WO2020155690A1 - 一种新型腈水合酶高效催化脂肪二腈水合反应的体系 - Google Patents

一种新型腈水合酶高效催化脂肪二腈水合反应的体系 Download PDF

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WO2020155690A1
WO2020155690A1 PCT/CN2019/113301 CN2019113301W WO2020155690A1 WO 2020155690 A1 WO2020155690 A1 WO 2020155690A1 CN 2019113301 W CN2019113301 W CN 2019113301W WO 2020155690 A1 WO2020155690 A1 WO 2020155690A1
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nitrile hydratase
reaction
ccm2595
rhodococcuserythropolis
culture
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French (fr)
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梁长海
王黎
刘胜先
窦同意
崔昌浩
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大连理工大学
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Priority to US16/772,063 priority Critical patent/US11421214B2/en
Priority to ZA2020/03389A priority patent/ZA202003389B/en
Publication of WO2020155690A1 publication Critical patent/WO2020155690A1/zh

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/002—Nitriles (-CN)
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    • 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
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01084—Nitrile hydratase (4.2.1.84)

Definitions

  • the present invention belongs to the technical field of green chemistry, and specifically relates to the application of a nitrile hydratase derived from Rhodococcuserythropolis CCM2595 in the aspect of regioselectively catalyzing fatty dinitrile to generate cyanoamide.
  • Nitrile hydratase (EC4.2.1.84, nitrile hydratase, NHase for short) is a type of metalloenzyme that catalyzes the formation of amides from nitriles.
  • Nitrile hydratase can catalyze the production of acrylamide, nicotinamide, 5-cyanovaleramide, etc. from nitriles, and is widely used in the production of fine chemicals, especially pesticides and organic solvents.
  • Nitriles enter people's lives mainly through sewage discharge from their production units, herbicides or accidental leakage. Many nitriles are neurotoxic and can cause cancer and teratogenesis in humans or animals. Therefore, nitrile hydratase also plays an important role in the field of environmental protection, which can degrade nitriles in the environment.
  • 5-Cyanovaleramide can be used to synthesize fentrazone and 6-aminocaproamide.
  • zonflufenazone can be used as a herbicide before plant seeds are unearthed.
  • 6-aminocaproamide is important for the synthesis of caprolactam.
  • the intermediate, caprolactam is an important chemical raw material.
  • 5-cyanovaleramide can be prepared by chemical catalysis or biocatalysis of adiponitrile hydration reaction. Adiponitrile contains 2 cyano groups, and the catalyst is required to regioselectively hydrate 1 cyano group to form 5-cyanovaleramide.
  • biocatalysis Compared with the harsh reaction conditions of chemical catalysis (high temperature and high pressure copper catalysis, etc.) and the formation of a large number of by-products, biocatalysis has the advantages of rapid and mild reaction, strong specificity, high selectivity, economy and environmental protection.
  • Rhodococcuserythropolis selected in the present invention CCM2595 belongs to Rhodococcus and has only been reported to have the ability to degrade phenol derivatives [Strnad H, Patek M, Fousek J, Szokol J, Ulbrich P, Nesvera J, Paces V, Vlcek C: Genome Sequence of Rhodococcuserythropolis Strain CCM2595, a Phenol Derivative-Degrading Bacterium. Genome announcements 2014, 2(2)], and its catalytic reaction of nitrile hydration of aliphatic dinitriles has not been reported yet.
  • the present invention provides Rhodococcuserythropolis Application of CCM2595 nitrile hydratase in catalyzing fatty dinitrile to cyanoamide.
  • a novel nitrile hydratase system that efficiently catalyzes the hydration reaction of fatty dinitrile.
  • concentration of nitrile hydratase bacteria is 1-3g/L
  • the final concentration of the substrate fatty dinitrile is 20-50mM/L
  • the conversion system is pH 7-8 PBS buffer solution, 25°C, 200
  • the reaction was oscillated at rpm. After 5 minutes of reaction, an equal volume of methanol was added to terminate the reaction. After centrifugation, the supernatant was taken. After filtration, it was tested by high performance liquid chromatography. The selectivity to the cyanoamide product was greater than 90%. Specific steps are as follows:
  • Plasmid construction The nucleotide sequence of the nitrile hydratase derived from RhodococcuserythropolisCCM2595 is:
  • the sequence contains 2596 nucleotides.
  • the plasmid pET-24a(+) is used as the expression vector. According to its restriction site characteristics, NdeI and HindIII restriction sites are selected to insert the ReNHase gene fragment.
  • the ReNHase gene fragment is derived from polymerase. The purified product obtained by polymerase chain reaction (PCR); after digestion, the corresponding DNA fragments are recovered and purified. The lacI promoter is selected, the KanR resistance gene fragment is added, and the T7 terminator is selected for transformation. Escherichia coli TOP10, the recombinant plasmid was named G0130349-1 after enzyme digestion verification;
  • IPTG isopropylthiogalactoside
  • the cell concentration is 1-3g/L
  • the final concentration of the substrate fatty dinitrile is 20-50mM/L
  • the conversion system is pH 7-8 PBS buffer solution, 25°C, 200
  • the reaction was oscillated at rpm, and the reaction time was controlled to be 5min-24h.
  • the reaction was terminated by adding equal reaction volume of methanol. The supernatant was taken after centrifugation, and the sample was filtered and sent for high performance liquid chromatography.
  • the present invention has discovered that it is derived from Rhodococcuserythropolis CCM2595's nitrile hydratase is a brand-new application in catalyzing aliphatic dinitriles.
  • This enzyme can selectively catalyze aliphatic dinitriles to form cyanoamides with high reaction rate and simple and mild reaction conditions, providing a method for the industrial production of cyanoamides.
  • Figure 1 is a map of recombinant plasmid pET-24a(+)-ReNHase.
  • Figure 2 is an SDS-PAGE electrophoresis diagram of ReNHase protein induced expression.
  • LaneA negative control
  • Lane B 37°C induced fragmentation (BL21(DE3));
  • Lane C Induced and disrupted supernatant at 37°C (BL21(DE3));
  • Lane D 16°C induced fragmentation (Arctic Expression (DE3));
  • Lane E 16°C induced fragmentation of the supernatant (Arctic Expression (DE3)).
  • Figure 3 shows the HPLC spectra of samples and products.
  • Figure 4 shows the enzyme activity of product 5-CVAM over time.
  • the OD value is about 0.6.
  • One tube of BL21 (DE3) without IPTG was used as a negative control, and one tube was added with IPTG to a final concentration of 1 mM/L, and induced at 37°C for 3 hours.
  • centrifuge at 12000 rpm, 1min to remove the supernatant to collect the bacteria, add buffer (20mM PB, 150mM NaCl, pH7.4) at 300W power, 4s, 6s interval, a total of 30 cycles to disrupt the bacteria.
  • the target protein subunit (about 27KDa) has soluble expression in the supernatant.
  • Seed culture Pick a single Arctic Expression (DE3) colony and inoculate it in a 50 ⁇ g/ml Kan 4ml LB broth shaker tube, shake culture at 37°C 220rpm for 24h.
  • Induction culture Inoculate 2ml of bacterial solution into an Erlenmeyer flask containing 50 ⁇ g/ml Kan of 200ml LB culture solution, shake culture at 37°C 220rpm for about 3h until the OD value is 0.6-0.8, add IPTG to the end The concentration was 0.1mM/L, and the induction was carried out at 16°C and 220rpm for 24h.
  • Liquid phase detection add 150 ⁇ l of bacterial solution to 300 ⁇ l of PBS buffer, then add 50 ⁇ l of 200mM adiponitrile substrate, and shake the reaction at 25°C at 200 rpm. Control the reaction time to 5min, 10min, 15min, 30min, For 1h, 2h, 3h, 6h, 15h, 24h, 500 ⁇ l methanol was added to stop the reaction after the reaction, and the supernatant was centrifuged at 13000rpm for 10min, filtered through a 0.22 ⁇ m membrane filter and sent for HPLC detection.
  • HPLC detection method Ultimate 5 ⁇ m 4.6 ⁇ 250mm LP-C18 column, mobile phase is 25mM phosphoric acid aqueous solution and methanol (89:11, vol:vol), detection wavelength is 200nm, column temperature is 25°C, flow rate is 1ml/min.
  • Example 2 step (4) refer to the above-mentioned Example 2 step (4) conditions for the reaction.
  • the final concentration of adiponitrile is 50mM
  • the initial total enzyme activity of the product 5-cyanovaleramide can reach 4269U.
  • the enzyme activity Gradually decreases. It shows that the enzyme can efficiently and shortly catalyze the formation of 5-cyanovaleramide from adiponitrile.

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Abstract

提供了一种新型腈水合酶高效催化脂肪二腈水合反应的体系。提供了来自红球菌Rhodococcus erythropolis CCM2595的腈水合酶在催化脂肪族二腈方面的全新应用。尤其该酶可选择性催化己二腈生成5-氰基戊酰胺，反应速率高，反应条件简单温和，为5-氰基戊酰胺的工业生产提供方法。

Description

一种新型腈水合酶高效催化脂肪二腈水合反应的体系 技术领域
本发明属于绿色化学技术领域，具体涉及来源于红球菌RhodococcuserythropolisCCM2595的腈水合酶在区域选择性催化脂肪二腈生成氰基酰胺方面的应用。
 背景技术
腈水合酶(EC4.2.1.84, nitrile hydratase, 简称NHase) 是一类催化腈类化合物生成酰胺类化合物的金属酶。腈水合酶可催化腈类物质生产丙烯酰胺，烟酰胺、5-氰基戊酰胺等，在精细化学品尤其是农药和有机溶剂生产中应用广泛。腈类物质主要通过其生产单位的污水排放，除草剂或是意外泄露等方式进入人们生活。许多腈类都具有神经毒性，可使人或动物致癌、致畸。因此腈水合酶在环保领域也有重要作用，可以降解环境中的腈类物质。
5-氰基戊酰胺（5-CVAM）可用于合成唑啶草酮和6-氨基己酰胺，其中唑啶草酮可用于植物种子出土前的除草剂，6-氨基己酰胺是合成己内酰胺的重要中间体，己内酰胺是重要的化工原料。5-氰基戊酰胺可通过化学催化或生物催化己二腈发生水合反应予以制备。己二腈中含有2个氰基，要求催化剂能够区域选择性水合1个氰基生成5-氰基戊酰胺。相比于化学催化法反应条件苛刻（高温高压铜催化等）同时伴有大量副产物生成，生物催化具有反应快速温和、专一性强、选择性高、经济环保等优点。
目前根据权威生物合成催化酶数据库BRENDA表明，具有区域选择性能够部分催化己二腈生成5-氰基戊酰胺的微生物非常少，仅有假单胞菌属Pseudomonas chlororaphis。因此发现可用于工业快速高效生产5-氰基戊酰胺的新型腈水合酶是十分重要的。
本发明选用的Rhodococcuserythropolis CCM2595属红球菌，目前仅被报道具有降解苯酚衍生物的能力[Strnad H, Patek M, Fousek J, Szokol J, Ulbrich P, Nesvera J, Paces V, Vlcek C: Genome Sequence of Rhodococcuserythropolis Strain CCM2595, a Phenol Derivative-Degrading Bacterium. Genome announcements 2014, 2(2)]，而其对脂肪族二腈的腈水合催化反应尚未有报道。
 技术问题
本发明提供了来源于红球菌Rhodococcuserythropolis CCM2595的腈水合酶在催化脂肪二腈生成氰基酰胺方面的应用。
 技术解决方案
本发明的技术方案：
一种新型腈水合酶高效催化脂肪二腈水合反应的体系，腈水合酶菌体浓度1-3g/L,底物脂肪二腈终浓度20-50mM/L,转化体系为pH 7-8 PBS缓冲溶液,25℃、200 rpm振荡反应,反应5min后加入等反应体积甲醇终止反应，离心后取上清液，经过滤后进行高效液相色谱检测, 对氰基酰胺产物生成的选择性大于90%。具体步骤如下：
（1）  质粒构建：来源于红球菌RhodococcuserythropolisCCM2595的腈水合酶的核苷酸序列为：
1CATATGTCAG TAACGATCGA CCACACAACG GAGAACGCCG CACCGGCCCA
51 GGCGCCGGTC TCCGATCGCG CGTGGGCCCT GTTCCGCGCA CTCGACGGTA
101 AGGGATTGGT ACCCGACGGT TACGTCGAGG GATGGAAGAA GACCTTCGAG
151 GAGGACTTCA GTCCAAGGCG CGGAGCGGAA TTGGTCGCGC GGGCTTGGAC
201 CGACCCCGAT TTCCGGCAAC TGCTTCTCAC CGACGGTACC GCCGCGGTTG
251 CCCAGTACGG ATATCTGGGC CCCCAGGGCG AATACATCGT GGCAGTCGAA
301 GACACCCCGA CCCTCAAGAA CGTGATCGTG TGCTCGCTGT GTTCATGCAC
351 CGCGTGGCCC ATCCTCGGTC TGCCGCCGAC CTGGTACAAG AGTTTCGAAT
401 ACCGTGCACG CGTGGTCCGC GAGCCACGGA AGGTTCTCTC CGAGATGGGA
451 ACCGAGATCG CGTCGGACGT CGAGATCCGC GTCTACGACA CCACCGCCGA
501 AACTCGGTAC ATGGTCCTAC CGCAACGTCC CGCAGGCACC GAAGGCTGGA
551 GCCAGGAACA ACTGCAGGAA ATCGTCACCA AGGACTGCCT GATCGGCGTC
601 GCAGTCCCGC AGGTCCCCAC CGTCTGACCA CCCCGACAAG AAAGAAGCAC
651 ACCATGGATG GAGTACACGA TCTTGCCGGA GTTCAAGGCT TCGGCAAAGT
701 CCCGCATACC GTCAACGCCG ACATCGGCCC CACCTTCCAC GCCGAGTGGG
751 AACACCTGCC GTACAGCCTG ATGTTCGCCG GTGTCGCCGA ACTCGGGGCC
801 TTCAGCGTCG ACGAAGTTCG ATACGTCGTC GAGCGGATGG AGCCCCGCCA
851 CTACATGATG ACCCCGTACT ACGAGCGGTA CGTCATCGGC GTCGCGGCGC
901 TGATGGTCGA AAAGGGAATC CTGACGCAGG AAGAGCTCGA AAGCCTTGCA
951 GGAGGACCGT TCCCACTCTC ACGGCCAAGC GAATCCGAAG GCCGACCGGC
1001 TCGCGTCGAC ACAACCACCT TCGAGGTCGG TCAGCGAGTA CGTGTGCGAG
1051 ACGAATACGT TCCCGGGCAT ATTCGAATGC CTGCTTACTG CCGAGGACGG
1101 GTGGGGACCA TCGCTCACCG GACCACCGAG AAGTGGCCGT TCCCCGACGC
1151 AATCGGTCAC GGCCGCAACG ACGCCGGCGA AGAACCCACC TACCACGTGA
1201 CGTTCGCTGC GGAGGAATTG TTCGGCAGCG ACACCGACGG CGGAAGCGTC
1251 GTTGTCGACC TCTTCGAGGG TTACCTCGAG CCTGCGGCCT GATCTTCCAG
1301 CATTCCAGGC GGCGGTCACG CGATCGCAGC GGTTCGCGTG ACCGCCGCCT
1351 GATCACAACG ATTCACTCAT TCGGAAGGAC ACTGGAAATC ATGGTCGACA
1401 CACGACTTCC GGTCACGGTG CTGTCAGGTT TCCTGGGCGC CGGGAAGACG
1451 ACGCTACTCA ACGAGATCCT GCGCAATCGG GAGGGCCGCC GGGTTGCGGT
1501 GATCGTCAAC GACATGAGCG AAATCAACAT CGACAGTGCA GAAGTCGAGC
1551 GTGAGATCTC GCTCAGTCGC TCCGAGGAGA AACTGGTCGA GATGACCAAC
1601 GGCTGCATCT GCTGCACTCT GCGAGAGGAT CTTCTTTCCG AGATAAGCGC
1651 CTTGGCCGCC GATGGCCGAT TCGACTACCT TCTCATCGAA TCTTCGGGCA
1701 TCTCCGAACC GCTGCCCGTC GCGGAGACGT TCACCTTCAT CGATACCGAC
1751 GGCCATGCCC TGGCCGACGT CGCCCGACTC GACACCATGG TCACAGTCGT
1801 CGACGGCAAC AGTTTTCTGC GCGACTACAC GGCTGGAGGT CGCGTCGAAG
1851 CCGATGCCCC GGAAGATGAA CGCGACATCG CGGATCTGCT TGTCGATCAG
1901 ATCGAGTTTG CCGACGTCAT CCTGGTGAGC AAGGCCGATC TCGTCTCGCA
1951 CCAGCACCTG GTCGAATTGA CTTCGGTCCT AAGATCTTTG AACGCAACTG
2001 CTGCGATAGT TCCGATGACT CTCGGCCGTA TCCCACTCGA CACGATTCTC
2051 GATACCGGCT TGTTCTCGCT CGAGAAAGCT GCTCAGGCCC CTGGATGGCT
2101 ACAAGAACTC CAAGGTGAAC ACACCCCCGA AACCGAGGAG TACGGAATCG
2151 GTTCGGTGGT GTACCGCGAG CGCGCGCCCT TCCACCCACA ACGCCTGCAT
2201 GATTTCCTGA GCAGCGAGTG GACCAACGGA AAGTTACTTC GGGCCAAGGG
2251 CTACTACTGG AATGCCGGCC GGTTCACCGA GATCGGGAGT ATTTCTCAGG
2301 CCGGTCATCT CATTCGCCAC GGATACGTCG GCCGTTGGTG GAAGTTTCTA
2351 CCCCGTGACG AGTGGCCGGC CGACGACTAC CGTCGCGACG GAATCCTCGA
2401 CAAGTGGGAA GAACCTGTCG GTGACTGCCG ACAAGAACTC GTCTTCATCG
2451 GCCAATCCAT CGACCCATCT CGACTGCACC GAGAACTCGA CGCGTGTCTA
2501 CTCACCACAG CCGAGATCGA ACTCGGGCCA GACGTGTGGA CCACCTGGAG
2551 CGACCCCCTG GGCGTCGGCT ATACCGACCA GACCGTTTGA AAGCTT
该序列含有2596个核苷酸，以质粒pET-24a(+)为表达载体，根据其酶切位点特性，选择NdeI和HindIII酶切位点插入ReNHase基因片段，ReNHase基因片段来源于经过聚合酶链式反应简称 (Polymerase Chain Reaction，PCR)得到的纯化产物；经过酶切后对相应DNA片段进行回收纯化，选用lacI启动子，加入卡那霉素KanR抗性基因片段，选用T7终止子，转化大肠杆菌TOP10，重组质粒经过酶切验证后命名为G0130349-1；
（2）蛋白表达验证：将步骤（1）中得到的重组质粒平行转化入BL21(DE3)和Arctic Expression(DE3)两种大肠杆菌感受态细菌中，加入LB液体培养基扩增培养后分别涂布于含50μg/ml 卡那霉素（Kan）的LB固体平板，37℃倒置培养24h；分别挑取平板上单菌落接种于LB液体培养基中，培养至OD值为0.6-0.8时加入终浓度为0.1-1mM/L的异丙基硫代半乳糖苷(IPTG)进行诱导3-24h；诱导完成后，离心收集菌体，洗涤后超声破碎菌体，进行SDS-PAGE分析，验证NHase蛋白表达情况；
（3）制备菌液：挑取步骤(2)中Arctic Expression(DE3)平板上单菌落接种于少量含50μg/ml Kan的LB液体培养基中，37℃、220rpm振荡培养12-18h得种子液；将种子液按体积1%接种量继续接种到含50μg/ml Kan的LB液体培养基中，37℃振荡培养至OD值为0.6-0.8时，加入IPTG使其终浓度为0.1mM/L，16℃、220rpm振荡培养24h得发酵液；离心去上清收集菌体，用pH7-8的PBS缓冲液洗涤2-3次后重新悬浮，得待用菌液；
（4）催化脂肪二腈水合反应：菌体浓度1-3g/L,底物脂肪二腈终浓度20-50mM/L,转化体系为pH 7-8 PBS缓冲溶液,25℃、200 rpm振荡反应,控制反应时间为5min-24h,加入等反应体积的甲醇终止反应，离心后取上清液，经过滤后送样进行高效液相色谱检测。
 有益效果
本发明的有益效果：本发明发现了来自红球菌Rhodococcuserythropolis CCM2595的腈水合酶在催化脂肪族二腈方面的全新应用，该酶可选择性催化脂肪二腈生成氰基酰胺，反应速率高，反应条件简单温和，为氰基酰胺的工业生产提供方法。
 附图说明
图1为重组质粒pET-24a(+)-ReNHase的图谱。
图2为ReNHase蛋白诱导表达SDS-PAGE电泳图。
图中：LaneA: 阴性对照；
Lane B: 37℃诱导破碎沉淀（BL21(DE3)）；
Lane C: 37℃诱导破碎上清（BL21(DE3)）；
Lane D: 16℃诱导破碎沉淀（Arctic Expression(DE3)）；
Lane E:16℃诱导破碎上清（Arctic Expression(DE3)）。
Figure dest_path_image001
图3 为样品及产物的HPLC谱图。
图中：（a）己二酰二胺标样；（b）5-氰基戊酰胺标样；（c）ReNHase催化己二腈反应5min的产物；（d）ReNHase催化己二腈反应30min的产物。
图4 为产物5-CVAM的酶活随时间变化曲线。
 本发明的实施方式
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。
实施例1ReNHase蛋白表达验证
将质粒1μl分别加入100μl BL21(DE3)和Arctic Expression(DE3)感受态细菌中，置于冰上20min，42℃热激90 sec后迅速置冰中3 min，加入600μl LB液体培养液，37℃220 rpm振摇培养1 h。取200μl菌液涂布于含50 μg/ml Kan的LB平板，37℃倒置培养24h。次日，挑取2个BL21(DE3)和1个Arctic Expression(DE3)的单菌落分别接种于含50μg/ml Kan的4 ml LB培养液的摇菌管中，37℃ 220 rpm振摇培养至OD值约0.6。BL21(DE3)一管不加IPTG做阴性对照，一管加IPTG至终浓度为1mM/L，37℃诱导3小时。Arctic Expression(DE3)的单管加IPTG至终浓度0.1mM/L，16℃诱导24h。次日，12000 rpm，1min离心去上清收集菌体，加入缓冲液（20mM PB，150mM NaCl，pH7.4）以300W功率，破碎4s，间隔6s，共30个循环破碎菌体。进行SDS-PAGE分析，选用12%分离胶及5%浓缩胶，电泳条件为先80V运行20min后160V 运行100min。如图2所示，目的蛋白亚基（约27KDa）在上清液中有可溶性表达。
实施例2腈水合酶催化己二腈反应
（1）种子培养：挑1个Arctic Expression(DE3)单菌落接种于50μg/ml Kan的4ml LB培养液的摇菌管中，37℃ 220rpm振摇培养24h。
（2）诱导培养：取2ml菌液接种到装有50μg/ml Kan的200ml LB培养液的锥形瓶中，37℃ 220rpm振摇培养约3h至OD值为0.6-0.8，加入IPTG至其终浓度为0.1mM/L, 16℃ 220rpm诱导24h。
（3）收集菌体：3000rpm10min离心去上清培养液，用pH7.4的PBS缓冲液洗涤菌体2次，最终用10ml PBS缓冲液重悬菌体。
（4）液相检测：将150μl菌液加入300μl PBS缓冲液中，然后加入50μl 浓度为200mM己二腈底物，25℃200 rpm振荡反应，控制反应时间分别为5min、10min、15min、30min、1h、2h、3h、6h、15h、24h，反应结束后加入500μl甲醇终止，13000rpm10min离心取上清液，经0.22μm滤膜过滤后送样进行高效液相色谱检测。HPLC检测方法：Ultimate 5μm 4.6Í250mm 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。
如图3所示，（a）己二酰二胺标准品出峰时间为4.4min；（b）5-氰基戊酰胺标准品出峰时间为7.1min；（c）按照上述实例2步骤（4）条件反应5min时，己二腈转化率为100%，5-氰基戊酰胺选择性≥90%；（d）随着反应时间增加，5-氰基戊酰胺含量降低，己二酰二胺含量上升，说明该酶可以分步催化己二腈。
如图4所示，参考上述实例2步骤（4）条件进行反应，当己二腈终浓度50mM时,产物5-氰基戊酰胺初始总酶活可达4269U，随着反应时间增长，酶活逐渐降低。说明该酶可以高效短时催化己二腈生成5-氰基戊酰胺。

Claims (6)

  1. 一种新型腈水合酶高效催化脂肪二腈水合反应的体系，其特征在于，来源于红球菌Rhodococcuserythropolis CCM2595的腈水合酶菌体浓度1-3g/L,底物脂肪族二腈终浓度20-50mM/L,转化体系为pH 7-8 PBS缓冲溶液,25℃、200 rpm振荡反应，反应5min后加入等反应体积甲醇终止反应，离心后取上清液，经过滤后进行高效液相色谱检测,氰基酰胺选择性大于90%。
  2. 根据权利要求1所述的体系，其特征在于，所述的腈水合酶菌体制备步骤如下：
    （1）质粒构建：来源于红球菌Rhodococcuserythropolis CCM2595的腈水合酶的基因序列含有2596个核苷酸，以质粒pET-24a(+)为表达载体，根据其酶切位点特性，选择NdeI和HindIII酶切位点插入ReNHase基因片段，ReNHase基因片段来源于经过PCR得到的纯化产物；经过酶切后对相应DNA片段进行回收纯化，选用lacI启动子，加入卡那霉素KanR抗性基因片段，选用T7终止子，转化大肠杆菌TOP10，重组质粒经过酶切验证后命名为G0130349-1；
    （2）蛋白表达验证：将步骤（1）中得到的重组质粒平行转化入BL21(DE3)和Arctic Expression(DE3)两种大肠杆菌感受态细菌中，加入LB液体培养基扩增培养后分别涂布于含50μg/ml 卡那霉素（Kan）的LB固体平板，37℃倒置培养24h；分别挑取平板上单菌落接种于LB液体培养基中，培养至OD值为0.6-0.8时加入终浓度为0.1-1mM/L的异丙基硫代半乳糖苷(IPTG)进行诱导3-24h；诱导完成后，离心收集菌体，洗涤后超声破碎菌体，进行SDS-PAGE分析，验证NHase蛋白表达情况；
    （3）制备菌液：挑取步骤(2)中Arctic Expression(DE3)平板上单菌落接种于少量含50μg/ml Kan的LB液体培养基中，37℃、220rpm振荡培养12-18h得种子液；将种子液按体积1%接种量继续接种到含50μg/ml Kan的LB液体培养基中，37℃振荡培养至OD值为0.6-0.8时，加入IPTG使其终浓度为0.1mM/L，16℃、220rpm振荡培养24h得发酵液；离心去上清收集菌体，用pH7-8的PBS缓冲液洗涤2-3次后重新悬浮，得待用菌液。
  3. 根据权利要求1或2所述的体系，其特征在于，所述的脂肪族二腈包括己二腈、丙二腈、丁二腈和癸二腈。
  4. 来源于红球菌Rhodococcuserythropolis CCM2595的腈水合酶在生产方面的应用。
  5. 来源于红球菌Rhodococcuserythropolis CCM2595的腈水合酶在催化脂肪族二腈反应中的应用。
  6. 来源于红球菌Rhodococcuserythropolis CCM2595的腈水合酶在制备5-氰基戊酰胺中的应用。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101619299A (zh) * 2009-07-08 2010-01-06 天津科技大学 一种赤红球菌以及利用该菌制备5-氰基戊酰胺的方法
CN109797164A (zh) * 2019-01-31 2019-05-24 大连理工大学 一种新型腈水合酶高效催化脂肪二腈水合反应的体系

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067738A2 (en) * 2003-01-27 2004-08-12 Degussa Ag Nitrile hydratases from rhodococcus erythropolis and their application
CN106715702B (zh) * 2014-09-30 2021-07-02 巴斯夫欧洲公司 制备具有低丙烯酸浓度的丙烯酰胺水溶液的方法
CA2962325A1 (en) * 2014-09-30 2016-04-07 Basf Se Means and methods for producing amide compounds with less acrylic acid
US10975177B2 (en) * 2016-03-29 2021-04-13 Solenis Technologies, L.P. Process for producing a polyacrylamide solution with increased viscosity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101619299A (zh) * 2009-07-08 2010-01-06 天津科技大学 一种赤红球菌以及利用该菌制备5-氰基戊酰胺的方法
CN109797164A (zh) * 2019-01-31 2019-05-24 大连理工大学 一种新型腈水合酶高效催化脂肪二腈水合反应的体系

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALENA ČEJKOVÁ ET AL: "Potential of Rhodococcus erythropolis as a bioremediation organism", WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, vol. 21, no. 3, 1 April 2005 (2005-04-01), pages 317 - 321, XP019271522 *
KAMBLE, A. L.: "Nitrile hydratase of Rhodococcus erythropolis:characterization of the enzyme and the use of whole cells for biotransformation of nitriles", 3 BIOTECH., vol. 3, no. 4, 16 December 2012 (2012-12-16), pages 319 - 330, XP055726220 *
KUBAC, D.: "Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4", JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC, vol. 50, 11 September 2007 (2007-09-11), pages 107 - 113, XP022387858 *
MOREAU, J. L.: "Application of High-performance liquid chromatography to the study of the biological transformation of adiponitrile by nitrile hydra- tase and amidase", ANALYST., vol. 116, no. 12, 31 December 1991 (1991-12-31), pages 1381 - 1383, XP055726221 *
RUCKA, L.: "Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes", ANTONIE VAN LEEUWENHOEK, vol. 105, no. 6, June 2014 (2014-06-01), pages 1179 - 1190, XP055726219 *
STRNAD, H.: "Genome sequence of Rhodococcus erythropolis strain CCM2595, a phenol derivative-degrading bacterium", GENOME ANNOUNCEMENTS, vol. 2, no. 2, 27 March 2014 (2014-03-27), pages e00208-14-1 - e00208-14-2, XP002731744 *

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