CN116694589A - 一种7β-羟基类固醇脱氢酶的突变体及其应用 - Google Patents
一种7β-羟基类固醇脱氢酶的突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种7β‑羟基类固醇脱氢酶的突变体及其应用。所述的7β‑HSDH突变体在SEQ ID NO.1所示的野生型上进行单点突变、两两复合突变和三点复合突变,经筛选获得复合突变体T189V/V207M,在此基础上进一步筛选出25个氨基酸残基位点,通过定点饱和突变,提高7β‑HSDH的催化效率以及热稳定性的突变体T189V/V207M/V91P、T189V/V207M/M144H、T189V/V207M/G146A、T189V/V207M/A158S、T189V/V207M/V183I。本发明构建的突变后的7β‑HSDH突变体相较于出发7β‑HSDH酶活力提高了3.8倍,突变后的7β‑HSDH突变体在催化100g·L‑1的7‑酮基‑石胆酸转化率可达到91%,具有工业化生产前景。
Description
技术领域
本发明涉及一种7β-羟基类固醇脱氢酶的突变体及其应用,属于生物酶工程技术领域。
背景技术
熊去氧胆酸(Ursodeoxycholic acid,UDCA)是临床上广泛使用的药物成分,作为胆汁酸它能溶解胆固醇结石,在胆汁淤积性疾病时,改善肝功能。
熊去氧胆酸的制备方法主要是化学合成和生物转化,其中通过化学合成有效的获得的熊去氧胆酸仅占总合成路线的30%。由于化学合成步骤繁琐,伴随着保护和去保护步骤,这些步骤中往往有危险和有毒试剂的参与,同时还会排放许多污染环境的废物。由于以上种种原因,研究人员将目光放在了生物转化法研究上,以胆酸和鹅去氧胆酸为原料通过微生物转化或化学酶法来生产熊去氧胆酸,利用微生物酶的高区域和立体选择性,作用在甾体骨架的特定位点上进行氧化还原反应以及构象的转换来实现熊去氧胆酸绿色、高效的生产。
7β-羟基类固醇脱氢酶(7β-HSDH)作为酶法合成熊去氧胆酸的关键酶,可将C-7位点上酮羰基还原成β取向的羟基。国内外报道来源的7β-HSDH有Clostridium absonum、Eubacterium aerofaciens、Ruminococcus torques、Ruminococcusgnavus、Collinsellaaerofaciens、Xanthomonas maltophilia。但这些酶的热稳定性和酶活力偏低,存在着很大改造空间。
近代生物学的发展使人们认识到蛋白质进化大多是由于某些点突变或修饰的积累,而在自然条件下,蛋白质性质或功能的改变通常需要很长时间。对7β-HSDH在近几年的得到了很大的关注。许建和团队通过多目标定向进化策略提高了7β-HSDH酶活力及其热稳定性。宋鹏等在发明专利中对7β-HSDH其底物耐受性进行研究,通过易错PCR确定影响7β-HSDH活性的关键氨基酸并进行定点饱和突变,提高7β-HSDH底物耐受浓度。王峰等发明了一种7β-HSDH与DPS形成的融合蛋白,融合蛋白的酶活力相较于野生酶有所提高。然而,目前已报道的催化生产熊去氧胆酸的7β-HSDH酶数量甚少,即使通过酶分子改造后7β-HSDH突变体的酶催化效率及转化率仍然偏低,这主要对7β-HSDH酶没有进行系统的酶分子改造,不能高效满足实际生产需求。
发明内容
为解决上述问题,本发明提供了一种提高酶催化效率及提高底物耐受浓度的7β-HSDH酶突变体,双突变体T189V/V207M、三突变体T189V/V207M/V91P、T189V/V207M/M144H、T189V/V207M/G146A、T189V/V207M/A158S、T189V/V207M/V183I的酶活力相对于野生型至少提高了2倍,且最优突变体T189V/V207M/G146A在底物7-oxo-LCA浓度为100g/L时转化率高达91.2%,解决了7β-HSDH催化效率及转化率偏低的问题,同时热稳定性有明显提升。
本发明的第一个目的是提供一种7β-羟基类固醇脱氢酶突变体,所述的突变体是对氨基酸序列如SEQ ID NO.1所示的7β-羟基类固醇脱氢酶进行突变:
将第189位苏氨酸突变为缬氨酸,并将第207位缬氨酸突变为甲硫氨酸(T189V/V207M);
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第91位缬氨酸突变为脯氨酸(T189V/V207M/V91P);
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第144位甲硫氨酸突变为组氨酸(T189V/V207M/M144H);
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第146位甘氨酸突变为丙氨酸(T189V/V207M/G146A);
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第158位丙氨酸突变为丝氨酸(T189V/V207M/A158S);
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第183位缬氨酸突变为异亮氨酸(T189V/V207M/V183I)。
本发明的第二个目的是提供编码所述7β-羟基类固醇脱氢酶突变体的基因。
进一步地,所述7β-羟基类固醇脱氢酶突变体的基因的核苷酸序列如SEQ ID NO.2所示(T189V/V207M)。
进一步地,所述7β-羟基类固醇脱氢酶突变体的基因的核苷酸序列如SEQ ID NO.3所示(T189V/V207M/V91P)。
进一步地,所述7β-羟基类固醇脱氢酶突变体的基因的核苷酸序列如SEQ ID NO.4所示(T189V/V207M/M144H)。
进一步地,所述7β-羟基类固醇脱氢酶突变体的基因的核苷酸序列如SEQ ID NO.5所示(T189V/V207M/G146A)。
进一步地,所述7β-羟基类固醇脱氢酶突变体的基因的核苷酸序列如SEQ ID NO.6所示(T189V/V207M/A158S)。
进一步地,所述7β-羟基类固醇脱氢酶突变体的基因的核苷酸序列如SEQ ID NO.7所示(T189V/V207M/V183I)。
本发明的第三个目的是提供携带所述基因的重组质粒。
本发明的第四个目的是提供表达所述7β-羟基类固醇脱氢酶突变体的宿主细胞。
进一步地,所述宿主细胞为细菌、真菌、植物细胞或动物细胞。
本发明的第五个目的是提供上述7β-羟基类固醇脱氢酶突变体、基因、表达载体或宿主细胞在制备熊去氧胆酸中的应用。
进一步地,以7-酮基-石胆酸为底物。
进一步地,所述应用为将上述7β-羟基类固醇脱氢酶突变体或含有该突变体编码基因的表达系统添加至反应体系中。
进一步地,所述反应体系中还包括葡萄糖脱氢酶。
进一步地,所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO.8所示。
本发明的有益效果:
本发明以来源于Olsenella sp.An188的7β-HSDH出发,通过定点突变筛选获得酶活力和催化效率kcat/Km值相较于野生酶提高了3倍和7.1倍的7β-HSDH突变体T189V/V207M。并进一步对7β-HSDH突变体T189V/V207M进行3D结构同源建模,经可视化软件分析,选取活性位点周围范围内氨基酸残基,确定了25个关键氨基酸残基位点,对这些关键氨基酸残基位点进行定点饱和突变,筛选获得5个具有高酶活力的突变体:T189V/V207M/V91P、T189V/V207M/M144H、T189V/V207M/G146A、T189V/V207M/A158S、T189V/V207M/V183I,其比酶活力分别提高至野生酶7β-HSDH_Osp的3.5、3.6、3.6、3.5、3.8倍。其中,突变体T189V/V207M/G146A的kcat/Km值显著提高,由T189V/V207M的352.63L·mol-1·min-1提高至654.42L·mol-1·min-1。其中,采用两步法催化时,突变体T189V/V207M/G146A和T189V/V207M/A158S催化100g/L底物生产熊去氧胆酸的转化率分别提高至91.2%和81.3%。
附图说明
图1为不同来源的7β-HSDH氨基酸序列比对;
图2为定点突变的突变体酶活力;
图3为定点饱和突变的突变体酶活力;
图4为两步法催化7-酮基-石胆酸生产熊去氧胆酸;
图5为7β-HSDH及其突变体的热稳定性测定结果。
具体实施方式
下面结合附图和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
1、定点突变——单点突变
基于对多种来源(R.gnavus、R.torques、C.absonum、E.aereofaciens和C.aerofaciens)的7β-HSDH氨基酸序列比对分析(图1)。并参考郑采用多目标进化策略通过首轮易错PCR(epPCR)和第二轮的DNA改组(DNA shuffling)获得了高酶活力和高热稳定性的潜在位点,并通过第三轮定点突变获酶催化效率提高的正向突变体T189V和V207M。以及Hummel在专利中对C.aerofaciens来源的7β-HSDH进行了分子改造,发现突变体G39A的比酶活力从15U·mg-1提高至21U·mg-1。基于上述文献结果和序列比对结果,确定7β-HSDH的3种提高其酶催化效率的定点突变方式:G39A、T189V、V207M。以pRSFDuet-1-7β-HSDH_Osp重组质粒为模板,对上述三个位点分别进行定点突变。PCR扩增的条件依据Prime STAR HS DNA聚合酶的说明书进行设置:预变性98℃,5min;变性95℃,30s;退火55℃,30s;延伸72℃(酶的延伸速度为1kb·min-1,具体时间根据扩增片段的长度来设置);设置循环30次;再延伸72℃,10min,完成PCR扩增过程,PCR扩增体系如表1所示。
表1PCR扩增体系
其中:
39位点突变
正向引物:GGCGGAATGAGCGTCGTAATGGTTGCTCGCCGCGAAG
反向引物:TTCGCGCAGTTTTTCTTCGCGGCGAGCAACCATTACG
189位点突变
正向引物:GTTGAGGTCATTACCCTTGGTACTGTTTTGACGCCGT
反向引物:ATTAGAAAGAAGAGACGGCGTCAAAACAGTACCAAGG
207位点突变
正向引物:CCGGGGGGTCCACAAGGAGAAGCTATGATGAAGATTG
反向引物:TTCGGGGGTTTGAGCAATCTTCATCATAGCTTCTCCT
将上述突变得到的质粒转化入E.coli BL21(DE3)感受态细胞中涂布于含50μg·L-1的卡那霉素的LB固体培养基上,37℃过夜培养,测序挑选正确的转化子,获得7β-HSDH突变体G39A、T189V、V207M。
2、定点突变——复合突变
对上述位点进行复合突变(方法同上),构建获得4个复合突变体G39A/T189V、G39A/V207M、T189V/V207M、G39A/T189V/V207M。其中突变体T189V/V207M在与文献(Zheng,Ming Min,et al.Two-step enzymatic synthesis ofursodeoxycholic acid with a new7β-hydroxysteroid dehydrogenase from Ruminococcus torques.ProcessBiochemistry 50(4):598-604.)相同的方式检测酶活力时,酶活力达到了58.6U·mg-1,高于文献中的突变体46.8U·mg-1。随后将本发明的突变体T189V/V207M应用到两步反应高效催化生产从熊去氧胆酸中发现在催化10mM鹅去氧胆酸最终收率可达到99%。
酶活力的测定方法:
酶反应体系:100mM磷酸钾缓冲液(pH=7.0)、10mM 7-酮基-石胆酸(7-oxo-LCA)、3mM NADPH、30℃。7β-HSDH酶活力定义单位为:在上述反应条件下,酶催化底物7-酮基-石胆酸每分钟消耗1μmol NAD(P)H所需的酶量,为一个酶活力单位(U)。
具体酶活力比较如图2所示。
实施例2定点饱和突变体文库的构建及高催化效率突变体的筛选
利用在线软件SWISS-MODEL(http://swissmodel.expasy.org/),以来源于C.aerofaciens的7β-HSDH的晶体结构(PDB ID:5GT9)为模板,对7β-HSDH突变体T189V/V207M进行3D结构同源建模。采用PyMOL软件分析7β-HSDH突变体活性位点周围范围内氨基酸残基,确定了25个关键氨基酸残基位点:V91、A92、C93、H95、N140、V141、S143、M144、T145、G146、S150、N153、G154、Q155、G157、A158、G159、A161、Y162、I163、L164、V183、I184、T185、L186。对上述关键氨基酸残基位点进行定点饱和突变,分别构建突变体文库,筛选具有高催化效率的酶突变体。
其中:
91位点突变
正向引物:CATGTCTTATNNKGCCTGTCTGC
反向引物:GCAGACAGGCMNNATAAGACATG
92位点突变
正向引物:GTCTTATGTCNNKTGTCTGCATA
反向引物:TATGCAGACAMNNGACATAAGAC
93位点突变
正向引物:TTATGTCGCCNNKCTGCATAGTT
反向引物:AACTATGCAGMNNGGCGACATAA
95位点突变
正向引物:CGCCTGTCTGNNKAGTTTCGGGA
反向引物:TCCCGAAACTMNNCAGACAGGCG
140位点突变
正向引物:CGCTGTGATTNNKGTTAGTAGTA
反向引物:TACTACTAACMNNAATCACAGCG
141位点突变
正向引物:TGTGATTAATNNKAGTAGTATGA
反向引物:TCATACTACTMNNATTAATCACA
143位点突变
正向引物:TAATGTTAGTNNKATGACCGGCG
反向引物:CGCCGGTCATMNNACTAACATTA
144位点突变
正向引物:TGTTAGTAGTNNKACCGGCGTTT
反向引物:AAACGCCGGTMNNACTACTAACA
145位点突变
正向引物:TAGTAGTATGNNKGGCGTTTCAT
反向引物:ATGAAACGCCMNNCATACTACTA
146位点突变
正向引物:TAGTATGACCNNKGTTTCATCAT
反向引物:ATGATGAAACMNNGGTCATACTA
150位点突变
正向引物:CGTTTCATCANNKCCATGGAATG
反向引物:CATTCCATGGMNNTGATGAAACG
153位点突变
正向引物:ATCTCCATGGNNKGGGCAGTATG
反向引物:CATACTGCCCMNNCCATGGAGAT
154位点突变
正向引物:TCCATGGAATNNKCAGTATGGAG
反向引物:CTCCATACTGMNNATTCCATGGA
155位点突变
正向引物:ATGGAATGGGNNKTATGGAGCAG
反向引物:CTGCTCCATAMNNCCCATTCCAT
157位点突变
正向引物:TGGGCAGTATNNKGCAGGTAAGG
反向引物:CCTTACCTGCMNNATACTGCCCA
158位点突变
正向引物:GCAGTATGGANNKGGTAAGGCGT
反向引物:ACGCCTTACCMNNTCCATACTGC
159位点突变
正向引物:GTATGGAGCANNKAAGGCGTACA
反向引物:TGTACGCCTTMNNTGCTCCATAC
161位点突变
正向引物:AGCAGGTAAGNNKTACATTCTGA
反向引物:TCAGAATGTAMNNCTTACCTGCT
162位点突变
正向引物:AGGTAAGGCGNNKATTCTGAAAA
反向引物:TTTTCAGAATMNNCGCCTTACCT
163位点突变
正向引物:TAAGGCGTACNNKCTGAAAATGA
反向引物:TCATTTTCAGMNNGTACGCCTTA
164位点突变
正向引物:GGCGTACATTNNKAAAATGACAG
反向引物:CTGTCATTTTMNNAATGTACGCC
183位点突变
正向引物:CGACGTTGAGNNKATTACCCTTG
反向引物:CAAGGGTAATMNNCTCAACGTCG
184位点突变
正向引物:CGTTGAGGTCNNKACCCTTGGTA
反向引物:TACCAAGGGTMNNGACCTCAACG
185位点突变
正向引物:TGAGGTCATTNNKCTTGGTACTG
反向引物:CAGTACCAAGMNNAATGACCTCA
186位点突变
正向引物:GGTCATTACCNNKGGTACTGTTT
反向引物:AAACAGTACCMNNGGTAATGACC
采用NNK简并密码子分别设计突变引物,对T189V/V207M进行定点饱和突变(SDM),分别构建了25个突变体文库。挑取单菌落接种于96深孔板中。使用300μL含50μg·mL-1的卡那霉素液体LB培养基37℃、250rpm、培养8h。将所有挑选的克隆的100μL培养物转移到新的96孔板上,其中含有30%的甘油作为副本,并将其保存在-20℃下。复制完成后添加800μL含50μg·mL-1的卡那霉素的液体TB培养基37℃、250rpm、培养3h后,加入终浓度为0.5mM的IPTG诱导后25℃、250rpm培养24h。使用孔板离心机收集菌体,加入500μL含5mg·mL-1的溶菌酶的100mM磷酸钾缓冲液(pH=7.0),37℃放置1h,随后-80℃下低温处理15min,并在37℃融化,如此反复冻融三次。4,500rpm离心20min,收集上清液,使用酶标仪(Molecular devices,SpectraMax 190,USA)对细胞破碎液酶活力进行检测。在340nm处30℃和300s检测其还原活性,不同活性的突变体在340nm处表现出不同的吸光变化率。反应体系(200μL):1mM 7-oxo-LCA、0.5mM NADPH和10μL细胞破碎液。
筛选获得5个突变体T189V/V207M/V91P、T189V/V207M/M144H、T189V/V207M/G146A、T189V/V207M/A158S、T189V/V207M/V183I的酶活力如图3所示。
实施例37β-HSDH及其突变体的表达
7β-HSDH的编码基因核苷酸序列通过天霖生物科技有限公司基因合成所得,在编码区两端分别添加BamHI和HindⅢ限制性酶内切酶位点。目的基因片段通过限制性酶内切酶BamHⅠ和HindⅢ双酶切后,与经相同方式酶切处理的pRSFDuet-1载体进行连接,转入大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞当中,筛选获得含有7β-HSDH基因的阳性重组质粒pRSFDuet-1-7β-HSDH_Osp,从而构建7β-HSDH的体外异源表达体系。
将含有编码重组基因SEQ ID NO.2-7的重组质粒接种到含50μg·mL-1的卡那霉素
的LB液体培养基中,37℃、200rpm条件下培养12h。按1%接种量转接于含50μg·mL-1的卡那霉素的TB液体培养基中,37℃、200rpm条件下培养至OD600=0.6-0.8,加入终浓度0.5mM的IPTG,25℃诱导表达24h。
发酵结束后,采用7,000rpm离心10min收集菌体,用生理盐水洗涤细胞2次,将菌体浓缩5倍悬浮于100mM磷酸钾缓冲液(pH=7.0)。将收集到的菌体经洗涤悬浮处理后,采用超声波破碎仪(UCD-650B)超声破碎30min(变幅杆6,功率比20%,工作2s,停止4s),12,000rpm离心30min,收集上清液(即粗酶液)。
利用蛋白纯化柱(Ni2+柱)对含有HIS标签的粗酶液进行纯化,获得目的蛋白。
实施例4突变体的生物催化
利用7β-HSDH及其突变体T189V/V207M、T189V/V207M/V91P、T189V/V207M/M144H、T189V/V207M/G146A、T189V/V207M/A158S、T189V/V207M/V183I和来源于Bacillussubtilis的葡萄糖脱氢酶(GDH)联合催化生产UDCA,催化示意图如图4所示。
将10g底物7-oxo-LCA溶解于100mL,100mM的磷酸钾缓冲液中,利用NaOH溶液对底物进行溶解后调节pH值至7.0,随后添加终浓度500mM的葡萄糖和10mM的NADPH。将7β-HSDH突变体和GDH按200U·mL-1的添加量,加入反应液当中。30℃反应24h,通过HPLC检测计算其转化率。转化率结果如表2所示。
表2突变体催化7-oxo-LCA的转化率
实施例5突变体的热稳定性
将7β-HSDH及其突变体T189V/V207M、T189V/V207M/V91P、T189V/V207M/M144H、T189V/V207M/G146A、T189V/V207M/A158S、T189V/V207M/V183I置于50℃保温15min,对其剩余酶活力进行检测,结果如图5所示。
突变体T189V/V207M/V91P和T189V/V207M/M144H的热稳定性相较于突变体T189V/V207M均有显著提高。其中,突变体T189V/V207M/V91P的残留酶活力由T189V/V207M的72.0%提高至91.2%。相较于T189V/V207M,突变体T189V/V207M/G146A的热稳定提高不显著。相较于突变体T189V/V207M,突变体T189V/V207M/A158S和T189V/V207M/V183I的热稳定性均降低。其中,突变体T189V/V207M/V183I热稳定性低于野生酶7β-HSDH_Osp;突变体T189V/V207M/A158S热处理后已完全失活。上述研究结果表明,突变方式V91P对提高T189V/V207M的热稳定性具有显著作用;突变方式A158S和V183I对T189V/V207M的热稳定性具有显著抑制作用。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种7β-羟基类固醇脱氢酶突变体,其特征在于,所述的突变体是对氨基酸序列如SEQ ID NO.1所示的7β-羟基类固醇脱氢酶进行突变:
将第189位苏氨酸突变为缬氨酸,并将第207位缬氨酸突变为甲硫氨酸;
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第91位缬氨酸突变为脯氨酸;
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第144位甲硫氨酸突变为组氨酸;
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第146位甘氨酸突变为丙氨酸;
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第158位丙氨酸突变为丝氨酸;
或将第189位苏氨酸突变为缬氨酸,将第207位缬氨酸突变为甲硫氨酸,并将第183位缬氨酸突变为异亮氨酸。
2.编码权利要求1所述的7β-羟基类固醇脱氢酶突变体的基因。
3.根据权利要求2所述的基因,其特征在于:核苷酸序列如SEQ ID NO.2~7所示。
4.携带权利要求2或3所述的基因的重组质粒。
5.表达权利要求1所述的7β-羟基类固醇脱氢酶突变体的宿主细胞。
6.根据权利要求5所述的宿主细胞,其特征在于:所述宿主细胞为细菌、真菌、植物细胞或动物细胞。
7.权利要求1所述的7β-羟基类固醇脱氢酶突变体、权利要求2或3所述的基因、权利要求4所述的重组质粒或权利要求5或6所述的宿主细胞在制备熊去氧胆酸中的应用。
8.根据权利要求7所述的应用,其特征在于:以7-酮基-石胆酸为底物。
9.根据权利要求7所述的应用,其特征在于:所述应用为将所述7β-羟基类固醇脱氢酶突变体或含有该突变体编码基因的表达系统添加至反应体系中。
10.根据权利要求9所述的应用,其特征在于:所述反应体系中还包括葡萄糖脱氢酶。
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