CN114214308B - 一种经半理性改造提升活性的腈水解酶突变体 - Google Patents
一种经半理性改造提升活性的腈水解酶突变体 Download PDFInfo
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Abstract
本发明公开了一种经半理性改造提升活性的腈水解酶突变体，属于酶工程领域。本发明为了提高腈水解酶PCC6803酶活，通过底物对接确定了催化口袋附近与催化活性相关的位点，利用CAST建库NNK突变，通过已构建的烟酸传感器作为初筛工具，利用荧光强度作为筛选依据，将获得的有效果(荧光强度低的)的两个库的位点进行组合突变，再利用传感器进行筛选，然后将荧光强度低的突变体重新构建至表达载体及宿主中表达及纯化，利用HPLC测定纯酶比酶活，最终获得比酶活比野生酶WT提高了57％、180％、555％的突变体。
Description
技术领域
本发明涉及一种经半理性改造提升活性的腈水解酶突变体，属于酶工程领域。
背景技术
腈水解酶属于腈水解酶超家族，是一种重要的工业用酶，可以将腈类化合物一步反应生成羧酸类物质和氨。羧酸类物质在大宗化学品、医药中间体等有广泛的应用价值，目前烟酸和扁桃酸可以在工业上进行规模化生产，烟酸可以作为食品添加剂、维生素及医药中间体存在。酶法合成相比化学法具有反应条件温和、高立体选择性和不需要添加昂贵的催化剂等优势，既可以产生巨大的经济效益，也可以减轻对环境的污染。但是天然的腈水解酶酶活较低使得其在工业应用中存在弊端，因此通过蛋白质工程改造腈水解酶提高酶活可以为其工业应用做出贡献。
由于野生型腈水解酶通常难以适应工业环境需求，因此通过理性和非理性蛋白质改造方法提高腈水解酶催化性能成为研究热点。DeSantis等通过点饱和突变技术改造野生型腈水解酶，获得的突变体A190H能够催化3 M 3-羟基戊二腈完全水解合成(R)-4-氰基-3-羟基丁酸，产物ee值高达99％(J.Am.Chem.Soc.,2003,125:11476-11477)。Schreiner等利用易错PCR技术改造Arabidopsis thaliana腈水解酶AtNIT2，筛选获得催化苯乙腈水解活力提高4倍的突变体(ChemCatChem,2010,2:263-267)。
发明内容
为提高对腈类物质的催化能力，本发明选用来源于集胞藻（Synechocystis sp.PCC6803）的腈水解酶Nit6803（氨基酸序列的NCBI登录号为：AGF53008.1），通过分析酶结构上的潜在突变位点，选择一个或多个突变位点，应用分子生物学技术，筛选出腈类物质催化能力提高的突变体进一步推进催化腈类物质的腈水合酶的优良改造，为工业化生产奠定基础。
本发明的目的在于提供一种腈类物质催化能力提高的腈水解酶的突变体及其应用。
本发明的第一个目的是提供一种腈水解酶突变体，所述突变体是在氨基酸序列如SEQ ID NO.1的腈水解酶的第170位、第197位、第198位中的两个以上的位点进行突变得到的。
在一种实施方式中，所述突变体为一下（a）~（c）任一：
（a）将氨基酸序列SEQ ID NO.1的第170位的蛋氨酸突变成甘氨酸，将第197位的蛋氨酸突变成苯丙氨酸，将第198位的缬氨酸突变成异亮氨酸；
（b）将氨基酸序列SEQ ID NO.1的第170位的蛋氨酸突变成甘氨酸，将第198位的缬氨酸突变成天冬氨酸；
（c）将氨基酸序列SEQ ID NO.1的第170位的蛋氨酸突变成甘氨酸，将第197位的蛋氨酸突变成苯丙氨酸，将第198位的缬氨酸突变成亮氨酸。
在本发明的一种实施方式中，所述突变体的氨基酸序列如SEQ ID NO.2~SEQ IDNO.4所示。
本发明的第二个目的是提供一种编码上述腈水解酶突变体的基因。
本发明的第三个目的是提供一种携带上述基因的重组载体。
在本发明的一种实施方式中，所述重组载体以pET-24a(+)为表达载体。
本发明的第四个目的是提供一种携带上述基因，或上述重组载体的微生物细胞。
在本发明的一种实施方式中，所述微生物细胞以细菌或真菌为表达宿主。
在本发明的一种实施方式中，所述微生物细胞以E. coliER2566为表达宿主。
本发明的第五个目的是提供一种烟酸的制备方法，所述方法为，将上述腈水解酶突变体，或上述微生物细胞添加至含有烟腈的培养基中，进行反应制备得到的。
本发明还提供上述腈水解酶突变体，或上述基因，或上述重组载体，或上述微生物细胞在制备含有羧酸类物质中的应用。
本发明还提供上述腈水解酶突变体，或上述基因，或上述重组载体，或上述微生物细胞在制备烟酸、2-吡啶甲酸、苯甲酸或含有烟酸、2-吡啶甲酸、苯甲酸的产品中的应用。
本发明还提供一种上述腈水解酶突变体的构建方法，所述方法具体为通过计算模拟获得与酶活改造相关的位点作为突变库，利用烟酸传感器作为筛选工具，利用报告基因绿色荧光蛋白的表达水平反映平板上突变体的酶活高低；将获得的荧光效果明显的突变体利用同源重组方式构建至pET质粒载体形成突变体质粒，在宿主中表达后，通过纯化得到突变体的纯酶，测定比酶活并与野生酶进行比较。
有益效果：
1、本发明提供了以野生型腈水解酶Nit6803的氨基酸序列为出发序列，通过对酶活催化口袋进行突变建库，结合烟酸传感器和绿色荧光蛋白进行催化烟腈能力提高的突变体的筛选，最终获得腈水解酶突变体C57-C3、C57-D3、C57-E10。突变体C57-C3、C57-D3、C57-E10在37℃下反应比酶活相比野生型提高了57%、180%、555%，这有利于利用此酶催化腈类物质产生羧酸类物质在工业上的应用。
2、本发明提供的突变体C57-E10在2-氰基吡啶和苯甲腈的比酶活较野生酶都有所提升，分别提高至1.8U/mg、0.5U/mg。
附图说明
图1：Nit-PCC6803的氨基酸序列，标红处为选择突变位点。
图2：PCC6803的晶体结构及催化口袋示意图。
图3：烟酸传感器筛选平台示意图。
图4：CAST库筛选相对荧光强度。
图5：组合突变筛选相对荧光强度。
图6：组合突变体比酶活。
图7：突变体和野生型酶在其他底物催化活性验证。
具体实施方式
腈水解酶的酶活力（U）：单位酶活力定义为37℃下，每分钟催化烟腈生成1 μmol烟酸所需要的酶量。
腈水解酶的比酶活（U/mg）：每毫克腈水解酶所具有的酶活力。
LB培养基(1L)：蛋白胨10g，酵母提取物5g，NaCl 10g。
筛选培养基A（1L）：蛋白胨10g，酵母提取物5g，NaCl 10g，1 mM Ara，20 mM 3-氰基吡啶，50 μg/mL卡那霉素，50 μg/mL氯霉素，琼脂2g。
筛选培养基B（1L）：蛋白胨10g，酵母提取物5g，NaCl 10g，0.1 mM Ara，20 mM 3-氰基吡啶，50 μg/mL卡那霉素，50 μg/mL氯霉素）。
筛选培养基C（1L）：蛋白胨10g，酵母提取物5g，NaCl 10g，0.05mM Ara，20mM 3-氰基吡啶，50μg/mL卡那霉素，50μg/mL氯霉素。
筛选培养基D（1L）：蛋白胨10g，酵母提取物5g，NaCl 10g，0.01mM Ara，20mM 3-氰基吡啶，50μg/mL卡那霉素，50μg/mL氯霉素。
相对荧光值表示方法：FI（测定荧光值）/OD600
实施例1 Nit6803单点突变体构建
将集胞藻（Synechocystis sp. PCC6803）来源的腈水解酶（Nit6803）与底物3-氰基吡啶对接后，选择催化口袋附近可能与酶催化能力有关的位点进行突变建库筛选（图1和图2）。利用CAST方法一共设计八个库CAST1~CAST8（表1）。对每个库进行NNK突变设计引物，引物序列6803-CAST1-F/R~6803-CAST8-F/R如表1所示。
合成Synechocystis sp. NitPCC6803基因（氨基酸序列的NCBI登录号为：AGF53008.1），并将该基因克隆于pET24a（+）质粒的NdeI和EcoRI酶切位点处，由苏州金唯智公司完成，获得重组质粒pET24a-Nit6803。以重组质粒pET24a-Nit6803为模板，利用引物6803-CAST1-F/R~6803-CAST8-F/R进行全质粒PCR，引物如表2所示，扩增体系如表3所示，PCR扩增反应条件为98℃预变性3 min，98℃变性15 s，55℃退火30 s，72℃延伸1 min45 s，72℃延伸5 min，共30个循环。将PCR产物用DpnI消化酶消化2-3 h，纯化获得8个突变库CAST1~CAST8的单一片段。
表1 获得的突变库设计位点
表2 引物
表3 全质粒PCR扩增反应体系
实施例2 利用烟酸传感器对所构建的CAST库进行筛选
（1）含有烟酸传感器的感受态细胞的构建
烟酸传感器的构建已公开于专利CN 112501193 A中，烟酸传感器的作用原理如图3所示。
将烟酸传感器pENAsensor转化至JM109中，并使用常规的CaCl2法制备含有烟酸传感器的感受态细胞。
（2）CAST库的筛选
（a）将实施例1中纯化得到的CAST1~CAST8的单一片段转入步骤（1）中制备的含有烟酸传感器的感受态细胞中，涂布于筛选培养基A中，37℃培养12-18 h后，在蓝光仪下对菌落进行选择，选择荧光较弱的单菌落接至每孔含有500 μL LB培养基（卡那霉素终浓度50 μg/mL，氯霉素终浓度34 μg/mL）的96孔板中，在37℃、300 rpm条件下培养7-8 h，获得种子液。
将种子液按2%（v/v）转接至每孔含有500 μL 筛选培养基B的96孔板中，37℃、300rpm条件下培养24 h，随后吸取200 μL菌液至酶标板中，用酶标仪进行荧光检测（检测条件：激发波长495 nm，发射波长525 nm）。
（b）吸取10 μL步骤（a）中检测到的相对荧光值低于野生型的突变体的菌液接种至3 mL LB培养基中（卡那霉素终浓度50 μg/mL，氯霉素终浓度34 μg/mL），在37℃、200 rpm条件下培养7-8 h，获得种子液。
将种子液按2%（v/v）转接至5 mL筛选培养基C中，37℃、200 rpm条件下培养24 h，随后吸取200 μL菌液至酶标板中，用酶标仪进行荧光检测（检测条件：激发波长495 nm，发射波长525 nm）。
检测结果如图4所示，将检测得到的相对荧光值低于2.0×105的突变体保存并提取质粒进行测序。
表5 CAST库突变位点总结
实施例3 Nit-6803组合突变体构建及烟酸传感器筛选
（a）根据实施例2中CAST库筛选得到的荧光结果，选择其中相对荧光值最低的两个库（CAST5和CAST7）进行组合突变筛选。以实施例1中的pET24a-Nit6803质粒作为模板，利用引物进行全质粒PCR，所用引物序列如表6所示（6803-170-i1、6803-170-i2、6803-197/198-v1、6803-197/198-v2），扩增体系如表3所示，PCR扩增反应条件为98℃预变性3 min，98℃变性15 s，55℃退火30 s，72℃延伸1 min30 s/8s，72℃延伸5 min，共30个循环。
（b）将PCR产物用DpnI消化酶消化2-3h，纯化组合突变体片段。将组合突变体片段转入含有烟酸传感器的感受态细胞中，并涂布于筛选培养基A，37℃培养12-18h后，在蓝光仪下对菌落进行选择，选择荧光较弱的单菌落接至每孔含有500 μL LB培养基（卡那霉素终浓度50 μg/mL，氯霉素终浓度34 μg/mL）的96孔板中，在37℃、300 rpm条件下培养7-8 h，获得种子液。
将种子液按2%（v/v）转接至每孔含有500 μL 筛选培养基B的96孔板中，37℃、300rpm条件下培养24 h，随后吸取200μL菌液至酶标板中，用酶标仪进行荧光检测（检测条件：激发波长495 nm，发射波长525 nm）。
（c）吸取10 μL步骤（b）中检测得到的相对荧光值低于野生型的突变体的菌液接种至3 mL LB培养基中（卡那霉素终浓度50 μg/mL，氯霉素终浓度34 μg/mL），在37℃、200 rpm条件下培养7-8 h，获得种子液。
将种子液按2%（v/v）转接至5mL筛选培养基D中，37℃、200 rpm条件下培养24 h，随后吸取200 μL菌液至酶标板中，用酶标仪进行荧光检测（检测条件：激发波长495 nm，发射波长525 nm）。
检测结果如图5所示。将检测得到的相对荧光值低的突变体保存及提取质粒。
表6 引物
表7 组合突变获得突变体突变位点总结
实施例4 组合突变体及野生型纯酶酶活检测
（1）重组菌的构建
将实施例3获得的突变体质粒(C57-C3、C57-D3、C57-E10)作为模板，用表6所示引物（P15A-i1、P15A-i2）进行PCR，扩增体系如表3所示。扩增反应条件为95℃预变性3 min，95℃变性15 s，55℃退火30 s，72℃延伸1 min20 s，72℃延伸5 min，共30个循环。将获得的PCR片段进行核酸胶进行大小验证，然后由苏州金唯智公司进行测序。
以测序正确的突变体质粒为模板，利用引物pET24a-6803-i1、pET24a-6803-i2进行全质粒PCR，引物序列如表6所示，扩增体系如表3所示，PCR扩增反应条件为98℃预变性3min，98℃变性15 s，55℃退火30 s，72℃延伸30 s，72℃延伸5 min，共30个循环。将PCR产物用DpnI消化酶消化2-3 h，纯化获得单一片段p24a-6803-i。
以pET24a（+）为模板，利用引物P24a-TONGYONG-v1、P24a-TONGYONG-v2进行PCR，获得pET24a骨架p24a-V，引物序列如表6所示。
将单一片段p24a-6803-i与pET24a骨架p24a-V进行组装，组装体系为4 μL 2×MultiF Seamless Aaaembly Mix/ 2 μL p24a-V /2 μL p24a-6803-i，50℃下孵育30 min，然后转化至感受态细胞E. coliER2566，涂布至LB培养基中，37℃培养12-18 h后，挑取单菌落至3 mL LB培养基（卡那霉素终浓度50 μg/mL）中，在37℃、200 rpm条件下培养7-8 h，获得种子液。
将种子液按2%（v/v）转接至100 mL LB培养基（卡那霉素终浓度50 μg/mL），在37℃、200 rpm条件下培养至OD600至0.6-0.8，加入终浓度为0.5 mM的异丙基硫代半乳糖苷（IPTG），改变培养温度为24℃，诱导表达12-16 h，获得菌液。
（2）蛋白纯化：
在10000 rpm条件下离心菌液3 min收集菌体细胞，用20mL PBS缓冲液（pH 7.4）重悬，于冰水混合物中超声破碎。破碎液在4℃、12000 rpm条件下离心30 min，取上清液过0.22 μm有机滤膜。
采用亲和层析的方法纯化野生型WT及突变体C57-C3、C57-D3、C57-E10，纯化柱为GE公司的HisTrap HP 5 mL柱。纯化柱在用结合缓冲液(Binding buffer)（0.2M 磷酸二氢钠，0.2M 磷酸氢二钠，调pH为7.4，加入20mM 咪唑）平衡后，进行上样，然后用结合缓冲液洗去杂蛋白，目的蛋白用洗脱缓冲液（Washing buffer）（0.2M 磷酸二氢钠，0.2M 磷酸氢二钠，调pH为7.4，加入500mM 咪唑）梯度洗脱并收集。蛋白浓度使用Bradford蛋白浓度检测试剂盒进行定量。采用SDS-PAGE检测目的蛋白的纯化质量，可见野生型及其突变体所表达的蛋白在纯化后蛋白条带单一，纯化质量高。
（3）酶活测定：
纯酶反应：用磷酸盐缓冲液（pH 7.4）将WT及其突变体纯酶的浓度稀释至0.5 mg/mL，取10 μL至1.5 mL离心管中，置于37℃金属浴上。向离心管中加入490 μL底物（100 mM烟腈溶液），充分涡旋混匀，37℃下反应10 min，然后加入500 μL 纯乙腈溶液进行终止。过0.22 μm滤膜。
腈水解酶的测定：用HPLC检测体系中烟酸产量，流动相为乙腈：水=1：2，检测波长为210 nm，流速为0.6mL/min，柱温为40℃，色谱柱为C18柱。
WT及突变体C57-C3、C57-D3、C57-E10的比酶活结果如图6所示：野生酶WT的比酶活为 4.93±0.48 U/mg，突变体C57-C3、C57-D3、C57-E10的比酶活分别为 7.75±0.41 U/mg、13.82±0.22 U/mg、32.3±0.11 U/mg，分别比野生酶WT提高了57%、180%、555%，可见，本发明通过建库筛选得到的荧光强度低的突变体的比酶活有不同程度提升，筛选得到的突变位点对于提高腈水解酶的催化活性具有很大的作用。
实施例5 突变体和野生型对于其他底物催化酶活验证
2-氰基吡啶： 将WT及其突变体C57-E10纯酶的浓度稀释至1mg/mL，取10 μL至1.5mL离心管中，置于37℃金属浴上。向离心管中加入490 μL底物（200 mM2-氰基吡啶溶液），充分涡旋混匀，37℃下反应30 min，然后加入500 μL 纯乙腈溶液进行终止。过0.22 μm滤膜。
2-氰基吡嗪：将WT及其突变体C57-E10纯酶的浓度稀释至0.5mg/mL，取10 μL至1.5mL离心管中，置于37℃金属浴上。向离心管中加入490 μL底物（100 mM2-氰基吡嗪溶液），充分涡旋混匀，37℃下反应10 min，然后加入500 μL 纯乙腈溶液进行终止。过0.22 μm滤膜。
苯甲腈：将WT及其突变体C57-E10纯酶的浓度稀释至1mg/mL，取20 μL至1.5 mL离心管中，置于37℃金属浴上。向离心管中加入480 μL底物（50 mM苯甲腈溶液），充分涡旋混匀，37℃下反应30 min，然后加入500 μL 纯乙腈溶液进行终止。过0.22 μm滤膜。
结果如图7所示：2-氰基吡啶野生型比酶活为：0.1U/mg，突变体比酶活为1.8U/mg；
2-氰基吡嗪野生型比酶活为35.5U/mg，突变体比酶活为18.7U/mg；苯甲腈野生型比酶活为0.3U/mg，突变体比酶活为0.5U/mg。
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。
SEQUENCE LISTING
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Claims (8)
1.一种腈水解酶突变体，其特征在于，所述突变体是将氨基酸序列SEQ ID NO.1的第170位的色氨酸突变成甘氨酸，将第197位的蛋氨酸突变成苯丙氨酸，将第198位的缬氨酸突变成异亮氨酸；或，将氨基酸序列SEQ ID NO.1的第170位的色氨酸突变成甘氨酸，将第197位的蛋氨酸突变成苯丙氨酸，将第198位的缬氨酸突变成亮氨酸。
2.一种编码权利要求1所述腈水解酶突变体的基因。
3.一种携带权利要求2所述基因的重组载体。
4.根据权利要求3所述的重组载体，其特征在于，所述重组载体以pET-24a(+)为表达载体。
5.一种携带权利要求2所述基因，或权利要求3或4所述重组载体的微生物细胞。
6.根据权利要求5所述的微生物细胞，其特征在于，所述微生物细胞以细菌或真菌为表达宿主。
7.一种烟酸的制备方法，其特征在于，所述方法为，将权利要求1所述腈水解酶突变体，或权利要求5或6所述微生物细胞添加至含有烟腈的培养基中，进行反应制备得到的。
8.权利要求1所述腈水解酶突变体，或者权利要求2所述基因，或者权利要求3或4所述重组载体，或者权利要求5或6所述微生物细胞在制备烟酸、2-吡啶甲酸、苯甲酸或含有烟酸、2-吡啶甲酸、苯甲酸的产品中的应用。
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| 腈水解酶在医药中间体生物催化研究中的最新进展;龚劲松等;《化学进展》;第27卷(第4期);第450页右栏第1段和第453页左栏第2-3段 * |
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