CN103540574B - 一种提高谷氨酰胺转胺酶比酶活及活化效率的方法 - Google Patents
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Abstract
本发明公开了一种提高谷氨酰胺转氨酶的比酶活及活化效率的方法，在谷氨酰胺转胺酶C端插入融合蛋白中常用的连接肽，上述连接肽的基因序列设计在引物上，利用定点突变技术插入到前导肽C端，其优选GS或PT。采用本发明提供的方法在不改变谷氨酰胺转氨酶分泌效率的前提下，显著提高谷氨酰胺转氨酶的比酶活，具有重要的意义。此外，本发明还发现使用本发明提供的短肽能显著提高TGase前导肽切割效率，缩短反应时间2/3。
Description
技术领域
本发明涉及一种提高谷氨酰胺转氨酶比酶活及活化效率的方法，特别是一种通过使用短肽提高谷氨酰胺转氨酶比酶活及活化效率的方法。
背景技术
微生物谷氨酰胺转胺酶（蛋白质-谷氨酸-谷氨酰胺转胺酶，MicrobialTransglutaminase，EC2.3.2.13简称MTG）能够催化蛋白质肽链中谷氨酰胺残基的γ-羧酰胺基与赖氨酸ε-酰基或其他酰基反应，形成ε-(γ-谷氨酰基)赖氨酸共价键。特殊的催化能力使TGase广泛应用于食品工程、纺织与皮革加工、材料工程、生物医药等领域。但由于MTG异源表达分泌量低等缺陷，限制了MTG的应用范围。
前导肽C端虽非TGase必需区域，但可影响TGase分泌和催化活性。目前TGase的改造限于成熟酶分子内部，忽略了前导肽对TGase催化性能的影响。实验室前期研究发现，共表达TGase前导肽和成熟酶可以实现TGase在E.coli中的活性表达，但TGase不能够分泌到胞外。共表达结果表明，前导肽和成熟酶之间的共价连接对TGase折叠没有影响，但是促进TGase分泌的必要条件。因此，为不影响pro-TGase分泌，又可改善TGase酶学性质，在不影响前导肽和成熟酶共价相连前提下，选取前导肽C端为改造区域。
发明内容
本发明所要解决的问题提供一种提高谷氨酰胺转氨酶的比酶活的方法，在谷氨酰胺转胺酶C端插入融合蛋白中常用的连接肽。
所述连接肽的基因序列设计在引物上，利用定点突变技术插入到前导肽C端。
所述连接肽优选GS或PT，插入位点选定在前导肽切割位点(L53-F54)前，即L53之前。
谷氨酰胺转胺酶活力的测定方法：
比色法测定酶活：以N-α-CBZ-GLN-GLY为作用底物，L-谷氨酸-γ单羟胺酸做标准曲线。1个单位谷氨酰胺转胺酶酶活定义为：37℃时每分钟催化形成1μmolL-谷氨酸-γ单羟胺酸的酶量(U/mL)。
试剂A：100mg的Nα-CBZ-GLN-GLY溶解于2mL0.2moL/L的NaOH溶液中，加入0.2mol/LpH6.0的Tris-HC缓冲液4mL，0.1mol/L羟胺2mL，0.01mol/L的还原型谷胱甘肽2mL，并调节pH至6.0。
试剂B：3mol/L的HCL，12%TCA，5%FeCL3按1：1：1混合。
配制0-4μmol/mL的L-谷氨酸-γ-单异羟肟酸标准溶液。取1mL试剂A与0.4mL不同浓度的L-谷氨酸-γ-单异羟肟酸标准溶液混合，37℃水浴10分钟。加0.4mL试剂B终止反应，在525nm比色，绘制出标准曲线。以0.4mL经适当稀释的酶液代替标准溶液，在相同条件下保温和比色，从标准曲线求出酶活。以100℃加热10分钟的离心后的上清液为空白。
酶活力(u/mL)=(6.8548×OD525-0.0164)×稀释倍数
采用本发明提供的方法在不改变谷氨酰胺转氨酶分泌效率的前提下，显著提高谷氨酰胺转氨酶的比酶活，具有重要的意义。此外，本发明还发现使用本发明提供的短肽能显著提高TGase前导肽切割效率，缩短反应时间2/3。
附图说明
图1突变体及其表达
a:pro-TGase结构示意图及插入短肽位序列；b：野生酶及突变酶胞外蛋白电泳；c：野生酶及突变酶全细胞蛋白电泳；0：野生酶；1：pro-52GG；2：pro-52GGG；3：pro-52GGGG；4：pro-52GS；5：pro-52PT：M：蛋白质标准分子量
图2SDS-PAGE分析纯化TGase
1：WT；2：pro-52GG；3：pro-52GGG；4：pro-52GGGG，5：pro-52GS，4：pro-52PT
图3突变酶pro-52GS和pro-52PT活化过程
●：WT；▲：pro-52GS；■：pro-52GS
图4蛋白电泳检测突变酶pro-52GS和pro-52PT活化过程
a：野生酶；b：pro-52GS；c：pro-52PT
图5前导肽突变酶结构模拟
a：野生酶；b：pro-52GGG；c：pro-52GS；d：pro-52-PT
具体实施方式
培养基
LB培养基：胰蛋白胨10g/L、酵母粉5g/L、NaCl10g/L，pH7.0；
TB培养基：蛋白胨12g/L，酵母膏24g/L，甘油8g/L，17mmol/LKH2PO4，72mmol/LK2HPO4。
实施例1：吸水链霉菌来源MTG晶体结构模拟
以已报道的S.mobaraensispro-TGase(PDBcode:3IU0)为模板(两者氨基酸相似度为73.1%)，利用在线模拟软件SWISS-MODEL，模拟S.hygroscopicusTGase的晶体结构。
实施例2：突变体的获得
将短肽的基因序列设计在引物上，利用定点突变技术插入到前导肽C端。以S.hygroscopicuspro-TGase表达质粒pBB1-1011为模板（在前期研究中，本研究室筛选出一株新的产谷氨酰胺转胺酶的菌株（StreptomyceshygroscopicusCCTCCM203062），通过基因克隆方法，得到了MTG基因序列及其上下游序列，含MTG自身的启动子和终止子（Genbank：EU477523），具体文献为LiuS,ZhangD,WangM,CuiW,ChenK,LiuY,DuG,ChenJ,ZhouZ(2011)Thepro-regionofStreptomyceshygroscopicustransglutaminaseaffectsitssecretionbyEscherichiacoli.FEMSMicrobiolLett324(2):98-105），进行全质粒PCR。引物如表1所示，由上海生工生物工程公司合成。其中引物Pro-52-R是构建所有突变酶的下游引物，而其余引物为构建对应突变酶的上游引物，对应的突变体命名见表1。
表1
PCR反应条件为：95℃5min、24个循环（95℃5min、65℃30s、72℃7min）、72℃10min
实施例3：突变体酶学性质检测
为使前导肽突变后仍能被正常切割，将短肽插入位点选定在前导肽切割位点(L53-F54)前，即L53之前(图1a)。选择GG、GGG、GGGG、GGGGS和PTPPTTPT为插入短肽，对应突变体分别为pro-52GG、pro-52GGG、pro-52GGGG、pro-52GS和pro-52-PT。将上述突变体进行发酵，利用SDS-PAGE检测胞内及胞外pro-TGase，所有突变酶都可分泌到胞外(图1b)，且胞内无积累(图1c)，表明前导肽C端插入上述短肽对其分泌并无明显影响。
纯化上述突变酶(图2)，检测酶学性质。与野生酶相比，前导肽插入不同长度短肽可显著提高突变酶的催化活性，突变酶pro-52G、pro-52GG、pro-52GGG和pro-52GGGG比酶活提高了14%，pro-52GS和pro-52PT比酶活分别增加28%和35%。比较突变酶的Km和kcat，突变酶pro-52GS和pro-52PT的Km下降，且kcat也分别是野生酶2倍和2.6倍(表2)，表明前导肽C端插入不同短肽可提高TGase催化性能。尽管突变体TGase比酶活显著增加，但其热稳定性并无提高(数据未显示)。
表2前导肽C端插入短肽突变酶酶学性质
由于插入短肽紧邻前导肽切割位点，可能会影响活化蛋白酶dispase对前导肽的切割，因此选取比酶活最高的突变酶pro-52GS和pro-52PT为研究对象，检测其活化过程。如图3所示，突变酶pro-52GS和pro-52PT活化曲线基本一致，活化5min后酶活达到最大值，表明pro-TGase已被完全活化，而野生酶则需15min。同时对活化样品进行SDS-PAGE检测，突变酶pro-TGase条带向成熟酶转化较快，5min后基本已完全活化(图4)，与酶活检测结果一致。上述结果表明，C端插入短肽GGGGS和PTPPTTPT可提高TGase前导肽切割效率。
实例4：突变酶pro-52GGGG，pro-52GS和pro-52PT结构模拟
为分析前导肽C端插入短肽对TGase结构影响，对催化活性提高较明显的突变酶pro-52GGGG，pro-52GS及pro-52PT进行结构模拟。如图5所示，与野生酶相比，成熟酶部分并无明显变化，但前导肽C端结构变化明显。突变酶pro-52GGGG前导肽C端形成了β-折叠结构(图5b)，而突变酶pro-52GS和pro-52PT前导肽C端均为α-螺旋结构(图5c和d)。而插入短肽GGGG，GGGGS和PTPPTTPT则在对应突变酶中分别形成了loop、loop及转角结构。在突变酶pro-52GS和pro-52PT中，由于前导肽C端结构及位置发生变化，使与其共价相连的成熟酶N端区域形成了螺旋结构，且偏离活性中心(箭头标注)。在野生TGase酶中，其N端为柔性较高的loop结构，位于活性裂缝的上面，对底物与催化活性区域的结合具有阻碍作用。因此，突变酶pro-52GS和pro-52PT表现出较高的催化活性可能是由于其成熟酶N端氨基酸偏移，进而降低了底物和活性中心的结合位阻。
除影响TGase酶N端区域，突变酶pro-52GS和pro-52PT中插入短肽靠近成熟酶活性裂缝左侧，可能在蛋白折叠过程中与活性区域发生相互作用，帮助蛋白折叠成具有较高的活性状态。此外，突变酶前导肽C端结构变化使其切割位点也发生了偏移，有助于活化蛋白酶dispase的识别和切割，进而使前导肽切割效率提高。GS和PT连接肽一般用于蛋白融合，使两个具有不同功能得到蛋白连接在一起，且不影响蛋白原来的结构和活性，还可帮助多聚体蛋白折叠。在本研究中，将GS和PT连接肽插入到前导肽C端不仅可以使突变酶正常表达分泌，还可提高蛋白催化活性，表明通过在前导肽C端插入短肽可作为TGase分子改造的有效方法。
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。
Claims (2)
1.一种提高谷氨酰胺转氨酶的比酶活及活化效率的方法，其特征在于在谷氨酰胺转胺酶C端插入融合蛋白中常用的连接肽；所述连接肽为GGGGS或PTPPTTPT；插入位点选定在前导肽切割位点L53-F54前，即L53之前。
2.权利要求1所述的方法，其特征在于所述连接肽的基因序列设计在引物上，利用定点突变技术插入到前导肽C端。
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| CN1796561A (zh) * | 2004-12-20 | 2006-07-05 | 南京大学 | 一种重组微生物谷氨酰胺转氨酶基因及其制备方法 |
| CN102174456A (zh) * | 2011-02-09 | 2011-09-07 | 天津科技大学 | 产谷氨酰胺转氨酶的基因工程菌及其构建方法 |
| CN102660515A (zh) * | 2012-05-10 | 2012-09-12 | 江南大学 | 一种酶活性及热稳定性提高的谷氨酰胺转氨酶 |
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| CN1796561A (zh) * | 2004-12-20 | 2006-07-05 | 南京大学 | 一种重组微生物谷氨酰胺转氨酶基因及其制备方法 |
| CN102174456A (zh) * | 2011-02-09 | 2011-09-07 | 天津科技大学 | 产谷氨酰胺转氨酶的基因工程菌及其构建方法 |
| CN102660515A (zh) * | 2012-05-10 | 2012-09-12 | 江南大学 | 一种酶活性及热稳定性提高的谷氨酰胺转氨酶 |
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| Title |
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| Enhancement of Streptomyces transglutaminase activity and pro-peptide cleavage efficiency by introducing linker peptide in the C-terminus of the pro-peptide;Kangkang Chen, et al;《J Ind Microbiol Biotechnol》;20130124;317-325 * |
| 谷氨酰胺转氨酶在豆渣制品中的应用;侯瑞雪等;《食品科技》(第08期);全文 * |
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