino acids together as substrate, but all corresponding alcohol metabolites were detected when three branched-chain amino acids were used separately. What’s more, the production content of three alcohol metabolites were not accord with the results of BCAT substrate specificity tests. The results of branched-chain amino acids metabolite influencing conditions experiments showed that 3-methyl-1-butanol conversion ratio increased with time going during 6-72h, but the increasing was not significantly (P>0.05). And 3-methyl-l-butanol conversion ratio was maximum when culture temperature was 45℃, culture pH was 6.4. NaCl concentration was 2.5%. |
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