黑曲霉金属蛋白酶的克隆表达与性质鉴定

Cloning,Expression and Characterization of Metalloproteinase MPase from Aspergillus Niger

在毕赤酵母中克隆表达黑曲霉CGMCC 3.7193中一个疑似金属蛋白酶基因MPase,分离纯化后对重组酶MPase进行理化特征分析。结果显示,纯化后的MPase:比酶活达到1859.2 U/mg,最适反应温度35℃,最适反应pH7.0;在低于40℃,pH5.0~8.0之间稳定;偏好水解大豆分离蛋白,K_(m)和V_(max)分别为3.9 mg/mL、892.7 mg/(mL·min);金属离子Co^(2+)和Zn^(2+)对其有激活作用,Cu^(2+)和Fe^(2+)对其有抑制作用;酶解大豆分离蛋白水解度可达到14.7%,并且产物分子量大小均匀;可耐受5000 mmol/L NaCl,在50℃保持30 min即可完全失活。其酶学特征表明:MPase在食品工业尤其在发酵食品加工中具有较大应用潜力。

A predicted metalloproteinase gene,MPase,from Aspergillus niger CGMCC 3.7193 was cloned and expressed in Pichia pastoris.The physicochemical characteristics of recombinant MPase were investigated after purification.The specific activity of the purified MPase reached 1859.2 U/mg.Optimum temperature and pH for MPase activity were 35℃ and 7.0,respectively.MPase remained stable up to 40℃ and within the pH range of 5.0 to 8.0.MPase preferred soybean protein isolate as substrate,and K_(m) and V_(max) were 3.9 mg/mL and 892.7 mg/(mL·min),respectively.MPase was activated by Co^(2+)and Zn^(2+),and inhibited by Cu^(2+)and Fe^(2+).The degree of hydrolysis of soybean protein isolate reached 14.7%,and hydrolysates were uniform in molecular weight.MPase could tolerate 5000 mmol/L NaCl,and lost its activity completely after 30min at 50℃.These enzymological characterizations indicated that MPase had great application potential in the food industry,especially in fermented food processing.