CN116042555B - 一种乙醇脱氢酶突变体及其应用 - Google Patents
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Abstract
本发明公开了一种乙醇脱氢酶突变体及其应用,涉及蛋白质工程技术领域,所述乙醇脱氢酶突变体由乙醇脱氢酶突变序列和N端依次融合HRV3C酶切位点和6His标签构成,所述突变序列中的突变为V63Y、T122L、G162Y、Q202M、A219M、D268N、N291M和A385M。本发明使其与野生型的乙醇脱氢酶相比具有更高的热稳定性,野生型Tm值为61.25℃、突变体Tm值为66.1℃,较野生型增加了近5℃,为该酶在工业生物催化制药过程中的应用提供了良好的应用场景。该突变体的蛋白产量也比野生型高出2倍。
Description
技术领域
本发明涉及蛋白质工程技术领域,具体涉及一种乙醇脱氢酶突变体及其应用。
背景技术
(磷酸)酰胺腺嘌呤二核苷酸(NAD(P)H)是生物体内一种常见的辅酶因子,参与了广泛的代谢反应,并应用于酶催化氧化还原反应和生物燃料电池中。但是由于(NAD(P)H)成本较高,很多时候限制了其应用。为了缓解这一限制因素,一些NAD(P)H再生系统,如甲酸脱氢酶、乙醇脱氢酶和葡萄糖脱氢酶等,已经被设计和研究来帮助解决这一问题。其中乙醇脱氢酶(ADH)是一类以价格便宜的乙醇作为底物,并将其氧化成乙醛,将电子提供给第二底物NAD(P)+从而生成NAD(P)H,因此备受关注。
蛋白质的三维结构决定了蛋白质的功能,了解蛋白质的信息对于蛋白功能的提升具有很好的指导意义。已报道的乙醇脱氢酶的种类和数量很多,一些乙醇脱氢酶的酶活性质已经被报道,但是其结构功能的缺失对于蛋白质的改造造成了阻碍,从而对于开发具有更高稳定性、更高活性的乙醇脱氢酶受到了限制。
发明内容
为了解决上述问题,本发明的首要目的在于提供一种高表达的乙醇脱氢酶突变体、蛋白质晶体三维结构和应用。
本发明通过以下技术方案来实现上述目的:
本发明提供了一种乙醇脱氢酶突变体,所述乙醇脱氢酶突变体由乙醇脱氢酶突变序列和N端依次融合HRV3C酶切位点和6His标签构成。
进一步改进在于,所述突变序列中的突变为V63Y、T122L、G162Y、Q202M、A219M、D268N、N291M和A385M。
进一步改进在于,所述乙醇脱氢酶突变体的氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种多核苷酸,所述多核苷酸编码上述所述的乙醇脱氢酶突变体。
进一步改进在于,所述多核苷酸的序列如SEQ ID NO.2所示。
本发明还提供了一种重组质粒,所述重组质粒为含有上述所述的多核苷酸且能够翻译表达出上述所述乙醇脱氢酶突变体的表达载体。
进一步改进在于,所述表达载体为pET-28a。
本发明还提供了一种乙醇脱氢酶突变体表达系统,为转入上述所述重组质粒的大肠杆菌BL21。
本发明还提供了一种复合蛋白质晶体,所述复合蛋白质晶体是将纯化得到的上述所述的乙醇脱氢酶突变体采用坐滴法进行结晶后获得。
进一步改进在于,所述复合蛋白质晶体的出晶条件为0.1M柠檬酸钠,pH4.5,20%PE G4000。
本发明还提供了乙醇脱氢酶突变体的三维结构信息。分辨率为其晶体结构空间群为C2。晶胞参数为:/>
进一步改进在于,一个晶体学不对称单位内有1个分子;该结构由17个α螺旋和8个β折叠片的组成,还可以清晰看见NADP与乙醇脱氢酶突变体结合。
本发明还提供了一种上述所述的复合蛋白质晶体在改造高活性高稳定性的乙醇脱氢酶中的应用。
一种辅酶因子NADPH的获得方法,以NADP+为底物,利用上述所述的乙醇脱氢酶突变体将NADP+转化成NADPH。
进一步改进在于,所述乙醇脱氢酶突变体参与NADP相互作用的氨基酸包括G39、G40、S41、N102、T138、P170、Y179和T182。
本发明具有如下有益效果:
本发明提供了一种以乙醇为底物的来源于芽孢杆菌的乙醇脱氢酶突变体,该突变体通过将乙醇脱氢酶蛋白序列的第63位的缬氨酸突变成酪氨酸,第122位的苏氨酸突变成亮氨酸,第162位的甘氨酸突变成酪氨酸,第202位的谷氨酰胺突变成甲硫氨酸,第219位的丙氨酸突变成甲硫氨酸,第268位的天冬氨酸突变成天冬酰胺,第291位的天冬酰胺突变成甲硫氨酸,第385位的丙氨酸突变成甲硫氨酸,使其与野生型的乙醇脱氢酶相比具有更高的热稳定性,野生型Tm值为61.25℃、突变体Tm值为66.1℃,较野生型增加了近5℃,为该酶在工业生物催化制药过程中的应用提供了良好的应用场景。此外,该突变体的蛋白产量也比野生型高出2倍;
最后,本发明还提供了该酶的蛋白质晶体三维结构,通过其结构可以清晰可见该酶与NADP小分子的结合的氨基酸包括G39、G40、S41、N102、T138、P170、Y179和T182;为进一步改造高活性、高稳定性的酶提供了研究基础。
附图说明
图1为野生型ADH2及ADH2突变体蛋白小量表达SDS-PAGE检测结果;
图2为野生型ADH2及ADH2突变体蛋白亲和纯化结果;
图3为ADH2突变体经3C酶酶切后的蛋白反向亲和纯化结果;
图4为ADH2突变体的蛋白质量检测结果;
图5为野生型ADH2及ADH2突变体热稳定性检测结果;
图6为ADH2突变体蛋白质晶体照片;
图7位ADH2突变体晶体三维结构图。
具体实施方式
下面结合附图对本申请作进一步详细描述,有必要在此指出的是,以下具体实施方式只用于对本申请进行进一步的说明,不能理解为对本申请保护范围的限制,该领域的技术人员可以根据上述申请内容对本申请作出一些非本质的改进和调整。
1、材料
本发明所用方法如无特别说明均为本领域的技术人员所知晓的常规方法,未注明具体条件者,按照常规条件或者制造商建议的条件进行,所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
2、方法
2.1重组质粒的构建及表达
(1)野生型乙醇脱氢酶ADH2(氨基酸序列如SEQ ID NO.3所示)及乙醇脱氢酶ADH2突变体的基因序列均是通过基因合成获得(以下简称乙醇脱氢酶为ADH2),分别在N端带有6His和HRV3C酶酶切位点,表达载体为pET-28a,重组质粒均经测序验证与目标序列完全一致,ADH2突变体是在野生型ADH2的原始序列基础上将第63位的缬氨酸突变成酪氨酸,第122位的苏氨酸突变成亮氨酸,第162位的甘氨酸突变成酪氨酸,第202位的谷氨酰胺突变成甲硫氨酸,第219位的丙氨酸突变成甲硫氨酸,第268位的天冬氨酸突变成天冬酰胺,第291位的天冬酰胺突变成甲硫氨酸,第385位的丙氨酸突变成甲硫氨酸,ADH2突变体的氨基酸序列如SEQ ID NO.1所示,其编码基因如SEQ ID NO.2所示。
将野生型ADH2和ADH2突变体两种类型的重组质粒分别按照常规分子生物学手段分别转化BL21(DE3)大肠杆菌感受态细胞,挑取单克隆菌斑至5mL LB液体培养基中,37℃培养,待菌液OD600至0.6-0.8时取少量菌液用loading buffer进行固定,并取少量菌液加入甘油冻至-80℃,剩余菌液加入0.5mM IPTG诱导4个小时后,收集菌体并取诱导后菌液进行SDS-PAGE检测。根据图1中SDS-PAGE结果可知,野生型ADH2和ADH2突变体在BL21(DE3)大肠杆菌中均明显表达。
2.1.2诱导表达融合蛋白
将明显表达的两种类型的菌株分别接种至50mL LB液体培养基中37℃培养过夜,将过夜培养的细菌按1:100的比例接至1L LB液体培养基中,37℃培养至菌液OD600为0.6-0.8时加入0.5mM IPTG 15℃培养过夜,5000rpm离心收集菌体。
2.1.3蛋白纯化
将收集的两种类型的菌块进行称重,再分别按照1:10比例加入相应体积的裂解缓冲液(50mM Tris-HCl(pH 8.0),500mM NaCl,5%glycerol),使用高压均质机破碎菌体,16000rpm高速离心收集上清。再使用亲和层析柱His FF富集纯化蛋白,纯化前先用裂解buffer平衡His FF柱,将所有细胞上清挂柱后,用不同梯度的咪唑溶液洗脱,收集不同梯度咪唑洗脱下的蛋白进行SDS-PAGE检测,收集纯度较好的蛋白用Nanodrop测定蛋白浓度,计算蛋白产量。
根据图2中SDS-PAGE结果可知,通过亲和纯化获得了纯度较高的野生型ADH2蛋白和ADH2突变体蛋白。再根据Nanodrop测定的蛋白浓度计算得到的野生型ADH2的产量为46.89mg/L,ADH2突变体的蛋白产量高达95.10mg/L。证明通过将野生型ADH2蛋白序列第63位的缬氨酸突变成酪氨酸,第122位的苏氨酸突变成亮氨酸,第162位的甘氨酸突变成酪氨酸,第202位的谷氨酰胺突变成甲硫氨酸,第219位的丙氨酸突变成甲硫氨酸,第268位的天冬氨酸突变成天冬酰胺,第291位的天冬酰胺突变成甲硫氨酸,第385位的丙氨酸突变成甲硫氨酸,ADH2突变体的蛋白产量相比于野生型ADH2提高了2倍。
为了避免N端标签影响ADH2突变体的晶体生长,将His FF亲和后的蛋白用3C酶将其标签去除,去除标签的蛋白重新进行His FF亲和层析。根据图3中SDS-PAGE结果可知,切除标签后的ADH2突变体蛋白纯度更高。
2.1.4蛋白质量检测
为了获得均一性好的蛋白,将切除标签后的ADH2突变体蛋白进行凝胶过滤层析,凝胶层析柱的型号为:HiLoad 16/600Superdex 200pg,凝胶层析的buffer为:50mM Tris-HCl(pH 8.0),500mM NaCl,5%glycerol。收集凝胶过滤层析后的样品,进行蛋白质量检测,即分别进行SDS-PAGE纯度检测、质谱分析和分析型分子筛的检测;
根据图4中SDS-PAGE结果可知,ADH2突变体蛋白纯度大于95%。质谱检测结果显示ADH2突变体蛋白检测的分子量为43350Da,与理论分子量43346Da非常接近,说明纯化后的蛋白为目标蛋白。此外,分析型分子筛的结果显示,ADH2突变体蛋白在溶液中的状态接近二聚体。
2.2野生型ADH2和ADH2突变体重组蛋白的热稳定性检测
2.2.1热稳定性检测
野生型ADH2和ADH2突变体重组蛋白热稳定性的检测技术选用微量差示扫描荧光技术(nano Differential Scanning Fluorimetry,nanoDSF)。该技术通过检测色氨酸自发荧光的微小变化来进行蛋白质稳定性的研究,通过检测蛋白内源荧光的变化来跟踪其折叠状态,荧光信号的比值会随温度的增加而变化,从而测定蛋白稳定性参数Tm值,实现在非标记环境下检测蛋白的热稳定性或化学稳定性,具体的实验方法如下:
分别取20μL浓度为0.5mg/ml的野生型ADH2和ADH2突变体蛋白加到384孔实验板中,震荡离心后(避免样品不均匀或吸样过程中吸进气泡),将实验板置于取样架上,使用Nano DSF毛细管吸样,保证样品充满整个毛细管。将毛细管放入nanoDSF仪器中,设置初始温度为20℃,以每分钟升温2.0℃的速度最终上升到90℃终止。仪器会依照设置好的参数进行升温和实时检测,Tm值测试的结果见图5。
2.2.2结果分析
野生型ADH2的Tm值为61.25℃,ADH2突变体的Tm值为66.1℃。当野生型ADH2蛋白序列的第63位的缬氨酸(V)突变成酪氨酸(Y),第122位的苏氨酸(T)突变成亮氨酸(L),第162位的甘氨酸(G)突变成酪氨酸(Y),第202位的谷氨酰胺(Q)突变成甲硫氨酸(M),第219位的丙氨酸(A)突变成甲硫氨酸(M),第268位的天冬氨酸(D)突变成天冬酰胺(N),第291位的天冬酰胺(N)突变成甲硫氨酸(M),第385位的丙氨酸(A)突变成甲硫氨酸(M)后,Tm值增加了近5℃。蛋白的热稳定性有所提高,已知在工业生物催化制药过程中,存在一些酶因为在高温下不稳定而限制了其应用,通过定向进化筛选出具有热稳定性的突变株已经成为一种常规的生物学手段,而本发明通过对野生型ADH2蛋白序列的第63位氨基酸、第122位氨基酸、第162位氨基酸、第202位氨基酸、第219位氨基酸、第268位氨基酸、第291位氨基酸、第385位氨基酸进行突变,提高了蛋白的热稳定性,也为ADH2在工业生物催化制药中的应用提供了更好的应用场景。
2.3ADH2突变体重组蛋白结晶
将纯化得到的ADH2突变体蛋白用超滤浓缩管在4℃浓缩至16.26mg/mL,用坐滴法进行结晶。选取Hampton Research公司的Crystal Screen、Index及QIAGEN公司生产的JCSGI、JCSGⅡ、JCSGⅢ等试剂盒进行结晶筛选。因为NADP+为ADH2突变体的底物,因此在结晶时,在ADH2突变体蛋白中加入4mM NADP+,4℃孵育1小时,进行晶体生长。具体操作为:取15μL结晶试剂于96孔结晶板中作为缓冲液,使用Mosquito LCP蛋白结晶筛选仪点样,按照蛋白:结晶缓冲液1:1(体积均为200μL)的比例,将蛋白与结晶缓冲液混合均匀,将MicroAmpOptical Adhesive膜密封后的96孔板放置在20℃的恒温培养箱进行培养,定期观察晶体生长情况。
通过对ADH2突变体蛋白的结晶筛选,在16.26mg/ml的蛋白浓度下,约3天获得了ADH2突变体晶体,结晶条件为0.1M柠檬酸钠,pH4.5,20%PEG4,000,晶体照片如图6所示。捞取ADH2突变体的晶体至防冻保护液(0.1M柠檬酸钠,pH4.5,20%PEG4,000,10%甘油)中浸泡数秒后,快速在液氮中冷冻,冷冻后的晶体送至同步辐射光源进行X射线衍射及晶体数据收集,最终收集到一套分辨率为的数据。晶胞参数为/> 空间群为C2,一个晶体学不对称单位内有1个分子,将处理好的数据进行结构解析,以AlphaFold预测的乙醇脱氢酶的结构作为模板,使用分子置换的方法解析了ADH2突变体的晶体结构。
蛋白质的三维结构决定了蛋白质的功能,了解蛋白质的信息对于蛋白功能的提升具有很好的指导意义。本发明提供的ADH2突变体晶体为使用X射线解析ADH2突变体的结构提供了基础,提供了基于结构定向改造ADH2突变体,为设计具有更高活性、更高稳定性的乙醇脱氢酶提供分子基础。
2.4结论
本发明提供了一种以乙醇为底物的来源于芽孢杆菌的ADH2突变体,该突变体与野生型ADH2相比具有更高的热稳定性,野生型ADH2的Tm值为61.25℃、突变体的Tm值为66.1℃,较野生型增加了近5℃。此外,突变体的蛋白产量也较野生型高出2倍。
本发明解析了一种已知活性的来源于芽孢杆菌的乙醇脱氢酶2(ADH2)的突变体与其底物NADP+的高分辨率的复合物晶体结构。如图6-7所示,一个晶体学不对称单位内有1个分子;该结构由17个α螺旋和8个β折叠片的组成,还可以清晰看见NADP与乙醇脱氢酶突变体结合。该晶体结构报道了ADH2突变体与NADP相互作用细节揭示了ADH2突变体将NADP+转化成NADPH的原理,ADH2突变体的G39、G40、S41、N102、T138、P170、Y179和T182氨基酸参与了与NADP相互作用,并为进一步开发具有更高活性且更高热稳定性的ADH2蛋白提供了结构信息的支持。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (8)
1.一种乙醇脱氢酶突变体,其特征在于,所述乙醇脱氢酶突变体由乙醇脱氢酶突变序列和N端依次融合HRV3C酶切位点和6His标签构成,所述突变序列中的突变为V63Y、T122L、G162Y、Q202M、A219M、D268N、N291M和A385M,所述乙醇脱氢酶突变体的氨基酸序列如SEQ IDNO.1所示。
2.一种多核苷酸,其特征在于,所述多核苷酸编码如权利要求1所述的乙醇脱氢酶突变体。
3.根据权利要求2所述的一种多核苷酸,其特征在于,所述多核苷酸的序列如SEQ IDNO.2所示。
4.一种重组质粒,其特征在于,所述重组质粒为含有如权利要求2-3任一所述的多核苷酸且能够翻译表达出如权利要求1所述乙醇脱氢酶突变体的表达载体。
5.根据权利要求4所述的一种重组质粒,其特征在于,所述表达载体为pET-28a。
6.一种乙醇脱氢酶突变体表达菌株,其特征在于,为转入权利要求4-5任一所述重组质粒的大肠杆菌BL21。
7.一种辅酶因子NADPH的获得方法,其特征在于,以NADP+为底物,利用权利要求1所述的乙醇脱氢酶突变体将NADP+转化成NADPH。
8.根据权利要求7所述的方法,其特征在于,所述乙醇脱氢酶突变体参与NADP相互作用的氨基酸包括G39、G40、S41、N102、T138、P170、Y179和T182。
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