安卡红曲霉酸性蛋白酶分离纯化及部分酶学性质分析

安卡红曲霉(Monascus anka)CICC 40806培养液经硫酸铵分级沉淀和CM Sepharose Fast Flow阴离子交换柱层析两步分离纯化,得到纯度较高的胞外酸性蛋白酶,然后对其性质和动力学进行研究。结果表明M.anka酸性蛋白酶的最适反应pH值为3.0,pH值稳定范围为3.0~7.0,最适反应温度为50℃,低于50℃时酶具有较好的稳定性,50℃时的衰减常数为0.569。M.anka酸性蛋白酶以酪蛋白为底物时反应活化能为28.85 k J/mol,相对较低。M.anka酸性蛋白酶具有一定的耐盐性,当NaCl质量分数为7%时,最适条件下反应时相对酶活力为12%。以酪蛋白、米渣蛋白、牛血清蛋白为底物时,K_m值分别为12.48、14.77、20.05 mg/mL,对米渣蛋白和酪蛋白的催化效率相对较高。M.anka酸性蛋白酶可能在米渣蛋白发酵降解中发挥积极作用。

Purification and Some Enzymatic Properties of Acid Protease from Monascus anka

Extracellular acid protease was obtained from Monascus anka CICC 40806 by ammonium sulfate precipitation, followed by anion exchange column chromatography with CM Sepharose, and then enzymatic and kinetic properties were determined. The results showed that the optimal pH value for the enzyme activity was 3.0 and the enzyme was stable in the pH range of 3.0–7.0. Its optimal reaction temperature was 50 ℃ and the enzyme was stable at temperature below 50 ℃; the attenuation constant at 50 ℃ was 0.569. The activation energy (Ea) was 28.85?kJ/mol when the substrate was casein. The protease had good salt resistance, and its residual enzymatic activity was 12% at a salt concentration of 7%. When the substrates were casein, rice dreg protein (RDP) and bovine serum albumin, the Km values were 12.48, 14.77 and 20.05?mg/mL, respectively, and it had higher catalytic efficiency for RDP and casein. The extracellular acid protease could play a positive role in fermentation and degradation of RDP.