CN116891843B - 一种提高高温碱性蛋白酶AprThc的酶活的方法及突变体 - Google Patents
一种提高高温碱性蛋白酶AprThc的酶活的方法及突变体 Download PDFInfo
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Abstract
本发明涉及基因工程领域,具体涉及一种提高高温碱性蛋白酶AprThc的酶活的方法及突变体。本申请对高温碱性蛋白酶AprThc进行S105A、I151V、I297T或F366Y突变,对高温碱性蛋白酶AprThc进行酶活的提升,对降低蛋白酶的使用成本具有重要意义,为蛋白酶的性质改良提供了有效的技术方法。
Description
技术领域
本发明涉及基因工程领域,具体涉及一种提高高温碱性蛋白酶AprThc的酶活的方法及突变体。
背景技术
蛋白酶在洗涤、饲料、食品、酿造以及医药行业具有重要的应用价值,其产量占酶制剂市场的65%以上。
在饲料行业,科学补充外源性蛋白酶对于控制饲料配方成本、提高蛋白质消化率、促进动物肠道健康、提高动物生产性能、减少环境污染等方面都有积极作用。在洗涤剂中添加蛋白酶可以提高洗涤剂的效果,制革工业中蛋白酶的利用可以水解角蛋白和一些其他球蛋白等杂蛋白,还会分解毛皮与表皮间连接的类粘蛋白,使鞣制后的皮革松软。蛋白酶在奶制品加工业、焙烤业豆制品以及去除杂味等领域也有着重要的作用。如在葡萄酒的生产过程中,通常加入酸性蛋白酶来水解酒中的蛋白质,可以大大减少蛋白质沉淀浑浊的现象。在奶酪的生产过程中,也同样需要蛋白酶来发酵使其具有特定的香味。蛋白酶在医学方面同样具有非常高的贡献,在用于治疗的酶中,蛋白酶几乎达到了一半以上,比如蛋白酶在体外的涂抹可以用于消炎等皮肤症状,服用蛋白酶类的药物可以治疗消化不良。血栓是人类以及动物中非常危险的一类疾病,具有纤维活性的蛋白酶不但可以在体外降解纤维蛋白,在体内也可以治疗静脉血栓。
虽然蛋白酶的应用空间十分广阔,但常温中性蛋白酶是目前国内外市场中主要蛋白酶类,高温碱性蛋白酶种类较少,价格较高,提高高温碱性蛋白酶的表达量,对于降低高温碱性蛋白酶的使用成本具有重要意义。
发明内容
本发明的目的是提供提高高温碱性蛋白酶AprThc的酶活的方法。
本发明的另一目的是提供酶活提高的高温碱性蛋白酶突变体。
本申请的另一目的是提供上述酶活提高的高温碱性蛋白酶突变体的应用。
根据本申请的提高高温碱性蛋白酶AprThc的酶活的方法,包括对高温碱性蛋白酶AprThc的氨基酸序列进行S105A或I151V或I297T或F366Y突变的步骤,其中,所述高温碱性蛋白酶AprThc的氨基酸序列如SEQ ID NO:1所示。
根据本申请的稳定性改良的高温碱性蛋白酶突变体,所述高温碱性蛋白酶突变体具有对高温碱性蛋白酶AprThc的氨基酸序列进行S105A或I151V或I297T或F366Y突变后的氨基酸序列,所述高温碱性蛋白酶AprThc的氨基酸序列如SEQ ID NO:1所示。所述突变体S105A的氨基酸序列如SEQ ID NO:2所示,所述突变体I151V的氨基酸序列如SEQ ID NO:3所示,所述突变体I297T的氨基酸序列如SEQ ID NO:4所示,所述突变体F366Y的氨基酸序列如SEQ ID NO:5所示。
本申请对高温碱性蛋白酶AprThc进行分子改良,以提高其酶活,降低蛋白酶的使用成本具有重要意义。
附图说明
图1显示高温碱性蛋白酶AprThc与其单点突变体的表达水平检测 M:蛋白Marker;1: AprThc野生型蛋白;2:S105A;3: I151V;4: I297T;5:F366Y。
具体实施方式
1.以下实施例所用培养基配方为:
LB 液体培养基:蛋白胨 10 g/L,酵母粉 5 g/L,NaCl 10 g/L;
LB 固体培养基:蛋白胨 10 g/L,酵母粉 5 g/L,NaCl 10 g/L, 琼脂粉15 g/L;
发酵培养基:豆粕 10 g,玉米粉 5 g,K2HPO3 1g,明胶 10 g,总体积 300 mL;
脱脂奶粉固体培养基:4%脱脂奶粉,2%琼脂粉;
LBS培养基:蛋白胨 10 g/L,酵母粉 5 g/L,NaCl 10 g/L,山梨醇91.1 g/L;
电转洗涤培养基(SMG):山梨醇91.1 g/L,甘露醇91.1 g/L,甘油100 g/L;
复苏培养基(LBSM):蛋白胨 10 g/L,酵母粉 5 g/L,NaCl 10 g/L,山梨醇91.1 g/L,甘露醇69.2 g/L。
2.高温碱性蛋白酶酶活力的测定
高温碱性蛋白酶酶活力测定按照中华人民共和国国家标准 GB/T 23527–2009 中所描述的福林法进行。在试管中加入 pH 10.5、1%酪蛋白溶液0.5 mL,40℃预热3 min,加入0.5 mL 适当稀释的酶液,混合均匀后,40℃条件下反应 10 min,加 0.4 M三氯乙酸溶液1mL终止反应。将该反应液转移至 2 m L EP管中,12,000rpm 离心 10min。取上清液1 m L,依次加入碳酸钠溶液5 m L 和福林试剂使用溶液1 mL,振荡混匀后,置于 40℃ 水浴锅中显色 20 min。680 nm条件下测定吸光度。
实施例1AprThc酶活提升突变体的设计
为了提升高温碱性蛋白酶AprThc在芽孢杆菌表达系统中的蛋白表达水平,以AprThc野生型蛋白表达载体为模板,通过重叠PCR(Overlap PCR)法依据单点突变的设计对相应位置氨基酸进行突变,构建AprThc的单点突变体。将转化平板长出的单克隆借助灭菌过后的牙签分别点在脱脂奶粉固体培养基上,37oC培养 30h 后,观察是否有透明圈的产生,并比较转化子与对照菌株的透明圈的直径,筛选阳性转化子。分别将阳性转化子和对照菌株即SCK6菌株接种至LB培养基中,阳性转化子的培养基中加入四环素,SCK6培养基不加四环素,200 rpm、37oC振荡培养。过夜培养后按 4%的接种量转接到发酵培养基中进行发酵培养,培养三天后收集发酵液并采用SDS-PAGE对发酵上清中的目的蛋白表达量进行分析。结果显示,4个单点突变体蛋白S105A、I151V、I297T和F366Y的表达水平较野生型AprThc蛋白具有明显的提升(图1),而另外2个单点突变体(T280V和A324P)的表达水平较野生型蛋白并无改善甚至变低。野生型AprThc蛋白表达菌株发酵粗酶液中蛋白酶活力为10889 U/mL,S105A、I151V、I297T和F366Y 表达菌株发酵粗酶液中蛋白酶活力分别为16533 U/mL,17200U/mL,23867 U/mL,18302 U/mL,较野生型AprThc蛋白的酶活分别提高了51.83%,57.95%,119.18%和68.08%。
以上实施例仅用于解释本申请的技术方案,不限定本申请的保护范围。
Claims (4)
1.一种提高高温碱性蛋白酶AprThc的酶活的方法,其特征在于,所述方法包括对高温碱性蛋白酶AprThc的氨基酸序列进行S105A或I151V或I297T或F366Y的突变的步骤,其中,所述高温碱性蛋白酶AprThc的氨基酸序列如SEQ ID NO:1所示。
2.一种酶活提高的高温碱性蛋白酶突变体,其特征在于,所述酶活提高的高温碱性蛋白酶突变体的氨基酸序列如SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4所示。
3.权利要求2所述酶活提高的高温碱性蛋白酶突变体用于水解蛋白的应用。
4.权利要求2所述酶活提高的高温碱性蛋白酶突变体作为饲料添加剂或洗涤剂制备原料的应用。
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