CN113005109B - 一种d-2-卤代酸脱卤酶突变体及其应用 - Google Patents
一种d-2-卤代酸脱卤酶突变体及其应用 Download PDFInfo
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Abstract
本发明属于酶工程领域，涉及突变体，特别是指一种D‑2‑卤代酸脱卤酶突变体及其应用。所述卤代酸脱卤酶突变体的氨基酸序列，是在SEQ IDNO.1所示的序列上进行单点突变，所述单点突变位于底物结合口袋入口处。本发明获得的突变体能够催化卤代酰胺的脱卤反应制备光学纯卤代酰胺或者D‑2‑羟基酰胺，其作用于氯乙酰胺、2‑氯丙酰胺与2‑溴丙酰胺的最大催化活力分别为4.43 U/mg蛋白、3.57 U/mg蛋白与0.36 U/mg蛋白，在化工合成中具有巨大的应用潜力。
Description
技术领域
本发明属于酶工程领域，涉及突变体，特别是指一种D-2-卤代酸脱卤酶突变体及其应用。
背景技术
2-卤代酸脱卤酶（2-haloacid dehalogenases，2-HADs）是催化2-卤代酸脱卤水解产生相应2-羟基酸的一类酶，包括具有立体选择性的L-与D-2-卤代酸脱卤酶（L-DEX与D-DEX）和无立体选择性的DL-2-卤代酸脱卤酶（DL-DEX）。其中D-DEX酶类（D-DEXs）是专一性地催化D-2-卤代酸脱卤水解产生L-2-羟基酸的一类酶。2-卤代酸脱卤酶的立体选择性使其在化工合成领域倍受青睐。具有较高立体选择性的2-卤代酸脱卤酶，可通过对酶不对称催化选择性的定向调控，控制反应程度，获得高光学纯的目标产物。如利用2-卤代酸脱卤酶生产小分子的手性羟基酸与卤代酸，这些小分子有机酸通常是农药、医药及化工合成的中间体，有着广泛的用途和巨大的经济价值。
目前虽然已通过多种策略获得多种不同来源的2-卤代酸脱卤酶，然而这些酶具有很强的底物选择性，仅对于碳链长度为2和3的氯代，溴代底物表现出高的催化活性，而对于氟代或碳链更长的底物的催化活性很低，对卤代酰胺仅有很低或者甚至无催化活性。2-羟基酰胺及其衍生物在医药化学中是重要的生物活性分子，如用于合成蛋白质及 DNA 研究。另外，手性2-羟基酰胺在有机合成中是重要的合成砌块，因此2-羟基酰胺类化合物的合成备受关注。目前2-羟基酰胺的获得主要是通过化学方法合成，通过氨酰基锂试剂在醛、酮的羰基碳上进行亲核加成反应引入氨酰基。其过程常常会涉及到刺激性、有毒试剂，反应效率低，副产物多，产物光学纯度低，而生物酶催化法不仅反应条件温和，对环境友好，且产率高，可精准地催化产生高度光学纯的手性化合物。
发明内容
为解决上述技术问题，本发明提出一种D-2-卤代酸脱卤酶突变体及其应用。
本发明的技术方案是这样实现的：
一种D-2-卤代酸脱卤酶突变体，所述卤代酸脱卤酶突变体的氨基酸序列，是在SEQIDNO.1所示的序列上进行单点突变，所述单点突变位于底物结合口袋入口处。
所述突变体为将SEQ ID NO.1序列的第288位亮氨酸突变为丙氨酸，氨基酸序列如SEQ ID NO.3所示。
一种编码D-2-卤代酸脱卤酶突变体的基因，其序列如SEQ ID NO.2所示。
含有上述的基因的重组质粒或工程菌。
上述的突变体在催化卤代酰胺脱卤反应中的应用。
上述的重组质粒或工程菌在催化制备手性卤代酰胺或手性羟基酰胺中的应用。
所述手性羟基酰胺为D-2-羟基酰胺。
本发明具有以下有益效果：
1、本发明对野生型D-2-卤代酸脱卤酶进行突变，对于氯乙酰胺突变体N203A，L288I，L288S，L288A，F281Y/L288I均表现出催化活性，催化比活性范围为0.09-4.43 U/mg蛋白，其中L288A的催化比活性为4.43（U/mg蛋白）；对于2-氯丙酰胺突变体突变体N203A，L288I，L288S，L288A，F281Y/L288I均表现出催化活性，催化比活性范围为0.02-3.57 U/mg蛋白，其中L288A的催化比活性为3.57 U/mg；对于2-氯丙酰胺突变体突变体N203A，L288S，L288A，F281Y/L288I均表现出催化活性，催化比活性范围为0.03-0.36 U/mg。其中L288A在催化D-2-卤代酸脱卤反应中，表现出卓越的催化活性；在催化氯乙酰胺脱氯效率催化活性最高为4.43 U/mg蛋白；催化2-氯丙酰胺脱氯效率催化活性最高为3.57 U/mg蛋白；催化2-溴丙酰胺脱溴的酶比活力最高为0.36 U/mg蛋白。。
2、现有的D-2-卤代酸脱卤酶DehD、DehII、DehDIV-R以及本发明中的野生型酶均不能催化卤代酰胺的脱卤反应，本发明获得的突变体使原来对卤代酰胺无催化能力的野生型D-2-卤代酸脱卤酶实现了对卤代酰胺的催化脱卤能力，拓宽了D-2-卤代酸脱卤酶的底物谱。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为各个HadD AJ1突变体酶的SDS-PAGE图谱；其中M，marker；1，N203A；2，204A；3，M284C；4，L288I；5，L288S；6，L288A；7，F281Y/L288I。
图2为HadD AJ1突变体酶催化氯乙酰胺脱氯的动力学曲线。
图3为HadD AJ1突变体酶催化2-氯丙酰胺脱氯的动力学曲线。
图4为HadD AJ1野生型酶与L288A突变体酶结构比较，其中箭头指向为结合口袋入口。
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一、卤代酸脱卤酶活测定方法
在1.0 mL反应体系中，含有10 mmol/L底物，100 mmol/L，pH10.0甘氨酸-氢氧化钠及适量酶。30℃水浴中反应适当时间，以保证底物减少量在5%以上，加入1%（v/v）的浓磷酸（85% w/w）终止反应。14,000×g，离心10 min去除沉淀。
酶活性通过HPLC检测产物生成量来确定，HPLC分析条件如下：
流动相A：磷酸水溶液，1L 超纯水加入磷酸（85%）至终浓度 0.015 mol/L，并用KH2PO4溶液调 pH 值至2.2；流动相 B：乙腈；分析柱：C18 ODS 柱 4.6 mm×250 mm；流动相A：流动相 B=80:20等度洗脱；柱温：30 ℃；流速：1 mL/min；检测波长：210 nm；进样体积：10μL。
酶活单位（U）定义为：每分钟催化1 μmol底物转化为产物所需要的酶量。
实施例1
D-2-卤代酸脱卤酶突变体的构建：
来源于Pseudomonas putida AJ1 的D-2-卤代酸脱卤酶序列（即序列表中SEQ IDNO. 1）的第203、204、281、284与288位点进行突变，分别设计并合成引入定点突变的引物，引物序列如表1所示：
表1突变体引物设计
定点突变体编码基因的PCR扩增：利用PCR技术，以构建质粒pET28a-HadD AJ1为模板，采用Takara PrimeSTAR® Max DNA Polymerase进行全质粒突变扩增，针对选择位点构建单点突变体。
PCR扩增程序设定为：首先，98℃预变性5min；然后进入30个循环：98℃变性10 s，55℃退火5s，72℃延伸35 s；最后72℃延伸7 min，4℃保温。PCR产物用1％琼脂糖凝胶电泳进行检测。
使用TaKaRa限制性内切酶DpnI除去反应体系中的质粒模板。
反应体系：8.5 μL PCR产物，1 μL 10×buffer，0.5 μLDpnI
反应条件：37 ℃，保温30 min。
该限制性内切酶消化产物冷却后直接转化至E. coli Dh5α感受态细胞。
然后挑取阳性克隆进行质粒抽提和DNA测序。将测序成功的突变体质粒转化至E. coli BL21（DE3）感受态细胞，构建突变体基因重组菌，用于突变体酶的诱导表达。
二、HadD AJ1突变体酶的表达纯化
将含突变体目的基因的重组质粒转化至感受态细胞E. coli BL21（DE3），挑取单克隆于50 mL 含50 mg/mL卡那霉素的LB培养基中，37 ℃，220 r/min培养至OD600达到0.45~0.55，加入终浓度为0.5 mmol/L 的IPTG，37 ℃诱导4 h。收集菌体，-80 ℃保存。
取-80 ℃冻存的菌体，重悬于预冷的含13.7 mmol/L β-ME与1 mmol/L PMSF的裂解缓冲液（50 mmol/LTris-HCl pH8.0，5%甘油，0.3 mol/LNaCl）中，置于冰浴中超声，条件为300 W，工作5 s，间隙5 s，120个循环。14,000 × g，离心30 min，收集上清。
准备镍离子亲和层析柱，向柱子里加入去离子水冲洗柱子（约8～12倍柱体积），然后用平衡缓冲液（50 mmol/L Tris-HCl pH8.0，5%甘油，0.3 mol/L NaCl，5 mmol/L咪唑）平衡柱子（约5倍柱体积缓冲液)，将粗酶液加入至柱子。先用含有低浓度咪唑的缓冲液（50mmol/L Tris-HCl pH8.0，5%甘油，0.3 mol/L NaCl，25 mmol/L咪唑）冲洗至杂蛋白，再用
含有高浓度咪唑的洗脱液（50 mmol/L Tris-HCl pH8.0，5%甘油，0.3 mol/LNaCl，350mmol/L咪唑）洗脱，收集洗脱的目的蛋白。
准备Superdex 200凝胶过滤层析柱，在4℃环境下利用恒流泵，向柱子里泵入平衡缓冲液（50 mmol/L KH2PO4-K2HPO4，pH8.0）平衡柱子，将镍柱亲和层析获得的目的蛋白打入进样器，使用洗脱缓冲液（50 mmol/L KH2PO4-K2HPO4，pH8.0）进行等度洗脱，检测波长为280nm，收集吸收峰处的洗脱液，用于后续酶活性分析及SDS-PAGE蛋白电泳。
如图1为纯化后HadD AJ1变体酶N203A、S204A、M284C、L288I、L288S、L288A、F281Y/L288I到电泳纯。
应用例1：HadD AJ1突变体酶催化氯乙酰胺脱氯效率测定
本应用例参照卤代酸脱卤酶酶活测定方法，将纯化后的野生型酶与突变体酶在标准反条件下进行催化反应，使用HPLC检测氯代乙酰胺含量，计算突变体酶活。
表2 野生型酶和突变体酶的催化比活性比较
如表2所示,发现突变体S204A与M284C无催化活性，突变体N203A、L288I、L288S、L288A、L288I/F281Y比酶活为0.09-4.43 U/mg蛋白，其中突变体L288A催化活性最高为4.43U/mg蛋白。
应用例2：HadD AJ1突变体酶催化2-氯丙酰胺脱氯效率测定
本应用例参照卤代酸脱卤酶酶活测定方法，将纯化后的野生型酶与突变体酶在标准反条件下进行催化反应，使用HPLC检测2-氯丙酰胺含量，计算突变体酶活。
表3 野生型酶和突变体酶的催化比活性比较
如表3所示，发现突变体S204A与M284C无催化活性，突变体N203A、L288I、L288S、L288A、L288I/F281Y比酶活为0.02-3.57 U/mg蛋白，其中突变体L288A催化活性最高为3.57U/mg蛋白。
应用例3 ：HadD AJ1突变体酶催化2-溴丙酰胺脱溴效率测定
本应用例参照卤代酸脱卤酶酶活测定方法，将纯化后的野生型酶与突变体酶在标准反条件下进行催化反应，使用HPLC检测2-溴丙酰胺含量，计算突变体酶活。
表4 野生型酶和突变体酶的催化比活性比较
如表4所示，发现突变体S204A、M284C、L288S与L288I无催化活性，突变体N203A、L288A、L288I/F281Y比酶活分别为0.03 U/mg蛋白、0.36 U/mg蛋白、0.15 U/mg蛋白。
应用例4： HadD AJ1突变体酶催化氯代酰胺脱氯动力学曲线
参照卤代酸脱卤酶酶活测定方法在标准反应体系中加入不同浓度的氯乙酰胺或者2-氯丙酰胺，30 ℃反应一段时间后，检测不同浓度底物下的反应速度。运用origin拟合反应速度与底物浓度的关系曲线计算突变体酶L288 A催化氯代酰胺脱氯的催化效率。如图2与图3所示，突变体酶L288 A催化氯乙酰胺与2-氯丙酰胺的K m值分别为8.31 mM与14.3mM，催化效率K cat/K m值分别为0.57 mM/s与0.16 mM/s。
本申请的突变体L288A的高级结构如图4所示，本发明中，以野生型 D-2-卤代酸脱卤酶晶体结构(PDB ID: 5H01)，通过Swiss-Model在线服务器构建了突变体酶结构。该突变位点位于底物结合口袋入口处。与野生型酶相比，突变体酶活性口袋入口无侧链异丙基的阻碍，为卤代酰胺进入酶活性中心创造空间。
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，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
<110> 商丘师范学院
<120> 一种D-2-卤代酸脱卤酶突变体及其应用
<141> 2021-03-30
<160> 3
<170> SIPOSequenceListing 1.0
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<213> 恶臭假单胞菌(Pseudomonas putida)
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atgaacctgc ctgacaattc catccatctt caactgccgc ggcccgtctg cgaggcaatc 60
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aagggccacg ttcggcgtgc cctgggttgg ggtcatcacg caggcggtcg cctactaccg 180
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cacgcgggcg gcaccctctc gcaggtttat gccgatatca agcagaccct gcaattgcct 600
ttcatcaaca gtgactacaa ggccatggcg cggtggccga gctacctgga gcaggcatgg 660
ggcgcgctga aaccctgtat cgacacaccg gcttatcagg cgggcaggtt cgacatcaat 720
gcgcgggcac tggctgcgct cgatgccttg ccgaccgctt accgaatgag ccgggacgat 780
gcgctacagg cgggcctcag cgaggcgcaa accgatgagc tcatacaggt catcagcttg 840
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aaataa 906
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Claims (3)
1.一种D-2-卤代酸脱卤酶突变体在催化制备手性羟基酰胺中的应用，其特征在于：所述卤代酸脱卤酶突变体的氨基酸序列，是在SEQ IDNO.1所示的序列上进行单点突变，将第288位亮氨酸突变为丙氨酸，所述单点突变位于底物结合口袋入口处。
2.根据权利要求1所述的应用，其特征在于：所述D-2-卤代酸脱卤酶突变体的基因序列如SEQ ID NO.2所示。
3.根据权利要求1或2所述的应用，其特征在于：所述手性羟基酰胺为D-2-羟基酰胺。
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| A new dl-2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism.;Chung Park等;《Journal of Molecular Catalysis B: Enzymatic》;20031231;第23卷;第329-336页 * |
| D-2-haloalkanoate dehalogenase [Pseudomonas putida];BARTH,P.T.等;《genbank：AAA25831.1》;19930426;ORIGIN * |
| Stereoselective catalysis controlled by a native leucine or variant isoleucine wing-gatekeeper in 2-haloacid dehalogenase.;Yanbin Feng等;《FEBS Letters》;20181228;第593卷;第308页摘要，第309页左栏倒数第1段-右栏第1段，第310页右栏第1段，第311页右栏倒数第1段-312页左栏第1段 * |
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