CN112391363A - 氨基酸脱氢酶突变体及其应用 - Google Patents
氨基酸脱氢酶突变体及其应用 Download PDFInfo
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- CN112391363A CN112391363A CN202110078514.9A CN202110078514A CN112391363A CN 112391363 A CN112391363 A CN 112391363A CN 202110078514 A CN202110078514 A CN 202110078514A CN 112391363 A CN112391363 A CN 112391363A
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- pet
- amino acid
- acid dehydrogenase
- ammonium
- mutation
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Abstract
本发明公开了一种氨基酸脱氢酶突变体及其应用。其中,该氨基酸脱氢酶突变体具有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K144G位点。本发明的上述氨基酸脱氢酶突变体是在SEQ ID NO:1所示的氨基酸脱氢酶基础上,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氨基酸脱氢酶,本发明的氨基酸脱氢酶突变体具有酶活性大幅度提高的优势,进而降低氨基酸脱氢酶的使用量,并且酶特异性也有相应提高,从而大幅度降低了氨基酸工业生产中的成本。
Description
技术领域
本发明涉及生物技术领域,具体而言,涉及一种氨基酸脱氢酶突变体及其应用。
背景技术
氨基酸脱氢酶(Amino acid dehydrogenase,简称AADH),酶分类号为EC(1.4.1.X),可以催化可逆的氨基酸氧化脱氨和酮酸还原胺化反应,是氨基酸合成和代谢途径中重要的酶类。其中,氨基酸脱氢酶在合成手性化合物方面,对称还原潜手性酮酸或酮制备手性胺具有高立体选择性、高效率、反应温和以及环境友好等优势。氨基酸脱氢酶催化的反应中,需要辅因子的参与,包括还原型的烟酰胺腺嘌呤二核苷酸(NADH)和氧化型的烟酰胺腺嘌呤二核苷酸(NAD+)。
在进行酮的还原反应过程中,一般需要还原型的辅因子 NADH 的参与,而在实际反应中,会添加氧化型的辅因子 NAD+,然后通过合适的辅因子再生体系再生为还原型的NADH。常用的辅因子再生体系包括葡萄糖和葡萄糖脱氢酶、甲酸盐和甲酸脱氢酶、仲醇和仲醇脱氢酶、亚磷酸盐和亚磷酸脱氢酶以及其它类似的系统。一般来说,辅酶再生体系的更换不会实质的影响氨基酸脱氢酶的功能。
虽然氨基酸脱氢酶具有广泛的商业价值,但其来源却较为有限,并且产酶菌株存在着产量低的问题,因此定向进化手段来获得具有优良特性的氨基酸脱氢酶菌株,对生产、应用氨基酸氢酶具有重要意义。
发明内容
本发明旨在提供一种氨基酸脱氢酶突变体及其应用,以解决现有技术中现有技术中野生型的氨基酸脱氢酶不适合应用于工业化生产的技术问题。
为了实现上述目的,根据本发明的一个方面,提供了一种氨基酸脱氢酶突变体。该氨基酸脱氢酶突变体具有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K144G位点。
进一步地,发生氨基酸突变的位点还包括如下任意一个或多个:L41I/L/T、G42V、G43N、T115I/L/V、M117L/Y、G118N、T119C/E/L/S、P121M/S/T/W、L135I/V、K184M/N/S、G293A以及G294I/V,其中“/”表示“或”。
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:K144G+L294I、K144G+L294I+L135V、K144G+L294I+T115I。
进一步地,氨基酸脱氢酶突变体具有K144G+L294I+L135V氨基酸突变,且与甲酸脱氢酶FDH共表达。
根据本发明的另一个方面,提供一种DNA分子。该DNA分子编码上述氨基酸脱氢酶突变体。
根据本发明的再一个方面,提供一种重组质粒。该重组质粒连接有上述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、pRSFDuet1或pUC-19。
根据本发明的又一个方面,提供一种宿主细胞。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;优选原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞,真核细胞为酵母。
根据本发明的再一个方面,提供一种生产氨基酸的方法。该方法包括采用氨基酸脱氢酶对酮类化合物进行催化还原氨化反应的步骤,氨基酸脱氢酶为上述任一种氨基酸脱氢酶突变体。
进一步地,酮类化合物为,还原氨化反应产物为,其中,R1代表-OH、-F、-Cl、-Br 或 -CH3,R2、R3、R4、R5 各自独立地代表-H、-OH、-F、-Cl、-Br 或 -CH3;优选的,酮类化合物为、、或。
进一步地,催化还原氨化反应中的氨基供体为甲酸铵、氯化铵、氨基甲酸铵、碳酸铵、碳酸氢铵、氨水、草酸铵、草酸氢铵、乳酸铵或磷酸铵;优选的,氨基酸脱氢酶催化还原氨化反应的条件为30~50℃,搅拌转速为50~250 rpm,催化还原氨化反应中还包括使用甲酸脱氢酶0.1 mg/mL ~1 mg/mL。
本发明的上述氨基酸脱氢酶突变体是在SEQ ID NO:1所示的氨基酸脱氢酶基础上,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氨基酸脱氢酶,本发明的氨基酸脱氢酶突变体具有酶活性大幅度提高的优势,进而降低氨基酸脱氢酶的使用量,并且酶特异性也有相应提高,从而大幅度降低了氨基酸工业生产中的成本。
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。
专利(CN108795893A)中来源于Thermoactinomyces intermedius ATCC33205的氨基酸脱氢酶突变体A135L(Template)可以催化目标底物得到产物,但是催化活性较差。本发明通过定向进化的方法提高其催化活性,降低氨基酸脱氢酶的使用量。本发明的模板氨基酸的序列为SEQ ID NO:1(MRDVFEMMDRYGHEQVIFCRHPQTGLKAIIALHNTTAGPALG GCRMIPYASTDEALEDVLRLSKGMTYKCSLADVDFGGGKMVIIGDPKKDKSPELFRVIGRFVGGLNGRFYTGTDMGTNPEDFVHAARESKSFLGLPKSYGGKGDTSIPTALGVFHGMRATARFLWGTDQLKGRVVAIQGVGKVGERLLQLLVEVGAYCKIADIDSVRCEQLKEKYGDKVQLVDVNRIHKESCDIFSPCAKGGVVNDDTIDEFRCLAIVGSANNQLVEDRHGALLQKRSICYAPDYLVNAGGLIQVADELEGFHEERVLAKTEAIYDMVLDIFHRAKNENITTCEAADRIVMERLKKLTDIRRILLEDPRNSARRLE*,*代表氨基酸终止密码子),对应的核苷酸序列为SEQ ID NO:2(ATGCGTGACGTATTCGAAATGATGGATCGCTACGGCCACGAGCAGGTGATTTTC TGT CGTCATCCGCAGACTGGCCTGAAAGCGATCATCGCTCTGCATAACACCACTGCCGGTCCGGCACTGGGCGGTTGTCGCATGATTCCATACGCAAGCACCGATGAAGCTCTGGAAGACGTTCTGCGTCTGAGCAAAGGTATGACCTATAAATGCTCTCTGGCGGATGTTGATTTCGGTGGCGGTAAAATGGTGATTATCGGCGATCCGAAAAAGGATAAAAGCCCAGAACTGTTCCGTGTTATCGGTCGCTTCGTTGGCGGCCTGAACGGTCGTTTCTATACCGGTACTGATATGGGCACCAATCCGGAAGATTTCGTGCACGCCGCTCGCGAAAGCAAATCTTTTCTAGGTCTGCCTAAATCTTACGGTGGTAAAGGTGACACTTCTATCCCGACCGCACTGGGTGTATTTCACGGCATGCGCGCGACCGCCCGCTTTCTGTGGGGCACCGATCAACTGAAAGGTCGTGTTGTTGCTATCCAGGGTGTTGGCAAAGTGGGTGAACGTCTGCTGCAGCTGCTGGTGGAAGTGGGTGCATACTGCAAAATTGCTGATATTGACTCTGTACGTTGTGAGCAGCTGAAAGAAAAGTACGGCGACAAAGTCCAGCTGGTAGACGTGAACCGTATCCACAAAGAGTCTTGTGACATCTTCTCCCCGTGCGCAAAAGGCGGCGTAGTCAACGACGACACTATTGACGAATTCCGCTGCCTGGCGATTGTTGGTTCCGCGAACAATCAGCTGGTTGAAGATCGTCATGGCGCGCTGCTGCAAAAACGCTCCATTTGCTATGCCCCGGATTATCTGGTTAACGCTGGCGGTCTGATCCAGGTCGCAGACGAACTGGAGGGTTTTCACGAGGAGCGTGTGCTGGCGAAAACGGAAGCCATCTACGACATGGTTCTGGACATCTTCCACCGCGCTAAGAACGAAAACATCACTACCTGCGAAGCAGCGGACCGTATCGTAATGGAACGTCTGAAGAAGCTGACGGACATCCGTCGTATCCTGCTGGAAGATCCGCGTAACTCCGCGCGTCGTCTCGAGTGA)。
首先通过定点突变的方式在氨基酸脱氢酶上引入突变位点,对突变体进行活性检测,挑选活性提高的突变体。其中突变体K144G相较于起始模板,活性提高5倍左右。后续,以K144G为模板继续进行突变,以期得到催化活性提高更为显著的突变体。
其中,定点突变:是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组,也可以是质粒)中引入所需变化(通常是表征有利方向的变化),包括碱基的添加、删除、点突变等。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 引入定点突变的方法简单有效,是目前使用比较多的手段。其原理是,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细胞内,然后通过超声破碎细胞的方法获得粗酶。
上述得将突变质粒转化至大肠杆菌细胞内,在大肠杆菌中过量表达。然后通过超声破碎细胞的方法获得粗酶。氨基酸脱氢酶诱导表达最佳条件:25 oC,0.1 mM IPTG 诱导16h。
通过采用软件对氨基酸脱氢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,从而提高了催化效率。根据本发明一种典型的实施方式,提供一种氨基酸脱氢酶突变体。该氨基酸脱氢酶突变体具有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K144G位点。
优选的,发生氨基酸突变的位点还包括如下任意一个或多个:L41I/L/T、G42V、G43N、T115I/L/V、M117L/Y、G118N、T119C/E/L/S、P121M/S/T/W、L135I/V、K184M/N/S、G293A以及G294I/V,其中“/”表示“或”;更优选的,发生氨基酸突变的位点包括如下任一种组合突变位点:K144G+L294I、K144G+L294I+L135V、K144G+L294I+T115I。
根据本发明一种典型的实施方式,氨基酸脱氢酶突变体具有K144G+L294I+L135V氨基酸突变,且与甲酸脱氢酶FDH共表达。
本发明的上述氨基酸脱氢酶突变体是在SEQ ID NO:1所示的氨基酸脱氢酶的基础上,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氨基酸脱氢酶,本发明的氨基酸脱氢酶突变体具有酶活性大幅度提高的优势,并且酶特异性也有相应提高,从而大幅度降低了工业生产的成本。
根据本发明一种典型的实施方式,提供一种DNA分子。上述DNA编码得到的氨基酸脱氢酶,提高了酶活性和酶的稳定性,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、pRSFDuet1或pUC-19。更优选,上述重组质粒是pET-22b(+)。
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。优选原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞,真核细胞为酵母。
根据本发明一种典型的实施方式,提供一种生产氨基酸的方法。该方法包括氨基酸脱氢酶对酮类化合物及氨基供体进行催化转氨基反应的步骤,氨基酸脱氢酶为上述任一种氨基酸脱氢酶突变体。由于本发明的上述氨基酸脱氢酶突变体具有更高的酶催化活性,因而利用本发明的氨基酸脱氢酶突变体制备的氨基酸不仅能够降低生产成本,而且所获得的氨基酸e.e.值(对映体过量)更高。
根据本发明一种典型的实施方式,酮类化合物为,还原氨化反应产物为,其中, R1代表-OH、-F、-Cl、-Br 或 -CH3,R2、R3、R4、R5 各自独立地代表-H、-OH、-F、-Cl、-Br 或 -CH3;
优选的,催化还原氨化反应中的氨基供体为甲酸铵、氯化铵、氨基甲酸铵、碳酸铵、碳酸氢铵、氨水、草酸铵、草酸氢铵、乳酸铵或磷酸铵;氨基酸脱氢酶催化还原氨化反应的条件为30~50℃,搅拌转速为50~250 rpm,催化还原氨化反应中还包括使用甲酸脱氢酶 0.1mg/mL~1 mg/mL。
下面将结合具体的实施例进一步说明本发明的有益效果。
底物1、2、3、4的反应验证:
底物1:
2-(1-金刚烷)-2-氧代乙酸
底物2:
2-(2-羟基-1-金刚烷)-2-氧代乙酸
底物3:
2-(3-氟基-1-金刚烷)-2-氧代乙酸
底物4:
2-(3-甲基-1-金刚烷)-2-氧代乙酸
实施例1
对A135L(模板Template,其中,A135L代表第135位的氨基酸A被替换为L)进行定点突变,在T115、L135、K144、T147、L294和V297共6个突变位点40个突变体,对底物1和底物2投反应验证,反应体系为:0.1 g 底物,0.001 g 氨基酸脱氢酶,0.001 g 甲酸脱氢酶,0.0256mg NAD+,0.056 g甲酸铵,0.1 M 磷酸钾溶液 pH 8.0,1.1 mL,40℃,16 h,其中K144G突变体转化率最高。具体结果见表1。
表1
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
实施例2
以 K144G 为模板,继续对 L41、G42、G43、G114、T115、M117、G118、T119、P121、L135、K184、G293 和 L294 的13个位点进行突变,得到共43个突变体,对底物1和底物2投反应验证,反应条件为:0.1 g底物,0.0005 g氨基酸脱氢酶,0.001 g 甲酸脱氢酶,0.0256 mg NAD+,0.056 g甲酸铵,0.1 M 磷酸钾溶液 pH 8.0,1 mL,40℃,16 h,其中,加 L135V、T115I 和L294I 位点后转化率提高。具体结果见表2。
表2
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
实施例3
组合饱和突变可以获得几个突变位点之间具有协同作用的突变体,而且可以对其氨基酸的组成进行优化组合。以K144G+L294I为模板,分别叠加反应较好的突变点L135V和T115I,反应条件为:0.1 g 底物,0.0005 g氨基酸脱氢酶,0.001 g 甲酸脱氢酶,0.0256 mgNAD+,0.056 g甲酸铵,0.1 M 磷酸钾溶液 pH 8.0,1 mL,40℃,100 rpm,16 h,其中活性最高为K144G+L294I+ L135V。结果见表3。
表3
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
实施例4
底物1、2、3和4反应放大:K144G+L294I+L135V投反应并检测e.e.,反应条件为:1 g 底物,0.005 g 氨基酸脱氢酶,0.01 g 甲酸脱氢酶,NAD+ 0.256 mg,0.056 g甲酸铵,0.1 M 磷酸钾溶液 pH 8.0,1 mL,40℃,100 rpm。
检测活性取样方法:取出反应体系0.5 mL,12000 rpm离心5 min,取上清0.050 mL加1 mL纯化水,混匀后送HPLC检测转化率。
检测e.e.取样方法:取反应转化完体系1 mL,用1 M NaOH调pH至10-11;加0.1 mL(BOC)2O,30℃,200 rpm,0.5 h,用1 M NaOH调pH至10-11,30℃,200 rpm,0.5 h;加等体积的乙酸异丙酯,用6 M HCl调pH至2.0,取0.5 mL体系加1 mL 乙酸乙酯振荡混匀;12000 rpm离心5 min,取上清吹干,加无水乙醇溶解送e.e.检测。结果见表4。
表4
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
实施例5
选择K144G+L294I+L135V突变体,对甲酸脱氢酶进行优化,反应条件为:0.1 g底物,氨基酸脱氢酶0.0005 g,NAD+ 0.0256 mg,0.056 g甲酸铵,0.1 M 磷酸钾溶液 pH 8.0,1 mL,40℃,100 rpm,其中甲酸脱氢酶在0.3 mg以下活性才有明显降低,说明反应过程中甲酸脱氢酶为非限制性因素。结果见表5。
表5
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
实施例6
甲酸脱氢酶0.3 mg条件下,对K144G+L294I+L135V酶量进行优化,反应条件为:0.1 g底物,NAD+ 0.0256 mg,0.056 g甲酸铵,0.1 M 磷酸钾溶液 pH 8.0,1.1 mL,40℃,100 rpm,随着酶量降低,转化率有明显下降,说明反应中氨基酸脱氢酶为限制性因素。结果见表6。
表6
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
实施例7
共表达菌株反应条件优化,构建K144G+L294I+L135V和甲酸脱氢酶FDH至共表达质粒pRSFDuet1,转化到E. coli BL21(DE3),投底物1反应验证,并对反应条件温度、转速优化,反应条件为:0.1 g底物,氨基酸脱氢酶为0.005g,NAD+ 0.0256 mg,0.1 M 磷酸钾溶液 pH8.0,1.1 mL。结果显示,最佳反应条件:40 ℃,100 rpm。结果见表7。
表7
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
实施例8
底物1、2、3和4反应放大:对共表达菌株K144G+L294I+L135V+FDH投反应并检测e.e.,反应条件为:1 g 底物,0.05 g 氨基酸脱氢酶,NAD+ 0.256 mg,0.1 M 磷酸钾溶液 pH 8.0,1mL ,40℃,100 rpm。
检测活性取样方法:取出反应体系0.5 mL,12000 rpm离心5 min,取上清0.050 mL加1 mL纯化水,混匀后送HPLC检测转化率。
检测e.e.取样方法:取反应转化完体系1 mL,用1 M NaOH调pH至10-11;加0.1 mL(BOC)2O,30℃,200 rpm,0.5 h,用1 M NaOH调pH至10-11,30℃,200 rpm,0.5 h;加等体积的乙酸异丙酯,用6 M HCl调pH至2.0,取0.5 mL体系加1 mL 乙酸乙酯振荡混匀;12000 rpm离心5 min,取上清吹干,加无水乙醇溶解送e.e.检测。结果见表8。
表8
注:-代表0.5-20%,+代表20-40%,++代表40-60%,+++代表60-80%,++++代表80-90%,+++++代表大于90%。
反应完全后,将体系降温至10-30℃,用10N氢氧化钠调节pH=9.8-10.2,加入boc酸酐,保温10-30℃反应(pH=9.8-10.2),反应毕,用35%硫酸调节pH=7.5-8.5,加入10体积的乙酸异丙酯,再用35%硫酸调节pH=1.8-2.2,搅拌5-10min,然后加入硅藻土,将体系过铺有硅藻土的压滤罐,滤饼用5体积乙酸异丙酯洗,合并后滤液在用10N氢氧化钠调节pH=8.2-8.5,搅拌1h后,搅拌静置分液,向下层水相中加入15体积的乙酸异丙酯,用35%硫酸调节pH=1.8-2.2,将体系升温至约40℃,保温搅拌3h后,静置1h分液,向水相中加入15V乙酸异丙酯,调节温度至约40℃保温3h后静置1h后分液,合并有机相,控温T≤50℃浓缩至约9体积,在10-15min内控温82-89℃加入20体积正庚烷,将体系逐渐降温,控制夹套温度在70℃保温1h,将体系继续降温至40℃,保温0.5h后继续降温至0-5℃,保温1h,将体系抽滤,用10v正庚烷淋洗滤饼,产品于55℃下干燥。称重得到产品0.8 g,收率为80%。
本发明中,突变位点的其它任意组合也可能有较好的效果,突变位点在其它同源性较高氨基酸脱氢酶上进行重复,也应该有比较好的效果。
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> 凯莱英生命科学技术(天津)有限公司
<120> 氨基酸脱氢酶突变体及其应用
<130> PN141852KLY
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 368
<212> PRT
<213> Thermoactinomyces intermedius ATCC33205
<400> 1
Met Arg Asp Val Phe Glu Met Met Asp Arg Tyr Gly His Glu Gln Val
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Ile Phe Cys Arg His Pro Gln Thr Gly Leu Lys Ala Ile Ile Ala Leu
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His Asn Thr Thr Ala Gly Pro Ala Leu Gly Gly Cys Arg Met Ile Pro
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Tyr Ala Ser Thr Asp Glu Ala Leu Glu Asp Val Leu Arg Leu Ser Lys
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Gly Met Thr Tyr Lys Cys Ser Leu Ala Asp Val Asp Phe Gly Gly Gly
65 70 75 80
Lys Met Val Ile Ile Gly Asp Pro Lys Lys Asp Lys Ser Pro Glu Leu
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Phe Arg Val Ile Gly Arg Phe Val Gly Gly Leu Asn Gly Arg Phe Tyr
100 105 110
Thr Gly Thr Asp Met Gly Thr Asn Pro Glu Asp Phe Val His Ala Ala
115 120 125
Arg Glu Ser Lys Ser Phe Leu Gly Leu Pro Lys Ser Tyr Gly Gly Lys
130 135 140
Gly Asp Thr Ser Ile Pro Thr Ala Leu Gly Val Phe His Gly Met Arg
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Ala Thr Ala Arg Phe Leu Trp Gly Thr Asp Gln Leu Lys Gly Arg Val
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Val Ala Ile Gln Gly Val Gly Lys Val Gly Glu Arg Leu Leu Gln Leu
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Leu Val Glu Val Gly Ala Tyr Cys Lys Ile Ala Asp Ile Asp Ser Val
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Arg Cys Glu Gln Leu Lys Glu Lys Tyr Gly Asp Lys Val Gln Leu Val
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Asp Val Asn Arg Ile His Lys Glu Ser Cys Asp Ile Phe Ser Pro Cys
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Ala Lys Gly Gly Val Val Asn Asp Asp Thr Ile Asp Glu Phe Arg Cys
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Leu Ala Ile Val Gly Ser Ala Asn Asn Gln Leu Val Glu Asp Arg His
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Val Asn Ala Gly Gly Leu Ile Gln Val Ala Asp Glu Leu Glu Gly Phe
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His Glu Glu Arg Val Leu Ala Lys Thr Glu Ala Ile Tyr Asp Met Val
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Leu Asp Ile Phe His Arg Ala Lys Asn Glu Asn Ile Thr Thr Cys Glu
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<210> 2
<211> 1107
<212> DNA
<213> Thermoactinomyces intermedius ATCC33205
<400> 2
atgcgtgacg tattcgaaat gatggatcgc tacggccacg agcaggtgat tttctgtcgt 60
catccgcaga ctggcctgaa agcgatcatc gctctgcata acaccactgc cggtccggca 120
ctgggcggtt gtcgcatgat tccatacgca agcaccgatg aagctctgga agacgttctg 180
cgtctgagca aaggtatgac ctataaatgc tctctggcgg atgttgattt cggtggcggt 240
aaaatggtga ttatcggcga tccgaaaaag gataaaagcc cagaactgtt ccgtgttatc 300
ggtcgcttcg ttggcggcct gaacggtcgt ttctataccg gtactgatat gggcaccaat 360
ccggaagatt tcgtgcacgc cgctcgcgaa agcaaatctt ttctaggtct gcctaaatct 420
tacggtggta aaggtgacac ttctatcccg accgcactgg gtgtatttca cggcatgcgc 480
gcgaccgccc gctttctgtg gggcaccgat caactgaaag gtcgtgttgt tgctatccag 540
ggtgttggca aagtgggtga acgtctgctg cagctgctgg tggaagtggg tgcatactgc 600
aaaattgctg atattgactc tgtacgttgt gagcagctga aagaaaagta cggcgacaaa 660
gtccagctgg tagacgtgaa ccgtatccac aaagagtctt gtgacatctt ctccccgtgc 720
gcaaaaggcg gcgtagtcaa cgacgacact attgacgaat tccgctgcct ggcgattgtt 780
ggttccgcga acaatcagct ggttgaagat cgtcatggcg cgctgctgca aaaacgctcc 840
atttgctatg ccccggatta tctggttaac gctggcggtc tgatccaggt cgcagacgaa 900
ctggagggtt ttcacgagga gcgtgtgctg gcgaaaacgg aagccatcta cgacatggtt 960
ctggacatct tccaccgcgc taagaacgaa aacatcacta cctgcgaagc agcggaccgt 1020
atcgtaatgg aacgtctgaa gaagctgacg gacatccgtc gtatcctgct ggaagatccg 1080
cgtaactccg cgcgtcgtct cgagtga 1107
Claims (14)
1.一种氨基酸脱氢酶突变体,其特征在于,所述氨基酸脱氢酶突变体具有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为K144G位点。
2.根据权利要求1所述的氨基酸脱氢酶突变体,其特征在于,所述发生氨基酸突变的位点为如下任一种组合突变位点:K144G+L294I、K144G+L294I+L135V、K144G+L294I+T115I、K144G+L41I、K144G+L41K、K144G+L41L、K144G+L41S、K144G+L41T、K144G+G42V、K144G+G43N、K144G+G114S、K144G+T115I、K144G+T115L、K144G+T115V、K144G+M117A、K144G+M117L、K144G+M117T、K144G+M117V、K144G+M117Y、K144G+G118A、K144G+G118H、K144G+G118N、K144G+G118Q、K144G+G118S、K144G+G118Y、K144G+T119A、K144G+T119C、K144G+T119E、K144G+T119L、K144G+T119N、K144G+T119S、K144G+P121M、K144G+P121S、K144G+P121T、K144G+P121W、K144G+L135I、K144G+L135V、K144G+K184H、K144G+K184M、K144G+K184N、K144G+K184S、K144G+G293A、K144G+G293P、K144G+G293S或K144G+L294V。
3.根据权利要求1所述的氨基酸脱氢酶突变体,其特征在于,所述氨基酸脱氢酶突变体具有K144G+L294I+L135V氨基酸突变,且与甲酸脱氢酶FDH共表达。
4.一种DNA分子,其特征在于,所述DNA分子编码权利要求1至3中任一项所述的氨基酸脱氢酶突变体。
5.一种重组质粒,其特征在于,所述重组质粒连接有权利要求4所述的DNA分子。
6.根据权利要求5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、pRSFDuet1或pUC-19。
7.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求5或6所述的重组质粒。
8.根据权利要求7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。
9.根据权利要求8所述的宿主细胞,其特征在于,所述原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞,所述真核细胞为酵母。
10.一种生产氨基酸的方法,包括采用氨基酸脱氢酶对酮类化合物进行催化还原氨化反应的步骤,其特征在于,所述氨基酸脱氢酶为权利要求1至3中任一项所述的氨基酸脱氢酶突变体。
13.根据权利要求11所述的方法,其特征在于,所述催化还原氨化反应中的氨基供体为甲酸铵、氯化铵、氨基甲酸铵、碳酸铵、碳酸氢铵、氨水、草酸铵、草酸氢铵、乳酸铵或磷酸铵。
14.根据权利要求11所述的方法,其特征在于,所述氨基酸脱氢酶催化还原氨化反应的条件为30~50℃,搅拌转速为50~250 rpm,所述催化还原氨化反应中还包括使用甲酸脱氢酶0.1 mg/mL~1 mg/mL。
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