天祝传统藏曲中蛋白酶产生菌的筛选与酶学特性研究

Research on Screening of Protease-producing Strains from Tianzhu Traditional Zangqu and Enzymatic Properties

采用酪蛋白平板对藏香型白酒大曲中高产蛋白酶菌株进行分离筛选,然后通过硫酸铵沉淀、透析和DEAE-Sepharose FF阴离子交换层析对其产生的蛋白酶进行纯化,并研究其酶学性质。结果表明:菌株ZP-8产酶活力最高,为197.6 U/mL,经鉴定为地衣芽孢杆菌(Bacillus licheniformis)。纯化后测得酶的分子量约为45.43 kDa,最适pH值为8.0,在pH 7.0~11.0的范围内稳定性好,属于碱性蛋白酶。该酶最适反应温度为50℃,在30~50℃范围内稳定性较好;Mg^(2^(+))、Na^(+)、Ca^(2^(+))和Fe^(2^(+))对蛋白酶相对酶活力无显著影响(P>0.05),Cu^(2^(+))和Mn^(2^(+))具有显著的激活作用(P<0.05)。本研究对酒曲中产蛋白酶微生物的开发利用具有重要的参考价值。

In the experiment,the casein plate was used to isolate and screen the high-protease-producing strains from Tibetan-flavor liquor Daqu.Then,ammonium sulfate precipitation,dialysis desalting and DEAE-Sepharose FF anion exchange chromatography were used to purify the protease,and its enzymatic properties were studied.The results showed that strain ZP-8 had the highest enzyme activity of 197.6 U/mL,which was identified as Bacillus licheniformis.The molecular weight of the purified enzyme is about 45.43 kDa.The optimal pH of this enzyme is 8.0,and it is relatively stable in the pH rang of 7.0~11.0,which belongs to alkaline protease.The optimal temperature of this enzyme is 50℃,and it is relatively stable at 30~50℃;Mg^(2^(+)),Na^(+),Ca^(2^(+))and Fe^(2^(+))have no significant effect on the relative activity of protease(P>0.05),while Cu^(2^(+))and Mn^(2^(+))have remarkable activation effects(P<0.05).This research has important reference value for the development and utilization of protease-producing microorganisms.