CN117402849A - 一种新型转氨酶突变体及其制备方法与应用 - Google Patents
一种新型转氨酶突变体及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供一种新型转氨酶突变体及其制备方法与应用。转氨酶突变体的氨基酸序列是与SEQ ID NO:2所示的氨基酸序列具有80%以上同源性的氨基酸序列且至少包括如下突变位点之一:第25位的谷氨酰胺突变为苯丙氨酸,第138位的酪氨酸突变为苯丙氨酸,第192位的脯氨酸突变为谷氨酰胺,第377位的苏氨酸突变为缬氨酸,第424位的精氨酸突变为亮氨酸,第448位的丙氨酸突变为精氨酸。本发明提供该突变体相比于野生型的酶在可以高活性、高选择性地方式对1‑萘乙酮进行转氨作用,适应了工业化生产的需求,具有广泛的应用价值。
Description
技术领域
本发明属于生物催化技术领域,具体一种新型转氨酶突变体及其制备方法与应用。
背景技术
(R)-1-萘乙胺是一种应用广泛的手性芳香胺,既是一种重要的医药中间体,又是一种常用的手性拆分剂,在医药化工领域具有重要的地位。目前,制备(R)-1-萘乙胺的方法主要包括化学拆分法和生物催化法。
化学拆分法主要包括两种方案,一是利用1-萘乙胺分子中胺基的弱碱性,向消旋物中加入酸性拆分剂中和形成盐,再利用他们溶解度的差异进行分离;二是选择特异性的酰基供体选择性地与其中一种构型的1-萘乙胺形成酰胺键,继而实现分离。虽然这两种方法都可以接近理论产率50%,但产品的手性纯度较差,且过程时间长,会产生大量化学废物。
生物催化法包括动力学拆分外消旋胺和不对称合成手性胺,虽然动力学拆分法可以得到手性,但最高得率仅为50%;另一方面,可以利用R-转氨酶对1-萘乙酮进行选择性转氨反应,实现(R)-1-萘乙胺的直接合成。就目前报道来看,虽有文献报道转氨酶催化不对称合成(R)-1-萘乙胺,但随着底物1-萘乙酮浓度的升高,转化率也显著降低,这严重限制了其工业化生产。
发明内容
本发明的目的是,提供一种催化活力提高的转氨酶突变体及其制备方法与应用。该转氨酶突变体可以高效、高选择性的实现(R)-1-萘乙胺高效合成,具有工业应用价值。
本发明为解决上述技术问题所采用的技术方案如下:
本发明提供一种新型的转氨酶突变体,所述转氨酶突变体的氨基酸序列是与SEQID NO:2所示的氨基酸序列具有80%以上同源性的氨基酸序列,且至少包括如下突变位点之一:第25位的谷氨酰胺突变为苯丙氨酸,第138位的酪氨酸突变为苯丙氨酸,第192位的脯氨酸突变为谷氨酰胺,第377位的苏氨酸突变为缬氨酸,第424位的精氨酸突变为亮氨酸,第448位的丙氨酸突变为精氨酸。
作为优选实施方式,所述转氨酶突变体的氨基酸序列为与SEQ ID NO:4、SEQ IDNO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14所示的氨基酸序列具有95%以上同源性,优选为96%的同源性,更优选为97%,98%,99%或100%的同源性的氨基酸系列。
本发明还提供一种转氨酶突变体基因,所述转氨酶突变体基因编码所述的转氨酶突变体。
作为优选实施方式,所述转氨酶突变体基因的序列为与SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13所示的序列具有95%以上同源性,优选为96%的同源性,更优选为97%,98%,99%或100%的同源性的氨基酸系列。
本发明还提供一种重组质粒,所述重组质粒含有所述的转氨酶突变体基因。
作为优选实施方式,所述重组质粒包括pET系列、pQE系列、pRSET系列、pGEX系列、pBV系列、pTrc系列、pTwin系列、pEZZ系列、pKK系列、pUC系列、pPIC系列、pHIL系列以及pYAM系列,优选pET-28a。
本发明还提供一种宿主细胞,所述宿主细胞含有所述的重组质粒。
作为优选实施方式,所述宿主细胞选自大肠杆菌基因工程菌或酵母菌基因工程菌。
本发明还提供一种生产(R)-1-萘乙胺的方法,包括转氨酶对1-萘乙酮进行转氨反应的步骤,其特征在于,所述转氨酶为所述的转氨酶突变体。
作为优选实施方式,所述1-萘乙酮的浓度为5~250g/L;所述转氨反应的产物为(R)-1-萘乙胺。
作为优选实施方式,所述1-萘乙酮的转化率大于88%;ee值为大于99%,优选ee值为99.5%。
作为优选实施方式,所述转氨酶为转氨酶突变体的酶粉、或含有所述转氨酶突变体的全细胞或细胞破碎液。
作为优选实施方式,所述转氨酶突变体的酶粉的浓度为1~10g/L;所述转氨酶细胞的浓度为5~50g/L。
作为优选实施方式,所述转氨反应还包括辅因子,所述辅因子选自磷酸吡哆醛,所述辅因子的浓度为0.05~1mM。
作为优选实施方式,所述转氨反应在缓冲液中进行,所述缓冲液选自磷酸盐缓冲液或乙醇胺缓冲液,所述缓冲液的浓度为50~500mmol/L。
作为优选实施方式,所述转氨反应在pH 5.5~8.5,温度30~50℃条件下进行。
与现有技术相比,本发明的有益效果如下:
本发明提供该突变体相比于野生型的酶在可以高活性、高选择性地方式对1-萘乙酮进行转氨作用,适应了工业化生产的需求,具有广泛的应用价值。
具体实施方式
为更确切说明本发明的技术方案和优化步骤更为明确,下面结合具体实施方式对本发明做进一步详细说明,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围内。
根据本发明一个实施例的转氨酶突变体基因来源于柄杆菌属(Caulobacter,CaATA),野生型模板的NCBI登录号为WP_110132869.1。
实施例1突变体的构建
为提高野生型CaATA转氨酶对该底物的活性和选择性,分别对其序列SEQ ID NO:2中的第25号残基、第138号残基、第192号残基、第377号残基、第424号残基以及第448号残基进行定点突变,具体突变位点为突变位点为第25位的谷氨酰胺突变为苯丙氨酸,第138位的酪氨酸突变为苯丙氨酸,第192位的脯氨酸突变为谷氨酰胺,第377位的苏氨酸突变为缬氨酸,第424位的精氨酸突变为亮氨酸,第448位的丙氨酸突变为精氨酸。所设计的引物如下表1所示:
表1
本实验使用PrimeSTAR max高保真DNA聚合酶(TAKARA,货号为R045A)对CaATA的表达质粒进行全质粒扩增,PCR体系为:1μLForward引物(20μM)、1μL Reverse引物(20μM)、1μL模板DNA(50μg/ml)、22μL ddH2O和25μL PrimeSTAR Max预混液(2×)。仪下表2所示的程序进行PCR实验程序如表2所示:
表2
将获得的全质粒突变片段经Dpn I酶切后转化进E.coli BL21(DE3)感受态细胞中,转化后挑取单菌落进行测序验证,选择正确的突变体培养并进行酶活力和选择性测试;并将性能提高的突变点进行叠加,突变方法同上。
实施例2转氨酶的表达及粗酶液制备
将野生型CaATA或其突变体对应的菌种在含有卡那抗生素的LB平板上划线培养,挑取单克隆接种至含有卡那抗生素的LB试管中,于37℃、200rpm条件下培养16h,获得一级种子液。然后将一级种子液以1%的接种量接种到含有卡那抗生素的TB摇瓶中,于37℃、200rpm条件下培养至OD600介于0.6~0.8之间,向各摇瓶中加入诱导剂IPTG(异丙基-β-D-硫代半乳糖苷)溶液(终浓度0.1mM)进行诱导,于25℃、200rpm条件下培养16h,离心收集菌体,保存至-20℃备用。
制备粗酶液时只需称取一定量冷冻保存的细胞,并将其悬浮于100mM,pH 7.0的磷酸盐缓冲液中,然后进行超声破碎,完成后于4℃、8000×g条件下离心25min,所得的上清液即为粗酶液。
实施例3酶活检测与分析级生物还原反应
基于实施例1,首先以目标产物(R)-1-萘乙胺对应的潜手性1-萘乙酮为底物,确定转氨酶突变体的催化活力,用转化率表示,再构建辅酶循环体系进行生物还原,检测产品的ee值。
分析级生物还原反应:具体操作步骤如下:
向2mL的离心管中加入500μL磷酸盐缓冲液(100mM、pH 8.0)、200μL异丙胺的水溶液(2.5M)、50μL辅酶磷酸吡哆醛的水溶液(10mM),100μL底物1-萘乙酮的DMSO溶液(500mM)以及野生型转氨酶或者其突变体的粗酶液150μL。然后将离心管放入恒温摇床中于35℃、200rpm条件下反应24h,反应结束后用稀盐酸淬灭,取样进行HPLC检测,确定各反应的转化率;再将剩余样品用氢氧化钠溶液调至pH=10,再用甲基叔丁基醚萃取后进行HPLC检测,以确定产品的ee值。各个突变体所对应的转化率和立体选择性ee值如下表3所示:
表3
突变体 | 转化率/% | ee值/% |
WT | 17.7 | >99.5 |
Q25F | 35.1 | >99.5 |
A448R | 43.4 | >99.5 |
Y138F/P192Q | 28.6 | >99.5 |
Q25F/T377V/R424L | 42.8 | >99.5 |
Q25F/Y138F/P192Q/T377V/R424L | 68.5 | >99.5 |
Q25F/Y138F/P192Q/T377V/R424L/A448R | 88.5 | >99.5 |
实施例4(R)-1-萘乙胺的酶法克级制备
向200mL反应体系中加入1M pH 8.0磷酸钾缓冲液20mL,底物1-萘乙酮2g,异丙胺6.95g,助溶剂DMSO 20mL,磷酸吡哆醛25mg,用稀盐酸将其pH调至8.0,再向其中加入转氨酶突变体Q25F/Y138F/P192Q/T377V/R424L/A448R的酶粉0.2g,于40℃水浴搅拌条件下反应48h。经提纯检测,反应转化率为89.6%,产品ee值>99.5%。
上述仅为本发明的部分优选实施例,本发明并不仅限于实施例的内容。对于本领域中的技术人员来说,在本发明技术方案的构思范围内可以有各种变化和更改,所作的任何变化和更改,均在本发明保护范围之内。
Claims (10)
1.一种新型的转氨酶突变体,其特征在于,所述转氨酶突变体的氨基酸序列是与SEQID NO:2所示的氨基酸序列具有80%以上同源性的氨基酸序列,且至少包括如下突变位点之一:第25位的谷氨酰胺突变为苯丙氨酸,第138位的酪氨酸突变为苯丙氨酸,第192位的脯氨酸突变为谷氨酰胺,第377位的苏氨酸突变为缬氨酸,第424位的精氨酸突变为亮氨酸,第448位的丙氨酸突变为精氨酸。
2.根据权利要求1所述的转氨酶突变体,其特征在于,所述转氨酶突变体的氨基酸序列为与SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14所示的氨基酸序列具有95%以上同源性,优选为96%的同源性,更优选为97%,98%,99%或100%的同源性的氨基酸系列。
3.一种转氨酶突变体基因,其特征在于,所述转氨酶突变体基因编码权利要求1或2所述的转氨酶突变体。
4.根据权利要求3所述的转氨酶突变体基因,其特征在于,所述转氨酶突变体基因的序列与SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13所示的序列具有95%以上同源性,优选为96%的同源性,更优选为97%,98%,99%或100%的同源性的氨基酸系列。
5.一种重组质粒,其特征在于,所述重组质粒含有权利要求3或4所述的转氨酶突变体基因;所述重组质粒包括pET系列、pQE系列、pRSET系列、pGEX系列、pBV系列、pTrc系列、pTwin系列、pEZZ系列、pKK系列、pUC系列、pPIC系列、pHIL系列以及pYAM系列。
6.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求5所述的重组质粒;所述宿主细胞选自大肠杆菌基因工程菌或酵母菌基因工程菌。
7.一种生产(R)-1-萘乙胺的方法,包括转氨酶对1-萘乙酮进行转氨反应的步骤,其特征在于,所述转氨酶为权利要求1或2所述的转氨酶突变体;所述1-萘乙酮的浓度为5~250g/L;所述转氨反应的产物为(R)-1-萘乙胺;所述1-萘乙酮的转化率大于88%;ee值为大于99%,优选ee值为99.5%。
8.根据权利要求7所述的方法,其特征在于,所述转氨酶为权利要求1或2所述的转氨酶突变体的酶粉、或含有所述转氨酶突变体的全细胞或细胞破碎液;所述转氨酶突变体的酶粉的浓度为1~10g/L;所述转氨酶细胞的浓度为5~50g/L。
9.根据权利要求7所述的方法,其特征在于,所述转氨反应还包括辅因子,所述辅因子选自磷酸吡哆醛,所述辅因子的浓度为0.05~1mM。
10.根据权利要求7所述的方法,其特征在于,所述转氨反应在pH 5.5~8.5,温度30~50℃条件下进行。
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