摘要
酸性蛋白酶在食品、酿造、饲料和皮革等行业具有重要的应用价值。然而,现有的酸性蛋白酶在50℃以上或pH> 3. 0时不稳定,限制了其应用范围。该研究通过分子克隆技术将黑曲霉CICIM F0510的酸性蛋白酶基因exp A在毕赤酵母中进行了克隆表达,构建获得了重组菌GS115 (p PIC-expA)。摇瓶发酵条件下,重组酶EXPA的酶活为257 380 RFU/h。生物信息学分析的结果显示,该酶属于天冬氨酸蛋白酶A1A家族。酶学性质的研究表明,该重组酶的最适反应温度和pH分别为50℃和3. 0;分别在40~50℃或pH 2. 5~3. 5孵育1 h后,仍能保留80%左右的活力。Zn~(2+)、Ca~(2+)、Fe~(2+)和Mn~(2+)对其活性有一定的促进作用;而Cu~(2+)、Co~(2+)、Fe~(3+)、EDTA和SDS则对其活性有显著的抑制作用。此外,重组酶EXPA对大豆分离蛋白、水溶性玉米蛋白和小麦水解蛋白均具有较好的水解作用。较好的耐热性和pH稳定性为EXPA在食品、饲料等领域的应用奠定了基础。
Acid protease has wide applications in foods,brewing,feeds,and leather industries. However,current acid proteases are unstable when the temperature or pH values are higher than 50 ℃ or 3. 0,respectively,which restricts their applications. Thus,acid protease encoding gene expA from Aspergillus niger CICIM F0510 was successfully cloned and expressed in Pichia pastoris,and the recombinant strain GS115(p PIC-expA) was generated in this study. In shaking flask experiments,the activity of recombinant enzyme EXPA was 257 380 RFU/h. Bioinformatics analysis showed that this enzyme belonged to family A1 A of aspartic proteases. The optimal temperature and pH of EXPA were 50 ℃ and 3. 0,respectively. After incubating at 40-50 ℃ or pH 2. 5-3. 5 for 1 h,the recombinant enzyme still retained more than 80% of its maximum activity. Zn~(2+),Ca~(2+),Fe~(2+),and Mn~(2+)enhanced EXPA activity,whereas Cu~(2+),Co~(2+),Fe~(3+),EDTA,and SDS had significant inhibitory effects on EXPA activity. Besides,EXPA also hydrolyzed soybean protein isolates,water-soluble corn protein,and wheat protein. Good thermostability and pH stability of EXPA provide a solid foundation for its applications in food and feed industries.
引文
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