CN109971733B - 一种热稳定性提高的谷氨酰胺转氨酶 - Google Patents

一种热稳定性提高的谷氨酰胺转氨酶 Download PDF

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CN109971733B
CN109971733B CN201910174239.3A CN201910174239A CN109971733B CN 109971733 B CN109971733 B CN 109971733B CN 201910174239 A CN201910174239 A CN 201910174239A CN 109971733 B CN109971733 B CN 109971733B
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刘松
童理明
陈坚
堵国成
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Abstract

一种谷氨酰胺转氨酶，特别是一种热稳定性提高的谷氨酰胺转氨酶。通过在成熟酶的N端的融合表达双亲短肤实现酶热稳定性的提高。

Description

一种热稳定性提高的谷氨酰胺转氨酶
技术领域
本发明涉及一种谷氨酰胺转氨酶，特别是一种热稳定性提高的谷氨酰胺转氨酶。
背景技术
谷氨酰胺转胺酶(Transglutaminase,EC 2.3.2.13,TGase)，是一种能够发生酰胺基转移反应，最后形成ε-(γ-谷氨酰基)赖氨酸共价键的蛋白。基于上述催化反应的存在，TGase能够促使各种蛋白质分子间、分子内交联以及谷氨酰胺残基的水解，从而提高蛋白质的各种功能性质，比如乳化性、溶解性等；同时，TGase能够通过将一些小分子物质比如赖氨酸等引入蛋白质，来增加蛋白质的营养价值。因此，市场上广泛应用TGase作为别具特色的食品添加剂于面制品、乳制品、碚烤制品、肉制品及水产品等食品的加工处理，市场需求量极其巨大。TGase特殊的催化能力使其在食品、纺织等工业生产中具有广泛应用，但其催化活性较低、热稳定性较差，使得TGase作为优良的催化剂在工业中的应用受到了严重限制。因此，目前的研究的热点大部分都集中于如何提高TGase的热稳定性。因此基于已得到的在大肠杆菌中的表达平台,通过在成熟酶的N端插入短肽,对进行分子改造,以期得到酶学性质更适合工业应用的TGase。
自组装双亲短肽(SAPs)通过疏水亲水氨基酸交替分布，自发组装成特殊短肽，形成的水凝胶能够对蛋白等大分子进行固定化，在分子电子学、细胞培养、纳米生物技术和生物医药等方面具有巨大的应用潜力。SAPs与蛋白末端融合，能提高酶的催化效率和热稳定性。
发明内容
本发明要解决的技术问题是得到一种热稳定性提高的谷氨酰胺转氨酶，通过在成熟酶的N端的融合表达双亲短肤实现酶热稳定性的提高，序列如SEQ ID NO.1所示或在SEQID NO.1基础上进行缺失、突变后谷氨酰胺转氨酶酶活性保持不变的氨基酸序列。
所述谷氨酰胺转氨酶的氨基酸序列还可以如SEQ ID NO.2所示，SEQ ID NO.3所示或SEQ ID NO.4所示。上述谷氨酰胺转氨酶均在成熟酶的N端融合了双亲短肽序列。
为解决上述问题技术问题，本发明采用以下示例：
1、双亲短肤基因的获得，根据双亲短肤的氨基酸序列，在引物上设计相应的DNA序列。
2、以S.hygroscopicus pro-TGase表达质粒pET-22b(+)/pro-TG(Liu S,Zhang D,Wang M,Cui W,Chen K,Du G,Chen J,Zhou Z.The order of expression is a keyfactor in the production of active transglutaminase in Escherichia coli byco-expression with its pro-peptide.Microb Cell Fact,2011,10(112):1-7)为模板，进行全质粒PCR。
3、将连接好的pET-22b(+)/pro-SAP-TG转进JM109，测序正确后转进E.coli BL21(DE3)。
培养基：
种子培养基(LB)：酵母粉5g/L，胰蛋白胨10g/L，NaCl 10g/L，(pH 7.0)。
发酵培养基(TB)：酵母粉24g/L，胰蛋白胨12g/L，甘油5g/L，K2HPO4 72mmol/L，KH2PO4 17mmol/L，(pH 7.0)。
培养方法：
种子培养条件：LB培养基，用250mL摇瓶培养，装液量为10％，培养温度为37℃，转速为220rpm，培养时间为10h。
摇瓶发酵条件：TB培养基，采用250mL摇瓶进行培养，装液量为10％，接种量为3％，培养温度为37℃，转速为220rpm，当OD600达到2.0时，终浓度为0.4mmol/mL的IPTG诱导，20℃诱导培养48h。
目的酶热稳定的检测方法：
将目的酶分别应用亲和层析等分离手段，得到电泳纯的目的酶。将目的酶在一定温度下保温，测定酶活相比初始未保温时损失所需要的时间。
酶活测定方法：
比色法测定酶活。L-谷氨酸-γ-单羟胺酸作为标准曲线，α-N-CBZ-GLN-GLY为底物。1个单位的TGase酶活定义为：在37℃的条件下，每分钟催化上述底物合成1μmol的L-谷氨酸-γ-单羟胺酸所用的酶量(U/mL)。酶活测定条件：在37℃条件下反应10min。
本发明以STG在大肠杆菌中的高效表达为平台，对在STG成熟酶的N端插入双亲短肽，得到了热稳定提高的突变株，热稳定提高了70％，改造后的酶更适合工业应用，可降低生产成本，提高生产效率。
具体实施方式
以下通过实施例来进一步阐明本发明,下列实施例中未注明具体条件的实验方法,基本上都按照常见的分子克隆手册所述的条件进行操作。
实施例1双亲短肽的获得
双亲短肤的氨基酸序列获得AEAEAKAKAEAEAKAK，将氨基酸的序列所对应的DNA序列设计在引物上：
上游引物M-F：
GCAGAAGCAGAAGCGAAAGCCAAAGCGGAGGCGGAAGCTAAGGCTAAACGGGCCCCCGACGCTGC
下游引物M-R：GAAGAGCGCACTGACGCTCGGC
实施例2在成熟酶N端插入双亲短肽的重组菌株的构建
以S.hygroscopicus pro-TGase表达质粒pET-22b(+)/pro-TG为模板，以上述实施例1中引物进行全质粒PCR。
实施例3重组菌株发酵生产TGase
将测序正确的质粒，命名为N，转化E.coli BL 21,挑选转化子接种到LB液体培养基中，37℃，培养12h，转接到TB培养基中，接种量为3％，当OD600达到2.0时，终浓度为0.4mmol/mL的IPTG诱导，20℃诱导培养48h。
实施例4比较重组菌株与野生TGase的热稳定
收集发酵上清，检测发酵上清酶活，并对样品进行His-镍柱纯化，对纯化后的TGase的Km值和t1/2进行测定，结果如表2所示，在成熟酶的N端插入双亲短肽其热稳定相比野生TGase提高了71％(表1)。
表1氨酰胺转氨酶酶学性质比较
Figure BDA0001988197120000031
实施例5在成熟酶N端插入双亲短肽的基础上对P132号位点做定点突变
利用定点突变试剂盒，在成熟酶N端插入双亲短肽的基础上对P132位点，S150位点，Y100位点P305位点做定点突变，分别突变为N-P132I、N-S150G、N-Y100M、N-P305Q(SEQID NO.2-5)转化子由上海生工测序。将测序正确的质粒，按照前文所述发酵纯化，并测其热稳定性(表2)。
表2氨酰胺转氨酶突变体酶学性质比较
Figure BDA0001988197120000041
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
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gcagaagcag aagcgaaagc caaagcggag gcggaagcta aggctaaacg ggcccccgac 60
gctgc 65
<210> 8
<211> 22
<212> DNA
<213> 人工序列
<400> 8
gaagagcgca ctgacgctcg gc 22

Claims (2)

1.一种热稳定提高的谷氨酰胺转氨酶，其特征在于，氨基酸序列如SEQ ID NO.4所示。
2.制备权利要求1所述谷氨酰胺转氨酶的方法，其特征在于，将双亲短肽融合表达到谷氨酰胺转氨酶成熟酶的N端，将其双亲短肽第100位的酪氨酸突变为蛋氨酸。
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