CN112048492A - 一种酯水解酶突变体 - Google Patents
一种酯水解酶突变体 Download PDFInfo
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- CN112048492A CN112048492A CN201910490170.5A CN201910490170A CN112048492A CN 112048492 A CN112048492 A CN 112048492A CN 201910490170 A CN201910490170 A CN 201910490170A CN 112048492 A CN112048492 A CN 112048492A
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Abstract
本发明涉及一种酯水解酶突变体,属于蛋白质工程技术领域。以酵母分泌表达获得的酯水解酶突变体作为生物催化剂，对高浓度底物3‑异丁基‑戊二酸二酯进行立体选择性水解，获得的手性产物(S)‑3‑异丁基‑戊二酸单酯的手性纯度大于98％，转化率达到100％。经过后期提取分离，收率达到97％，大幅度提高了合成效率，降低了生产成本，适合工业化生产。
Description
技术领域：本发明属于蛋白质工程技术领域，具体涉及一种高立体选择性的酯水解酶突变体。
背景技术：
普瑞巴林(Pregabalin，简称PGB)，商品名为乐瑞卡(Lyricca)，化学名为(S)-3-氨甲基-5-甲基己酸，是美国辉瑞(Pfizer)研制的一种γ-氨基丁酸类似物，是一种手性分子，S构型疗效高，主要用于治疗癫痫、广泛性焦虑症及糖尿病和疱疹后的神经痛。该药上市后反应良好，具有药效强、不良反应小、服用次数少等优点，受到全球各医药公司的高度关注。目前，市场上的普瑞巴林药物仍以化学法合成为主，主要使用手性拆分试剂进行化学拆分，或使用不对称催化剂、手性配体进行不对称合成。这些方法需要苛刻的高温高压等极端反应条件，同时伴随着副产物多、收率低、污染环境等缺点。
随着生物技术的发展，特别是生物酶催化技术在化学合成中的应用，使得一些难合成的手性分子，在酶的参与下变的非常容易，这也吸引了大量的科学人员对该领域进行深入研究。美国专利US2005/0283023中报道使用96孔板筛选可选择性水解(R/S)-2-羰乙基-3-氰基-5-甲基乙酸乙酯的水解酶方法，得到较好的商品酶。中国专利CN109503401，利用脂肪酶对普瑞巴林进行手性拆分，获得了高手性产物，但拆分的收率最高仅有50％，限制了其应用。辉瑞在专利CN107746834中报道可利用转氨酶或其突变体合成普瑞巴林，虽然能够得到高的手性产物，但转换底物浓度过低且转化率不高，限制了其工业化的应用。
大量文献报道采用脂肪酶制备普瑞巴林，专利CN1972904、CN103045559、CN103695385、CN103981160、CN104130987、CN104560912中均不同程度报道脂肪酶可以用来制备普瑞巴林中间体，但是转化率仅为40％左右，造成原料浪费。
文献(Organic&Biomolecular Chemistry，2013,11(22),3635)中报道合成普瑞巴林中间体的方法，如Scheme 2所示。
此路线中采用的脂肪酶为野生型，当R为甲基、乙基时，ee值为75％左右，当R为异丙基时，ee值也仅为93％，并不满足工业化生产的需要。
专利WO2009158343报道一种脂肪酶及其在普瑞巴林中间体制备中的应用。
通过酶法合成将3-异丁基戊二酸甲酯转化为S-3-异丁基戊二酸单甲酯，其中酶来自CalB(南极假丝酵母脂酶B)，收率为96％，ee为95.5％，但是实际上无法重现专利报道的结果。
由于目前报道的酯水解酶转化率较低，手性纯度不高，因此需要寻找一种高选择性酯水解酶用于制备普瑞巴林。
发明内容：
为了克服现有技术存在的上述缺陷，本发明提供一种用于制备普瑞巴林中间体的高选择性酯水解酶突变体。
本发明提供的酯水解酶突变体的氨基酸序列是以SEQ ID NO.1所示的野生型酯水解酶为参考序列发生突变的氨基酸序列，突变位点为第141位的丙氨酸突变为亮氨酸，第154位的缬氨酸突变为精氨酸和第157位的谷氨酰胺突变为色氨酸。
进一步，所述酯水解酶的氨基酸序列为SEQ ID No.2所示。
进一步，所述酯水解酶的基因核苷酸序列为SEQ ID No.3所示。
进一步，所述酯水解酶来源于Candida Antarctica。
进一步，所述酯水解酶用于制备普瑞巴林中间体。
更进一步，所述酯水解酶用于制备普瑞巴林中间体，具体反应如式I所示：
其中，R为甲基、乙基、异丙基、苄基，优选乙基。
有益效果：本发明发现一种区别于野生型的酯水解酶突变体，对高浓度底物3-异丁基-戊二酸二乙酯进行立体选择性水解，获得的产物(S)-3-异丁基-戊二酸单乙酯的手性纯度大于98％，转化率达到100％。经过后期提取分离，收率达到97％，大幅度提高了合成效率，降低了生产成本，适合工业化生产。
附图说明
图1不同构型与酯水解酶的对接图，其中A为S型结构对接，B为R型结构对接
图2突变体测序的比对图
图3酵母转化子在平板上的表达图
图4外消旋体3-异丁基-戊二酸单乙酯定位图
图5底物3-异丁基-戊二酸二乙酯的定位图
图6野生型固定化酶催化底物的HPLC手性分析图
图7突变体固定化酶催化100g/L底物的HPLC手性分析图
具体实施方式
下面结合具体实施例对本发明的技术内容作进一步的阐述，其目的是为了更好的理解本发明的内容，但本发明的保护范围不限于此。
实施例1酯水解酶突变体的理性构建
来自南极假丝酵母的酯水解酶CalB具有一定的手性选择性，对底物3-异丁基-戊二酸二乙酯水解后可以获得S手性单酯产物的纯度在70％，为了进一步提高CalB的手性选择性，对其结构进行理性分析。以PDB数据库中的野生型结构1TCA为参考，分别将S-3-异丁基-戊二酸单乙酯和R-3-异丁基-戊二酸单乙酯与其半柔性对接，对接结果见附图1。从对接的结果中发现，想要提高S手性选择性，则需要将S结构伸向的空间释放更大些，更有利于S结构的摆放。相反，对于R结构的对接，需要蛋白结构中存在更大阻力，不利于R结构的摆放。从对接中找到三个影响主要位点141、154和157，分别对141、154和157进行其余19个氨基酸的突变构建，通过同源建模获得其他突变体的结构，再分别与S-3-异丁基-戊二酸单乙酯和R-3-异丁基-戊二酸单乙酯进行半柔性对接，分析每个突变体对接后的能量比较值ES/ER，这个值越小则越有利于蛋白选择S结构。通过一系列分析，最终确定A141L/V154R/Q157W的能量比值最小0.0045。分别对以上3个位点设计突变引物，利用全质粒PCR迭代突变获得突变体，具体引物设计见表1。
表1定点突变的引物序列
表1中带下划线的序列为突变位点，利用Primer STAR max DNA聚合酶(TaKaRa公司)在正反向饱和引物的参与下，以重组质粒pPIC9K-CalB为模板，进行全质粒扩增反应，PCR反应程序参考表2。
表2全质粒扩增程序表
将获得的全质粒突变体转入DH5α感受态细胞中，涂布于含有30ug/mL的Kan+抗性LB平板上，37℃培养过夜，挑取单克隆进行测序鉴定位点是否突变，测序比对结果见附图2，显示三个位点逐步叠加，实现了同时突变。为了在酵母体系表达突变体，需要利用限制性内切酶BglⅡ消化突变体重组质粒，产生线性的质粒，结合电转仪，将突变体基因整合到毕赤酵母染色体上。转化液加入冰冷的1M山梨醇，在30℃震荡培养箱中孵育1h后，涂布在含有100ug/mL的G418抗性YPD平板上，30℃培养3天，挑取单克隆准备发酵分泌表达蛋白突变体。
实施例2酯水解酶突变体的分泌表达和制备
随机挑取部分在抗性平板上生长的转化子，利用牙签转接到含有三丁酸甘油酯的乳化平板上，并在其平板盖上添加200uL的甲醇(终浓度为1％)，30℃倒置培养诱导重组菌体产酶，在培养2天后，转化子周围会出现透明圈见附图3，挑选形成透明圈最大的转化子进行发酵。
挑取形成透明圈最大的转化子单菌落，接种于50mL BMGY培养基中，30℃，200rpm震荡培养。当OD600达到3～8时，在无菌的离心管中，4℃和4000rpm条件下，离心10min，弃去上清，用同样体积的BMMY培养基重悬细胞，置于28℃震荡培养，每隔24h向培养基中加入1％的纯甲醇，作为诱导剂。培养5天后，在4000rpm条件下离心30min，弃去沉淀，收集表达的上清液，也就是所谓的酶液。
为了更好的分离和储藏酶，可以将酶液进一步利用25％的饱和硫酸铵沉淀，获得纯度较高的酶液。再利用环氧化树脂LX-1000P作为载体，通过共价键的结合，使得酶蛋白固定到树脂载体上，从而获得固定化酶。
实施例3液相分析手性纯度的方法及标品定位
由于反应的产物具有手性，需要利用手性柱子分析，柱子型号为：Chiral IC-3，(150X4.6mm)。流动相为(n-Hexane：n-Butanol：TFA＝950:50:1)，流速为0.3mL/min，检测波长为212nm，注射样为3uL，柱子温度控制在25℃，分析仪器为Shimadzu 2010AHT。
称量3-异丁基-戊二酸单乙酯外消旋体0.5g，用2mL乙醇溶解，并且要超声震荡，在进样前采用0.22um的膜过滤，滤液转入分析进样瓶中，等待自动进样分析。结果见附图4，分别在17min和20min出现不同手性的峰图，两者峰面积的比为51:49，接近1:1。
称取3-异丁基-戊二酸二乙酯的底物0.2g，用1mL乙醇溶解，并且要超声震荡，在进样前采用0.22um的膜过滤，滤液转入分析进样瓶中，等待自动进样分析。结果见附图5，底物出峰时间在15.6min。
实施例4野生型固定化酶制备S手性产物
用标准pH 6.8和pH 9.2两种试剂校准pH计，将校准好的pH计插入250mL的三口反应釜中，向反应釜中加入50mM的磷酸盐缓冲液，并开启磁力搅拌，利用5M的HCl将pH调至8.0，依次再加入四氢呋喃助溶剂3mL(控制体积比在10％)和底物3-异丁基-戊二酸二乙酯0.3g，控制反应的体系在30mL，最后再投入野生型固定化CalB酶0.05g，在37℃下反应。反应过程中用1M的NaOH控制pH稳定在8.0，至反应体系的pH值不再下降，并出现向上反弹时，标志着反应结束。
反应液通过离心，获取上清液，利用2.5M的HCl将pH调至3.0，再加入25％的乙酸乙酯进行萃取。萃取后的产物旋蒸发除去乙酸乙酯有机溶剂，残留的油状物用乙醇溶解，再借助HPLC分析产物的手性、浓度和转化率，附图6显示S手性纯度为73％，底物无剩余，转化率达到100％，计算收率达到90％。
实施例5突变体固定化酶制备S手性产物
10g/L的底物浓度反应：利用标准pH 6.8和pH 9.2两种试剂校准pH计，将校准好的pH计插入250mL的三口反应釜中，向反应釜中加入50mM的Tris缓冲液，并开启磁力搅拌，用5M的HCl将pH调至8.0，依次再加入四氢呋喃助溶剂3mL(控制体积比在10％)和底物3-异丁基-戊二酸二乙酯0.3g，控制反应的体系在30mL，最后再投入固定化CalB酶0.05g，在37℃下反应。反应过程中用1M的NaOH控制pH稳定在8.0，至反应体系pH值不再下降，反而上升，标志着反应结束。
100g/L的底物浓度反应：向250mL反应釜中加入50mM的Tris缓冲液，并开启磁力搅拌，利用5M的HCL将pH调至8.0，依次再加入四氢呋喃助溶剂3mL(控制体积比在10％)和底物3-异丁基-戊二酸二乙酯3g，控制反应的体系在30mL，最后再投入突变体固定化CalB酶0.05g，在37℃下反应。反应过程中用1M的NaOH控制pH稳定在8.0，至反应体系的pH值不再下降，反而上升，标志着反应结束。
反应液通过离心，获取上清液，利用2.5M的HCl将pH调至3.0，再加入25％的乙酸乙酯进行萃取。萃取后的产物旋转蒸发除去乙酸乙酯，残留的油状物用乙醇溶解，再借助HPLC分析产物的手性、浓度和转化率，图7结果显示S手性纯度达到98.5％，底物无剩余，转化率达到100％，计算收率达到97％。
实施例6突变体酶液制备S手性产物
向250mL反应釜中加入50mM的Tris缓冲液，并开启磁力搅拌，利用5M的HCl将pH至8.0，依次再加入四氢呋喃助溶剂3mL(控制体积比在10％)和底物3-异丁基-戊二酸二乙酯3g，最后直接加入酵母分泌表达后的上清液4mL，控制反应的体系30mL，在37℃下反应。反应过程中用1M的NaOH控制pH稳定在8.0，至反应体系的pH值不再下降，反而上升，标志反应结束。
直接向反应液中加入2.5M的HCl，将其pH调至3.0(可以使酶蛋白变性失活)，再加入25％的乙酸乙酯进行萃取。萃取后的产物再旋转蒸发彻底除去乙酸乙酯，残留的油状物用乙醇溶解，再借助HPLC分析产物的手性、浓度和转化率，结果显示S手性纯度达到98.4％，底物也无剩余，转化率达到100％，计算收率也达到95％。
序列表
<110> 尚科生物医药（上海）有限公司
<120> 一种酯水解酶突变体
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Claims (6)
1.一种酯水解酶突变体，其特征在于，所述酯水解酶突变体的氨基酸序列是以SEQ IDNO.1所示的野生型酯水解酶为参考序列发生突变的氨基酸序列，突变位点为第141位的丙氨酸突变为亮氨酸，第154位的缬氨酸突变为精氨酸和第157位的谷氨酰胺突变为色氨酸。
2.如权利要求1所述的酯水解酶突变体，其特征在于，所述酯水解酶的氨基酸序列为SEQ ID No.2所示。
3.如权利要求1所述的酯水解酶突变体，其特征在于，所述酯水解酶的基因核苷酸序列为SEQ ID No.3所示。
4.如权利要求1所述的酯水解酶突变体，其特征在于，所述酯水解酶来源于CandidaAntarctica。
5.如权利要求1所述的酯水解酶突变体，其特征在于，所述酯水解酶用于制备普瑞巴林中间体。
6.如权利要求5所述的酯水解酶用于制备普瑞巴林中间体，具体反应如式I所示：
其中，R为甲基、乙基、异丙基、苄基。
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