CN111849933A - 一种亮氨酸脱氢酶突变体及其构建方法和应用 - Google Patents
一种亮氨酸脱氢酶突变体及其构建方法和应用 Download PDFInfo
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- CN111849933A CN111849933A CN202010800001.XA CN202010800001A CN111849933A CN 111849933 A CN111849933 A CN 111849933A CN 202010800001 A CN202010800001 A CN 202010800001A CN 111849933 A CN111849933 A CN 111849933A
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- leucine dehydrogenase
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Abstract
本发明提供了一种亮氨酸脱氢酶突变体及其构建方法和应用，属于基因工程技术领域。本发明所述突变体是由野生型亮氨酸脱氢酶氨基酸序列突变而来，所述野生型亮氨酸脱氢酶氨基酸序列第89位的赖氨酸饱和突变为NNN或第122位的丙氨酸定点突变为甘氨酸，所述野生型亮氨酸脱氢酶的氨基酸序列如SEQ ID NO.1所示。实验结果表明：K89T亮氨酸脱氢酶对Asn和Thr表现出一定活性，A122G亮氨酸脱氢酶对Phe、His、Met表现一定活性，而野生型亮氨酸脱氢酶对这些氨基酸完全无活性；并且，K89T亮氨酸脱氢酶对于Asp氧化脱氨反应的酶活是野生型亮氨酸脱氢酶的2倍。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及一种亮氨酸脱氢酶突变体及其构建方法和应用。
背景技术
亮氨酸脱氢酶(Leucine dehydrogenase,LeuDH)属于短链脱氢酶家族，催化天然的底物4-甲基-2-氧代戊酸还原氨化得到L-亮氨酸，能可逆催化L-型氨基酸氧化脱氨合成α-酮酸，目前亮氨酸脱氢酶已经广泛应用于工业生产手性氨基酸。亮氨酸脱氢酶在自然界生物体内广泛存在，NCBI数据库检索其来源，总来源有11万种，动物来源有1757种，细菌来源有108864种，真菌来源有2221种，其余来源共计5000种。目前亮氨酸脱氢酶已经广泛应用于手性氨基酸催化合成。
亮氨酸脱氢酶应用十分广泛，主要集中于生物酶催化合成医药中间体方面。据文献报道，非蛋白源氨基酸在药物合成中有非常重要的作用，因此亮氨酸脱氢酶在合成非蛋白源氨基酸有巨大潜力和经济价值。例如常用于非蛋白源氨基酸L-叔亮氨酸及其衍生物合成，由于其侧链叔丁基团的空间位阻及强疏水性，常用于分子结构的调控，在多肽类药物中常替代其他氨基酸，赋予多肽分子更强的疏水性和稳定性。应用亮氨酸脱氢酶生物酶催化合成L-叔亮氨酸已经实现产业化。此外，亮氨酸脱氢酶在酶诊断方面具有重要应用。亮氨酸脱氢酶不仅作为酶制剂可用于患枫糖尿症病患血液中L-亮氨酸含量的诊断，也可与脲酶进行偶联测定尿素，这种方法具有较强的抗内源性氨干扰的能力。另外，亮氨酸脱氢酶也可用于血液中肿瘤及血管生长的标志物LAP(leucine aminopeptidase)含量的诊断，对于早期及时发现和治疗病毒性肝病具有重大作用。
虽然亮氨酸脱氢酶的应用范围非常广，然而由于其自身底物谱范围的限制，导致亮氨酸脱氢酶只能够催化合成L-亮氨酸等少数几个手性氨基酸，不能催化合成其他手性氨基酸。所以需要开拓新的方法来拓宽亮氨酸脱氢酶在工业生产和医药等行业的应用范围。
发明内容
有鉴于此，本发明的目的在于提供一种亮氨酸脱氢酶突变体及其构建方法和应用。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种亮氨酸脱氢酶突变体，所述突变体是由野生型亮氨酸脱氢酶氨基酸序列突变而来，所述野生型亮氨酸脱氢酶氨基酸序列第89位的赖氨酸饱和突变为NNN或第122位的丙氨酸定点突变为甘氨酸。
优选的，所述野生型亮氨酸脱氢酶的氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种根据上述亮氨酸脱氢酶突变体的构建方法，包括：
1)将野生型亮氨酸脱氢酶DNA与表达载体连接获得重组载体；
2)以步骤1)获得的重组载体为模板，通过PCR突变得到PCR产物；
当突变位点为野生型亮氨酸脱氢酶氨基酸序列第89位点时，所述PCR突变用引物为K89饱-IR和K89饱-IF，所述K89饱-IR的序列如SEQ ID NO.2所示；所述K89饱-IF的序列如SEQ ID NO.3所示；
当突变位点为野生型亮氨酸脱氢酶氨基酸序列第122位点时，所述PCR突变用引物为A122G-IF和A122G-IR，所述A122G-IF的序列如SEQ ID NO.4所示；所述A122G-IR的序列如SEQ ID NO.5所示；
3)用DPN1酶将步骤2)获得的PCR产物消化处理，得到突变重组载体；
4)将步骤3)获得的突变重组载体转入表达菌株，获得重组菌株，对所述重组菌株进行培养，得到亮氨酸脱氢酶突变体。
优选的，步骤1)所述表达载体为pET28a质粒。
优选的，在步骤2)中，当突变位点为野生型亮氨酸脱氢酶氨基酸序列第89位点时，所述PCR突变程序为：94℃预变性4min；94℃变性20s，60℃退火20s，72℃延伸3min，35循环；72℃延伸10min；4℃保存。
优选的，在步骤2)中，当突变位点为野生型亮氨酸脱氢酶氨基酸序列第122位点时，所述PCR突变程序为：4℃预变性4min；94℃变性20s，62℃退火20s，72℃延伸3min，30循环；72℃延伸10min；4℃保存。
优选的，步骤3)所述消化处理的温度为36～38℃，时间为1.5～2.5h。
优选的，步骤4)所述表达菌株为大肠杆菌BL21(DE3)。
优选的，步骤4)所述培养后还包括以下步骤：将所述培养得到的重组菌株破碎，收集上清液，经镍柱纯化后获得亮氨酸脱氢酶突变体。
本发明还提供了上述亮氨酸脱氢酶突变体在改变亮氨酸脱氢酶底物谱中的应用。
相对于现有技术，本发明具有如下有益效果：
本发明提供的突变体是由野生型亮氨酸脱氢酶氨基酸序列突变而来，所述野生型亮氨酸脱氢酶氨基酸序列第89位的赖氨酸饱和突变为NNN或第122位的丙氨酸定点突变为甘氨酸，所述野生型亮氨酸脱氢酶的氨基酸序列如SEQ ID NO.1所示。实验结果表明：K89T亮氨酸脱氢酶对Asn和Thr表现出一定活性，A122G亮氨酸脱氢酶对Phe、His、Met表现出一定活性，而野生型亮氨酸脱氢酶对这些氨基酸完全无活性；并且，K89T亮氨酸脱氢酶对于Asp氧化脱氨反应的酶活是野生型亮氨酸脱氢酶的2倍。
附图说明
图1为突变89位点DNA凝胶电泳图，其中M为Marker，1、2、3、4、5均为重复样；
图2为突变122位点DNA凝胶电泳图，其中M为Marker，1、2、3、4均为重复样；
图3为蛋白SDS-PAGE图；
图4为野生型亮氨酸脱氢酶的催化底物谱；
图5为突变89位点和突变122位点的亮氨酸脱氢酶催化底物谱。
具体实施方式
本发明提供了一种亮氨酸脱氢酶突变体，所述突变体是由野生型亮氨酸脱氢酶氨基酸序列突变而来，所述野生型亮氨酸脱氢酶氨基酸序列第89位的赖氨酸饱和突变为NNN或第122位的丙氨酸定点突变为甘氨酸。
本发明中，所述野生型亮氨酸脱氢酶的氨基酸序列如SEQ ID NO.1所示，具体如下：
Met Val Glu Thr Asn Val Glu Ala Arg Phe Ser Ile Phe Glu Thr Met AlaMet Glu Asp Tyr Glu Gln Val Val Phe Cys His Asp Lys Val Ser Gly Leu Lys AlaIle Ile Ala Ile His Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Leu Arg Met TrpAsn Tyr Ala Ser Asp Glu Glu Ala Leu Ile Asp AlaLeuArgLeu Ala Lys Gly Met ThrTyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys Ala Val Ile Ile GlyAsp Ala Lys Thr Gln Lys Ser Glu Ala Leu Phe Arg Ala Phe Gly Arg Tyr Val GlnSer Leu Asn Gly Arg Tyr Ile Thr Ala Glu Asp Val Asn Thr Thr Val Ala Asp MetAsp Tyr Ile His Met Glu Thr Asp Phe Val Thr Gly Val Ser Pro Ala PheGlySer SerGly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala Ala AlaLys Glu Val Tyr Gly Thr Asp Ser Leu Gly Gly Lys Thr Val Ala Ile Gln Gly ValGly Asn Val Ala Phe Asn Leu Cys Arg His Leu His Glu Glu Gly Ala Lys Leu IleVal Thr Asp Ile Asn Gln Asp Ala Leu Arg Arg Ala Glu Glu Ala Phe Gly Ala LeuVal Val Gly Pro Asp Glu Ile Tyr Ser Val Asp Ala Asp Ile Phe Ala Pro Cys AlaLeu Gly Ala Thr Leu Asn Asp Glu Thr Ile Pro Gln Leu Lys Val Lys Ile Ile AlaGly Ala Ala Asn Asn Gln Leu Lys Glu Asp Arg His Gly Asp Met Leu Gln Glu ArgGly Ile Leu Tyr Thr Pro Asp Phe Val Ile Asn Ala Gly Gly Val Ile Asn Val AlaAsp Glu Leu Asp Gly Tyr Asn Arg Glu Arg Ala Met Lys Lys Val Glu Leu Val TyrAsp Ala Val Ala Lys Val Ile Glu Ile Ala Lys Arg Asp His Leu Pro Thr Tyr ArgAla Ala Glu Lys Met Ala Glu Glu Arg Ile Ala Thr Met Gly Ser Ala Arg Ser GlnPhe Leu Arg Arg Asp Lys Asn Ile Leu Gly Ser Arg。
本发明还提供了一种根据上述亮氨酸脱氢酶突变体的构建方法，包括：1)将野生型亮氨酸脱氢酶DNA与表达载体连接获得重组载体；2)以步骤1)获得的重组载体为模板，通过PCR突变得到PCR产物；当突变位点为野生型亮氨酸脱氢酶氨基酸序列第89位点时，所述PCR突变用引物为K89饱-IR和K89饱-IF，所述K89饱-IR的序列如SEQ ID NO.2所示；所述K89饱-IF的序列如SEQ ID NO.3所示；当突变位点为野生型亮氨酸脱氢酶氨基酸序列第122位点时，所述PCR突变用引物为A122G-IF和A122G-IR，所述A122G-IF的序列如SEQ ID NO.4所示；所述A122G-IR的序列如SEQ ID NO.5所示；3)用DPN1酶将步骤2)获得的PCR产物消化处理，得到突变重组载体；4)将步骤3)获得的突变重组载体转入表达菌株，获得重组菌株，对所述重组菌株进行培养，得到亮氨酸脱氢酶突变体。
在本发明中，将野生型亮氨酸脱氢酶DNA与表达载体连接获得重组载体。在本发明中所述野生型亮氨酸脱氢酶DNA序列优选为短小芽孢杆菌(Exiguobacteriumsibiricum255-15)来源的亮氨酸脱氢酶DNA。在本发明中，所述表达载体优选为大肠杆菌表达载体，更优选为pET28a质粒。在本发明中，所述连接具体为将亮氨酸脱氢酶基因和原始质粒pET28a用限制性内切酶切割后用连接酶连接。在本发明中所述限制性内切酶优选为Xho1和BamH1，所述连接酶优选为T4连接酶。在本发明中所述连接的具体体系为野生型亮氨酸脱氢酶基因、载体质粒pET28a、10xT4 DNA ligase buffer、T4 DNA ligase、补充ddH2O至总体积达20μL。在本发明中所述连接的温度优选为15～20℃，更优选为16℃；所述连接的时间优选为10～15h；更优选为12h。
本发明在获得重组载体后，以获得的重组载体为模板，通过PCR突变得到PCR产物。在本发明中，当突变位点为野生型亮氨酸脱氢酶氨基酸序列第89位点时，所述PCR突变用引物为K89饱-IR和K89饱-IF，所述K89饱-IR的序列如SEQ ID NO.2所示，具体为：GATGATGACCGCNNNCCCG；所述K89饱-IF的序列如SEQ ID NO.3所示，具体为：GCGGGNNNGCGGTCATCAT；其中，K89饱-IR和K89饱-IF序列中的N代表A、C、G、T其中的一种；当突变位点为野生型亮氨酸脱氢酶氨基酸序列第122位点时，所述PCR突变用引物为A122G-IF和A122G-IR，所述A122G-IF的序列如SEQ ID NO.4所示，具体为：CGTCTTCGCCAGTGATGTAACGTC；所述A122G-IR的序列如SEQ ID NO.5所示，具体为：CATCACTGGCGAAGACGTCAACAC。
在本发明中所述PCR突变的体系优选包括：15ng/μL的野生型亮氨酸脱氢酶DNA 1μL，突变引物IF、IR(10μM)各1μL，2X Trans start fastPfu 15μL，ddH2O补充体系至30μL。
在本发明中，在步骤2)中，当突变位点为野生型亮氨酸脱氢酶氨基酸序列第89位点时，所述PCR突变程序为：94℃预变性4min；94℃变性20s，60℃退火20s，72℃延伸3min，35循环；72℃延伸10min；4℃保存；所述PCR突变后，获得的突变体名称为K89T。
在本发明中，在步骤2)中，当突变位点为野生型亮氨酸脱氢酶氨基酸序列第122位点时，所述PCR突变程序为：4℃预变性4min；94℃变性20s，62℃退火20s，72℃延伸3min，30循环；72℃延伸10min；4℃保存；所述PCR突变后，获得的突变体名称为A122G。
本发明在获得PCR产物后，用DPN1酶将获得的PCR产物消化处理，得到突变重组载体。在本发明中，所述消化处理的温度优选为36～38℃，更优选为37℃，时间优选为1.5～2.5h，更优选为2h。在本发明中，对PCR产物进行消化处理，能够消解非突变型基因。
本发明在获得突变重组载体后，将获得的突变重组载体转入表达菌株，获得重组菌株，对所述重组菌株进行培养，得到亮氨酸脱氢酶突变体。在本发明中，所述表达菌株优选为大肠杆菌，更优选为大肠杆菌BL21(DE3)。
在本发明中所述的转化方法优选采用热激法。具体为：向置于冰上已融化的大肠杆菌BL21(DE3)悬浮液中加入突变重组载体然后轻轻旋转离心管以混匀内容物，于冰上放置30min，接着用42℃水浴热激90s迅速置于冰上3min，然后在无菌条件下加入700μL无抗生素的LB液体培养基，混匀后置于37℃摇床培养1h，然后涂板。在本发明中所述大肠杆菌BL21(DE3)悬浮液与突变重组载体的体积之比不小于10:1。
本发明在获得重组菌株后，对获得的重组菌株进行诱导扩大培养获得大量重组菌株。在本发明中，所述诱导培养优选采用化学试剂IPTG(异丙基硫代半乳糖苷)诱导方法进行突变蛋白的诱导表达。本发明所述诱导扩大培养步骤具体为：获得重组菌株后将，取适量菌液置于200mLLB培养基进行液体培养，培养至OD600达到0.6～0.8后，在无菌条件下加入诱导剂IPTG，诱导培养。在发明中所述IPTG的浓度优选为80～150mM，更优选为100mM，所述IPTG终溶度优选的为0.2～0.5mM，更优选为0.4mM；所述IPTG诱导培养的时间优选为2～3h，更优选的为2.5h。本发明在所述IPTG诱导培养结束后获得大量表达亮氨酸脱氢酶突变体的重组菌株。
本发明在所述IPTG诱导扩大培养后，优选还包括收集重组菌株。在本发明中所述收集重组菌株的方法优选为离心。在本发明中所述离心的温度优选为3～5℃，更优选为4℃；所述离心的转速优选为7500～10000rpm，更优选为8500rpm；所述离心的时间优选为8～20min，更优选为10min。
本发明在收集获得重组菌株后，优选还包括以下步骤：将所述培养得到的重组菌株破碎，收集上清液，经镍柱纯化后获得亮氨酸脱氢酶突变体。本发明对破碎的方式没有特殊的限定，采用本领域技术人员常规方法即可。
本发明还提供了上述亮氨酸脱氢酶突变体在改变亮氨酸脱氢酶底物谱中的应用。
本发明中所涉及的原料及试剂的来源没有特殊的限定，采用本领域技术人员常规选用的产品即可。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
亮氨酸脱氢酶突变体的制备
以来源短小芽孢杆菌的亮氨酸脱氢酶为野生型，即WT EsiLeuDH。
(1)将WT EsiLeuDH DNA与质粒pET28a连接，得到重组载体。
(2)获得突变体K89T：
以重组载体的DNA为模板，以K89饱-IR(核苷酸序列如SEQ ID NO.2所示，具体为：GATGATGACCGCNNNCCCG)和K89饱-IF(序列如SEQ ID NO.3所示，具体为：GCGGGNNNGCGGTCATCAT)为引物，进行PCR定点饱和突变，得到PCR产物。其中，PCR反应体系如下：15ng/μL的WT EsiLeuDH DNA 1μL，突变引物IF、IR(10μM)各1μL，2X Trans startfastPfu 15μL，ddH2O补充体系至30μL。PCR反应程序：94℃预变性4min；94℃变性20s，60℃退火20s，72℃延伸3min，35循环；72℃延伸10min；4℃保存。
获得突变体A122G：
以重组载体的DNA为模板，以A122G-IF(核苷酸序列如SEQ ID NO.4所示，具体为：CGTCTTCGCCAGTGATGTAACGTC)和A122G-IR(核苷酸序列如SEQ ID NO.5所示，具体为：CATCACTGGCGAAGACGTCAACAC)为引物，进行PCR定点突变，得到PCR产物。其中，PCR反应体系如下：15ng/μL的WT EsiLeuDH DNA 1μL，突变引物IF、IR(10μM)各1μL，2X Trans startfastPfu 15μL，ddH2O补充体系至30μL。PCR反应程序：94℃预变性4min；94℃变性20s，60℃退火20s，72℃延伸3min，35循环；72℃延伸10min；4℃保存。4℃预变性4min；94℃变性20s，62℃退火20s，72℃延伸3min，30循环；72℃延伸10min；4℃保存。
(3)将PCR产物进行琼脂糖凝胶电泳判断是否突变基因构建成功
精确称取0.25g琼脂糖(Agarose)，用1×TAE Buffer加热溶解，冷却至60℃时加入2.5μL 4S Red Plus核酸染色剂，摇匀后缓慢倒入已经插好样品梳的制胶槽中，待其凝固，将胶连同托板放入电泳槽中，样品孔一端靠近电泳槽负极，添加1×TAE buffer使其没过凝胶。拔掉样品梳，向第一个孔中加入5μL DNA Marker，用于目的基因大小的比对。向样品中添加一定量的6×DNA loading buffer，混匀后从第二个孔开始依次加入处理后的样品，启动电泳仪，程序设置为120V，35min。将电泳后的凝胶用凝胶成像仪进行分析后，保存图片。
突变基因构建成功的标准：目标条带最亮的部分与Marker7000bp条带相比较，相近即为构建成功。
由图1和图2可知，突变体K89T基因和突变体A122G基因构建成功。
实施例2
突变体的表达及纯化
将实施例1中获得的PCR产物利用DPN1酶在37℃情况下进行消化处理2h。消化处理结束后采用热激法将产物直接转入大肠杆菌BL21(DE3)感受态细胞，得到重组菌株。将获得的重组菌株涂布至含Kana抗生素的平板当中，将平板至于37℃培养箱当中培养12h，待长出菌落后，挑取菌落进行PCR菌落验证和突变文库的筛选，随后进行液培，将部分菌液送去测序。PCR菌落验证以及测序结果表明突变体A122G和K89T基因已经构建好并成功导入大肠杆菌BL21(DE3)。
将获得的重组菌株扩大培养，采用化学试剂IPTG诱导方法进行突变蛋白的诱导表达，诱导表达结束后，经过离心、破碎、镍柱纯化等操作后，得到纯化蛋白，即亮氨酸脱氢酶，采取SDS-PAGE方法对其进行验证。
由图3可知，WT纯化蛋白、K89T纯化蛋白、A122G粗酶和A122G纯化蛋白均有明显条带，可见，突变体K89T和突变体A122G基因在大肠杆菌BL21(DE3)中成功表达出亮氨酸脱氢酶。
实施例3
亮氨酸脱氢酶的酶活测定
将实施例2中得到的WT纯化蛋白、K89T纯化蛋白和A122G纯化蛋白进行底物谱测定，底物谱测定体系：使用酶标仪测定，反应体系总体积220μL，光程为0.5cm，将亮氨酸脱氢酶适当稀释后，取10μL，4mM反应底物(实验室藏有的20种氨基酸中的任一种)50μL，20mM辅酶NAD+11μL，0.2M，pH9.5甘氨酸-氢氧化钠缓冲液149μL；在波长340nm处测定NADH浓度的增加量，一个单位的亮氨酸脱氢酶的酶活力定义为1min内还原1μmol NAD+所需的酶量。
酶活测定原理：
酶活(U/mL)＝ΔA/Δt×Vt/(Vs×L×ε)；
比酶活(U/mg)＝酶活(U/mL)/酶浓度(mg/mL)；
其中，Vs：反应总体积(mL)；Vt酶体积(mL)；ΔA：吸光度变化值；Δt：吸光度变化的时间(min)；L：光程(cm)；ε：摩尔吸光系数(6.22mL·(μmol·cm)-1)。
WT纯化蛋白、K89T纯化蛋白和A122G纯化蛋白的比酶活结果见表1。
表1 WT纯化蛋白、K89T纯化蛋白和A122G纯化蛋白的比酶活
由表1和图4-5可以得出，虽然K89T亮氨酸脱氢酶和A122G亮氨酸脱氢酶对于WTEsiLeuDH原有底物谱范围变窄，但是K89T亮氨酸脱氢酶对Asn和Thr表现出一定活性，A122G亮氨酸脱氢酶对Phe、His、Met表现出一定活性，而WT EsiLeuDH对这些氨基酸完全无活性。并且，K89T亮氨酸脱氢酶对于Asp氧化脱氨反应的酶活是WT EsiLeuDH的2倍。由此可见，采用本发明的技术方案成功改变了亮氨酸脱氢酶底物谱，成功合成类似Phe等其他手性氨基酸。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
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Claims (10)
1.一种亮氨酸脱氢酶突变体，其特征在于，所述突变体是由野生型亮氨酸脱氢酶氨基酸序列突变而来，所述野生型亮氨酸脱氢酶氨基酸序列第89位的赖氨酸饱和突变为NNN或第122位的丙氨酸定点突变为甘氨酸。
2.根据权利要求1所述的亮氨酸脱氢酶突变体，其特征在于，所述野生型亮氨酸脱氢酶的氨基酸序列如SEQ ID NO.1所示。
3.一种根据权利要求1或2所述亮氨酸脱氢酶突变体的构建方法，其特征在于，包括：
1)将野生型亮氨酸脱氢酶DNA与表达载体连接获得重组载体；
2)以步骤1)获得的重组载体为模板，通过PCR突变得到PCR产物；
当突变位点为野生型亮氨酸脱氢酶氨基酸序列第89位点时，所述PCR突变用引物为K89饱-IR和K89饱-IF，所述K89饱-IR的序列如SEQ ID NO.2所示；所述K89饱-IF的序列如SEQID NO.3所示；
当突变位点为野生型亮氨酸脱氢酶氨基酸序列第122位点时，所述PCR突变用引物为A122G-IF和A122G-IR，所述A122G-IF的序列如SEQ ID NO.4所示；所述A122G-IR的序列如SEQ ID NO.5所示；
3)用DPN1酶将步骤2)获得的PCR产物消化处理，得到突变重组载体；
4)将步骤3)获得的突变重组载体转入表达菌株，获得重组菌株，对所述重组菌株进行培养，得到亮氨酸脱氢酶突变体。
4.根据权利要求3所述的构建方法，其特征在于，步骤1)所述表达载体为pET28a质粒。
5.根据权利要求3所述的构建方法，其特征在于，在步骤2)中，当突变位点为野生型亮氨酸脱氢酶氨基酸序列第89位点时，所述PCR突变程序为：94℃预变性4min；94℃变性20s，60℃退火20s，72℃延伸3min，35循环；72℃延伸10min；4℃保存。
6.根据权利要求3所述的构建方法，其特征在于，在步骤2)中，当突变位点为野生型亮氨酸脱氢酶氨基酸序列第122位点时，所述PCR突变程序为：4℃预变性4min；94℃变性20s，62℃退火20s，72℃延伸3min，30循环；72℃延伸10min；4℃保存。
7.根据权利要求3所述的构建方法，其特征在于，步骤3)所述消化处理的温度为36～38℃，时间为1.5～2.5h。
8.根据权利要求3所述的构建方法，其特征在于，步骤4)所述表达菌株为大肠杆菌BL21(DE3)。
9.根据权利要求3所述的构建方法，其特征在于，步骤4)所述培养后还包括以下步骤：将所述培养得到的重组菌株破碎，收集上清液，经镍柱纯化后获得亮氨酸脱氢酶突变体。
10.权利要求1所述的亮氨酸脱氢酶突变体在改变亮氨酸脱氢酶底物谱中的应用。
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