CN114277011A - 转氨酶突变体及其应用 - Google Patents
转氨酶突变体及其应用 Download PDFInfo
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- CN114277011A CN114277011A CN202111645514.9A CN202111645514A CN114277011A CN 114277011 A CN114277011 A CN 114277011A CN 202111645514 A CN202111645514 A CN 202111645514A CN 114277011 A CN114277011 A CN 114277011A
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- Prior art keywords
- pet
- transaminase
- cell
- ketone compound
- reaction
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Abstract
本发明公开了一种转氨酶突变体及其应用。其中,该转氨酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列,发生氨基酸突变的位点包括V242W位点。本发明的上述转氨酶突变体是在SEQ ID NO:1所示的转氨酶的基础上,通过定点突变的方法进行突变,从而改变其氨基酸序列,实现蛋白质结构和功能的改变,再通过定向筛选的方法,得到具有上述突变位点的转氨酶,本发明获得的转氨酶催化活性较高、选择性专一且底物谱广泛,具有广泛的工业化前景。
Description
技术领域
本发明涉及生物技术领域,具体而言,涉及一种转氨酶突变体及其应用。
背景技术
手性胺类化合物是一种重要的手性药物中间体,在医药等领域具有广泛的应用。工业化生产主要依靠过渡金属催化剂不对称合成,但这个过程需要昂贵的过渡金属配合物作为催化剂,而且由于这些过渡金属资源有限、成本高,这种途径难以实现可持续性。生物酶催化方法由于底物酮的广泛性、反应条件温和和产品选择性高等优点而备受关注。
转氨酶(transaminase,TA,EC 2.6.1.X),又称氨基转移酶(aminotransferase),能够可逆催化酮基与氨基之间的氨基转移反应。其中,转氨酶具有优良的立体选择性高、辅因子可再生、反应活性强及环境友好等特性,利用其进行生物催化生产手性胺,已被广泛地应用于合成医药和农药中间体。
转氨酶反应通常需要磷酸吡哆醛作为辅酶,磷酸吡哆醛共价结合到转氨酶活性中心赖氨酸残基的ε-氨基上,同时需要氨基供体参与反应,常用的氨基供体有异丙胺、苯乙胺等。
尽管使用转氨酶生产手性胺的进展已被高度关注,但酶促方法在放大生产应用中仍然存在很多问题,如现有的转氨酶选择性差,且存在可逆反应会导致转化率低,不利于工业化的生产。
发明内容
本发明旨在提供一种转氨酶突变体及其应用,以提高转氨酶的选择性。
为了实现上述目的,根据本发明的一个方面,提供了一种转氨酶突变体。该转氨酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列,发生氨基酸突变的位点包括V242W位点,或者转氨酶突变体包括上述突变位点,且与具有突变位点的氨基酸序列有80%以上,优选90%以上,更优选95%以上同一性的氨基酸序列。
进一步地,发生氨基酸突变的位点包括如下任一种组合突变位点:V242W+L59Q、V242W+F164C、V242W+F164Q、V242W+F164W、V242W+F164Y、V242W+L272G、V242W+L272I、V242W+L272K、V242W+L272M、V242W+L272P、V242W+L272V、V242W+L272Y、V242W+V328C、V242W+V328I、V242W+V328L、V242W+V328M、V242W+V328Q、V242W+V328S、V242W+V328T、V242W+V328W、V242W+T330F、V242W+T330I、V242W+T330S、V242W+A436H、V242W+A436K、V242W+A436L、V242W+A436N、V242W+A436P、V242W+A436Q、V242W+A436S、V242W+A436Y、V242W+R442A、V242W+R442C、V242W+R442F、V242W+R442G、V242W+R442H、V242W+R442N、V242W+R442Q、V242W+R442S、V242W+R442T、V242W+F164Q+V328A、V242W+F164Q+V328C、V242W+F164Q+V328D、V242W+F164Q+V328E、V242W+F164Q+V328F、V242W+F164Q+V328G、V242W+F164Q+V328H、V242W+F164Q+V328I、V242W+F164Q+V328L、V242W+F164Q+V328M、V242W+F164Q+V328P、V242W+F164Q+V328Q、V242W+F164Q+V328R、V242W+F164Q+V328S、V242W+F164Q+V328W、V242W+F164Q+V328T、V242W+F164Q+V328Y、V242W+F164Q+R442T、V242W+F164Q+V328I+G2S、V242W+F164Q+V328I+T46M、V242W+F164Q+V328I+G48D、V242W+F164Q+V328I+C185Y、V242W+F164Q+V328I+S186N、V242W+F164Q+V328I+S194P、V242W+F164Q+V328I+T197M、V242W+F164Q+V328I+N202D、V242W+F164Q+V328I+Y205L、V242W+F164Q+V328I+T245A、V242W+F164Q+V328I+V252I、V242W+F164Q+V328I+S268N、V242W+F164Q+V328I+L353F、V242W+F164Q+V328I+N359D、V242W+F164Q+V328I+R409T、V242W+F164Q+V328I+E424K、V242W+F164Q+V328I+A436V、V242W+F164Q+V328I+R442T、V242W+F164Q+V328I+R442T+G48D、V242W+F164Q+V328I+R442T+S194P、V242W+F164Q+V328I+R442T+V252I、V242W+F164Q+V328I+R442T+S194P+V252I、V242W+F164Q+V328I+R442T+V252I+G48D、V242W+F164Q+V328I+R442T+S194P+G48D或V242W+F164Q+V328I+R442T+S194P+V252I+G48D,或者转氨酶突变体包括上述突变位点,且与具有突变位点的氨基酸序列有80%以上,优选90%以上,更优选95%以上同一性的氨基酸序列。
根据本发明的另一个方面,提供一种DNA分子。该DNA分子编码上述任一种转氨酶突变体。
根据本发明的再一个方面,提供一种重组质粒。该重组质粒含有上述任一种DNA分子。
进一步地,重组质粒为pET-22a(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18或pUC-19。
根据本发明的另一方面,提供了一种宿主细胞。该宿主细胞含有上述任一种重组质粒。
进一步地,宿主细胞包括原核细胞或真核细胞;优选原核细胞为BL21-DE3细胞或大肠杆菌Rosetta-DE3细胞;真核细胞为酵母细胞。
根据本发明的再一方面,提供了一种生产手性胺的方法。该方法包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤,转氨酶为上述任一种转氨酶突变体。
进一步地,氨基供体为异丙胺或丙氨酸,优选为异丙胺。
进一步地,在转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中,所用酶量为1.5~6.6mg/mL;优选为1.7mg/mL;优选的,转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系的温度为20℃~45℃,更优选为30℃。
本发明的上述转氨酶突变体是在SEQ ID NO:1所示的转氨酶的基础上,通过定点突变的方法进行突变,从而改变其氨基酸序列,实现蛋白质结构和功能的改变,再通过定向筛选的方法,得到具有上述突变位点的转氨酶,本发明获得的转氨酶催化活性较高、选择性专一且底物谱广泛,具有广泛的工业化前景。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
转氨酶是一类以蛋白质为主体的生物催化剂,其催化的反应可以通过如下反应式表示:
n=0,1,2或3;X=C,N,O或S;R=H,F,Cl,Br,CH3或CH2CH3
现有的转氨酶选择性差,且存在可逆反应会导致转化率低,不利于工业化的生产。针对此技术问题,本发明在SEQ ID NO:1所示的转氨酶基础上通过定向进化的方法提高转氨酶的选择性及活性,获得催化活性较高、选择性专一的转氨酶。
首先通过定点突变的方式在转氨酶上引入突变位点,对突变体进行选择性检测,挑选选择性提高的突变体。其中,突变体V242W相较于起始模板,选择性提高2倍左右,但其活性较差。后续,以V242W为模板继续进行突变,以期得到选择性提高和活性提高更为显著的突变体。
定点突变:是指通过聚合酶链式反应(PCR)等方法在DNA片段(可以是基因组,也可以是质粒)的特定位点引入碱基改变,或片段的插入以及删除。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征,是基因研究工作中一种非常有用的手段。
利用全质粒PCR引入单个或多个定点突变具有简单高效等优点。其原理是,将一对包含突变位点的引物(正、反向)和模板质粒退火后用聚合酶“循环延伸”,(所谓的循环延伸是指聚合酶按照模版延伸引物,一圈后回到引物5’端终止,再经过反复加热退火延伸的循环,这个反应区别于滚环扩增,不会形成多个串联拷贝。正反向引物的延伸产物退火后配对成为带缺刻的开环质粒。Dpn I酶切延伸产物,由于原来的模版质粒来源于常规大肠杆菌,是经dam甲基化修饰的,对Dpn I敏感而被切碎,而体外合成的带突变序列的质粒由于没有甲基化而不被切开,因此在随后的转化中得以成功转化,即可得到突变质粒的克隆。
将突变质粒转化至大肠杆菌宿主细胞内,然后通过超声破碎细胞的方法获得粗酶用于反应验证。转氨酶诱导表达最佳条件:20℃,0.06mM IPTG诱导16h。
根据本发明一种典型的实施方式,提供一种转氨酶突变体。该转氨酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列,发生氨基酸突变的位点包括V242W位点。
优选的,发生氨基酸突变的位点包括如下任一种组合突变位点:V242W+L59Q、V242W+F164C、V242W+F164Q、V242W+F164W、V242W+F164Y、V242W+L272G、V242W+L272I、V242W+L272K、V242W+L272M、V242W+L272P、V242W+L272V、V242W+L272Y、V242W+V328C、V242W+V328I、V242W+V328L、V242W+V328M、V242W+V328Q、V242W+V328S、V242W+V328T、V242W+V328W、V242W+T330F、V242W+T330I、V242W+T330S、V242W+A436H、V242W+A436K、V242W+A436L、V242W+A436N、V242W+A436P、V242W+A436Q、V242W+A436S、V242W+A436Y、V242W+R442A、V242W+R442C、V242W+R442F、V242W+R442G、V242W+R442H、V242W+R442N、V242W+R442Q、V242W+R442S、V242W+R442T、V242W+F164Q+V328A、V242W+F164Q+V328C、V242W+F164Q+V328D、V242W+F164Q+V328E、V242W+F164Q+V328F、V242W+F164Q+V328G、V242W+F164Q+V328H、V242W+F164Q+V328I、V242W+F164Q+V328L、V242W+F164Q+V328M、V242W+F164Q+V328P、V242W+F164Q+V328Q、V242W+F164Q+V328R、V242W+F164Q+V328S、V242W+F164Q+V328W、V242W+F164Q+V328T、V242W+F164Q+V328Y、V242W+F164Q+R442T、V242W+F164Q+V328I+G2S、V242W+F164Q+V328I+T46M、V242W+F164Q+V328I+G48D、V242W+F164Q+V328I+C185Y、V242W+F164Q+V328I+S186N、V242W+F164Q+V328I+S194P、V242W+F164Q+V328I+T197M、V242W+F164Q+V328I+N202D、V242W+F164Q+V328I+Y205L、V242W+F164Q+V328I+T245A、V242W+F164Q+V328I+V252I、V242W+F164Q+V328I+S268N、V242W+F164Q+V328I+L353F、V242W+F164Q+V328I+N359D、V242W+F164Q+V328I+R409T、V242W+F164Q+V328I+E424K、V242W+F164Q+V328I+A436V、V242W+F164Q+V328I+R442T、V242W+F164Q+V328I+R442T+G48D、V242W+F164Q+V328I+R442T+S194P、V242W+F164Q+V328I+R442T+V252I、V242W+F164Q+V328I+R442T+S194P+V252I、V242W+F164Q+V328I+R442T+V252I+G48D、V242W+F164Q+V328I+R442T+S194P+G48D、或V242W+F164Q+V328I+R442T+S194P+V252I+G48D。
本发明的上述转氨酶突变体是在SEQ ID NO:1所示的转氨酶的基础上,通过定点突变的方法进行突变,从而改变其氨基酸序列,实现蛋白质结构和功能的改变,再通过定向筛选的方法,得到具有上述突变位点的转氨酶,本发明获得的转氨酶催化活性较高、选择性专一且底物谱广泛,具有广泛的工业化前景。
根据本发明一种典型的实施方式,提供一种DNA分子。上述DNA编码得到的转氨酶突变体,提高了选择性和活性,在氨基酸的工业生产中可以减少加入的酶量,降低后处理难度。
本发明的上述DNA分子还可以以“表达盒”的形式存在。“表达盒”是指线性或环状的核酸分子,涵盖了能够指导特定核苷酸序列在恰当宿主细胞中表达的DNA和RNA序列。一般而言,包括与目标核苷酸有效连接的启动子,其任选的是与终止信号和/或其他调控元件有效连接的。表达盒还可以包括核苷酸序列正确翻译所需的序列。编码区通常编码目标蛋白,但在正义或反义方向也编码目标功能RNA,例如反义RNA或非翻译的RNA。包含目标多核苷酸序列的表达盒可以是嵌合的,意指至少一个其组分与其至少一个其他组分是异源的。表达盒还可以是天然存在的,但以用于异源表达的有效重组形成获得的。
根据本发明一种典型的实施方式,提供一种重组质粒。该重组质粒含有上述任一种DNA分子。上述重组质粒中的DNA分子置于重组质粒的适当位置,使得上述DNA分子能够正确地、顺利地复制、转录或表达。
虽然本发明在限定上述DNA分子时所用限定语为“含有”,但其并不意味着可以在DNA序列的两端任意加入与其功能不相关的其他序列。本领域技术人员知晓,为了满足重组操作的要求,需要在DNA序列的两端添加合适的限制性内切酶的酶切位点,或者额外增加启动密码子、终止密码子等,因此,如果用封闭式的表述来限定将不能真实地覆盖这些情形。
本发明中所使用的术语“质粒”包括双链或单链线状或环状形式的任何质粒、粘粒、噬菌体或农杆菌二元核酸分子,优选为重组表达质粒,可以是原核表达质粒也可以是真核表达质粒,但优选原核表达质粒,在某些实施方案中,重组质粒所用的载体选自pET-22a(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18或pUC-19。
根据本发明一种典型的实施方式,提供一种宿主细胞,宿主细胞含有上述任一种重组质粒。适用于本发明的宿主细胞包括但不仅限于原核细胞或真核细胞。优选原核细胞为BL21-DE3细胞或大肠杆菌Rosetta-DE3细胞,真核细胞为酵母。
根据本发明一种典型的实施方式,提供一种生产手性胺的方法。该方法包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤,转氨酶为本发明的转氨酶突变体。优选的,酮类化合物为n=0,1,2或3;X=C,N,O或S;R=H,F,Cl,Br,CH3或CH2CH3。
应用本发明的转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中,pH为7~11,优选为7.5。转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系的温度为20~45℃,更优选为30℃,也就是说温度的取值可以任选为20~45℃中的值,例如20、21、22、25、27、28、29、20、31、32、35、37、38、39、40、42、45等。在转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中,所用酶量为1.5~6.6mg/mL;优选为1.7mg/mL;也就是说酶量的取值可以任选为1.5~6.6mg/mL中的值,例如1.5,1.6,1.7,1.8,1.9,2.0,2.5,3.0,3.6,3.8,4.0,4.4,4.5,4.9,5.1,5.5,6.6等。
在本发明一实施方式中,本发明的转氨酶突变体的底物如下:
底物1:
Tetrahydrofuran-3-one
四氢呋喃-3-酮
底物2:
2-甲基四氢噻吩-3-酮
2-Methylthiolan-3-one
底物3:
2-Chlorocyclopentanone
2-氯环戊酮
底物4:
Cyclopentanone
环戊酮
底物5:
3-Methylcyclobutan-1-one
3-甲基环丁酮
根据本发明一种典型的实施方式,底物1、底物2、底物3、底物4、底物5的反应验证方法如下:
向5mL离心管中分别加入10mg底物,1mg酶,磷酸吡哆醛0.1mg,6M异丙胺盐酸盐20mg,补加0.1M磷酸缓冲液pH 7.5至总体积0.5mL,45℃,200rpm,16h。
反应结束后,底物1、2和3的选择性方法检测:取出反应体系0.06mL加0.04mL 0.1M磷酸缓冲液pH 7.5,取稀释过体系0.1mL加0.3mL乙腈、水和碳酸氢钠等体积溶液,混匀取出0.1mL加0.9mL 5mg/mL的Nα-(2,4-二硝基-5-氟苯基)-L-丙氨酰胺试剂,50℃金属浴放置3h,取出衍生体系12000rpm离心5min,取0.5mL加0.5mL乙腈和水等体积溶液,混匀送e.e.检测。在本申请中,活性检测方法(通过底物转化率表示):酶催化底物反应后的反应体系取100μL,加900μL甲醇,震荡离心取上清送样;采用HPLC(高效液相色谱法)检测出的产品所占的相对峰面积即代表酶的活性。
在本发明一种典型的实施方式中,氨基供体为异丙胺或丙氨酸,优选为异丙胺。
下面将结合实施例进一步说明本发明的有益效果。
实施例1
对C60Y(Template,如SEQ ID NO:1,MGLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLITPGGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSFRSGLVGENSESFSAQIPGSSCSSAVLMAPSSNTFQDSNGNYLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGVGSTMPPYEYVPQIRKMTKELGVLWISDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVVVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENLVEQAKNSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFRHGMNPNQIPTQIIMEKALEKGVLIGGAMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQQSLEHHHHHH;对应的核苷酸序列为SEQ ID NO:2,atgggcctgaccgtacagaagatcaactgggaacaagtaaaggagtgggaccgcaagtacctgatgcgcactttttccactcagaacgaataccagccggtaccgattgaatctacggaaggtgattatctgatcaccccgggtggcacccgtctgctggacttcttcaaccaactgtattgcgttaacctgggccagaaaaaccagaaagtcaacgccgcgattaaagaggcactggaccgctacggcttcgtatgggacacttatgctaccgactacaaggcgaaagccgcgaaaatcattatcgaggatattctgggtgacgaggattggcctggtaaagttcgttttgttagcactggctccgaagccgtggaaaccgcgctgaatatcgcgcgtctgtataccaatcgtccgctggttgtgacccgtgaacacgactatcacggttggaccggcggcgctgcaaccgtcactcgtctgcgtagcttccgttctggtctggtgggtgaaaactccgaaagcttctctgctcagatcccgggttcttcttgtagctctgcagtcctgatggccccgtcttccaacaccttccaggactctaacggcaactacctgaaagacgaaaacggtgaactgctgtccgttaaatacacccgtcgcatgattgaaaactacggcccggagcaggtagccgcagtgattacggaagttagccagggtgttggttctacgatgccgccatacgaatatgttccacagatccgtaaaatgactaaagagctgggcgttctgtggatctccgacgaggtcctgaccggttttggtcgtaccggtaaatggttcggttaccagcattatggcgttcaaccagatatcattactatgggcaagggcctgtcttcttcctctctgccggcgggcgcagtagttgtgtccaaagaaatcgctgcgtttatggacaaacaccgctgggaatctgtttccacctacgcgggtcacccggtggcgatggcagctgtgtgcgctaacctggaagtgatgatggaagaaaacctggtggaacaagctaaaaatagcggcgaatacatccgtagcaaactggaactgctgcaggaaaagcataaaagcatcggcaacttcgacggttacggtctgctgtggattgttgatatcgtgaatgcgaaaactaaaaccccgtatgtcaaactggatcgcaacttccgccacggcatgaacccgaaccagatcccgacgcagattatcatggaaaaagctctggaaaaaggcgttctgatcggtggtgcaatgcctaacactatgcgtatcggcgcatctctgaacgtatctcgtggtgatatcgataaagctatggatgctctggattacgcgctggattacctggagtccggtgaatggcagcagagcctcgagcaccaccaccaccaccactga)进行定点突变,在L59、Y60、Y148、H149、F164、E237、V242、V271、L272和V328进行突变,共10个突变位点72个突变体。
对底物1进行反应验证,反应体系为:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,1mg酶,补0.1M磷酸缓冲液pH 7.5至总体积0.5mL,反应条件为:45℃,200rpm,16h。其中,突变体L59Q、L59W、H149D、H149I、H149R、F164W、V242W、V242H、V242Q、V328I提高明显,其中e.e.最高为V242W较Template提高了2倍左右,但活性(底物转化率)有所降低。结果如表1所示。
表1
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例2
以V242W为模板,继续对L59、F164、L272、V328、T330、A436和R442的6个位点进行突变,得到共40个突变体。对底物1进行反应验证,反应体系为:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,1mg酶,补0.1M磷酸缓冲液pH 7.5至总体积0.5mL,反应条件为:45℃,200rpm,16h。结果见表2,V242W+F164Q、V242W+L272K、V242W+V328M、V242W+V328I和V242W+R442T等的e.e.较V242W提高较多,由于其中V242W+F164Q的活性(转化率)和选择性较模板V242W均提高较多,因此选择V242W+F164Q作为后续模板。
表2
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例3
选择以V242W+F164Q为模板,构建V328的17种氨基酸位点及R442T共18个单点突变位点。对底物1进行反应验证,反应体系为:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,1mg酶,补0.1M磷酸缓冲液pH7.5至总体积0.5mL,反应条件为:45℃,200rpm,16h。结果如表3所示,筛选到2个活性(以转化率体现)提高明显突变体分别为V242W+F164Q+V328I和V242W+F164Q+R442T。选择V242W+F164Q+V328I作为下一步模板。
表3
突变位点 | e.e.% | 转化率% |
Template | ++ | *** |
V242W | +++++ | * |
V242W+F164Q | ++++++ | *** |
V242W+F164Q+V328A | ++++ | *** |
V242W+F164Q+V328C | ++++ | *** |
V242W+F164Q+V328D | +++++ | *** |
V242W+F164Q+V328E | ++++++ | *** |
V242W+F164Q+V338F | +++++ | *** |
V242W+F164Q+V328G | +++++ | *** |
V242W+F164Q+V328H | +++++ | *** |
V242W+F164Q+V328I | ++++++ | **** |
V242W+F164Q+V328L | ++++ | *** |
V242W+F164Q+V328M | +++++ | *** |
V242W+F164Q+V328P | +++++ | *** |
V242W+F164Q+V328Q | ++++++ | *** |
V242W+F164Q+V328R | +++++ | *** |
V242W+F164Q+V328S | +++++ | *** |
V242W+F164Q+V328W | - | *** |
V242W+F164Q+V328T | +++++ | *** |
V242W+F164Q+V328Y | ++++ | *** |
V242W+F164Q+R442T | ++++++ | **** |
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例4
选取V242W+F164Q+V328I为模板,构建G2S、T46M、G48D、C185Y、S186N、S194P、T197M、N202D、Y205L、T245A、V252I、S268N、L353F、N359D、R409T、E424K、A436V和R442的18个单点突变位点。对底物1进行反应验证,反应条件:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,0.25mg酶,补0.1M磷酸缓冲液pH7.5至总体积0.5mL,45℃,200rpm,16h。V242W+F164Q+V328I+R442T的活性提高较多,以此进行下一步模板,表4为对应e.e.和活性结果。
表4
突变位点 | e.e.% | 转化率% |
Template | ++ | ** |
V242W | +++++ | * |
V245W+F164Q | ++++++ | ** |
V242W+F164Q+V328I | ++++++ | *** |
V242W+F164Q+V328I+G2S | ++++++ | ** |
V242W+F164Q+V328I+T46M | ++++++ | *** |
V242W+F164Q+V328I+G48D | ++++++ | *** |
V242W+F163Q+V328I+C185Y | ++++++ | ** |
V242W+F164Q+V328I+S186N | ++++++ | *** |
V242W+F164Q+V328I+S194P | ++++++ | *** |
V242W+F164Q+V328I+T197M | ++++++ | ** |
V242W+F164Q+V328I+N202D | ++++++ | ** |
V242W+F164Q+V328I+Y205L | ++++++ | ** |
V242W+F163Q+V328I+T245A | ++++++ | ** |
V242W+F164Q+V328I+V252I | ++++++ | *** |
V242W+F163Q+V328I+S268N | ++++++ | *** |
V242W+F164Q+V328I+L353F | ++++++ | *** |
V242W+F164Q+V328I+N359D | ++++++ | *** |
V242W+F164Q+V328I+R409T | ++++++ | *** |
V242W+F164Q+V328I+E324K | ++++++ | *** |
V242W+F164Q+V32SI+A436V | +++++ | ** |
V242W+F163Q+V328I+R442T | ++++++ | *** |
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例5
选取V242W+F164Q+V328I+R442T为模板进行单点突变,引物G48D、S194P和V252T。对底物1进行反应验证,反应体系为:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,0.25mg酶,补0.1M磷酸缓冲液pH 7.5至总体积0.5mL,反应条件为:45℃,200rpm,16h。V242W+F164Q+V328I+R442T+S194P和V242W+F164Q+V328I+R442T+V252I的活性最高,蛋白电泳结果突变体的蛋白表达明显在上清。
表5
突变位点 | e.e.% | 转化率% | 蛋白表达上清 | 蛋白表达沉淀 |
Template | ++ | ** | # | #### |
V242W+F1G4Q+V328I | ++++++ | *** | #### | ### |
V242W+F164Q+V325I+R442T | ++++++ | *** | ##### | ### |
V242W+F164Q+V328I+R442T+G48D | ++++++ | *** | ### | ## |
V242W+F164Q+V328I+R442T+S194P | ++++++ | **** | ###### | # |
V242W+F164Q+V328I+R442T+V252I | ++++++ | **** | ###### | # |
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
#代表SDS-PAGE条带百分比为10~25%之间,##代表SDS-PAGE条带百分比为25%~40%之间,###代表SDS-PAGE条带百分比为40%~55%之间,####代表SDS-PAGE条带百分比为55%~70%之间,#####代表SDS-PAGE条带百分比为70%~85%之间,######代表SDS-PAGE条带百分比为85%~90%之间。
实施例6
选取V242W+F164Q+V328I+R442T+S194P为模板单点突变,突变位点V252I和G48D;选取V242W+F164Q+V328I+R442T+V252I为模板,突变位点G48D。对底物1进行反应验证,反应体系为:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,0.25mg酶,补0.1M磷酸缓冲液pH 7.5至总体积0.5mL,反应条件为:45℃,200rpm,16h。结果如表6所示,突变体V242W+F164Q+V328I+R442T+V252I+G48D的活性有所提高,蛋白表达明显在上清。
表6
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
#代表SDS-PAGE条带百分比为10~25%之间,##代表SDS-PAGE条带百分比为25%~40%之间,###代表SDS-PAGE条带百分比为40%~55%之间,####代表SDS-PAGE条带百分比为55%~70%之间,#####代表SDS-PAGE条带百分比为70%~85%之间,######代表SDS-PAGE条带百分比为85%~90%之间。
实施例7
选取V242W+F164Q+V328I+R442T+S194P+V252I为模板单点突变,突变位点G48D。投底物1的反应验证,反应体系为:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,0.25mg酶,补0.1M磷酸缓冲液pH 7.5至总体积0.5mL,反应条件为:45℃,200rpm,40h。结果见表7,突变体V242W+F164Q+V328I+R442T+S194P+V252I+G48D的活性最高。
表7
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例8
在酶量固定1mg条件下,对底物1不同的底物浓度进行验证。反应条件:0.1mg磷酸吡哆醛,不同浓度6M异丙胺盐酸盐,总体积为2mL,0.1M磷酸缓冲液pH 7.5,45℃,200rpm,40h。结果见表8,e.e.会随着底物浓度提高而提高,但底物转化率会随着底物浓度提高而降低,优选底物浓度为30mg/mL。
表8
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例9
固定底物1浓度为30mg/mL条件下,对不同酶的使用量进行验证。反应体系为:0.5mg磷酸吡哆醛,90mg 6M异丙胺盐酸盐,0.1M磷酸缓冲液pH 7.5,总体积1.5mL;反应条件为:45℃,200rpm,40h。结果见表9:随着酶量降低,e.e.稍有提高,突变体活性变化不大,优选1.7mg/mL酶量。
表9
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例10
对底物1优化反应温度。反应体系为:60mg底物,3mg酶,0.6mg磷酸吡哆醛,120mg6M异丙胺盐酸盐,补0.1M磷酸缓冲液pH 7.5至总体积1.8mL;反应条件为:45℃,200rpm,40h。结果见表10:e.e.随着温度降低无明显变化,但活性在20℃时降低明显,优选30℃反应。
表10
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
实施例11
对底物1反应进行放大,反应体系:1g底物,0.05g酶,10mg磷酸吡哆醛,2g 6M异丙胺盐酸盐,补加0.1M磷酸缓冲液pH 7.5至总体积30mL;反应条件:30℃,200rpm,60h,于250mL三角瓶中反应。结果见表11,突变体较母本的e.e.和活性均提高较多。
表11
突变体 | e.e.(%) | 活性(%) |
Template | ++ | **** |
V242W+F164Q+V328I+R442T+S194P+V252I+G48D | ++++++ | ******* |
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
将反应结束体系进行处理,具体步骤如下:去杂质,将体系转移至50mL离心管中。加体系一半体积的乙酸乙酯振荡,分装至两个50mL离心管中,4000rpm离心10min。弃上清,取下层体系加体系一半体积的乙酸乙酯振荡,4000rpm离心10min。重复一次。取下层体系加体系一半体积的乙酸乙酯振荡,4000rpm离心10min。重复一次。取下层体系转移至250mL圆底烧瓶,称重。为除去底物,加氢氧化钠干粉,加入量为剩余体系重量的一半,至圆底烧瓶中的体系中,全程保证体系低于40℃。加入0.5倍体系(体系体积的一半)的甲叔醚,转移至两个50mL离心管中,4000rpm离心10min。将上清加0.5倍体系(上清体积的一半)的甲叔醚振荡,4000rpm离心10min。重复一次。上清转移至圆底烧瓶,旋蒸后产品收率为53%。
实施例12
使用优化后的反应条件,对底物2、底物3、底物4、底物5进行验证,反应体系:10mg底物,0.1mg磷酸吡哆醛,20mg 6M异丙胺盐酸盐,0.5mg酶,补0.1M磷酸缓冲液pH7.5至总体积0.3mL,反应条件为:30℃,200rpm,60h。结果见表12,对于不同的底物,突变体活性均有提高。
表12
注:-代表-50%<e.e.<1%,+代表1-30%,++代表30-60%,+++代表60-80%,++++代表80-90%,+++++代表90-95%,++++++代表大于95%。
注:*代表活性1-30%,**代表30-60%,***代表60-70%,****代表70-80%,*****代表80-90%,******代表90-95%,*******代表大于95%。
另外,通过采用软件对转氨酶的三维结构进行计算机模拟分析,发现突变的位点大部分位于活性中心附近,突变后有可能增强了底物和酶的结合,从而提高了选择性和催化效率。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,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之内。
序列表
<110> 凯莱英医药集团(天津)股份有限公司
<120> 转氨酶突变体及其应用
<130> PN157561KLY
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 484
<212> PRT
<213> Arthrobacter citreus
<400> 1
Met Gly Leu Thr Val Gln Lys Ile Asn Trp Glu Gln Val Lys Glu Trp
1 5 10 15
Asp Arg Lys Tyr Leu Met Arg Thr Phe Ser Thr Gln Asn Glu Tyr Gln
20 25 30
Pro Val Pro Ile Glu Ser Thr Glu Gly Asp Tyr Leu Ile Thr Pro Gly
35 40 45
Gly Thr Arg Leu Leu Asp Phe Phe Asn Gln Leu Tyr Cys Val Asn Leu
50 55 60
Gly Gln Lys Asn Gln Lys Val Asn Ala Ala Ile Lys Glu Ala Leu Asp
65 70 75 80
Arg Tyr Gly Phe Val Trp Asp Thr Tyr Ala Thr Asp Tyr Lys Ala Lys
85 90 95
Ala Ala Lys Ile Ile Ile Glu Asp Ile Leu Gly Asp Glu Asp Trp Pro
100 105 110
Gly Lys Val Arg Phe Val Ser Thr Gly Ser Glu Ala Val Glu Thr Ala
115 120 125
Leu Asn Ile Ala Arg Leu Tyr Thr Asn Arg Pro Leu Val Val Thr Arg
130 135 140
Glu His Asp Tyr His Gly Trp Thr Gly Gly Ala Ala Thr Val Thr Arg
145 150 155 160
Leu Arg Ser Phe Arg Ser Gly Leu Val Gly Glu Asn Ser Glu Ser Phe
165 170 175
Ser Ala Gln Ile Pro Gly Ser Ser Cys Ser Ser Ala Val Leu Met Ala
180 185 190
Pro Ser Ser Asn Thr Phe Gln Asp Ser Asn Gly Asn Tyr Leu Lys Asp
195 200 205
Glu Asn Gly Glu Leu Leu Ser Val Lys Tyr Thr Arg Arg Met Ile Glu
210 215 220
Asn Tyr Gly Pro Glu Gln Val Ala Ala Val Ile Thr Glu Val Ser Gln
225 230 235 240
Gly Val Gly Ser Thr Met Pro Pro Tyr Glu Tyr Val Pro Gln Ile Arg
245 250 255
Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Ser Asp Glu Val Leu
260 265 270
Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly
275 280 285
Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser
290 295 300
Leu Pro Ala Gly Ala Val Val Val Ser Lys Glu Ile Ala Ala Phe Met
305 310 315 320
Asp Lys His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val
325 330 335
Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn
340 345 350
Leu Val Glu Gln Ala Lys Asn Ser Gly Glu Tyr Ile Arg Ser Lys Leu
355 360 365
Glu Leu Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Tyr
370 375 380
Gly Leu Leu Trp Ile Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro
385 390 395 400
Tyr Val Lys Leu Asp Arg Asn Phe Arg His Gly Met Asn Pro Asn Gln
405 410 415
Ile Pro Thr Gln Ile Ile Met Glu Lys Ala Leu Glu Lys Gly Val Leu
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Ile Gly Gly Ala Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn
435 440 445
Val Ser Arg Gly Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala
450 455 460
Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Gln Ser Leu Glu His His
465 470 475 480
His His His His
<210> 2
<211> 1455
<212> DNA
<213> Arthrobacter citreus
<400> 2
atgggcctga ccgtacagaa gatcaactgg gaacaagtaa aggagtggga ccgcaagtac 60
ctgatgcgca ctttttccac tcagaacgaa taccagccgg taccgattga atctacggaa 120
ggtgattatc tgatcacccc gggtggcacc cgtctgctgg acttcttcaa ccaactgtat 180
tgcgttaacc tgggccagaa aaaccagaaa gtcaacgccg cgattaaaga ggcactggac 240
cgctacggct tcgtatggga cacttatgct accgactaca aggcgaaagc cgcgaaaatc 300
attatcgagg atattctggg tgacgaggat tggcctggta aagttcgttt tgttagcact 360
ggctccgaag ccgtggaaac cgcgctgaat atcgcgcgtc tgtataccaa tcgtccgctg 420
gttgtgaccc gtgaacacga ctatcacggt tggaccggcg gcgctgcaac cgtcactcgt 480
ctgcgtagct tccgttctgg tctggtgggt gaaaactccg aaagcttctc tgctcagatc 540
ccgggttctt cttgtagctc tgcagtcctg atggccccgt cttccaacac cttccaggac 600
tctaacggca actacctgaa agacgaaaac ggtgaactgc tgtccgttaa atacacccgt 660
cgcatgattg aaaactacgg cccggagcag gtagccgcag tgattacgga agttagccag 720
ggtgttggtt ctacgatgcc gccatacgaa tatgttccac agatccgtaa aatgactaaa 780
gagctgggcg ttctgtggat ctccgacgag gtcctgaccg gttttggtcg taccggtaaa 840
tggttcggtt accagcatta tggcgttcaa ccagatatca ttactatggg caagggcctg 900
tcttcttcct ctctgccggc gggcgcagta gttgtgtcca aagaaatcgc tgcgtttatg 960
gacaaacacc gctgggaatc tgtttccacc tacgcgggtc acccggtggc gatggcagct 1020
gtgtgcgcta acctggaagt gatgatggaa gaaaacctgg tggaacaagc taaaaatagc 1080
ggcgaataca tccgtagcaa actggaactg ctgcaggaaa agcataaaag catcggcaac 1140
ttcgacggtt acggtctgct gtggattgtt gatatcgtga atgcgaaaac taaaaccccg 1200
tatgtcaaac tggatcgcaa cttccgccac ggcatgaacc cgaaccagat cccgacgcag 1260
attatcatgg aaaaagctct ggaaaaaggc gttctgatcg gtggtgcaat gcctaacact 1320
atgcgtatcg gcgcatctct gaacgtatct cgtggtgata tcgataaagc tatggatgct 1380
ctggattacg cgctggatta cctggagtcc ggtgaatggc agcagagcct cgagcaccac 1440
caccaccacc actga 1455
Claims (17)
1.一种转氨酶突变体,其特征在于,所述转氨酶突变体具有SEQ ID NO:1所示序列发生氨基酸突变的序列,所述发生氨基酸突变的位点包括V242W位点。
2.根据权利要求1所述的转氨酶突变体,其特征在于,所述发生氨基酸突变的位点包括如下任一种组合突变位点:V242W+L59Q、V242W+F164C、V242W+F164Q、V242W+F164W、V242W+F164Y、V242W+L272G、V242W+L272I、V242W+L272K、V242W+L272M、V242W+L272P、V242W+L272V、V242W+L272Y、V242W+V328C、V242W+V328I、V242W+V328L、V242W+V328M、V242W+V328Q、V242W+V328S、V242W+V328T、V242W+V328W、V242W+T330F、V242W+T330I、V242W+T330S、V242W+A436H、V242W+A436K、V242W+A436L、V242W+A436N、V242W+A436P、V242W+A436Q、V242W+A436S、V242W+A436Y、V242W+R442A、V242W+R442C、V242W+R442F、V242W+R442G、V242W+R442H、V242W+R442N、V242W+R442Q、V242W+R442S、V242W+R442T、V242W+F164Q+V328A、V242W+F164Q+V328C、V242W+F164Q+V328D、V242W+F164Q+V328E、V242W+F164Q+V328F、V242W+F164Q+V328G、V242W+F164Q+V328H、V242W+F164Q+V328I、V242W+F164Q+V328L、V242W+F164Q+V328M、V242W+F164Q+V328P、V242W+F164Q+V328Q、V242W+F164Q+V328R、V242W+F164Q+V328S、V242W+F164Q+V328W、V242W+F164Q+V328T、V242W+F164Q+V328Y、V242W+F164Q+R442T、V242W+F164Q+V328I+G2S、V242W+F164Q+V328I+T46M、V242W+F164Q+V328I+G48D、V242W+F164Q+V328I+C185Y、V242W+F164Q+V328I+S186N、V242W+F164Q+V328I+S194P、V242W+F164Q+V328I+T197M、V242W+F164Q+V328I+N202D、V242W+F164Q+V328I+Y205L、V242W+F164Q+V328I+T245A、V242W+F164Q+V328I+V252I、V242W+F164Q+V328I+S268N、V242W+F164Q+V328I+L353F、V242W+F164Q+V328I+N359D、V242W+F164Q+V328I+R409T、V242W+F164Q+V328I+E424K、V242W+F164Q+V328I+A436V、V242W+F164Q+V328I+R442T、V242W+F164Q+V328I+R442T+G48D、V242W+F164Q+V328I+R442T+S194P、V242W+F164Q+V328I+R442T+V252I、V242W+F164Q+V328I+R442T+S194P+V252I、V242W+F164Q+V328I+R442T+V252I+G48D、V242W+F164Q+V328I+R442T+S194P+G48D或V242W+F164Q+V328I+R442T+S194P+V252I+G48D。
3.一种DNA分子,其特征在于,所述DNA分子编码权利要求1或2所述的转氨酶突变体。
4.一种重组质粒,其特征在于,所述重组质粒含有权利要求3所述的DNA分子。
5.根据权利要求4所述的重组质粒,其特征在于,所述重组质粒为pET-22a(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18或pUC-19。
6.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求4或5所述的重组质粒。
7.根据权利要求6所述的宿主细胞,其特征在于,所述宿主细胞包括原核细胞或真核细胞。
8.根据权利要求7所述的宿主细胞,其特征在于,所述原核细胞为BL21-DE3细胞或大肠杆菌Rosetta-DE3细胞;所述真核细胞为酵母细胞。
9.一种生产手性胺的方法,包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤,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。
12.根据权利要求9所述的方法,其特征在于,所述氨基供体为异丙胺或丙氨酸。
13.根据权利要求12所述的方法,其特征在于,所述氨基供体为异丙胺。
14.根据权利要求9所述的方法,其特征在于,在转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中,所用酶量为1.5~6.6mg/mL。
15.根据权利要求14所述的方法,其特征在于,在转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中,所用酶量为1.7mg/mL。
16.根据权利要求14所述的方法,其特征在于,转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系的温度为20℃~45℃。
17.根据权利要求16所述的方法,其特征在于,转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系的温度为30℃。
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