CN117431226A - 解木糖赖氨酸芽孢杆菌内消旋-二氨基庚二酸脱氢酶突变体及其应用 - Google Patents
解木糖赖氨酸芽孢杆菌内消旋-二氨基庚二酸脱氢酶突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种源于解木糖赖氨酸芽孢杆菌(Lysinibacillus xylanilyticus)的内消旋‑二氨基庚二酸脱氢酶(LxDAPDH)突变体及其应用，属于生物工程技术领域。该突变体是将解木糖赖氨酸芽孢杆菌内消旋‑二氨基庚二酸脱氢酶的第94位天冬氨酸氨酸突变为丙氨酸。以大肠杆菌BL21为宿主，突变体LxDAPDHD94A基因和源于巨大芽孢杆菌的葡萄糖脱氢酶(BmGDH)基因采用同源重组技术共表达。以共表达菌株的全细胞为生物催化剂，4‑溴苯丙酮酸和氯化铵为底物，葡萄糖为辅底物制备4‑溴‑D‑苯丙氨酸。4‑溴苯丙酮酸的转化率为95％。D‑4‑溴苯丙氨酸的分离收率为80％，ee>99％。
Description
技术领域 本发明涉及一种解木糖赖氨酸芽孢杆菌内消旋-二氨基庚二酸脱氢酶突变体及其应用，属于生物工程技术领域。
背景技术
诺欣妥(Entresto)是诺华公司研发的脑啡肽酶抑制剂沙库必曲(Sacubitril)和血管紧张素Ⅱ受体拮抗剂缬沙坦(Valsartan)复方制剂(Entresto)，用于射血分数降低的成人慢性心衰患者，以降低其心血管死亡和心衰住院的风险。该药物于2015年7月由FDA批准上市，2017年7月经NMPA批准在中国上市。2021年NMPA批准了诺欣妥的新适应症，用于治疗原发性高血压。
D-4,4’-联苯丙氨酸是沙库必曲的关键手性中间体，可以通过4-溴-D-苯丙氨酸与苯硼酸偶联制备(Ahmed ST.ACS Catal，2015,5(9),5410–5413)。以4-溴-L-苯丙氨酸或4-溴-DL-苯丙氨酸为原料，通过L-氨基酸脱氨酶和D-氨基酸转氨酶的级联催化手性反转和去消旋化合成4-溴-D-苯丙氨酸，ee>99％，但该方法的缺点是需要化学计量的D-谷氨酸(Walton CJW et al.ChemCatChem,2018.10(2),470-474.)。以内消旋-二氨基庚二酸脱氢酶(meso-Diaminopimelate dehydrogenase,DAPDH)为生物催化剂催化4-溴苯丙酮酸不对称还原胺化是合成4-溴-D-苯丙氨酸的最好方法。以共表达CgDAPDHBC621-GDH的全细胞为生物催化剂，40mmol/L 4-溴苯丙酮酸转化为4-溴-D-苯丙氨酸，转化率>95％。4-溴-D-苯丙氨酸分离收率69％，ee>99％(Parmeggiani F et al.Adv Synth Catal,2016,358(20),3298-3306.)。该方法的缺点是DAPDH的酶活低，热稳定性差，不能实现4-溴-D-苯丙氨酸的高浓度合成。
源于嗜热球型芽孢杆菌(Ureibacillus thermosphaericus)的内消旋-二氨基庚二酸脱氢酶(UtDAPDH)，其突变体UtDAPDHD94A对苯丙酮酸的比活力相对于野生型提高了8倍，且具有很高的热稳定性(Hayashi J et al.App Environ Microb,2017,83(11),e00491-17.)。
解木糖赖氨酸芽孢杆菌(Lysinibacillus xylanilyticus)XX-2中的L-氨基酸脱氢酶已用于系列酮酸的制备(夏仕文等.ZL201510697875.6,2016-02-10.)和L-2-氨基己二酸的生物合成(刘娇等.分子催化,2023,37(3),285-292.)。通过全基因组测序和基因注释发现该菌株中存在内消旋-二氨基庚二酸脱氢酶基因(LxDAPDH)。通过序列比对发现LxDAPDH基因与UtDAPDH有很高的相似性(89％)，其突变体LxDAPDHD94A对苯丙酮酸的活性相较于野生型提高了37倍。
发明内容
为解决内消旋-二氨基庚二酸脱氢酶催化制备4-溴-D-苯丙氨酸过程中存在酶活低，转化强度低的技术问题，本发明提供了一种解木糖赖氨酸芽孢杆菌内消旋-二氨基庚二酸脱氢酶突变体LxDAPDHD94A以及利用该突变体催化4-溴苯丙酮酸不对称还原胺化合成4-溴-D-苯丙氨酸的方法。
本发明的第一个目的是提供一种解木糖赖氨酸芽孢杆菌内消旋-二氨基庚二酸脱氢酶(LxDAPDH)突变体，所述突变体是将氨基酸序列如SEQ ID NO.1所示的野生型内消旋-二氨基庚二酸脱氢酶的第94位天冬氨酸突变为丙氨酸。突变体命名为LxDAPDHD94A，其氨基酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供一种LxDAPDHD94A和巨大芽孢杆菌葡萄糖脱氢酶(BmGDH)共表达的重组菌株。LxDAPDHD94A和BmGDH采用同源重组技术共表达在大肠杆菌BL21中。
本发明的第三个目的是利用LxDAPDHD94A和BmGDH共表达的重组菌细胞，以4-溴苯丙酮酸为原料制备4-溴-D-苯丙氨酸。在催化反应体系中，共表达细胞的浓度为20-50g(湿重)/L，4-溴苯丙酮酸浓度为50-300mmol/L，氯化铵浓度为100-300mmol/L，碳酸钠浓度为50-200mmol/L，甲醇的体积百分比为2-10％，NADP+浓度为0.2-1.0mmol/L。反应温度20-50℃，pH8-11，反应时间10-50h。
优选地，共表达细胞的浓度为30g(湿重)/L，4-溴苯丙酮酸浓度为200mmol/L，氯化铵浓度为200mmol/L，碳酸钠浓度为100mmol/L，甲醇的体积百分比为4％，NADP+浓度为0.5mmol/L。反应温度30℃，pH9.0，反应时间24h。
4-溴-苯丙酮酸的转化率，4-溴-D-苯丙氨酸的产率、化学纯度和光学纯度采用高效液相色谱(HPLC)检测。
非手性HPLC。色谱柱：Sepax MAH-C18(250×4.6mm，5μm)；流动相：0.1％三氟乙酸水溶液/甲醇(49/51，v/v)；流速：1mL/min；检测波长：254nm；柱温：25℃。4-溴苯丙酮酸和4-溴-D-苯丙氨酸的保留时间分别为12.3min和4.2min。
手性HPLC。色谱柱：Crownpak CR(+)(150×4.0mm，5μm)，流动相：HClO4溶液(pH1.3)，流速：1.0mL/min，检测波长：200nm，柱温：30℃。4-溴苯丙氨酸的L-和D-对映体保留时间分别为12.3min和7.1min。
本发明的有益效果
本发明构建的LxDAPDHD94A-BmGDH共表达菌株，稳定性好，耐热性强，催化活性高。建立的基于LxDAPDHD94A-BmGDH的制备方法，适合4-溴-D-苯丙氨酸的工业化生产。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：LxDAPDHD94A-BmGDH共表达工程菌构建
目的基因扩增。以源于解木糖赖氨酸芽孢杆菌(Lysinibacillus xylanilyticus)的内消旋-二氨基庚二酸脱氢酶突变体D94A基因(如SEQ ID NO.2所示)的pET-28a重组质粒作为模板，设计PCR扩增引物。扩增引物既包含用于延伸的引物序列，5'端根据所连表达载体添加相应的同源臂，即D94A-F和D94A-R引物对。具体引物如下(加粗及下划线为突变位点))：
D94AF ccacagccaggatccgaattccATGGGCATGAGCGCGATT
D94AR gcattatgcggccgcaagcttTTACAGCAGTTCTTTGCGCAGT
按照下表所示的扩增体系，获取目的基因。PCR反应结束后，目的基因采用胶回收的方式进行纯化。
PCR扩增体系
载体的线性化。选取EcoRⅠ和HindⅢ酶切位点，采用双酶切的方式使pETDuet-1载体线性化，酶切体系如下表所示。酶切结束后溶液胶回收，得到载体片段。
双酶切反应体系
目的基因连接表达载体。两端具有同源臂的目的基因同源重组酶的作用下，与酶切线性化后的载体两端发生同源重组，目的基因与表达载体进行连接。重组体系如下表所示。
同源重组体系
连接验证及测序验证。将连接产物转化感受态细胞，并通过菌落PCR验证转化子是否含有相应大小的条带(900bp)。菌落PCR验证体系如下表所示。选取阳性转化子(即含有相应的条带)测序。
菌落PCR体系
LxDAPDHD94A-BmGDH共表达。选择含有两个多克隆位点的蛋白双表达载体pETDuet-1，每一个多克隆位点通过T7启动子/Lac操纵子和一个核糖体结合位点组成。构建的质粒转化至大肠杆菌BL21，得到共表达重组菌。SDS-PAGE验证，两个酶在大肠杆菌中实现了共表达。
实施例2：LxDAPDHD94A-BmGDH共表达工程菌培养
携带pACYC-LxDAPDHD94A-BmGDH质粒的大肠杆菌BL21(DE3)的单个菌落接种于添加氨苄青霉素(100μg/mL)的LB培养基(10mL)中，37℃、220rpm下生长过夜，获得种子液。种子液按1％接种量接种于添加氨苄青霉素(100μg/mL)的LB培养基中。37℃、160rpm下培养至OD600达到0.6-0.8，加入IPTG(终浓度为1mmol/L)诱导表达3h。离心(4℃，8000rpm，20min)收集湿菌体。湿菌体用磷酸盐缓冲液(100mmol/L，pH 8.0)洗涤2次，-20℃保存备用。
实施例3：4-溴-D-苯丙氨酸制备
共表达工程菌细胞(5g)悬浮在150ml水中，加入1.59g Na2CO3，1.63g氯化铵，2.7g葡萄糖，6ml甲醇和56mg NADP+，7.32g 4-溴苯丙酮酸，30℃下磁力搅拌反应24h。反应过程中，反应液pH用6mol/L NaOH溶液控制在9.0-9.2。反应完成后，离心去除细胞。上清液用6mol/L盐酸调pH至5.5，4℃下放置过夜，抽滤，固体分别用20ml乙酸乙酯和20ml冰水洗涤。固体烘干，得4-溴-D-苯丙氨酸5.93g，分离收率80％，ee>99％。1H NMR(400MHz,D2O+NaOH):d＝7.34(d,2H,ArH,J＝8Hz),7.00(d,2H,ArH,J＝8Hz),3.28-3.33(m,1H,CHNH2),2.75(dd,1H,CHH,J＝16,8Hz),2.65(dd,1H,CHH,J＝16,8Hz)；13C NMR(100MHz,D2O+NaOH):d＝182.13,137.39,131.32,131.22,119.73,57.28,40.21.
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是SDS-PAGE结果示意图。
Claims (6)
1.一种内消旋-二氨基庚二酸脱氢酶突变体，其特征在于，所述内消旋-二氨基庚二酸脱氢酶来源于解木糖赖氨酸芽孢杆菌，野生酶的氨基酸序列和编码该酶的核苷酸序列如SEQ ID NO.1所示(Genbank号为OQ559328)。
2.一种内消旋-二氨基庚二酸脱氢酶突变体，其特征在于，SEQ ID NO.1中的氨基酸序列的第94位天冬氨酸突变为丙氨酸。
3.一种内消旋-二氨基庚二酸脱氢酶突变体，其特征在于，突变酶的氨基酸序列和编码该突变酶的核苷酸序列如SEQ ID NO.2所示。
4.一种内消旋-二氨基庚二酸脱氢酶突变体应用，其特征在于，突变酶LxDAPDHD94A基因和巨大芽孢杆菌葡萄糖脱氢酶基因(BmGDH，如SEQ ID NO.3所示)通过同源重组共表达在大肠杆菌BL21中。
5.如权利要求4所述的内消旋-二氨基庚二酸脱氢酶突变体应用，其特征在于，共表达菌株于30-37℃下培养至OD600为0.6-0.8，加入诱导剂IPTG(终浓度为0.05-1mmol/L)于30-40℃诱导表达2-5h。
6.如权利要求4所述的内消旋-二氨基庚二酸脱氢酶突变体应用，其特征在于，在制备4-溴-D-苯丙氨酸的催化反应体系中，共表达细胞的浓度为20-50g(湿重)/L，4-溴苯丙酮酸浓度为50-300mmol/L，氯化铵浓度为100-300mmol/L，碳酸钠100-300mmol/L，甲醇的体积百分比为2-10％，NADP+浓度为0.05-0.2mmol/L。反应温度20-50℃，反应时间10-50h。
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