CN114250217B - 一种理性设计提升腈水解酶活性的方法及应用 - Google Patents
一种理性设计提升腈水解酶活性的方法及应用 Download PDFInfo
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Abstract
本发明公开了一种理性设计提升腈水解酶活性的方法及应用,属于酶工程领域。本发明通过腈水解酶的晶体结构进行分析,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,对每个点做饱和突变后,通过两种计算方法Rosetta‑Cartesian和FEP对获得的突变体进行稳定性和结合自由能分析,对满足要求的突变体做酶活测定。将单点突变酶活高于野生型的位点进行组合突变再进行计算,最终获得符合要求的突变体进行酶活测定。通过这种方法我们最后获得了两个单点突变体,比酶活为野生型的2、1.5倍,组合突变体F64YW170G比酶活为野生型的4.56倍。
Description
技术领域
本发明涉及一种理性设计提升腈水解酶活性的方法及应用,属于酶工程领域。
背景技术
腈水解酶(Nitrilase,EC 3.5.5.1)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。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、稳定性和耐受性差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可以为其工业应用做出贡献。
目前常用的酶改造方法有定向进化、半理性设计和理性设计,大多数酶的改造基本上都可以通过对其催化口袋进行设计改造的方法进行,大多数可以取得较好的效果(提高酶的稳定性、耐受性或酶活)。Syechocystis sp.PCC6803的腈水解酶具有较宽广的底物谱,对脂肪族、芳香族等腈类物质都存在一定的催化能力,有很大的应用前景。因此通过对Nit-PCC6803晶体结构进行分析并进行理性设计以期提高腈水解酶的酶活或稳定性。这对于利用Nit6803催化腈类物质生产羧酸类物质具有重要的意义。
发明内容
针对现有的技术难点及存在的问题,本发明旨在提供一种来源于Syechocystissp.的催化烟腈能力提高的腈水解酶突变体(Nit-PCC6803-F64YW170G)。
为提高对腈类物质的催化能力,本发明选用来源于集胞藻(Syechocystissp.PCC6803)的腈水解酶Nit6803(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64位、第170位的氨基酸中的一个或多个进行突变得到的。
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64位的苯丙氨酸氨酸突变成酪氨酸;
(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;
(c)将氨基酸序列SEQ ID NO.1的第64位的苯丙氨酸氨酸突变成酪氨酸,将第170位的蛋氨酸突变成甘氨酸。
在本发明的一种实施方式中,所述腈水解酶突变体的氨基酸序列如SEQ ID NO.2~SEQ ID NO.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.coli ER2566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或含有烟酸的产品中的应用。
本发明还提供一种上述腈水解酶突变体的构建方法,所述方法具为通过底物对接确定催化口袋周围的相关位点作为计算位点,通过利用Rosetta-Cartesian软件对选择的位点进行饱和突变后选择分析结果中对酶稳定性影响小的突变体,再通过FEP方法对这些突变体突变后与底物3-氰基吡啶之间自由能变化的高低进行筛选,最后选择△△Gbinding小于-1的突变体进行质粒构建并表达测定其全细胞酶活和纯酶比酶活。选择其中效果好的两个位点进行组合设计,再利用Rosetta-Cartesian和FEP进行分析,最后获得较好突变体进行构建、表达并测定其全细胞酶活和纯酶比酶活。
有益效果:
本发明提供了腈水解酶Nit-PCC6803的氨基酸序列,并且通过对晶体结构进行分析后对所选择的位点进行稳定性和自由能的计算,再通过表达、纯化并检测酶活,最终获得腈水解酶突变体F64Y、W170G在37℃下反应比酶活为野生型的2、1.5倍,组合突变体F64YW170G在37℃下反应比酶活为野生型的4.56倍且其稳定性与野生型相比并未下降。所以本发明提供了一个理性设计方案在不影响酶稳定性的前提下提高其催化活性;且获得了高活性突变体,这有利于利用腈水解酶PCC6803催化腈类物质产生羧酸类物质在工业上的应用。
附图说明
图1:PCC6803催化口袋示意图。
图2:酶催化能力相关位点的预测;A:单点突变的Rosetta-Cartesian分析结果,B:单点突变的FEP分析结合自由能结果。
图3:单点突变的全细胞相对酶活及纯酶比酶活结果;A:全细胞相对酶活,B:纯酶比酶活。
图4:A:组合突变的Rosetta-Cartesian分析结果,B:组合突变的FEP分析结合自由能结果。
图5:组合突变的全细胞相对酶活及纯酶比酶活结果;A:全细胞相对酶活,B:纯酶比酶活。
图6:野生型与组合突变体F64YW170G的稳定性比较。
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1μmol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。
相对酶活的定义(%):以野生型在37℃下反应10min的酶活作为100%。
LB培养基(1L):胰蛋白胨10g,酵母提取物5g,NaCl 10g。
2×YT培养基(1L):胰蛋白胨16.0g,酵母提取物10.0g,NaCl 5.0g。
实施例1 Nit6803各突变体的构建
将集胞藻(Syechocystis 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作为突变位点如图1所示。然后利用Rosetta-Cartesian软件对每个突变位点进行饱和突变后产生的突变体进行稳定性的分析,选择△△G小于5的突变体(认为突变后对酶的结构影响小)进行下一步分析;再利用FEP对其结合自由能进行计算,最终选择△△Gbinding小于-1的突变体进行质粒构建及表达验证(图2)。
合成Syechocystis 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pET24a-Nit6803重组质粒。
以pET24a-Nit6803质粒作为模板,利用突变位点相应的引物进行全质粒PCR,构建了重组质粒F64Y、T139K、Y140H、Y140A、H141K、W170G、M197Y、M197V、M197I、V198D。
所用引物序列如表1所示,扩增体系如表2所示,PCR扩增反应条件为98℃预变性3min,98℃变性15s,55℃退火30s,72℃延伸1min45s,72℃延伸5min,共30个循环。
将PCR产物用DpnI消化酶消化2-3h,纯化获得各个突变体单一片段。将获得的单一片段转化至E.coli JM109,阳性转化子的基因序列由苏州金唯智公司进行测序验证。
表1引物
表2全质粒PCR扩增反应体系
实施例2野生酶WT与各突变体的表达及酶活检测
(1)重组菌株的构建:
将Nit6803的野生型WT及实施例1测序正确的突变体质粒F64Y、T139K、Y140H、Y140A、H141K、W170G、M197Y、M197V、M197I、V198D分别转化至感受态细胞E.coli ER2566,涂布至LB培养基中,37℃培养12-18h后,挑取单菌落至3mL LB培养基(卡那霉素终浓度50μg/mL),37℃、200rpm条件下培养7-8h,获得种子液。
将种子液按2%(v/v)转接至5mL LB培养基(卡那霉素终浓度50μg/mL),在37℃、200rpm条件下培养至OD600至0.6-0.8,加入终浓度为0.5mM的异丙基硫代半乳糖苷(IPTG),改变培养温度为25℃,诱导表达12-16h,获得菌液。
(2)全细胞酶活测定:
收1mL菌液12000rpm离心1min,用1mL PBS缓冲液(pH7.4)重悬,用紫外分光光度计测定其在OD600下的吸光度,然后调OD600至2。取100μL菌液12000rpm离心1min,再用500μLPBS缓冲液(pH7.4)重悬,加入500μL 100mM 3-氰基吡啶底物,37℃、200rpm反应10min,取出后1200rpm离心10min,取上清过0.22μm的滤膜后作为液相检测样品。
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210nm,流速为0.6mL/min,柱温为40℃,色谱柱为C18柱。
野生型和突变体的相对酶活结果如图3A所示,在所构建的突变体中F64Y和W170G的相对酶活为200%、150%,全细胞催化活性显著高于野生型酶WT。
(3)蛋白纯化:
将突变体F64Y和W170G挑取单菌落至3mL LB培养基(卡那霉素终浓度50μg/mL),37℃、200rpm条件下培养7-8h,获得种子液。将种子液按2%(v/v)转接至100mL 2×YT培养基(卡那霉素终浓度50μg/mL),在37℃、200rpm条件下培养至OD600至0.6-0.8,加入终浓度为0.5mM的异丙基硫代半乳糖苷(IPTG),改变培养温度为25℃,诱导表达12-16h,获得菌液。
在10000rpm条件下离心菌液3min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℃、12000rpm条件下离心30min,取上清液过0.22μm有机滤膜。
采用亲和层析的方法纯化野生型WT及突变体F64Y、W170G,纯化柱为GE公司的HisTrap HP 5mL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。
(4)纯酶酶活测定:
纯酶反应:用磷酸盐缓冲液(pH 7.4)将WT及其突变体F64Y、T139K、Y140H、Y140A、H141K、W170G、M197Y、M197V、M197I、V198D纯酶的浓度稀释至0.5mg/mL,取10μL至1.5mL离心管中,置于37℃金属浴上。向离心管中加入490μL底物(100mM烟腈溶液),充分涡旋混匀,37℃下反应10min,然后加入500μL纯乙腈溶液进行终止。然后离心去除沉淀,取上清过0.22μm的滤膜后作为液相测定的样品。
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210nm,流速为0.6mL/min,柱温为40℃,色谱柱为C18柱。
野生型及突变体的纯酶比酶活结果如图3所示,其中野生型比酶活为4.93±0.48U/mg,F64Y的比酶活为10.04±0.24U/mg,W170G的比酶活为7.1±0.41U/mg。相对于野生型来说分别提高了103.6%和44%。
实施例3 Nit-6803组合突变体构建及表达
(1)重组菌的构建
由实施例2可知64和170两个位点对于腈水解酶的酶活有很大作用,所以选择这两个位点做组合突变以期获得酶活提升更多的突变体。将两个点同时做饱和突变然后利用Rosetta-Cartesian软件对每个突变体进行稳定性的分析,选择△△G小的突变体(认为突变后对酶的结构影响小)进行下一步分析;再利用FEP对其结合自由能进行计算,最终发现F64YW170G的组合突变体的效果最好,所以选择这个突变体进行质粒构建及表达验证(图4)。
以pET24a-Nit6803质粒作为模板,利用引物6803-64Y-F、6803-64Y-R扩增得到片段p24a-6803-64i,利用引物6803-170G-F、6803-170G-R扩增得到片段p24a-6803-170v,所用引物序列如表1所示,扩增体系如表2所示,PCR扩增反应条件为98℃预变性3min,98℃变性15s,55℃退火30s,72℃延伸1min10s/8s,72℃延伸5min,共30个循环。将PCR产物用DpnI消化酶消化2-3h,纯化获得单一片段。
将纯化后的片段p24a-6803-64i和p24a-6803-170v进行组装,组装体系为4μL 2×MultiF Seamless Aaaembly Mix/2μL p24a-6803-64i/2μL p24a-6803-170v,50℃下孵育30min,转化至E.coli JM109,阳性转化子的基因序列由苏州金唯智公司进行测序验证。
将正确的质粒转化至感受态细胞E.coli ER2566,涂布至LB培养基中,37℃培养12-18h后,挑取单菌落至3mL LB培养基(卡那霉素终浓度50μg/mL)中,在37℃、200rpm条件下培养7-8h,获得种子液。
将种子液按2%(v/v)转接至5mL LB培养基(卡那霉素终浓度50μg/mL),在37℃、200rpm条件下培养至OD600至0.6-0.8,加入终浓度为0.5mM的异丙基硫代半乳糖苷(IPTG),改变培养温度为25℃,诱导表达12-16h,获得菌液。
(2)全细胞酶活测定
收1mL菌液12000rpm离心1min,用1mLPBS缓冲液(pH7.4)重悬,用紫外分光光度计测定其在OD600下的吸光度,然后调OD至2。取100μL菌液12000rpm离心1min,再用500μL PBS缓冲液(pH7.4)重悬,加入500μL 100mM 3-氰基吡啶底物,37℃、200rpm反应10min,取出后1200rpm离心10min,取上清过0.22μm的滤膜后作为液相检测样品。
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210nm,流速为0.6mL/min,柱温为40℃,色谱柱为C18柱。
结果如图5所示,突变体F64YW170G的相对酶活为野生型的476%,相对于野生型来说有明显的提升。
实施例4组合突变体及野生型纯酶比酶活检测及稳定性分析
(1)蛋白纯化:
将野生型与突变体F64YW170G挑取单菌落至3mL LB培养基(卡那霉素终浓度50μg/mL),37℃、200rpm条件下培养7-8h,获得种子液。将种子液按2%(v/v)转接至100mL 2×YT培养基(卡那霉素终浓度50μg/mL),在37℃、200rpm条件下培养至OD600至0.6-0.8,加入终浓度为0.5mM的异丙基硫代半乳糖苷(IPTG),改变培养温度为25℃,诱导表达12-16h,获得菌液。
在10000rpm条件下离心菌液3min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℃、12000rpm条件下离心30min,取上清液过0.22μm有机滤膜。
采用亲和层析的方法纯化野生型WT及突变体F64YW170G,纯化柱为GE公司的HisTrap HP 5mL柱。纯化柱在用结合缓冲液(Binding buffer)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)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。
(2)纯酶酶活测定:
纯酶反应:用磷酸盐缓冲液(pH 7.4)将WT及其突变体F64YW170G纯酶的浓度稀释至0.5mg/mL,取10μL至1.5mL离心管中,置于37℃金属浴上。向离心管中加入490μL底物(100mM烟腈溶液),充分涡旋混匀,37℃下反应10min,然后加入500μL纯乙腈溶液进行终止。然后离心去除沉淀,取上清过0.22μm的滤膜后作为液相测定的样品。
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210nm,流速为0.6mL/min,柱温为40℃,色谱柱为C18柱。
野生型及突变体的纯酶比酶活结果如图5所示,其中野生型比酶活为4.93±0.48U/mg,突变体F64YW170G的比酶活为22.48±0.64U/mg,为野生型的4.56倍。
然后我们在40℃和50℃下分别孵育4h,取0min、30min、1h、2h、4h的样品进行反应,反应方法如上述所示。最终结果如图6所示,突变体的稳定性与野生型几乎没有差别,在40℃下热稳定性略好于野生型。
本发明通过对腈水解酶的晶体结构进行分析,利用两种不同计算方法Rosetta-Cartesian和FEP,在不影响稳定性的前提下提高酶活,最终我们获得了单点突变体F64Y和W170G的酶活高于野生型,组合突变体F64YW170G的比酶活为野生型的4.56倍,且其稳定性不低于野生型。
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64位的苯丙氨酸氨酸突变成酪氨酸,将第170位的蛋氨酸突变成甘氨酸。
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,或权利要求2所述基因,或权利要求3或4所述重组载体,或权利要求5或6所述微生物细胞在制备烟酸或含有烟酸的产品中的应用。
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