CN108823186B - 一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体及其制备方法和应用 - Google Patents
一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种玉米淀粉降解能力提高的嗜热酸性生淀粉α‑淀粉酶突变体及其制备方法和应用,属于基因工程和酶工程领域。本发明对生淀粉α‑淀粉酶GTamy的SBD结构域进行环化重排突变,再将GTamy中SBD替换为SBD环化重排突变体,构建GTamy环化重排突变体。通过比较GTamy环化重排突变体对玉米淀粉的酶活力,筛选出玉米淀粉降解能力显著提高的生淀粉α‑淀粉酶突变体GTamy‑S498。本发明提供的生淀粉α‑淀粉酶突变体GTamy‑S498对玉米淀粉的酶活力由对照(突变前)的21.08U/mg提高到114.77U/mg,提高了5.44倍。生淀粉α‑淀粉酶突变体GTamy‑S498的玉米淀粉降解能力显著提高,并且其酶学性质符合淀粉液化工艺的需要,更适用于淀粉液化工艺。
Description
技术领域
本发明属于基因工程和酶工程领域,具体涉及一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体及其制备方法和应用。
背景技术
天然生淀粉是一种结构复杂而致密的颗粒,在应用前往往需要经过强酸、强碱、高温或者酶法处理来破坏生淀粉颗粒的结构。酶法降解生淀粉能够简化现代发酵工业中的生淀粉前处理过程,更加环保并节约能耗,因此生淀粉降解酶的研究一直受到高度关注。生淀粉酶是指能对不经过蒸煮糊化的生淀粉颗粒表现出强水解活性的酶类。在α-淀粉酶、β-淀粉酶、葡萄糖淀粉酶、异淀粉酶中均有部分酶具有水解生淀粉的能力。
生淀粉α-淀粉酶可以在低于糊化温度条件下直接作用于未经蒸煮糊化的生淀粉颗粒,在淀粉液化过程中能够省去生淀粉糊化步骤,有利于节约能源和简化工艺,因此生淀粉α-淀粉酶在酿造、食品、造纸、纺织等领域具有巨大的应用潜力。在淀粉双酶法水解工艺中为了防止杂菌污染需要适当加温,至少要达到60℃。此外,生淀粉加水形成的乳浊液的pH值一般为5.8~6.5,由于淀粉工业中循环工艺的应用,淀粉水解过程中的pH通常在5.0左右。因此,获得嗜热酸性和不依赖于Ca2+的生淀粉α-淀粉酶,有利于对现行淀粉加工工业的工艺体系进行技术升级和改造。
来源于嗜热菌Geobacillus thermoleovorans的α-淀粉酶GTamy属于嗜热酸性生淀粉α-淀粉酶(Mehta D,Satyanarayana T,Biochemical and molecularcharacterization of recombinant acidic and thermostable raw-starchhydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans[J].Journal of Molecular Catalysis B:enzymatic,2013,85:229-238.),具有优良的高温活性和热稳定性,其最适反应温度为80℃,最适反应pH为5.0,于80℃的可溶性淀粉酶活达1723U/mg,于80℃的半衰期为184min,其活性和热稳定性均不依赖于Ca2+。GTamy是目前报道的玉米淀粉降解率最高的生淀粉α-淀粉酶,使用0.1U/(mg淀粉)的酶液,于80℃水解30%玉米淀粉3h(pH 5.0),玉米淀粉降解率达54%。并且GTamy的酶学性质(如热稳定、低pH、不依赖于Ca2+)使得其在淀粉液化工艺中具有巨大的应用潜力。
GTamy应用于淀粉液化工艺中,可以在满足工业生产要求的前提下(30%玉米淀粉乳、反应时间3h、pH 5.0),在低于淀粉糊化温度的条件下液化玉米淀粉,省去传统双酶法水解工艺中的糊化步骤,有利于简化工艺、节约能源。但是GTamy的玉米淀粉降解能力距离工业应用仍有一定的距离,例如0.1U/(mg淀粉)的酶液在以上条件下80℃处理玉米淀粉的降解率为54%,因此GTamy的玉米淀粉降解能力有待进一步提高。本发明拟采用基于蛋白质分子结构的环化重排方法对GTamy进行分子改造,来获得玉米淀粉降解能力提高的GTamy突变体。
发明内容
为解决生淀粉α-淀粉酶GTamy的玉米淀粉降解能力不能满足淀粉液化工艺的要求这个问题,本发明提供了一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体GTamy-S498,其氨基酸序列如SEQ ID NO:1所示。
本发明还提供一种编码上述的生淀粉α-淀粉酶突变体GTamy-S498的基因;所述基因的核苷酸序列如SEQ ID NO:2所示。
本发明还提供一种能表达生产上述生淀粉α-淀粉酶突变体GTamy-S498的载体。
本发明还提供一种能表达生产上述生淀粉α-淀粉酶突变体GTamy-S498的基因工程菌。
本发明还提供一种上述的生淀粉α-淀粉酶突变体GTamy-S498的制备方法,以氨基酸序列如SEQ ID NO:3所示的生淀粉α-淀粉酶GTamy为出发序列,将GTamy中SBD结构域的氨基酸序列替换为突变体SBD-S498的氨基酸序列。其中SBD的氨基酸序列如SEQ ID NO:5所示,SBD-S498的氨基酸序列如SEQ ID NO:19所示。
上述的制备方法,具体步骤如下:
1)根据Geobacillus thermoleovorans的生淀粉α-淀粉酶GTamy的基因序列,其基因序列如SEQ ID NO:4所示,采用化学全合成的方法合成优化的基因后,将其克隆到重组质粒pSTOP1622中,构建重组质粒pSTOP1622-gtamyh;
2)合成核苷酸序列“5’-ATCGCGCGCAGGGAT-SEQ ID NO:20-CACCATCACCATCAC-3’”,采用无缝克隆方法将重组质粒pSTOP1622-gtamyh中编码SBD的核苷酸序列(SEQ ID NO:6)替换为SBD环化重排突变体SBD-S498的核苷酸序列(SEQ ID NO:20),获得GTamy环化重排突变体的表达载体pSTOP1622-gtamyh/S498;
3)将重组质粒pSTOP1622-gtamyh/S498转化枯草芽孢杆菌Bacillus subtilisWB600,获得枯草芽孢杆菌基因工程菌,诱导表达,获得生淀粉α-淀粉酶突变体GTamy-S498。
上述生淀粉α-淀粉酶突变体GTamy-S498在纺织、洗涤剂、制革、造纸、食品领域的应用。
本发明对生淀粉α-淀粉酶GTamy的SBD结构域进行环化重排突变,再将GTamy中SBD替换为SBD环化重排突变体,构建GTamy环化重排突变体。通过比较GTamy环化重排突变体对玉米淀粉的酶活力,筛选出玉米淀粉降解能力显著提高的生淀粉α-淀粉酶突变体GTamy-S498。
本发明的优点:本发明提供的生淀粉α-淀粉酶突变体GTamy-S498对玉米淀粉的酶活力由对照(突变前)的21.08U/mg提高到114.77U/mg,提高了5.44倍。以玉米淀粉为底物,生淀粉α-淀粉酶突变体GTamy-S498最适反应温度为80℃,最适反应pH为5.0,于80℃的半衰期为180min,并且其活性和热稳定性均不依赖于Ca2+。生淀粉α-淀粉酶突变体GTamy-S498的玉米淀粉降解能力显著提高,更适用于淀粉液化工艺,有利于简化工艺、节约能源。
附图说明
图1为SBD环化重排突变体的示意图;
图2为GTamy和GTamy-S498的最适反应温度;
图3为GTamy和GTamy-S498的最适反应pH;
图4为GTamy和GTamy-S498于80℃的热稳定性。
具体实施方式
下面结合具体实施例对本发明一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体及其制备方法和应用作进一步的详细说明。
实验条件:
1、菌株与载体
大肠杆菌Escherichia coli JM109(本实验室保存),枯草芽孢杆菌Bacillussubtilis WB600(本实验室保存),枯草芽孢杆菌表达载体pSTOP1622(购自MoBiTec公司)。
2、酶类及其他生化试剂
KOD DNA聚合酶及KOD-Plus-neo DNA聚合酶购自Toyobo公司,DNA限制性内切酶、T4DNA连接酶购自Fermentase公司,细菌基因组提取试剂盒、DNA胶回收试剂盒、质粒抽提试剂盒E.Z.N.A.购自Omega Bio-tek公司,In-Fusion HD Cloning kit购自日本Takara公司,玉米淀粉购自百威灵科技有限公司,其它化学试剂均为国产或进口分析纯。
3、培养基
LB培养基(g/L):胰蛋白胨10、酵母提取物5、NaCl 10,pH 7.0。筛选培养基采用含50μg/mL氨苄青霉素的LB培养基。
本发明中所用到的分子克隆技术和蛋白质检测技术均为本领域中的常规技术。在以下实施例中未作详细介绍的技术,均按照如下实验手册中的相关部分来进行。Green MR,Sambrook J.Molecular cloning:a laboratory manual[M].New York:Cold SpringHarbor Laboratory Press,2012。
实施例1:GTamy环化重排突变体的初步筛选
(1)基因gtamy的合成
根据来源于嗜热菌Geobacillus thermoleovorans的嗜热酸性生淀粉α-淀粉酶GTamy的NCBI登录号JQ409473.1,搜索获得其基因序列,如SEQ ID NO:4所示,交由上海博益生物科技有限公司进行α-淀粉酶GTamy的全基因合成。
(2)表达载体pSTOP1622-gtamyh的构建
根据GTamy的基因序列设计PCR引物P1、P2(表1),以合成基因gtamy为模板,以P1、P2为引物,进行PCR扩增。PCR扩增条件为:98℃5min;98℃20sec,60℃40sec,74℃2min,30个循环;74℃,10min。扩增产物经Spe I和Sac I双酶切,连接至载体pSTOP1622,构建重组质粒pSTOP1622-gtamyh。
表1构建重组质粒所用引物
注:下划线标注的部分为限制性酶切割位点。
(3)GTamy环化重排突变体的表达载体的构建
以来源于Bacillus stearothermophilus的α-淀粉酶的三级结构(PDB ID:1hvxA)为模板,采用Swiss-Model(http://swissmodel.expasy.org)对GTamy-SBD进行三维模建,获得SBD的三级结构。将SBD的蛋白质分子结构信息(模拟得到的三级结构信息)输入到在线软件Cpred(http://sarst.life.nthu.edu.tw/CPred)中,得出SBD结构中每一个氨基酸残基作为环化重排突变的概率,选择环化重排突变概率高的氨基酸残基位点进行突变。根据以上信息,SBD结构域中可以作为环化重排突变位点的氨基酸残基如下:第432位丝氨酸(T432)、第441位天冬氨酸(D441)、第454位脯氨酸(P454)、第465位甘氨酸(G465)、第479位丙氨酸(A479)、第491位丝氨酸(S491)、第498位丝氨酸(S498)、第508位甘氨酸(G508),对应的SBD环化重排突变体的氨基酸序列和核苷酸序列如表2所示。SBD环化重排突变体的示意图如图1所示。
表2 SBD环化重排突变体的氨基酸序列和核苷酸序列
由上海博益生物科技有限公司合成包含同源臂和编码SBD环化重排突变体的核苷酸序列的DNA片段(5’-ATCGCGCGCAGGGAT-核苷酸序列-CACCATCACCATCAC-3’)。以重组质粒pSTOP1622-gtamyh为模板,以P3、P4为引物,进行PCR扩增得到不包含编码SBD的核苷酸序列的线性化载体片段。PCR扩增条件为:98℃5min;98℃20sec,60℃20sec,74℃5min,30个循环;74℃,10min。将PCR扩增的载体片段与合成的DNA片段混合,采用In-Fusion HD Cloningkit进行无缝克隆,获得GTamy环化重排突变体(见表3)的表达载体。
表3 SBD环化重排突变体和对应的GTamy环化重排突变体
(4)GTamy及其环化重排突变体在枯草芽孢杆菌中的表达
将构建成功的GTamy及其环化重排突变体的表达载体分别转化至枯草芽孢杆菌Bacillus subtilis WB600感受态细胞,同时转化pSTOP1622作为阴性对照Contr.,获得重组枯草芽孢杆菌。
重组枯草芽孢杆菌的种子培养条件为:采用LB液体培养基,用250mL三角瓶进行培养,其中培养基的装液量为20mL,培养温度为37℃,转速为200rpm,培养时间为10h。重组枯草芽孢杆菌的发酵培养条件为:采用LB液体培养基,用250mL三角瓶进行培养,其中培养基的装液量为25mL,接种量为3%,培养温度为37℃,转速为200rpm。当培养至菌体OD600nm达到1时,添加终浓度为0.5%的木糖,诱导时间为30h。
采用Ni2+亲和层析柱对发酵上清液中目的蛋白质进行纯化,用250mmol/L咪唑洗脱缓冲液洗脱,即得到纯化后的重组α-淀粉酶。利用SDS-PAGE检测重组α-淀粉酶的纯度,并采用Bradford法测定重组α-淀粉酶的浓度。
(5)重组α-淀粉酶的酶活力测定
重组α-淀粉酶对可溶性淀粉的酶活力测定:将10μL酶液与490μL含1%(m/V)可溶性淀粉的50mmol/L MES,pH 5.0缓冲液混合,于80℃反应30min后,迅速放入冰水浴中终止反应,然后采用3,5-二硝基水杨酸(3,5-Dinitrosalicylic acid,DNS)法测定反应体系中还原糖量。酶活力单位(U)定义:在一定反应条件下,每分钟催化产生1μmol还原糖的酶量为一个酶活力单位(U)。重组α-淀粉酶对可溶性淀粉的酶活力测定结果如表4所示。
重组α-淀粉酶对玉米淀粉的酶活力测定:将50μL酶液与450μL含1%(m/V)玉米淀粉的50mmol/L MES,pH 5.0缓冲液混合,于80℃反应30min后,迅速放入冰水浴中终止反应,将反应液于10000×g离心10min,然后采用3,5-二硝基水杨酸(3,5-Dinitrosalicylicacid,DNS)法测定反应液上清中还原糖量。酶活力单位(U)定义:在一定反应条件下,每分钟催化产生1μmol还原糖的酶量为一个酶活力单位(U)。重组α-淀粉酶对玉米淀粉的酶活力测定结果如表4所示。
表4重组α-淀粉酶的酶活力
与GTamy相比,突变体GTamy-S498对可溶性淀粉的酶活力基本不变,对玉米淀粉的酶活力提高了5.44倍。
实施例2:GTamy环化重排突变体GTamy-S498的酶学性质验证
重组α-淀粉酶的最适反应温度测定:重组α-淀粉酶对玉米淀粉的酶活力测定参照实施例1,参照实施例1混合酶液和底物,将混合物分别于30~100℃反应30min,测定不同温度条件下酶比活力,并以酶比活力对温度作图,确定其最适反应温度。重组α-淀粉酶GTamy和GTamy-S498的最适反应温度测定结果如图2所示。
重组α-淀粉酶的最适反应pH测定:将酶液与不同pH的1%(W/V)玉米淀粉溶液混合,于80℃下进行酶活测定。以酶比活力对pH作图,确定其最适反应pH。采用不同缓冲液配制不同pH的1%(W/V)玉米淀粉溶液:50mmol/L MES(pH 3.0~7.0)、50mmol/L MOPS(pH 7.0~11.0)。重组α-淀粉酶GTamy和GTamy-S498的最适反应pH测定结果如图3所示。
重组α-淀粉酶的热稳定性测定:将酶液于80℃保温,分时间梯度取出部分样品,以1%(W/V)玉米淀粉溶液为底物测定样品的酶活力。将未处理的酶液的酶活定义为100%,并以相对酶活的百分比对时间作图,评价酶的热稳定性。重组α-淀粉酶GTamy和GTamy-S498的热稳定性测定结果如图4所示。
以上酶学性质测定结果显示:突变体GTamy-S498的最适反应温度为80℃,最适反应pH为5.0,于80℃的半衰期为180min,突变体GTamy-S498的最适反应温度、最适反应pH和热稳定性与GTamy基本一致。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围的内。因此,本发明的保护范围应该以权利要求所界定的保护范围为准。
Claims (8)
1.一种玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体,其特征在于,其氨基酸序列如SEQ ID NO:1所示。
2.一种编码权利要求1所述的玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体的基因。
3.根据权利要求2所述的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO:2所示。
4.一种能表达生产权利要求1所述玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体的载体。
5.一种能表达生产权利要求1所述玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体的基因工程菌。
6.根据权利要求1所述的玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体的制备方法,其特征在于,以氨基酸序列如SEQ ID NO:3所示的生淀粉α-淀粉酶GTamy为出发序列,将GTamy中SBD结构域的氨基酸序列替换为突变体SBD-S498的氨基酸序列,其中SBD的氨基酸序列如SEQ ID NO:5所示,SBD-S498的氨基酸序列如SEQ ID NO:19所示。
7.根据权利要求6所述的制备方法,其特征在于,具体步骤如下:
1)根据Geobacillus thermoleovorans的生淀粉α-淀粉酶GTamy的基因序列,其基因序列如SEQ ID NO:4所示,采用化学全合成的方法合成优化的基因后,将其克隆到重组质粒pSTOP1622中,构建重组质粒pSTOP1622-gtamyh;
2)合成核苷酸序列“5’-ATCGCGCGCAGGGAT-SEQ ID NO:20-CACCATCACCATCAC-3’”,采用无缝克隆方法将重组质粒pSTOP1622-gtamyh中编码SBD的核苷酸序列SEQ ID NO:6替换为SBD环化重排突变体SBD-S498的核苷酸序列SEQ ID NO:20,获得GTamy环化重排突变体的表达载体pSTOP1622-gtamyh/S498;
3)将重组质粒pSTOP1622-gtamyh/S498转化枯草芽孢杆菌Bacillus subtilis WB600,获得枯草芽孢杆菌基因工程菌,诱导表达,获得生淀粉α-淀粉酶突变体GTamy-S498。
8.根据权利要求1所述玉米淀粉降解能力提高的嗜热酸性生淀粉α-淀粉酶突变体在纺织、洗涤剂、制革、造纸、食品领域的应用。
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