CN116574750B - 一种提高腈类化合物生物转化效率的腈水合酶重组质粒及其构建方法与应用 - Google Patents
一种提高腈类化合物生物转化效率的腈水合酶重组质粒及其构建方法与应用 Download PDFInfo
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Abstract
本发明属于绿色化学技术领域,具体涉及一种提高腈类化合物生物转化效率的腈水合酶重组质粒及其构建方法与应用。所述的构建方法是对腈水合酶重组质粒ReNHase‑AC‑His的β亚基序列上游的核糖体结合位点RBS进行序列优化。新构建的腈水合酶重组质粒可应用于催化腈类化合物反应中。与优化前相比,明显改善了腈水合酶α亚基与β亚基表达量之间的平衡,并且显著提高了催化腈类化合物的转化效率,为腈类化合物的生物转化工业化应用提供了新的理论依据。
Description
技术领域
本发明属于绿色化学技术领域,具体涉及一种提高腈类化合物生物转化效率的腈水合酶重组质粒及其构建方法与应用,所述的腈水合酶重组质粒可以促进重组腈水合酶蛋白亚基平衡表达并提高腈类化合物催化效率。
背景技术
大肠杆菌(Escherichia coli)作为一种被人们广泛熟知的革兰氏阴性细菌,因其具备清晰的基因组信息,基因技术操作简单易行,环境适应能力强且易于大规模发酵培养,早在几十年前便成为公认的基因工程研究首选菌株。通过对大肠杆菌进行基因改造,商业化的大肠杆菌基因工程菌目前已广泛应用于分子研究、发酵合成等众多领域。在将外源基因导入大肠杆菌宿主之后,便可以利用大肠杆菌自身携带的基因元件快速高效地合成并分泌大量外源蛋白。然而,虽然大肠杆菌这种原核生物表达系统在表达外源蛋白的过程中具有多种不可比拟的优势,在进行外源蛋白表达的过程中亦会受到启动子强度、密码子种类、mRNA的稳定性、核糖体结合效率与翻译速率等诸多因素的影响。由于来源不同的外源蛋白结构组成各不相同,许多蛋白质需要由多个亚基组合才可组装成结构完整且具备正常功能的四级结构,这种多亚基的表达对于大肠杆菌表达系统是一种艰巨挑战——不同亚基在表达的过程中存在时间和空间上的先后差异,这种差异导致核糖体对于各亚基的翻译强度不同,最终导致各亚基表达不平衡,很大程度上影响了酶结构的完整性,导致酶活性的缺失,也限制了大肠杆菌在表达蛋白产物及发酵生产方面的工业化应用。
在基因指导合成蛋白质的过程中,需要经历转录、翻译、新生肽链折叠与相互作用,最终形成亚基并组合形成具备完整结构蛋白质的过程。在翻译的过程中,需要由核糖体结合位于信使RNA(messager RNA,简称mRNA)上的核糖体结合位点(Ribosome bind site,简称RBS)来引导翻译的起始。研究发现,根据核糖体结合位点碱基序列种类及排布的不同,其对核糖体的结合能力也存在差异,这种结合强度的强弱直接影响到位于RBS后面基因的表达,一段能够强效结合核糖体的基因序列往往可以大幅度强化核糖体翻译的效率。因此,通过对核糖体结合位点序列进行碱基序列优化,从而提高核糖体结合与翻译效率,被认为有利于改善蛋白质表达量低及多亚基表达不平衡的问题。
腈水合酶(EC4.2.1.84,Nitrile Hydratase,简称NHase),是腈转化酶中的一种酶,依据活性中心金属离子种类不同,分为Fe型腈水合酶与Co型腈水合酶,并由α亚基与β亚基结合金属离子,以多聚体的形式构成完整且具备催化功能的腈水合酶。因其可以将腈类化合物催化转化为具有高附加值的酰胺类化合物,近年来引起了人们广泛的关注。利用腈水合酶生成的丙烯酰胺、烟酰胺等酰胺类化合物也被广泛应用于化工、医药、农药等领域。
本发明在研究前期,发现了一种腈水合酶基因并制备了腈水合酶基因工程菌,发现其有显著的区域选择性。然而由于成熟的腈水合酶由α亚基与β亚基构成,在使用大肠杆菌进行外源表达时,位于前段的α亚基序列被优先表达,β亚基随后表达,二者在表达的过程中存在时空上的差异,导致两种亚基之间表达量严重不平衡,同时酶活力受到了严重抑制。因此,本发明通过对核糖体结合位点序列进行碱基序列优化,提高核糖体与RBS序列的结合强度,促进β亚基的翻译效率,增加β亚基的表达量,对于改善腈水合酶α亚基与β亚基间平衡表达有良好的借鉴意义,也为提高腈水合酶催化活性、增强腈水合酶的工业化应用价值带来希望。
发明内容
本发明提供了一种通过优化核糖体结合位点(RBS)序列促进重组腈水合酶蛋白亚基平衡表达及提高二腈化合物催化效率的腈水合酶重组质粒的构建方法与应用。
本发明的技术方案:
一种提高腈类化合物生物转化效率的腈水合酶重组质粒及其构建方法,所述的构建方法是对腈水合酶重组质粒ReNHase-AC-His的β亚基序列上游的核糖体结合位点RBS进行序列优化,构建提高腈类化合物生物转化效率的腈水合酶重组质粒βWT-ReNHase-AC-His。具体方法如下:
对腈水合酶重组质粒ReNHase-AC-His的β亚基序列上游的核糖体结合位点RBS进行序列优化,将腈水合酶重组质粒ReNHase-AC-His作为克隆载体,利用基因搭桥PCR技术,对位于腈水合酶β亚基基因序列上游的核糖体结合序列RBS进行序列改造并扩增,使用T4连接酶将RBS序列改造后的片段以及经限制性内切酶进行双酶切后的线性质粒进行连接反应,得到提高腈类化合物生物转化效率的腈水合酶重组质粒βWT-ReNHase-AC-His。腈水合酶重组质粒ReNHase-AC-His核苷酸序列如SEQ ID NO.1所示,提高腈类化合物生物转化效率的腈水合酶重组质粒βWT-ReNHase-AC-His核苷酸序列如SEQ ID NO.2所示,优化前的RBS的序列如SEQ ID NO.3所示,优化后的RBS的序列如SEQ ID NO.4所示。
SEQ ID NO.1:
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCAGTAACGATCGACCACACAACGGAGAACGCCGCACCGGCCCAGGCGCCGGTCTCCGATCGCGCGTGGGCCCTGTTCCGCGCACTCGACGGTAAGGGATTGGTACCCGACGGTTACGTCGAGGGATGGAAGAAGACCTTCGAGGAGGACTTCAGTCCAAGGCGCGGAGCGGAATTGGTCGCGCGGACTTCATACCCCGGAACATACCGGCAACTGCTTCTCACCGACGGTACCGCCGCGGTTGCCCAGTACGGATATCTGGGCCCCCAGGGCGAATACATCGTGGCAGTCGAAGACACCCCGACCCTCAAGAACGTGATCGTGTGCTCGCTGTGTTCATGCACCGCGTGGCCCATCCTCGGTCTGCCGCCGACCTGGTACAAGAGTTTCGAATACCGTGCACGCGTGGTCCGCGAGCCACGGAAGGTTCTCTCCGAGATGGGAACCGAGATCGCGTCGGACGTCGAGATCCGCGTCTACGACACCACCGCCGAAACTCGGTACATGGTCCTACCGCAACGTCCCGCAGGCACCGAAGGCTGGAGCCAGGAACAACTGCAGGAAATCGTCACCAAGGACTGCCTGATCGGCGTCGCAGTCCCGCAGGTCCCCACCGTCCACCACCACCACCACCACTGACCACCCCGACAAGAAAGAAGCACACCATGGATGGAGTACACGATCTTGCCGGAGTTCAAGGCTTCGGCAAAGTCCCGCATACCGTCAACGCCGACATCGGCCCCACCTTCCACGCCGAGTGGGAACACCTGCCGTACAGCCTGATGTTCGCCGGTGTCGCCGAACTCGGGGCCTTCAGCGTCGACGAAGTTCGATACGTCGTCGAGCGGATGGAGCCCCGCCACTACATGATGACCCCGTACTACGAGCGGTACGTCATCGGCGTCGCGGCGCTGATGGTCGAAAAGGGAATCCTGACGCAGGAAGAGCTCGAAAGCCTTGCAGGAGGACCGTTCCCACTCTCACGGCCAAGCGAATCCGAAGGCCGACCGGCTCGCGTCGACACAACCACCTTCGAGGTCGGTCAGCGAGTACGTGTGCGAGACGAATACGTTCCCGGGCATATTCGAATGCCTGCTTACTGCCGAGGACGGGTGGGGACCATCGCTCACCGGACCACCGAGAAGTGGCCGTTCCCCGACGCAATCGGTCACGGCCGCAACGACGCCGGCGAAGAACCCACCTACCACGTGACGTTCGCTGCGGAGGAATTGTTCGGCAGCGACACCGACGGCGGAAGCGTCGTTGTCGACCTCTTCGAGGGTTACCTCGAGCCTGCGGCCTGATCTTCCAGCATTCCAGGCGGCGGTCACGCGATCGCAGCGGTTCGCGTGACCGCCGCCTGATCACAACGATTCACTCATTCGGAAGGACACTGGAAATCATGGTCGACACACGACTTCCGGTCACGGTGCTGTCAGGTTTCCTGGGCGCCGGGAAGACGACGCTACTCAACGAGATCCTGCGCAATCGGGAGGGCCGCCGGGTTGCGGTGATCGTCAACGACATGAGCGAAATCAACATCGACAGTGCAGAAGTCGAGCGTGAGATCTCGCTCAGTCGCTCCGAGGAGAAACTGGTCGAGATGACCAACGGCTGCATCTGCTGCACTCTGCGAGAGGATCTTCTTTCCGAGATAAGCGCCTTGGCCGCCGATGGCCGATTCGACTACCTTCTCATCGAATCTTCGGGCATCTCCGAACCGCTGCCCGTCGCGGAGACGTTCACCTTCATCGATACCGACGGCCATGCCCTGGCCGACGTCGCCCGACTCGACACCATGGTCACAGTCGTCGACGGCAACAGTTTTCTGCGCGACTACACGGCTGGAGGTCGCGTCGAAGCCGATGCCCCGGAAGATGAACGCGACATCGCGGATCTGCTTGTCGATCAGATCGAGTTTGCCGACGTCATCCTGGTGAGCAAGGCCGATCTCGTCTCGCACCAGCACCTGGTCGAATTGACTTCGGTCCTAAGATCTTTGAACGCAACTGCTGCGATAGTTCCGATGACTCTCGGCCGTATCCCACTCGACACGATTCTCGATACCGGCTTGTTCTCGCTCGAGAAAGCTGCTCAGGCCCCTGGATGGCTACAAGAACTCCAAGGTGAACACACCCCCGAAACCGAGGAGTACGGAATCGGTTCGGTGGTGTACCGCGAGCGCGCGCCCTTCCACCCACAACGCCTGCATGATTTCCTGAGCAGCGAGTGGACCAACGGAAAGTTACTTCGGGCCAAGGGCTACTACTGGAATGCCGGCCGGTTCACCGAGATCGGGAGTATTTCTCAGGCCGGTCATCTCATTCGCCACGGATACGTCGGCCGTTGGTGGAAGTTTCTACCCCGTGACGAGTGGCCGGCCGACGACTACCGTCGCGACGGAATCCTCGACAAGTGGGAAGAACCTGTCGGTGACTGCCGACAAGAACTCGTCTTCATCGGCCAATCCATCGACCCATCTCGACTGCACCGAGAACTCGACGCGTGTCTACTCACCACAGCCGAGATCGAACTCGGGCCAGACGTGTGGACCACCTGGAGCGACCCCCTGGGCGTCGGCTATACCGACCAGACCGTTTGAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
SEQ ID NO.2:
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCAGTAACGATCGACCACACAACGGAGAACGCCGCACCGGCCCAGGCGCCGGTCTCCGATCGCGCGTGGGCCCTGTTCCGCGCACTCGACGGTAAGGGATTGGTACCCGACGGTTACGTCGAGGGATGGAAGAAGACCTTCGAGGAGGACTTCAGTCCAAGGCGCGGAGCGGAATTGGTCGCGCGGACTTCATACCCCGGAACATACCGGCAACTGCTTCTCACCGACGGTACCGCCGCGGTTGCCCAGTACGGATATCTGGGCCCCCAGGGCGAATACATCGTGGCAGTCGAAGACACCCCGACCCTCAAGAACGTGATCGTGTGCTCGCTGTGTTCATGCACCGCGTGGCCCATCCTCGGTCTGCCGCCGACCTGGTACAAGAGTTTCGAATACCGTGCACGCGTGGTCCGCGAGCCACGGAAGGTTCTCTCCGAGATGGGAACCGAGATCGCGTCGGACGTCGAGATCCGCGTCTACGACACCACCGCCGAAACTCGGTACATGGTCCTACCGCAACGTCCCGCAGGCACCGAAGGCTGGAGCCAGGAACAACTGCAGGAAATCGTCACCAAGGACTGCCTGATCGGCGTCGCAGTCCCGCAGGTCCCCACCGTCCACCACCACCACCACCACTGACCACCCCGAAAGAAGGAGATATACATATGGATGGAGTACACGATCTTGCCGGAGTTCAAGGCTTCGGCAAAGTCCCGCATACCGTCAACGCCGACATCGGCCCCACCTTCCACGCCGAGTGGGAACACCTGCCGTACAGCCTGATGTTCGCCGGTGTCGCCGAACTCGGGGCCTTCAGCGTCGACGAAGTTCGATACGTCGTCGAGCGGATGGAGCCCCGCCACTACATGATGACCCCGTACTACGAGCGGTACGTCATCGGCGTCGCGGCGCTGATGGTCGAAAAGGGAATCCTGACGCAGGAAGAGCTCGAAAGCCTTGCAGGAGGACCGTTCCCACTCTCACGGCCAAGCGAATCCGAAGGCCGACCGGCTCGCGTCGACACAACCACCTTCGAGGTCGGTCAGCGAGTACGTGTGCGAGACGAATACGTTCCCGGGCATATTCGAATGCCTGCTTACTGCCGAGGACGGGTGGGGACCATCGCTCACCGGACCACCGAGAAGTGGCCGTTCCCCGACGCAATCGGTCACGGCCGCAACGACGCCGGCGAAGAACCCACCTACCACGTGACGTTCGCTGCGGAGGAATTGTTCGGCAGCGACACCGACGGCGGAAGCGTCGTTGTCGACCTCTTCGAGGGTTACCTCGAGCCTGCGGCCTGATCTTCCAGCATTCCAGGCGGCGGTCACGCGATCGCAGCGGTTCGCGTGACCGCCGCCTGATCACAACGATTCACTCATTCGGAAGGACACTGGAAATCATGGTCGACACACGACTTCCGGTCACGGTGCTGTCAGGTTTCCTGGGCGCCGGGAAGACGACGCTACTCAACGAGATCCTGCGCAATCGGGAGGGCCGCCGGGTTGCGGTGATCGTCAACGACATGAGCGAAATCAACATCGACAGTGCAGAAGTCGAGCGTGAGATCTCGCTCAGTCGCTCCGAGGAGAAACTGGTCGAGATGACCAACGGCTGCATCTGCTGCACTCTGCGAGAGGATCTTCTTTCCGAGATAAGCGCCTTGGCCGCCGATGGCCGATTCGACTACCTTCTCATCGAATCTTCGGGCATCTCCGAACCGCTGCCCGTCGCGGAGACGTTCACCTTCATCGATACCGACGGCCATGCCCTGGCCGACGTCGCCCGACTCGACACCATGGTCACAGTCGTCGACGGCAACAGTTTTCTGCGCGACTACACGGCTGGAGGTCGCGTCGAAGCCGATGCCCCGGAAGATGAACGCGACATCGCGGATCTGCTTGTCGATCAGATCGAGTTTGCCGACGTCATCCTGGTGAGCAAGGCCGATCTCGTCTCGCACCAGCACCTGGTCGAATTGACTTCGGTCCTAAGATCTTTGAACGCAACTGCTGCGATAGTTCCGATGACTCTCGGCCGTATCCCACTCGACACGATTCTCGATACCGGCTTGTTCTCGCTCGAGAAAGCTGCTCAGGCCCCTGGATGGCTACAAGAACTCCAAGGTGAACACACCCCCGAAACCGAGGAGTACGGAATCGGTTCGGTGGTGTACCGCGAGCGCGCGCCCTTCCACCCACAACGCCTGCATGATTTCCTGAGCAGCGAGTGGACCAACGGAAAGTTACTTCGGGCCAAGGGCTACTACTGGAATGCCGGCCGGTTCACCGAGATCGGGAGTATTTCTCAGGCCGGTCATCTCATTCGCCACGGATACGTCGGCCGTTGGTGGAAGTTTCTACCCCGTGACGAGTGGCCGGCCGACGACTACCGTCGCGACGGAATCCTCGACAAGTGGGAAGAACCTGTCGGTGACTGCCGACAAGAACTCGTCTTCATCGGCCAATCCATCGACCCATCTCGACTGCACCGAGAACTCGACGCGTGTCTACTCACCACAGCCGAGATCGAACTCGGGCCAGACGTGTGGACCACCTGGAGCGACCCCCTGGGCGTCGGCTATACCGACCAGACCGTTTGAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
SEQ ID NO.3:CAACAAAGAAGGACACC
SEQ ID NO.4:AAGAAGGAGATATACAT
上述构建的腈水合酶重组质粒在催化腈类化合物反应中的应用,具体如下:
蛋白表达纯化:将提高腈类化合物生物转化效率的腈水合酶重组质粒βWT-ReNHase-AC-His转化入ArcticExpress(DE3)大肠杆菌感受态细胞,涂板后挑取单克隆进行培养,后加入IPTG进行低温诱导蛋白表达;结束诱导后离心菌液,用PB缓冲液进行清洗并重悬得到细菌悬浊液,超声破碎后进行离心,上清液经滤膜过滤后,使用AKTA pure进行蛋白纯化,得到腈水合酶纯酶;
催化反应:在腈类化合物反应中,将得到的腈水合酶纯酶作为催化剂,进行催化反应。
所述的腈类化合物为脂肪族腈类化合物或芳香族腈类化合物。
所述的催化腈类化合物反应包括催化己二腈合成5-氰基戊酰胺。
本发明的有益效果:
(1)腈水合酶重组质粒,经RBS序列改造,可以优化多亚基蛋白的亚基表达,使各亚基之间的蛋白表达量趋于平衡;
(2)本发明经RBS序列优化后表达的腈水合酶对腈类化合物的催化活性明显增加,大幅提高了腈水合酶重组蛋白催化腈类化合物转化反应效率,为改善蛋白质多亚基之间的平衡表达与腈水合酶催化腈类化合物的工业化应用提供了理论依据。
附图说明
图1为ReNHase-AC-His蛋白诱导表达SDS-PAGE电泳图,图中:
Lane M:蛋白Marker;
Lane 1:对照组PGB1-ReNHase-AC-His,其中31kDa条带为PGB1+α亚基,25kDa条带为β亚基;
Lane 2:实验组βPGB1-ReNHase-AC-His,其中31kDa条带为PGB1+α亚基,25kDa条带为β亚基。
图2为优化了β亚基表达的腈水合酶βWT-ReNHase-AC-His与对照组WT-ReNHase-AC-His分别在1/3/5/10/20/30min下催化己二腈生成的5-氰基戊酰胺浓度,每组实验重复三次。
具体实施方式
以下结合附图和技术方案,进一步说明本发明的具体实施方式。
本发明的一种提高腈类化合物生物转化效率的腈水合酶重组蛋白表达载体的构建方法,对腈水合酶重组质粒ReNHase-AC-His的β亚基序列上游的核糖体结合位点(RBS)进行序列优化,构建新的腈水合酶重组质粒并进行蛋白表达获得腈水合酶纯酶;具体步骤如下:
(1)RBS序列优化:将腈水合酶重组质粒ReNHase-AC-His作为克隆载体,利用基因搭桥PCR技术,对位于腈水合酶β亚基基因序列上游的核糖体结合序列(RBS)进行序列改造并扩增,使用T4连接酶将RBS序列改造后的片段以及经限制性内切酶进行双酶切后的线性质粒进行连接反应,得到RBS改造后的重组质粒。为便于区分改造前后,改造前的重组质粒命名为WT-ReNHase-AC-His,改造后的重组质粒命名为βWT-ReNHase-AC-His。
(2)蛋白表达纯化:将提高腈类化合物生物转化效率的腈水合酶重组质粒βWT-ReNHase-AC-His转化入ArcticExpress大肠杆菌感受态细胞,涂板后挑取单克隆进行培养,后加入IPTG进行低温诱导蛋白表达;结束诱导后离心菌液,用PB缓冲液进行清洗并重悬得到细菌悬浊液,超声破碎后进行离心,上清液经滤膜过滤后,使用AKTA pure进行蛋白纯化,得到腈水合酶纯酶;
(3)SDS-PAGE验证:
由于腈水合酶α亚基与β亚基蛋白分子量大小过于接近(均为25kDa),为便于区分α亚基与β亚基,同时观察优化RBS序列后β亚基表达量的变化,分别在步骤(1)改造前与改造后的腈水合酶重组质粒上,位于α亚基5’端前NdeⅠ酶切位点(即序列CATATG)至α亚基起始密码子(即序列ATGTCA)这段序列范围内,引入PGB1融合标签与一段柔性肽,构建得到新的重组质粒,并分别命名为PGB1-ReNHase-AC-His与βPGB1-ReNHase-AC-His。将上述两种重组质粒转化入ArcticExpress(DE3)大肠杆菌感受态细胞,涂板后挑取单克隆进行培养,后加入IPTG进行低温诱导蛋白表达。结束诱导后高速离心菌液,用PB缓冲液进行清洗并重悬得到细菌悬浊液,超声破碎后进行离心,同时对上清液取样,经SDS-PAGE检测腈水合酶蛋白表达情况。
其中,PGB1与柔性肽的氨基酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示。
经对照试验发现,经过RBS序列碱基优化的实验组腈水合酶α亚基与β亚基表达量趋于平衡,并且催化比酶活明显提升。
SEQ ID NO.5:
MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE
SEQ ID NO.6:GGGGSGGGGSGGGGS
实施例1βPGB1-ReNHase-AC-His蛋白表达验证
由于腈水合酶重组质粒ReNHase-AC-His的α亚基与β亚基分子量极为接近(均为25kDa左右),在SDS-PAGE蛋白表达验证时无法分辨二者条带大小及位置,影响对于不同亚基表达量的判断。因此在进行蛋白表达验证之前,在通过α亚基前加入PGB1蛋白融合标签,使α亚基和β亚基在进行蛋白表达验证时蛋白条带能够明显分开,然后再进行核糖体结合位点序列的优化。将序列优化后的腈水合酶重组基因βPGB1-ReNHase-AC-His与未优化的腈水合酶基因PGB1-ReNHase-His分别取2μL加入至50μL的感受态大肠杆菌Arctic Express(DE3),并在冰上静置30min,之后在42℃热激90秒后迅速置于冰上3min,加入500μL LB液体培养基,在37℃、220rpm条件下振荡活化1h。在提前制备的LB固体培养基分别吸取两支活化后的大肠杆菌100μL,并均匀涂布于平板上,在恒温摇床上倒置静止过夜培养,12h后平板上长有大量单克隆菌落。用移液枪头分别从两种不同平板上挑取明显的单克隆菌落打入含有50μg/mL卡那霉素的LB液体培养基试管中,37℃、220rpm振荡培养至OD600值在0.6-0.8之间,加入终浓度为1mM的IPTG,在200rpm的条件下过夜诱导18h。次日以5000rpm转速收集菌体,倒掉上清液,用20mM PB(pH=7)清洗两次并重悬菌体;使用超声波破碎仪300W,破碎1s,间隔9s将菌体破碎,然后在10000rpm条件下离心0.5h。将离心后的上清液制备SDS-PAGE蛋白电泳样品,并在100V条件下进行SDS-PAGE蛋白电泳,1h后使用考马斯亮蓝G250染液对蛋白胶进行染色和脱色,观察并比较实验组和对照组样品条带。如图1所示,对照组PGB1ReNHase-AC-His泳道内31kDa处出现明显的带有PGB1标签的α亚基条带,但在25kDa处的β亚基条带却远少于α亚基,证明β亚基表达量过低;而在实验组βPGB1-ReNHase-AC-His泳道内31kDa处出现连接有PGB1标签的α亚基,25kDa处出现明显且大小接近于连接有PGB1标签的α亚基条带,证明β亚基的表达量得到了明显改善。
实施例2βWT-ReNHase-AC-His/WT-ReNHase-AC-His纯酶催化己二腈反应及酶动力学参数测定
(1)菌液制备:将RBS序列优化的腈水合酶重组质粒βWT-ReNHase-AC-His与未优化的腈水合酶重组质粒WT-ReNHase-AC-His分别取2μL加入至50μL的感受态大肠杆菌ArcticExpress(DE3),并在冰上静置30min,之后在42℃热激90秒后迅速置于冰上3min,加入500μLLB液体培养基,在37℃、220rpm条件下振荡活化1h。在提前制备的LB固体培养基分别吸取两支活化后的大肠杆菌100μL,并均匀涂布于平板上,在恒温摇床上倒置静止过夜培养,12h后平板上长有大量单克隆菌落。挑取上述平板上的单克隆菌落打入含有50μg/mL卡那霉素的LB液体培养基试管中,37℃、220rpm振荡培养至OD600值在0.6-0.8之间,将试管中菌液转入500mL LB液体培养基中进行扩大培养,待其OD600值在0.6-0.8范围内加入终浓度为1mMIPTG,并于16℃、200rpm条件下进行低温诱导过夜培养18h。次日,在5000rpm、4℃条件下回收菌液并倒掉上清液,用蛋白纯化结合缓冲液(即pH=7,20mM的PB溶液)清洗两次并用20mL上述结合缓冲液重悬菌体;
(2)细胞破碎及酶纯化:将上述重悬细菌的悬浊液于超声破碎仪进行破碎,功率300W,破碎1s,间歇9s,共持续90个循环。将破碎后的悬浊液10000rpm条件下离心30min,然后将上清液经0.22μm的滤膜过滤,后通过AKTA pure蛋白纯化系统,使用离子交换层析进行酶的纯化,以此得到纯酶。通过Bradford蛋白质定量检测试剂盒测定纯酶浓度,并将其稀释至0.2mg/mL。
(3)腈水合酶催化己二腈转化反应过程:在两支EP管中分别依次加入250μL 200mM己二腈,200μL 20mM PB缓冲液(pH=7),最后分别加入0.2mg/mL经过碱基优化的βWT-ReNHase-AC-His纯酶稀释液与对照组WT-ReNHase-AC-His纯酶稀释液各50μL,放入摇床中在200rpm、30℃及光照条件下进行纯酶催化己二腈转化反应,体系中底物己二腈反应浓度为100mM。反应2/3/5/10/20/30/40min后,加入500μL甲醇终止反应;
(4)酶动力学参数测定:在若干支试管中分别加入不同体积不同浓度的己二腈,20mM PB缓冲液(pH=7),然后分别加入0.5mg/mL经过碱基优化的βWT-ReNHase-AC-His与对照组WT-ReNHase-AC-His的纯酶稀释液,最终反应体系的总体积为500μL,且不同试管中的己二腈浓度为5/10/20/40/60/80/100mM,酶反应浓度为0.05mg/mL。放入摇床中在200rpm、30℃及光照条件下进行纯酶催化己二腈转化反应,反应3min后,加入500μL甲醇终止反应;
(5)高效液相色谱(HPLC)分析:将上述两支试管在10000rpm转速下离心20min,过0.22μm滤膜后分别进行高效液相色谱检测。高效液相使用Ultimate 5μm 4.6×250mm LP-C18柱,流动相为25mM磷酸水溶液和甲醇(89:11,vol:vol),检测波长200nm,柱温25℃,流速1ml/min,检测并计算实验组和对照组生成5-氰基戊酰胺的含量,同时计算实验组和对照组的比酶活。
如图2所示,实验组βWT-ReNHase-AC-His在2min时便可以将底物100mM己二腈全部转化为5-氰基戊酰胺,并且在后续转化为己二酰二胺。而对照组WT-ReNHase-AC-His在30min时才能将底物己二腈完全转化,且后续进一步转化为副产物己二酰二胺的速率较慢。计算实验组和对照组纯酶的比酶活,对照组WT-ReNHase-AC-His比酶活为299.84±2.84U/mg,而实验组βWT-ReNHase-AC-His的比酶活为3679.06±11.95U/mg,相较于对照组提高了约12倍。该结果说明通过优化核糖体结合位点序列,可以显著提高重组腈水合酶的催化活性。
表1为腈水合酶重组质粒WT-ReNHase-AC-His/βWT-ReNHase-AC-His动力学参数测定结果,如表所示,βWT-ReNHase-AC-His的米氏常数Km值为15.7±1.96mmol/L,低于WT-ReNHase-AC-His的Km值,说明与底物的亲和力增强;而其酶催化常数Kcat为4004.93±63.08s-1,显著高于WT-ReNHase-AC-His的Kcat,说明在单位时间内,βWT-ReNHase-AC-His酶分子将底物转化为产物的效率明显高于WT-ReNHase-AC-His;通过计算酶催化效率参数Kcat/Km,βWT-ReNHase-AC-His的Kcat/Km值为255.09L/mmol.s,明显大于WT-ReNHase-AC-His,说明酶的催化能力有显著提升。
表1WT/βWT-ReNHase-AC-His纯酶动力学参数
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Claims (7)
1.一种提高腈类化合物生物转化效率的腈水合酶重组质粒,其特征在于,所述的腈水合酶重组质粒的核苷酸序列如SEQ ID NO.2所示。
2.权利要求1所述的提高腈类化合物生物转化效率的腈水合酶重组质粒的构建方法,其特征在于,对腈水合酶重组质粒ReNHase-AC-His的β亚基序列上游的核糖体结合位点RBS进行序列优化,构建提高腈类化合物生物转化效率的腈水合酶重组质粒;腈水合酶重组质粒ReNHase-AC-His核苷酸序列如SEQ ID NO.1所示,优化前的RBS的序列如SEQ ID NO.3所示。
3.根据权利要求2所述的提高腈类化合物生物转化效率的腈水合酶重组质粒的构建方法,其特征在于,优化后的RBS的序列如SEQ ID NO.4所示。
4.根据权利要求2所述的提高腈类化合物生物转化效率的腈水合酶重组质粒的构建方法,其特征在于,具体步骤如下:对腈水合酶重组质粒ReNHase-AC-His的β亚基序列上游的核糖体结合位点RBS进行序列优化,将腈水合酶重组质粒ReNHase-AC-His作为克隆载体,利用基因搭桥PCR技术,对位于腈水合酶β亚基基因序列上游的核糖体结合序列RBS进行序列改造并扩增,使用T4连接酶将RBS序列改造后的片段以及经限制性内切酶进行双酶切后的线性质粒进行连接反应,得到提高腈类化合物生物转化效率的腈水合酶重组质粒βWT-ReNHase-AC-His。
5.采用权利要求2或3或4所述的提高腈类化合物生物转化效率的腈水合酶重组质粒的构建方法构建得到的提高腈类化合物生物转化效率的腈水合酶重组质粒在催化己二腈反应中的应用。
6.根据权利要求5所述的应用,其特征在于,步骤如下:
蛋白表达纯化:将提高腈类化合物生物转化效率的腈水合酶重组质粒βWT-ReNHase-AC-His转化入ArcticExpress大肠杆菌感受态细胞,涂板后挑取单克隆进行培养,后加入IPTG进行低温诱导蛋白表达;结束诱导后离心菌液,用PB缓冲液进行清洗并重悬得到细菌悬浊液,超声破碎后进行离心,上清液经滤膜过滤后,使用AKTA pure进行蛋白纯化,得到腈水合酶纯酶;
催化反应:在己二腈反应中,将得到的腈水合酶纯酶作为催化剂,进行催化反应。
7.根据权利要求5或6所述的应用,其特征在于,所述的催化己二腈反应为催化己二腈合成5-氰基戊酰胺。
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