CN104450657B - 腈水合酶及其编码基因和应用 - Google Patents
腈水合酶及其编码基因和应用 Download PDFInfo
- Publication number
- CN104450657B CN104450657B CN201410621651.2A CN201410621651A CN104450657B CN 104450657 B CN104450657 B CN 104450657B CN 201410621651 A CN201410621651 A CN 201410621651A CN 104450657 B CN104450657 B CN 104450657B
- Authority
- CN
- China
- Prior art keywords
- pet
- coli
- nitrile hydratase
- nhasek
- chlorphenyls
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010024026 Nitrile hydratase Proteins 0.000 title claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 18
- 230000014509 gene expression Effects 0.000 claims abstract description 13
- 241001545494 Klebsiella michiganensis KCTC 1686 Species 0.000 claims abstract description 12
- 241000588724 Escherichia coli Species 0.000 claims description 34
- 241000894006 Bacteria Species 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 21
- 238000010353 genetic engineering Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- 238000006555 catalytic reaction Methods 0.000 claims description 16
- 239000012530 fluid Substances 0.000 claims description 14
- 238000006703 hydration reaction Methods 0.000 claims description 12
- 239000006285 cell suspension Substances 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 11
- 230000000284 resting effect Effects 0.000 claims description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 230000029087 digestion Effects 0.000 claims description 6
- 150000002825 nitriles Chemical class 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 238000011084 recovery Methods 0.000 claims description 6
- 108091008146 restriction endonucleases Proteins 0.000 claims description 6
- 230000009514 concussion Effects 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- 230000036571 hydration Effects 0.000 claims description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims 1
- 238000010422 painting Methods 0.000 claims 1
- 210000001082 somatic cell Anatomy 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 7
- 239000000758 substrate Substances 0.000 abstract description 6
- 238000001228 spectrum Methods 0.000 abstract description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 21
- 239000007788 liquid Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- JFDZBHWFFUWGJE-UHFFFAOYSA-N benzonitrile Chemical compound N#CC1=CC=CC=C1 JFDZBHWFFUWGJE-UHFFFAOYSA-N 0.000 description 9
- SUSQOBVLVYHIEX-UHFFFAOYSA-N phenylacetonitrile Chemical compound N#CCC1=CC=CC=C1 SUSQOBVLVYHIEX-UHFFFAOYSA-N 0.000 description 9
- 239000000725 suspension Substances 0.000 description 9
- LSBDFXRDZJMBSC-UHFFFAOYSA-N 2-phenylacetamide Chemical compound NC(=O)CC1=CC=CC=C1 LSBDFXRDZJMBSC-UHFFFAOYSA-N 0.000 description 8
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 6
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 6
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 6
- -1 carboxylic acid compound Chemical class 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 235000005152 nicotinamide Nutrition 0.000 description 6
- 239000011570 nicotinamide Substances 0.000 description 6
- 229960003966 nicotinamide Drugs 0.000 description 6
- 125000004432 carbon atom Chemical group C* 0.000 description 5
- DNSISZSEWVHGLH-UHFFFAOYSA-N butanamide Chemical compound CCCC(N)=O DNSISZSEWVHGLH-UHFFFAOYSA-N 0.000 description 4
- KVNRLNFWIYMESJ-UHFFFAOYSA-N butyronitrile Chemical compound CCCC#N KVNRLNFWIYMESJ-UHFFFAOYSA-N 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- GZPHSAQLYPIAIN-UHFFFAOYSA-N 3-pyridinecarbonitrile Chemical compound N#CC1=CC=CN=C1 GZPHSAQLYPIAIN-UHFFFAOYSA-N 0.000 description 3
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 3
- 108700023418 Amidases Proteins 0.000 description 3
- GYCMBHHDWRMZGG-UHFFFAOYSA-N Methylacrylonitrile Chemical compound CC(=C)C#N GYCMBHHDWRMZGG-UHFFFAOYSA-N 0.000 description 3
- 108010033272 Nitrilase Proteins 0.000 description 3
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 102000005922 amidase Human genes 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000036983 biotransformation Effects 0.000 description 2
- 230000007073 chemical hydrolysis Effects 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical class CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000588986 Alcaligenes Species 0.000 description 1
- 241000589518 Comamonas testosteroni Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108090001042 Hydro-Lyases Proteins 0.000 description 1
- 102000004867 Hydro-Lyases Human genes 0.000 description 1
- 241001006344 Microbacterium flavum Species 0.000 description 1
- 241000109365 Rosa arkansana Species 0.000 description 1
- 235000005066 Rosa arkansana Nutrition 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 150000007962 benzene acetonitriles Chemical class 0.000 description 1
- 150000008359 benzonitriles Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 125000002560 nitrile group Chemical group 0.000 description 1
- 238000010641 nitrile hydrolysis reaction Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229940080818 propionamide Drugs 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 125000004417 unsaturated alkyl group Chemical group 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01084—Nitrile hydratase (4.2.1.84)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种腈水合酶及其编码基因和应用,该腈水合酶由α亚基和β亚基组成,所述α亚基的氨基酸序列如SEQ ID NO.4所示,β亚基的氨基酸序列如SEQ ID NO.5所示。本发明从产酸克雷伯菌KCTC 1686中克隆到腈水合酶基因,该基因表达后成功获得具有高表达量和高活性而且底物谱广、具手性选择性的腈水合酶。
Description
技术领域
本发明涉及基因工程领域,尤其涉及一种腈水合酶及其编码基因和应用。
背景技术
腈是一类重要的化合物,它的水解反应被广泛应用于氨基酸、酰胺、羧酸及其衍生物的合成,在有机合成中占有及其重要的地位。腈水解的方法主要有化学水解法和生物转化法,与化学水解法相比生物转化法具有条件温和、环境污染小、并能实现化学、区域和对映体选择性等优点。生物转化法主要涉及腈水合酶、酰胺酶和腈水解酶。其中腈水合酶可以催化腈水合生成酰胺,酰胺酶进一步将酰胺催化水解生成羧酸化合物,而腈水解酶可以直接催化腈生成酰胺化合物。
产腈水合酶微生物的分布是相当广泛的,如红球菌、假单胞菌、诺卡氏菌、假诺卡氏菌、产碱杆菌、棒状杆菌等。其中一些微生物已经用于丙烯酰胺、烟酰胺的工业化生产中,如专利号为US007405064B2的专利公开了一种野生***丛毛单胞菌5-MGAM-4D;专利号ZL88106735的专利公开了一种野生玫瑰色红球菌J-1;专利号为ZL86100062的专利公开了一种红球菌S-6、氧化节杆菌和黄色微杆菌;专利号为ZL99106291.4的专利公开了一种野生嗜热假诺卡氏菌JCM3095;专利号为ZL03115536.7的专利公开了一种野生丙酸棒杆菌。
目前,利用微生物法生产丙烯酰胺、烟酰胺的方法主要从菌株的筛选和驯化以及生产工艺的建立和改造等方面开展工作。但是,其中普遍存在野生菌种的酶活稳定性差、对底物和产物的耐受性不高、产品质量不够稳定等问题,同时在野生菌株中除腈水合酶外,还存在酰胺酶和腈水解酶,其可以造成副产物烟酸和丙烯酸的生成,从而严重影响烟酰胺和丙烯酰胺的产量和质量,同时增加分离纯化的难度和生产成本。
随着生物技术的迅速发展,人们认识到用基因工程技术手段来构造具有腈水合酶活性的基因工程菌株可以用来解决上述的难题。用基因工程菌来表达腈水合酶有许多野生菌所不具有的优点,如可以单独克隆表达腈水合酶,阻断催化反应过程中副反应的发生,不会使酰胺在产生的同时又有部分被降解,从而提高产品的产量和质量;另外基因工程菌的适应性强、发酵周期短,有利于实现大规模培养和工业化生产。当前,重组腈水合酶的研究已经引起人们广泛的重视,经过众多科研工作者的多年努力也取得了相当多的成就,但是还存在着诸多问题,如酶的表达水平低、热稳定性不高、对底物和产物的耐受性差,这严重制约了腈水合酶的工业化应用。
发明内容
本发明提供了一种高表达量、高活性且底物谱广、具手性选择性的腈水合酶。
一种腈水合酶,由α亚基和β亚基组成,所述α亚基的氨基酸序列如SEQ ID NO.4所示,β亚基的氨基酸序列如SEQ ID NO.5所示。该腈水合酶的命名为NHaseK。
本发明还提供了一种编码所述的腈水合酶的基因。
优选的,所述的基因包含碱基序列如SEQ ID NO.1所示的编码α亚基的基因和如SEQ ID NO.2所示的编码β亚基的基因。该碱基序列来源于产酸克雷伯菌KCTC 1686(Klebsiella oxytoca KCTC 1686),由1278个碱基组成。自5’端第1位到609位碱基编码腈水合酶α亚基;自5’端第625位到1278位碱基编码腈水合酶β亚基。进一步,碱基序列如SEQID NO.7所示。
所述的基因还包含编码腈水合酶激活子的碱基序列。所述腈水合酶激活子的碱基序列如SEQ ID NO.3所示。为提高表达活性,所述基因经过修饰后连接有激活子的基因(SEQID NO.3)。进一步地,碱基序列如SEQ ID NO.8所示。
本发明还公开了一种腈水合酶激活子,氨基酸序列如SEQ ID NO.6所示。
本发明还公开了一种任一所述基因的表达盒、重组载体和转化子。
本发明又公开了一种所述的腈水合酶在制备酰胺化合物中的应用。
由于氨基酸序列的特殊性,任何含有SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示氨基酸序列的肽蛋白的片段或其变体,只要该肽蛋白的片段或肽蛋白变体与前述氨基酸序列同源性在90%以上,且具有相同的酶活力,均可实现本发明目的,属于本发明保护范围之列。
由于核苷酸序列的特殊性,任何含有SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示多核苷酸的变体,只要其与该多核苷酸具有90%以上同源性,均可实现本发明目的,属于本发明保护范围之列。
本发明提供了一种包含所述腈水合酶基因的重组载体和转化子。
本发明还提供了一种所述重组载体和转化子转化得到的基因工程菌。
本发明又提供了一种所述的腈水合酶在催化腈化合物生成酰胺中的应用。
所述的腈化合物为式(I)和(II)所示腈化合物生成对应酰胺中的应用:
式(I)~(II)中:
X:OH,H,NH2,烷基;
R:H,任选地被NH2取代的、分支的或不分支的、具有1-12个C原子的饱和的烷基;分子的或不分支的、具有双键和1-12个C原子的不饱和烷基;具有3-6个C原子的环烷基;
R′:H,具有1-3个C原子的烷基;
R″:单核或双核的不饱和环,其具有6-12个C原子,且其任选地被1个或2个Cl,Br,F取代;具有1-6个C原子的烷基腈基团。
本发明从产酸克雷伯菌KCTC 1686中克隆到腈水合酶基因,该基因表达后成功获得具有高表达量和高活性而且底物谱广、具手性选择性的腈水合酶。
附图说明
图1为本发明腈水合酶基因的PCR扩增电泳图;
M:核酸Marker;1和2:腈水合酶的PCR扩增产物。
图2为本发明重组质粒pET-30a(+)-NHaseK的图谱。
图3为本发明基因工程菌株E.coli BL21(DE3)/pET-30a(+)-NHaseK诱导表达产物的SDS-PAGE电泳图。
M:低分子量标准蛋白质;1:pET-30a(+)空载质粒对照破胞液;
2:基因工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK诱导后菌体破胞液;
3:基因工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK诱导菌体破胞上清液;
4:基因工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK诱导菌体破胞沉淀,箭头分别指示α亚基、β亚基和激活子P17K的位置。
图4为表达质粒pET-30a(+)-NHaseK的构建示意图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并非仅限于此。
实施例中的材料与方法如下:
本发明中的实验方法如无特别说明均为常规方法,具体可参见J.萨姆布鲁克等编写的《分子克隆实验指南》。
本发明实施例中所使用的限制性内切酶EcoRI、HindIII和T4 DNA连接酶购自TaKaRa,宝生物工程(大连)有限公司;基因组提取试剂盒、质粒提取试剂盒、DNA回收纯化试剂盒购自Axygen杭州有限公司;E.coli DH5α、E.coli BL21(DE3)、质粒pET-30a(+)购自Novagen公司;DNA marker、FastPfu DNA聚合酶、低分子量标准蛋白质、琼脂糖电泳试剂购自北京全式金生物技术有限公司;引物合成与序列测序工作由上海生工生物工程技术有限公司完成。以上试剂使用方法参考商品说明书。本发明采用的产酸克雷伯菌KCTC 1686(Klebsiella oxytoca KCTC 1686)购自韩国典型微生物保藏中心(Korean Collectionfor Type Cultures (KCTC))
实施例1
一、从产酸克雷伯菌KCTC 1686(Klebsiella oxytoca KCTC 1686)基因组中克隆腈水合酶和其激活子基因
根据Klebsiella oxytoca KCTC 1686基因组DNA序列(GenBank登录号:CP003218.1)设计引物NH-F和NH-R。
NH-F序列:5’-CCGGAATTCATGAGCCATAAACACGACCACG-3’
NH-R序列:5’-TTCCCAAGCTTGTTATGGTGTAACTCCATTATCG-3
在上、下游引物中分别加入限制性酶切位点EcoRI、HindIII(下划线所示)。以Klebsiella oxytoca KCTC 1686基因组DNA为模板,NH-F和NH-R为引物进行PCR扩增,PCR反应体系和反应条件如下:
PCR扩增体系:
PCR扩增条件:
1)预变性:95℃5min;
2)变性:98℃10s;退火:57℃15s;延伸:72℃60s;共循环30次;
3)延伸:72℃10min;
4)4℃保存2.0h。
用0.8%琼脂糖凝胶电泳检测PCR扩增产物,产物为单一条带,大小为1700bp左右(如图1所示)。用DNA回收纯化试剂盒对PCR扩增产物进行纯化回收,具体步骤参照该试剂盒说明书。
二、表达载体和工程菌的构建
先将纯化回收后的目的片段和提取的pET-30a(+)空质粒分别用限制性内切酶EcoRI和HindIII进行双酶切。之后用DNA回收纯化试剂盒对酶切产物进行纯化回收以去除限制性内切酶和酶切下来的核苷酸小片段。最后用T4 DNA连接酶将目的片段与pET-30a(+)质粒进行连接,连接体系如下表1所示:
表1 pET-30a(+)-NHaseK重组表达质粒连接体系
将上述各试剂进行轻轻混合,并放于16℃金属浴中连接12h。之后用连接产物转化E.coli DH5a感受态细胞,涂平板、挑单菌落进行LB液体培养,PCR法鉴定构建成功的阳性转化子。用Axygen质粒提取试剂盒从E.coli DH5a阳性转化菌株中提取重组质粒pET-30a(+)-NHaseK,并用其转化表达宿主E coli BL21(DE3)感受态细胞。用PCR法来验证转化的重组子,验证无误后的基因工程菌即为E.coli BL21(DE3)/pET-30a(+)-NHaseK。
三、重组腈水合酶的表达
将构建的基因工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK接于5mL含50μg/mlKan的LB液体培养基中,37℃震荡培养过夜。取1mL培养液转接至50mL同样含50μg/ml Kan的新鲜LB液体培养基中,37℃震荡培养至OD600达到0.8左右时,加入IPTG至其终浓度为0.5mM,20℃下诱导18h。其诱导表达产物的SDS-PAGE电泳图,如图3所示。
实施例2 基因工程菌催化丙烯腈生成丙烯酰胺
酶活力单位定义为:在反应条件下,每分钟催化底物反应产生1μmol产物的酶量。
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.5)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入1.5ml丙烯腈,35℃下进行水合反应,反应2小时。之后用气相色谱法检测反应体系内的丙烯腈和丙烯酰胺的含量。结果发现反应体系内已无丙烯腈残留,全部转化为丙烯酰胺。
实施例3 基因工程菌催化丁腈生成丁酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入7.0g丁腈,35℃下进行水合反应,反应2小时。之后用气相色谱法检测反应体系内的丁腈和丁酰胺的含量。结果发现反应体系内已无丁腈残留,全部转化为丁酰胺。
实施例4 基因工程菌催化甲基丙烯腈生成甲基丙烯酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入3.0g甲基丙烯腈,30℃下进行水合反应,反应2小时。之后用气相色谱法检测反应体系内的甲基丙烯腈和甲基丙烯酰胺的含量。结果发现反应体系内已无甲基丙烯腈残留,全部转化为甲基丙烯酰胺。
实施例5 基因工程菌催化3-氰基吡啶生成烟酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入5.0g 3-氰基吡啶,35℃下进行水合反应,反应2小时。之后用高效液相色谱法检测反应体系内的3-氰基吡啶和烟酰胺的含量。结果发现反应体系内已无3-氰基吡啶残留,全部转化为烟酰胺。所用高效液相色谱仪为Agilent 1100,色谱柱为C18柱(5μm,4.6×150mm)。
实施例6 基因工程菌催化苯甲腈生成苯甲酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入3.5g苯甲腈,35℃下进行水合反应,反应2小时。之后用高效液相色谱法检测反应体系内的苯甲腈和苯甲酰胺的含量。结果发现反应体系内已无苯甲腈残留,全部转化为苯甲酰胺。所用高效液相色谱仪为Agilent 1100,色谱柱为C18柱(5μm,4.6×150mm)。
实施例7 基因工程菌催化苯乙腈生成苯乙酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入3.0g苯乙腈,35℃下进行水合反应,反应2小时。之后用高效液相色谱法检测反应体系内的苯乙腈和苯乙酰胺的含量。结果发现反应体系内已无苯乙腈残留,全部转化为苯乙酰胺。所用高效液相色谱仪为Agilent 1100,色谱柱为C18柱(5μm,4.6×150mm)。
实施例8 基因工程菌催化α-甲基苯乙腈生成α-甲基苯乙酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入2.5g α-甲基苯乙腈,35℃下进行水合反应,反应30min。之后用高效液相色谱法来检测反应体系内的α-甲基苯乙腈和α-甲基苯乙酰胺的含量及其光学纯度。结果发现基因工程菌在催化外消旋α-甲基苯乙腈时,表现出S-异构体立体选择性。所用高效液相色谱仪为Agilent1100,色谱柱为AY-RH手性柱(5μm,4.6×150mm,CHIRALPAK)。
实施例9 基因工程菌催化2,2-二甲基环丙腈生成2,2-二甲基环丙酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入1.5g 2,2-二甲基环丙腈,35℃下进行水合反应,反应30min。之后用高效液相色谱法来检测反应体系内的2,2-二甲基环丙腈和2,2-二甲基环丙酰胺的含量及其光学纯度。结果发现基因工程菌在催化外消旋2,2-二甲基环丙腈时,表现出S-异构体立体选择性。所用高效液相色谱仪为Agilent 1100,色谱柱为AY-RH手性柱(5μm,4.6×150mm,CHIRALPAK)。
实施例10 基因工程菌催化2-(4-氯苯基)-3-甲基丁腈生成2-(4-氯苯基)-3-甲基丁酰胺
取25ml实施例1工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,然后用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液。向重悬液中加入1.0g 2-(4-氯苯基)-3-甲基丁腈,35℃下进行水合反应,反应30min。之后用高效液相色谱法来检测反应体系内的2-(4-氯苯基)-3-甲基丁腈和2-(4-氯苯基)-3-甲基丁酰胺的含量及其光学纯度。结果发现基因工程菌在催化外消旋2-(4-氯苯基)-3-甲基丁腈时,表现出S-异构体立体选择性。所用高效液相色谱仪为Agilent 1100,色谱柱为AY-RH手性柱(5μm,4.6×150mm,CHIRALPAK)。
对比例1
Fallon,R.d.(Applied Microbiology and Biotechnology,47,156-161,1997)等用恶臭假单胞菌NRRL-18668(Pseudomonas putida NRRL-18668)来催化2-(4-氯苯基)-3-甲基丁腈的水合反应。发现该野生菌含有的腈水合酶能催化2-(4-氯苯基)-3-甲基丁腈水合生成2-(4-氯苯基)-3-甲基丁酰胺,并表现出(S)型立体选择性,酶活力为7.1×10-3μmol/min/mg湿细胞。其最适反应温度为30℃,当温度超过30℃时酶蛋白开始快速失活。本发明的工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK催化2-(4-氯苯基)-3-甲基丁腈时,其酶活力达到40.0×10-3μmol/min/mg湿细胞。最适反应温度为35℃,在40℃以下酶蛋白表现出相当好的稳定性。
对比例2
Shun-Ichi Masutomo(Bioscience Biotechnology and Biochemistry,59(4),720-722,1995)等用假单胞菌B21C9(pseudomonas sp.B21C9)来催化2-(4-氯苯基)-3-甲基丁腈的水合反应。发现该野生菌含有的腈水合酶能催化2-(4-氯苯基)-3-甲基丁腈水合生成2-(4-氯苯基)-3-甲基丁酰胺,并表现出一定的(S)型立体选择性,但是其活性较低,仅为1.8×10-4μmol/min/mg干细胞。本发明的工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK催化2-(4-氯苯基)-3-甲基丁腈时,其酶活力达到7.8×10-2μmol/min/mg干细胞。
Claims (2)
1.一种利用腈水合酶催化2-(4-氯苯基)-3-甲基丁腈生成2-(4-氯苯基)-3-甲基丁酰胺的方法,其特征在于,所述腈水合酶的编码基因的碱基序列如SEQ ID NO.7所示;
所述方法,包括以下步骤:
(1)克隆腈水合酶和其激活子基因:根据Klebsiella oxytoca KCTC 1686基因组DNA序列设计引物NH-F和NH-R,所述DNA序列的GenBank登录号为CP003218.1;
NH-F序列:5’-CCGGAATTCATGAGCCATAAACACGACCACG-3’;
NH-R序列:5’-TTCCCAAGCTTGTTATGGTGTAACTCCATTATCG-3’;
以Klebsiella oxytoca KCTC 1686基因组DNA为模板,NH-F和NH-R为引物进行PCR扩增;对PCR扩增产物进行纯化回收;
(2)表达载体和工程菌的构建:将纯化回收后的目的片段和提取的pET-30a(+)空质粒分别用限制性内切酶EcoRI和HindIII进行双酶切;之后用DNA回收纯化试剂盒对酶切产物进行纯化回收以去除限制性内切酶和酶切下来的核苷酸小片段;再用T4DNA连接酶将目的片段与pET-30a(+)质粒进行连接;之后用连接产物转化E.coli DH5a感受态细胞,涂平板、挑单菌落进行LB液体培养,PCR法鉴定构建成功的阳性转化子;从E.coli DH5a阳性转化菌株中提取重组质粒pET-30a(+)-NHaseK,并用其转化表达宿主E coli BL21(DE3)感受态细胞;验证无误后的基因工程菌即为E.coli BL21(DE3)/pET-30a(+)-NHaseK;
(3)重组腈水合酶的表达:将构建的基因工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK接于5mL含50μg/ml Kan的LB液体培养基中,37℃震荡培养过夜;取1mL培养液转接至50mL同样含50μg/ml Kan的新鲜LB液体培养基中,37℃震荡培养至OD600达到0.8时,加入IPTG至其终浓度为0.5mM,20℃下诱导18h;
(4)基因工程菌催化2-(4-氯苯基)-3-甲基丁腈生成2-(4-氯苯基)-3-甲基丁酰胺:取25ml工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的发酵液,10000rpm,10min离心收集菌体,用250ml 50mM Tris-HCl(pH 7.0)缓冲液重悬菌体细胞,即得工程菌E.coli BL21(DE3)/pET-30a(+)-NHaseK的静息细胞悬液;向静息细胞悬液中加入1.0g 2-(4-氯苯基)-3-甲基丁腈,35℃下进行水合反应,反应30min。
2.如权利要求1所述的方法,其特征在于,所述腈水合酶的编码基因还包含编码腈水合酶激活子的序列,其碱基序列如SEQ ID NO.3所示。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410621651.2A CN104450657B (zh) | 2014-11-06 | 2014-11-06 | 腈水合酶及其编码基因和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410621651.2A CN104450657B (zh) | 2014-11-06 | 2014-11-06 | 腈水合酶及其编码基因和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104450657A CN104450657A (zh) | 2015-03-25 |
CN104450657B true CN104450657B (zh) | 2017-10-03 |
Family
ID=52897411
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201410621651.2A Active CN104450657B (zh) | 2014-11-06 | 2014-11-06 | 腈水合酶及其编码基因和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104450657B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021214283A1 (en) * | 2020-04-24 | 2021-10-28 | Pharmazell Gmbh | Enantioselective chemo-enzymatic synthesis of optically active amino amide compounds |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104862296B (zh) * | 2015-04-22 | 2017-12-12 | 江南大学 | 一种比酶活和稳定性提高的融合型腈水合酶 |
CN107881163A (zh) * | 2017-09-29 | 2018-04-06 | 浙江大学 | 一种耐热腈水合酶、工程菌及其在催化腈化合物水合反应生成酰胺中的应用 |
CN107916271B (zh) * | 2017-09-29 | 2018-09-28 | 浙江大学 | 一种重组腈水合酶的高效表达方法 |
CN110396494A (zh) * | 2019-08-01 | 2019-11-01 | 安徽瑞邦生物科技有限公司 | 一种低副产物吡啶甲酰胺转化微生物的制备及应用 |
CN112322607B (zh) * | 2020-11-20 | 2023-03-28 | 江南大学 | 一种融合型腈水合酶及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1584024A (zh) * | 2004-05-24 | 2005-02-23 | 清华大学 | 一种腈水合酶及其编码基因与应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040225116A1 (en) * | 2003-05-08 | 2004-11-11 | Payne Mark S. | Nucleic acid fragments encoding nitrile hydratase and amidase enzymes from comamonas testosteroni 5-MGAM-4D and recombinant organisms expressing those enzymes useful for the production of amides and acids |
-
2014
- 2014-11-06 CN CN201410621651.2A patent/CN104450657B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1584024A (zh) * | 2004-05-24 | 2005-02-23 | 清华大学 | 一种腈水合酶及其编码基因与应用 |
Non-Patent Citations (1)
Title |
---|
Klebsiella oxytoca KCTC 1686,complete genome;genbank;《genbank》;20140131;gene,cds * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021214283A1 (en) * | 2020-04-24 | 2021-10-28 | Pharmazell Gmbh | Enantioselective chemo-enzymatic synthesis of optically active amino amide compounds |
Also Published As
Publication number | Publication date |
---|---|
CN104450657A (zh) | 2015-03-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104450657B (zh) | 腈水合酶及其编码基因和应用 | |
Prasad et al. | Nitrile hydratases (NHases): at the interface of academia and industry | |
Sharma et al. | Amidases: versatile enzymes in nature | |
CN104561064B (zh) | 一种腈水合酶基因、编码酶、载体、工程菌及其制备酰胺化合物的应用 | |
Velankar et al. | Developments in nitrile and amide biotransformation processes | |
Jiao et al. | Advances in acrylamide bioproduction catalyzed with Rhodococcus cells harboring nitrile hydratase | |
CN107881163A (zh) | 一种耐热腈水合酶、工程菌及其在催化腈化合物水合反应生成酰胺中的应用 | |
Nigam et al. | Bioengineering of nitrilases towards its use as green catalyst: applications and perspectives | |
US11332731B2 (en) | Nitrile hydratase mutant, genetically engineered bacterium containing mutant and applications thereof | |
CN104830747B (zh) | 一种高效表达高分子量型腈水合酶的基因工程菌及其应用 | |
EP2893026B1 (en) | Process for preparing sebacic acid | |
Kang et al. | High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production | |
US20210388336A1 (en) | Mutant of Nitrile Hydratase Derived from Caldalkalibacillus thermarum | |
CN104293752A (zh) | 重组酰胺酶Dt-Ami 2、编码基因、载体、工程菌及应用 | |
CN104498466B (zh) | 腈水合酶及其应用 | |
WO1999055719A1 (en) | Method for producing amide compounds using a nitrile hydratase from a thermophilic bacillus | |
Kim et al. | Enhanced stability and reusability of marine epoxide hydrolase using ship-in-a-bottle approach with magnetically-separable mesoporous silica | |
Padmakumar et al. | Bioconversion of acrylonitruke to acrylamide using a thermostable nitrile hydratase | |
CN104404011B (zh) | 酰胺酶及其编码基因和应用 | |
CN106244569B (zh) | 一种酯酶EstC10及其编码基因和应用 | |
Ikemi | Industrial chemicals: enzymatic transformation by recombinant microbes | |
CN104774829B (zh) | 一种比酶活和稳定性提高的融合型腈水合酶 | |
JP3408737B2 (ja) | ニトリルヒドラターゼの活性化に関与するタンパク質及びそれをコードする遺伝子 | |
Fan et al. | Characterization of a novel nitrilase, BGC4, from Paraburkholderia graminis showing wide-spectrum substrate specificity, a potential versatile biocatalyst for the degradation of nitriles | |
Luo et al. | Gene cloning, overexpression, and characterization of the nitrilase from Rhodococcus rhodochrous tg1-A6 in E. coli |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |