WO2019154249A1 - 一种腈水解酶突变体及其构建方法和应用 - Google Patents

一种腈水解酶突变体及其构建方法和应用 Download PDF

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WO2019154249A1
WO2019154249A1 PCT/CN2019/074110 CN2019074110W WO2019154249A1 WO 2019154249 A1 WO2019154249 A1 WO 2019154249A1 CN 2019074110 W CN2019074110 W CN 2019074110W WO 2019154249 A1 WO2019154249 A1 WO 2019154249A1
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nitrilase
mutant
brnit
atnit
seq
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French (fr)
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郑仁朝
郑裕国
张琴
吴哲明
汤晓玲
卢夏峰
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浙江工业大学
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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • 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
    • 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
    • C12P13/002—Nitriles (-CN)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)

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  • the invention relates to the field of bioengineering technology, in particular to a nitrilase mutant with improved catalytic activity and stereoselectivity and application in the synthesis of a pregabalin chiral intermediate.
  • Nitrile hydrolase (Nitrilase EC 3.5.5.1) is a kind of biocatalyst for the hydrolysis of nitrile compounds (R-CN) to form carboxylic acids and ammonia. It is widely used in the synthesis of (chiral) amino acids, carboxylic acids and their derivatives.
  • the enzymatic hydrolysis of nitrile has attracted the attention of academic and industrial circles due to its mild reaction conditions, high process efficiency, environmental friendliness and high chemical, regional and stereoselective advantages.
  • wild-type nitrilase Since wild-type nitrilase is often difficult to adapt to industrial environmental requirements, it has become a research hotspot to improve the catalytic performance of nitrilase by rational and irrational protein modification methods.
  • DeSantis et al. modified the wild-type nitrilase by point saturation mutation technique, and the obtained mutant A190H was able to catalyze the complete hydrolysis of 3 M 3-hydroxyglutaronitrile to synthesize (R)-4-cyano-3-hydroxybutyric acid. The product ee value Up to 99% (J. Am. Chem. Soc., 2003, 125: 11476-11477). Schreiner et al.
  • Pregabalin chemically known as (3S)-3-aminomethyl-5-methylhexanoic acid, is a treatment for neuropathic pain, epilepsy, anxiety, and central nervous system pain caused by spinal cord injury, trauma, or multiple sclerosis.
  • First-line drugs Compared with traditional drugs of the same type, pregabalin has the advantages of low dosage, low frequency, long duration, small side effects and strong tolerance, and has become one of the best-selling drugs in the world.
  • the object of the present invention is to modify the wild-type nitrilase by genetic engineering means, so that the catalytic activity and stereoselectivity of the modified nitrilase mutant are significantly improved, and the requirements for industrial application are met.
  • the present invention adopts the following technical solutions:
  • the present invention relates to the amino acid sequences of Brassica rapa nitrilase (BrNIT) and Arabidopsis thaliana nitrilase (AtNIT), especially the polyamino acids in the region 225-285. Substitution to obtain a nitrilase mutant as shown in SEQ ID NO. 1 or SEQ ID NO. 3, the study demonstrates that the mutant is a substrate to racemic isobutyl butyl compared to the wild-type nitrilase. The catalytic activity and stereoselectivity of dinitrile are significantly improved.
  • the present invention also provides a gene encoding the nitrilase mutant, the nucleotide sequence of which is set forth in SEQ ID NO. 2 or SEQ ID NO.
  • the invention also provides a recombinant vector comprising the encoding gene.
  • the original vector is pET28b.
  • the invention also provides a recombinant genetically engineered bacteria comprising the recombinant vector.
  • the recombinant vector is transformed into a host cell to obtain a recombinant genetically engineered bacteria, and the host cell can be various conventional host cells in the art, and preferably, the host cell is Escherichia coli E. coli BL21.
  • Another object of the present invention is to provide a method of preparing the nitrilase mutant, the method comprising the steps of:
  • the BrNIT plasmid deleted from the nucleotide sequence 676-858 of the phthalocyanine hydrolytic enzyme was obtained by reverse PCR amplification. Fragment or AtNIT plasmid fragment deleted at position 673-855 of the Arabidopsis nitrilase nucleotide sequence;
  • Brassica rapa nitrilase (BrNIT), Arabidopsis thaliana nitrilase (AtNIT) and Arabis alpine nitrilase (AaNIT) have catalytic racemization Isobutylsuccinonitrile produces the activity of the pregabalin chiral intermediate (S)-3-cyano-5-methylhexanoic acid, but has advantages and disadvantages in terms of catalytic activity and stereoselectivity.
  • the phthalocyanine nitrilase BrNIT and the Arabidopsis nitrilase AtNIT are used as parents, and the peptides 225-285 of the Alpine Arabidopsis nitrilase AaNIT are substituted for the 226-286 of the BrNIT amino acid sequence by peptide replacement. And at positions 225-285 of the AtNIT amino acid sequence, the nitrilase mutants BrNIT 225-285 and AtNIT 225-285 having the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3 were obtained to enhance the catalysis of the nitrilase. Activity and stereoselectivity.
  • the original vector of the recombinant plasmid is pET28b.
  • the PCR primers required for the nitrilase mutant BrNIT 225-285 are prepared:
  • step (1) the primers required to amplify the DNA fragment I:
  • Upstream primer 5'-GAATGGCAGTCTTCTATGATGCACATCGC-3';
  • Downstream primer 5'-GAAGTTCGGACCAGCCAGAACCTGACCC-3'.
  • step (2) the primers required to amplify the BrNIT plasmid fragment:
  • Upstream primer 5'-GCGATGTGCATCATAGAAGACTGCCATTC-3';
  • Downstream primer 5'-GGGTCAGGTTCTGGCTGGTCCGAACTTC-3'.
  • step (1) the primers required to amplify the DNA fragment II:
  • Upstream primer 5'-CTAAAGAATGGCAGTCTTCTATGCTGCACATCGC-3';
  • Downstream primer 5'-GATTCGAAGTTCGGACCAGCCAGAACCTGACCCAGC-3'.
  • step (2) the primers required to amplify the AtNIT plasmid fragment:
  • Upstream primer 5'-GCGATGTGCAGCATAGAAGACTGCCATTCTTTAG-3';
  • Downstream primer 5'-GCTGGGTCAGGTTCTGGCTGGTCCGAACTTCGAATC-3'.
  • the host strain is Escherichia coli E. coli BL21.
  • Another object of the present invention is to provide the use of said nitrilase mutant for the catalysis of the preparation of (S)-3-cyano-5-methylhexanoic acid as racemic isobutylsuccinonitrile.
  • the application is a wet cell, a wet cell immobilized cell obtained by fermentation culture of a genetically engineered bacteria containing a nitrilase-containing mutant, or a pure enzyme extracted by ultrasonication of a wet cell as a catalyst, and racemization.
  • Isobutyl succinonitrile is used as a substrate, and a buffer solution having a pH of 5.0 to 10.0 is used as a reaction medium, and hydrolysis reaction is carried out at 25 to 45 ° C and 100 to 300 rpm.
  • (S)-3-cyanide is obtained.
  • a mixed liquid of 5-methylhexanoic acid was isolated and purified to obtain (S)-3-cyano-5-methylhexanoic acid.
  • the final concentration of the substrate is from 0.5 to 1.5 M, and the amount of the catalyst is from 10 to 30 g/L based on the weight of the wet cells. More preferably, the amount of wet cells required per 1 mol of the substrate is 20 g.
  • the reaction medium is a Tris-HCl buffer having a pH of 8.0.
  • the hydrolysis reaction is carried out at 30 to 35 ° C and 200 rpm.
  • the wet cell is a recombinant Escherichia coli BL21(DE3)/pET28b-BrNIT 225-285 or E.coli BL21(DE3)/pET28b-AtNIT 225-285 containing a nitrilase-encoding gene.
  • Propyl- ⁇ -D-thiogalactopyranoside (IPTG) was induced to culture at 28 ° C for 10-12 h, centrifuged, and the cells were collected to obtain the wet cells.
  • the invention has the beneficial effects:
  • the present invention provides an enzyme-directed transformation method for replacing the key peptide of the algal nitrilase into the corresponding region of the phthalocyanine hydrolytic enzyme or the Arabidopsis nitrilase, and rationally reforming the wild type nitrile hydrolysis Enzyme, a highly viable, highly stereoselective nitrilase mutant was constructed, which has good performance in the catalytic synthesis of (S)-3-cyano-5-methylhexanoic acid as racemic isobutylsuccinonitrile. Application prospects.
  • the nitrilase mutant BrNIT 225-285 provided by the present invention which catalyzes the hydrolysis of racemic isobutyl succinonitrile to be 2.5 times that of the wild type, and the enantioselectivity (E value) is increased by 200. Up to 500; the nitrilase mutant AtNIT 225-285 catalyzes the hydrolysis of racemic isobutyl succinonitrile to 1.9 times that of the wild type. While not attenuating the catalytic activity, the stereoselectivity is improved to meet the requirements of industrial applications.
  • Figure 1 is an electrophoresis pattern after purification of a nitrilase and its mutant protein.
  • Lane 1 is BrNIT
  • lane 2 is BrNIT 225-285
  • lane 3 is AtNIT
  • lane 4 is AtNIT 225-285 .
  • Figure 2 is a comparison of the stereoselective hydrolysis reaction of a 100 N/L racemic isobutyl succinonitrile catalyzed by a BrNIT mutant and a wild enzyme.
  • the main experimental material sources used in the following examples are:
  • E. coli BL21 (DE3) and E. coli BL21 (DH5 ⁇ ) were purchased from Transgen, the expression vector pET-28b (+) was purchased from Novagen, and Phanta Max Super-Fidelity DNA Polymerase was purchased from Vazyme, 2 ⁇ TsingKe Master Mix (blue) was purchased from TsingKe; kanamycin was purchased from Dalian Bao Biotech Co., Ltd.; IPTG was purchased from Promega.
  • the present invention employs a simple, fast and efficient DNA seamless cloning technique - Technique, the amplified peptide fragment is directionally cloned into a BrNIT plasmid fragment lacking the corresponding gene fragment.
  • Each peptide was determined by performing an alignment analysis on the nucleotide and amino acid sequences of the cruciferous plant nitrilase.
  • the amino acid sequence of the wild-type phthalocyanine nitrilase is SEQ ID No. 5, which is encoded by the nucleotide sequence of SEQ ID No. 6; the amino acid sequence of the wild-type Alpine Arabine nitrilase is SEQ ID No. .7, the protein is encoded by the nucleotide sequence of SEQ ID No. 8.
  • the AaNIT nucleotide sequence was used as a template to clone the 0-85, 85-175, 175-225, 225-285 and 285-342 peptides, respectively.
  • primers were designed to amplify BrNIT plasmid fragments lacking 0-86, 86-176, 176-226, 226-286 and 286-342 peptides. .
  • the BrNIT vector sequence lacking the corresponding peptide gene fragment was linearized, and the end sequence of the linearized vector was introduced at the 5' end of the insert forward/reverse PCR primer, so that the 5' and 3' end of the PCR product were linear and linear, respectively.
  • the primer sequences are shown in Table 1.
  • PCR amplification of each peptide PCR reaction system (50 ⁇ L): Template DNA ⁇ 1 ⁇ g, Master Mix, 0.2 ⁇ M each of the upstream and downstream primers, and the remaining ddH 2 O was added to the total volume.
  • PCR reaction parameters (1) pre-denaturation at 94 ° C for 5 min; (2) denaturation at 94 ° C for 30 s; (3) annealing at 58 ° C for 30 s; (4) extension at 72 ° C for 10 s, and steps (2) - (4) for 30 cycles; 5) Re-extend for 10 min at 72 ° C and store at 4 ° C.
  • the PCR product was analyzed by agarose gel electrophoresis, and then the gel was recovered, inactivated at 65 ° C for 10 min, and placed at 4 ° C for use.
  • Vector linearization was obtained by reverse PCR amplification.
  • PCR reaction system 50 ⁇ L: template DNA 0.1 ng-1 ng, 2 ⁇ Phanta Max Buffer, dNTPs (10 mM each) 0.2 mM, upstream and downstream primers 0.2 ⁇ M each, Phanta Max Super-Fidelity DNA Polymerase 1 U, and the remaining ddH 2 O was added to total capacity.
  • PCR reaction parameters (1) pre-denaturation at 95 °C for 30 s; (2) denaturation at 95 °C for 15 s; (3) annealing at 63 °C for 15 s; (4) extension at 72 °C for 6.0 min, and steps (2)-(4) for 30 cycles; (5) Thoroughly extend for 5 min at 72 ° C and store at 4 ° C.
  • the PCR product was analyzed by agarose gel electrophoresis, digested with endonuclease Dpn I for 3 h at 37 ° C, and inactivated at 65 ° C for 10 min.
  • the composition of the reaction reaction system was as shown in Table 2. After mixing the PCR samples, they were placed at 37 ° C for 30 min and lowered to 4 ° C.
  • a single colony was picked and placed in 5 mL of liquid LB medium, and the final concentration of kanamycin was 50 mg/L.
  • the culture conditions were 37 ° C, 200 rpm, and cultured for 6-8 h.
  • the above seed solution was transferred to a fresh LB liquid medium containing 50 mg/L of kanamycin at a volume ratio of 2%, and cultured at 37 ° C, 150 rpm until the OD 600 of the cells was about 0.6-0.8, IPTG (final concentration: 0.1 mM) was added to the LB liquid medium, and the culture was induced for 10-12 hours at 28 ° C, 150 rpm.
  • the fermentation broth was collected and centrifuged at 8000 rpm for 10 min at 4 ° C, then the physiological cells were added to wash the cells once and centrifuged.
  • the obtained cells were stored in a refrigerator at -20 °C. After the cells were disrupted, the nitrilase mutant BrNIT 225-285 was isolated and purified, and the electrophoresis was detected as shown in Fig. 1.
  • the recombinant Escherichia coli BL21(DE3)/pET28b-BrNIT 0-85 , E.coli BL21(DE3)/pET28b-BrNIT 85-175 , E.coli BL21 (DE3) containing the nitrilase mutant will be cultured.
  • /pET28b-BrNIT 175-225 , E. coli BL21(DE3)/pET28b-BrNIT 225-285 and E. coli BL21(DE3)/pET28b-BrNIT 285-342 were assayed for viability.
  • the reaction solution was reacted at 30 ° C, 200 rpm for 15 min. 500 ⁇ L was sampled and the reaction was stopped by the addition of 200 ⁇ L of 2 M HCl.
  • the enantiomeric excess of the substrate racemic isobutylsuccinonitrile and the product 3-cyano-5-methylhexanoic acid was determined by gas chromatography.
  • the gas chromatograph model number is 7890N (Agilent) and the capillary column model is BGB-174 (BGB Analytik Switzerland).
  • the chromatographic conditions were: injection volume of 1.0 ⁇ L, inlet and detector temperatures of 250 ° C, column temperature of 120 ° C for 15 min, and temperature of 10 ° C / min to 170 ° C for 9 min.
  • the carrier gas was high purity helium with a flow rate of 1.0 mL/min and a split ratio of 50:1.
  • ND No Detection
  • the recombinant Escherichia coli BL21(DE3)/pET28b-BrNIT 225-285 containing the nitrilase mutant and the recombinant Escherichia coli BL21(DE3)/pET28b-BrNIT containing the wild type nitrilase were cultured.
  • the effect of stereoselective hydrolysis of racemic isobutyl succinonitrile was compared.
  • the reaction system consisted of (100 mL): Tris-HCl buffer solution (50 mM, pH 8.0), 1.5 g of wet cells and 10 g of racemic isobutyl succinonitrile to form a reaction system.
  • the reaction was carried out at 35 ° C and 200 rpm, and samples were taken every 1 hour, 500 ⁇ L each time, and the reaction was terminated by adding 200 ⁇ L of 2 M HCl.
  • the hydrolysis process of the nitrilase mutant and the wild type catalyzed racemic isobutyl succinonitrile is shown in Figure 2.
  • amino acid sequence of the wild type Arabidopsis nitrilase is SEQ ID No. 9, which is encoded by the nucleotide sequence of SEQ ID No. 10.
  • the Arabidopsis nitrilase mutant AtNIT 225-285 was constructed following the method of Example 1. The primers used in the construction of the mutants are shown in Table 4.
  • Recombinant Escherichia coli E. coli BL21(DE3)/pET28b-AtNIT 225-285 containing Arabidopsis nitrilase mutant and recombinant Escherichia coli containing wild type Arabidopsis nitrilase were obtained by the method of Example 1. Coli BL21(DE3)/pET28b-AtNIT. Recombinant E. coli was induced to express whole cells and used as a biocatalyst. After the cells were disrupted, the nitrilase mutant AtNIT 225-285 was isolated and purified, and the electrophoresis was shown in Figure 1.
  • each resting cell containing Arabidopsis nitrilase mutant and wild type was suspended in 10 mL of Tris-HCl buffer (50 mM, pH 8.0), and 0.3 g of external reaction was added to the reaction system. Spinopyl butyl succinonitrile was reacted at 30 ° C, 200 rpm.
  • the nitrilase mutant AtNIT 225-285 was determined to catalyze the hydrolysis of racemic isobutyl succinonitrile by 1.9 times that of the wild type. After 24 h of reaction, the conversion rates of wild-type AtNIT and mutant AtNIT 225-285 were 25.64% and 48.76%, respectively, and the ee was greater than 98.5%.

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Abstract

提供了一种腈水解酶突变体及其构建方法和在普瑞巴林手性中间体合成中的应用。分别以芜菁腈水解酶BrNIT和拟南芥腈水解酶AtNIT为亲本，运用肽段替换，将高山南芥腈水解酶AaNIT的225-285位肽段替换BrNIT氨基酸序列的226-286位和AtNIT氨基酸序列的225-285位，获得氨基酸序列如SEQ ID NO.1或SEQ ID NO.3所示的腈水解酶突变体BrNIT225-285及AtNIT225-285。相比于野生型腈水解酶，所提供的腈水解酶突变体催化水解外消旋异丁基丁二腈的活力及产物的立体选择性显著提高，可以满足工业化应用要求，同时在高效催化外消旋异丁基丁二腈合成3-氰基-5-甲基己酸中具有良好的应用前景。

Description

一种腈水解酶突变体及其构建方法和应用 技术领域
本发明涉及生物工程技术领域，具体涉及一种催化活力及立体选择性提高的腈水解酶突变体及在普瑞巴林手性中间体合成中的应用。
背景技术
腈水解酶(Nitrilase EC 3.5.5.1)是一类催化腈类化合物(R-CN)水解生成羧酸和氨的生物催化剂，广泛应用于(手性)氨基酸、羧酸及其衍生物的合成。腈的酶法水解因其反应条件温和、过程高效、环境友好以及高度化学、区域和立体选择性等优势引起学术界和工业界的重视。
由于野生型腈水解酶通常难以适应工业环境需求，因此通过理性和非理性蛋白质改造方法提高腈水解酶催化性能成为研究热点。DeSantis等通过点饱和突变技术改造野生型腈水解酶，获得的突变体A190H能够催化3 M 3-羟基戊二腈完全水解合成(R)-4-氰基-3-羟基丁酸，产物ee值高达99％(J.Am.Chem.Soc.,2003,125:11476-11477)。Schreiner等利用易错PCR技术改造Arabidopsis thaliana腈水解酶AtNIT2，筛选获得催化苯乙腈水解活力提高4倍的突变体(ChemCatChem,2010,2:263-267)。
普瑞巴林，化学名为(3S)-3-氨甲基-5-甲基己酸，是治疗神经病理性疼痛、癫痫、焦虑和由脊髓损伤、外伤或多发性硬化症等引起的中枢神经疼痛的一线药物。与传统同类药物相比，普瑞巴林具有服用剂量低、次数少、持续时间长、副作用小、耐受性强等优点，已成为全球畅销药之一。
手性中心的构筑是普瑞巴林合成的关键，生物催化法合成普瑞巴林手性中间体的研究受到越来越多的关注。腈水解酶区域、立体选择性水解外消旋异丁基丁二腈合成普瑞巴林手性中间体(S)-3-氰基-5-甲基己酸路线具有原料廉价、原子经济性高等优势，但现有的腈水解酶催化活力低(J.Mol.Catal.B:Enzym.2006,41:75-80)，难以满足规模化应用要求。因此，开发腈水解酶突变体构建技术，获得高活力腈水解酶生物催化剂，对实现普瑞巴林的高效、绿色工业化生产具有重要意义。
发明内容
本发明的目的在于通过基因工程手段对野生型腈水解酶进行改造，使改造后腈水解酶突变体的催化活力和立体选择性等催化性能显著提高，达到工业化应用的要求。
为实现上述目的，本发明采用如下技术方案：
一种腈水解酶突变体，其氨基酸序列如SEQ ID NO.1或SEQ ID NO.3所示。
本发明分别对十字花科植物芜菁(Brassica rapa)腈水解酶(BrNIT)和拟南芥(Arabidopsis thaliana)腈水解酶(AtNIT)的氨基酸序列，尤其是225-285位区域中的多位氨基酸进行替换，获得如SEQ ID NO.1或SEQ ID NO.3所示的腈水解酶突变体，研究证明，相较于野生型腈水解酶，该突变体对底物外消旋异丁基丁二腈的催化活性、立体选择性显著提升。
对所述腈水解酶突变体的其他氨基酸位点的保守取代形式、增加或缺失一个或几个氨基酸的形式、氨基端截断的形式、羧基端截断的形式，这些突变体形式也包括在本发明的范围内。
本发明还提供了一种编码所述腈水解酶突变体的编码基因，其核苷酸序列如SEQ ID NO.2或SEQ ID NO.4所示。
本发明还提供了一种包含所述编码基因的重组载体。作为优选，原始载体为pET28b。
本发明还提供了一种包含所述重组载体的重组基因工程菌。所述重组载体转化宿主细胞获得重组基因工程菌，所述宿主细胞可以为本领域的各种常规宿主细胞，作为优选，宿主细胞为大肠杆菌E.coli BL21。
本发明的另一个目的是提供一种制备所述腈水解酶突变体的方法，所述方法，包括以下步骤：
(1)针对芜菁腈水解酶基因或拟南芥腈水解酶基因序列，设计PCR引物，以高山南芥cDNA为模板，利用所述PCR引物扩增获得含有高山南芥腈水解酶核苷酸序列673-855位的DNA片段Ⅰ或DNA片段Ⅱ；
(2)以携带有芜菁腈水解酶基因或拟南芥腈水解酶基因的重组质粒为模板，利用反向PCR扩增获得芜菁腈水解酶核苷酸序列676-858位缺失的BrNIT质粒片段或拟南芥腈水解酶核苷酸序列673-855位缺失的AtNIT质粒片段；
(3)将DNA片段Ⅰ和BrNIT质粒片段，或DNA片段Ⅱ和AtNIT质粒片段重组，再将重组产物转化至宿主菌，筛选获得腈水解酶突变体表达菌株；
(4)对腈水解酶突变体表达菌株进行诱导表达，获得所述的腈水解酶突变体。
十字花科植物芜菁(Brassica rapa)腈水解酶(BrNIT)、拟南芥(Arabidopsis thaliana)腈水解酶(AtNIT)和高山南芥(Arabis alpine)腈水解酶(AaNIT)均具有催化外消旋异丁基丁二腈生成普瑞巴林手性中间体(S)-3-氰基-5-甲基己酸的活力，但是在催化活性及立体选择性方面各有优缺点。本发明分别以芜菁腈水解酶BrNIT和拟南芥腈水解酶AtNIT为亲本，运用肽段替换，将高山南芥腈水解酶AaNIT的225-285位肽段替换BrNIT氨基酸序列的226-286位和AtNIT氨基酸序列的225-285位，获得氨基酸序列如SEQ ID NO.1或SEQ ID NO.3所示的腈水解酶突变体BrNIT 225-285及AtNIT 225-285，以提高腈水解酶的催化活性和立体选择性。
作为优选，步骤(2)中，所述重组质粒的原始载体为pET28b。
作为优选，制备腈水解酶突变体BrNIT 225-285所需的PCR引物：
步骤(1)中，扩增DNA片段Ⅰ所需引物：
上游引物：5’-GAATGGCAGTCTTCTATGATGCACATCGC-3’；
下游引物：5’-GAAGTTCGGACCAGCCAGAACCTGACCC-3’。
步骤(2)中，扩增BrNIT质粒片段所需引物：
上游引物：5’-GCGATGTGCATCATAGAAGACTGCCATTC-3’；
下游引物：5’-GGGTCAGGTTCTGGCTGGTCCGAACTTC-3’。
制备腈水解酶突变体AtNIT 225-285所需的PCR引物：
步骤(1)中，扩增DNA片段Ⅱ所需引物：
上游引物：5’-CTAAAGAATGGCAGTCTTCTATGCTGCACATCGC-3’；
下游引物：5’-GATTCGAAGTTCGGACCAGCCAGAACCTGACCCAGC-3’。
步骤(2)中，扩增AtNIT质粒片段所需引物：
上游引物：5’-GCGATGTGCAGCATAGAAGACTGCCATTCTTTAG-3’；
下游引物：5’-GCTGGGTCAGGTTCTGGCTGGTCCGAACTTCGAATC-3’。
作为优选，所述宿主菌为大肠杆菌E.coli BL21。
本发明的另一个目的是提供所述的腈水解酶突变体在催化外消旋异丁基丁二腈制备(S)-3-氰基-5-甲基己酸中的应用。
具体地，所述应用为以含腈水解酶突变体编码基因工程菌经发酵培养获得的湿菌体、湿菌体固定化细胞或者湿菌体超声破碎后提取的纯酶为催化剂，以外消旋异丁基丁二腈为底物，以pH 5.0～10.0的缓冲液为反应介质，在25～45℃、100～300rpm条件下进行水解反应，反应完全后，获得含(S)-3-氰基-5-甲基己酸的混合液，将混合液分离纯化，获得(S)-3-氰基-5-甲基己酸。
作为优选，反应体系中，底物的终浓度为0.5～1.5M，催化剂的用量以湿菌体重量计为10～30g/L。更为优选，每1mol的底物所需湿菌体的添加量为20g。
作为优选，所述反应介质为pH 8.0的Tris-HCl缓冲液。
作为优选，在30～35℃、200rpm条件下进行水解反应。
作为优选，所述湿菌体为含腈水解酶突变体编码基因的重组大肠杆菌E.coli BL21(DE3)/pET28b-BrNIT 225-285或E.coli BL21(DE3)/pET28b-AtNIT 225-285，发酵培养的方法为：含腈水解酶突变体编码基因的重组大肠杆菌接种于含卡那霉素的LB培养基中，培养至OD 600＝0.6～0.8时，加入终浓度为0.1mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，28℃诱导培养10-12h，离心，收集菌体得到所述湿菌体。
与现有技术相比，本发明具备的有益效果：
(1)本发明提供了一种酶定向改造方法，将高山南芥腈水解酶的关键性肽段替换至芜菁腈水解酶或拟南芥腈水解酶对应区域中，理性改造野生型腈水解酶，构建了高活力、高立体 选择性的腈水解酶突变体，在高效催化外消旋异丁基丁二腈合成(S)-3-氰基-5-甲基己酸中具有良好的应用前景。
(2)本发明提供的腈水解酶突变体BrNIT 225-285，其催化外消旋异丁基丁二腈水解的活力是野生型的2.5倍，对映体选择率(E值)由200提高到500以上；腈水解酶突变体AtNIT 225-285催化外消旋异丁基丁二腈水解的活力是野生型的1.9倍。在不减弱催化活力的同时，提高立体选择性，满足工业化应用要求。
附图说明
图1为腈水解酶及其突变体蛋白纯化后的电泳图。泳道1为BrNIT，泳道2为BrNIT 225-285，泳道3为AtNIT，泳道4为AtNIT 225-285。
图2为BrNIT突变体和野生酶催化100g/L外消旋异丁基丁二腈立体选择性水解反应进程比较。
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不仅限于此。
以下实施例所用主要实验材料来源为：
大肠杆菌宿主菌株E.coli BL21(DE3)和E.coli BL21(DH5α)购自Transgen公司，表达载体pET-28b(+)购自Novagen公司，Phanta Max Super-Fidelity DNA Polymerase购自Vazyme公司，2×TsingKe Master Mix(blue)购自TsingKe公司；卡那霉素购自大连宝生物有限公司；IPTG为Promega公司产品。
实施例1
1、腈水解酶突变体的构建
本发明采用一种简单、快速并且高效的DNA无缝克隆技术—
Figure PCTCN2019074110-appb-000001
技术，将扩增的肽段定向克隆至缺失对应基因片段的BrNIT质粒片段中。
通过对十字花科植物腈水解酶核苷酸和氨基酸序列进行比对分析，确定各个肽段。野生型芜菁腈水解酶的氨基酸序列为SEQ ID No.5，该蛋白是由SEQ ID No.6的核苷酸序列所编码的；野生型高山南芥腈水解酶的氨基酸序列为SEQ ID No.7，该蛋白是由SEQ ID No.8的核苷酸序列所编码的。
以AaNIT核苷酸序列为模板，分别克隆0-85位、85-175位、175-225位、225-285位和285-342位肽段。
同时，以包含BrNIT核苷酸序列的重组质粒为模板，设计引物扩增缺失0-86位、86-176 位、176-226位、226-286位和286-342位肽段的BrNIT质粒片段。
将缺失对应肽段基因片段的BrNIT载体序列进行线性化，在插入片段正/反向PCR引物5’端引入线性化载体的末端序列，使得PCR产物5’和3’最末端分别带有和线性化载体两末端一致的序列。其引物序列如表1所示。
对各个肽段进行PCR扩增，PCR反应体系(50μL):Template DNA<1μg，
Figure PCTCN2019074110-appb-000002
Master Mix，上下游引物各0.2μM，其余ddH 2O补充至总体积。PCR反应参数：(1)94℃预变性5min；(2)94℃变性30s；(3)58℃退火30s；(4)72℃延伸10s，步骤(2)-(4)循环30次；(5)72℃再次延伸10min，4℃保存。PCR产物经过琼脂糖凝胶电泳分析后切胶回收，65℃灭活10min，放置4℃备用。
载体线性化采用反向PCR扩增的方式获得。PCR反应体系(50μL):模板DNA 0.1ng-1ng，2×Phanta Max Buffer，dNTPs(10mM each)0.2mM，上下游引物各0.2μM，Phanta Max Super-Fidelity DNA Polymerase 1U，其余ddH 2O补充至总体积。PCR反应参数：(1)95℃预变性30s；(2)95℃变性15s；(3)63℃退火15s；(4)72℃延伸6.0min，步骤(2)-(4)循环30次；(5)72℃彻底延伸5min，4℃保存。PCR产物经过琼脂糖凝胶电泳分析后，加入内切酶Dpn I于37℃消化3h，65℃灭活10min。
表1：BrNIT嵌合酶引物设计表
Figure PCTCN2019074110-appb-000003
Figure PCTCN2019074110-appb-000004
将上述所获得的插入片段和线性化载体利用NanoDrop TM One/OneC超微量紫外分光光度计进行基因浓度测定，计算出各个插入肽段及对应缺失肽段的线性化载体的添加量。连接反应体系组成如表2，将PCR样品混匀后，放置37℃保温30min，降至4℃。
表2：重组反应体系
Figure PCTCN2019074110-appb-000005
将10μL重组产物加入到100μL E.coli BL21(DH 5α)感受态细胞中，涂布于含卡那霉素抗性(终浓度为50mg/L)的LB平板上，37℃培养10-12h。挑取单菌落培养于含卡那霉素抗性(终浓度为50mg/L)的LB液体培养基中，提取质粒测序。测序结果正确者转化入E.coli BL21(DE3)感受态细胞中，涂布在含卡那霉素的LB平板上培养过夜，得到腈水解酶突变体表达菌株。
2、腈水解酶突变体基因的表达
挑取单菌落接入5mL液体LB培养基中，卡那霉素的终浓度为50mg/L。培养条件为37℃，200rpm，培养6-8h。将上述种子液以2％的体积比转接至新鲜的含有50mg/L的卡那霉素的LB液体培养基中，37℃，150rpm培养至菌体OD 600约为0.6-0.8时，向上述LB液 体培养基中加入IPTG(终浓度为0.1mM)，28℃，150rpm诱导培养10-12h，收集发酵液于4℃，8000rpm的条件下离心10min，然后加入生理盐水洗涤菌体一次，将离心所得菌体置于-20℃冰箱保存。菌体破碎后，分离纯化获得腈水解酶突变体BrNIT 225-285，电泳检测如图1所示。
3、含腈水解酶突变体重组大肠杆菌活力测定
将培养得到的含有腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET28b-BrNIT 0-85、E.coli BL21(DE3)/pET28b-BrNIT 85-175、E.coli BL21(DE3)/pET28b-BrNIT 175-225、E.coli BL21(DE3)/pET28b-BrNIT 225-285以及E.coli BL21(DE3)/pET28b-BrNIT 285-342进行活力测定。含腈水解酶突变体的重组大肠杆菌活力检测反应体系(10mL)：Tris-HCl缓冲溶液(50mM，pH8.0)，外消旋异丁基丁二腈30g/L，湿菌体10g/L。反应液于30℃，200rpm反应15min。取样500μL，加入200μL 2 M HCl终止反应。
底物外消旋异丁基丁二腈和产物3-氰基-5-甲基己酸的对映体过量值由气相色谱测定。气相色谱型号为7890N(安捷伦)，毛细管柱型号为BGB-174(BGB Analytik Switzerland)。色谱条件为：进样量1.0μL，进样口、检测器温度均为250℃，柱温为120℃保持15min，10℃/min升温至170℃，保持9min。载气为高纯氦气，流速为1.0mL/min，分流比为50:1。
对映体过量值(ee)、转化率(c)的计算参考Rakels等的计算方法(Enzyme Microb.Technol.,1993,15:1051)。
经测定，各腈水解酶突变体活力如表3所示：
表3：含腈水解酶突变体重组大肠杆菌活力测定结果
Figure PCTCN2019074110-appb-000006
注：ND为No Detection(未检测)。
4、腈水解酶突变体BrNIT 225-285和野生型催化外消旋异丁基丁二腈水解比较
以培养得到的含有腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET28b-BrNIT 225-285以及含有野生型腈水解酶的重组大肠杆菌E.coli BL21(DE3)/pET28b-BrNIT为生物催化剂，比较其立体选择性水解外消旋异丁基丁二腈的效 果。
反应体系组成为(100mL):Tris-HCl缓冲溶液(50mM，pH 8.0)，1.5g湿菌体和10g外消旋异丁基丁二腈构成反应体系。35℃、200rpm条件下反应，每隔1h取样，每次取样500μL，加入200μL 2 M HCl终止反应。腈水解酶突变体和野生型催化外消旋异丁基丁二腈水解进程如图2所示。
5、重组大肠杆菌E.coli BL21(DE3)/pET28b-BrNIT 225-285催化合成(S)-3-氰基-5-甲基己酸
以重组大肠杆菌E.coli BL21(DE3)/pET28b-BrNIT 225-285为生物催化剂，在100mL Tris-HCl缓冲液(pH 8.0)中加入2.0g湿菌体(终浓度20g/L)和终浓度1.0M(136g/L)外消旋异丁基丁二腈构成反应体系。于30℃、200rpm条件下反应，每隔一定时间取样500μL，加入2M的HCl终止反应。样品检测方式参照步骤3。反应8h，转化率达到39.8％，产物(S)-3-氰基-5-甲基己酸的ee值大于99.3％。相对于已报道的催化工艺过程，菌体添加量减少了2.5倍。
实施例2
拟南芥腈水解酶突变体AtNIT 225-285的构建及其催化合成(S)-3-氰基-5-甲基己酸
野生型拟南芥腈水解酶的氨基酸序列为SEQ ID No.9，该蛋白是由SEQ ID No.10的核苷酸序列所编码的。
参照实施例1的方法构建拟南芥腈水解酶突变体AtNIT 225-285。突变体构建所用引物如表4所示。
表4AtNIT嵌合酶引物设计表
Figure PCTCN2019074110-appb-000007
参照实施例1的方法获得含有拟南芥腈水解酶突变体的重组大肠杆菌E.coli BL21(DE3)/pET28b-AtNIT 225-285和含有野生型拟南芥腈水解酶的重组大肠杆菌E.coli BL21(DE3)/pET28b-AtNIT。重组大肠杆菌经诱导表达后收集全细胞，作为生物催化剂。菌体破碎后，分离纯化获得腈水解酶突变体AtNIT 225-285，电泳检测如图1所示。
分别取含有拟南芥腈水解酶突变体和野生型的静息细胞各0.1g(湿重)，悬浮于10mL  Tris-HCl缓冲液中(50mM，pH 8.0)，反应体系中添加0.3g外消旋异丁基丁二腈，于30℃，200rpm条件下进行反应。经测定，腈水解酶突变体AtNIT 225-285催化外消旋异丁基丁二腈水解的活力是野生型的1.9倍。反应24h，野生型AtNIT和突变体AtNIT 225-285的转化率分别为25.64％和48.76％，ee均大于98.5％。

Claims (10)

  1. 一种腈水解酶突变体，其特征在于，其氨基酸序列如SEQ ID NO.1或SEQ ID NO.3所示。
  2. 一种编码如权利要求1所述的腈水解酶突变体的编码基因，其特征在于，其核苷酸序列如SEQ ID NO.2或SEQ ID NO.4所示。
  3. 一种包含如权利要求2所述的编码基因的重组载体。
  4. 一种包含如权利要求3所述的重组载体的重组基因工程菌。
  5. 如权利要求1所述的腈水解酶突变体的制备方法，其特征在于，包括以下步骤：
    (1)针对芜菁腈水解酶基因或拟南芥腈水解酶基因序列，设计PCR引物，以高山南芥cDNA为模板，利用所述PCR引物扩增获得含有高山南芥腈水解酶核苷酸序列673-855位的DNA片段Ⅰ或DNA片段Ⅱ；
    (2)以携带有芜菁腈水解酶基因或拟南芥腈水解酶基因的重组质粒为模板，利用反向PCR扩增获得芜菁腈水解酶核苷酸序列676-858位缺失的BrNIT质粒片段或拟南芥腈水解酶核苷酸序列673-855位缺失的AtNIT质粒片段；
    (3)将DNA片段Ⅰ和BrNIT质粒片段，或DNA片段Ⅱ和AtNIT质粒片段重组，再将重组产物转化至宿主菌，筛选获得腈水解酶突变体表达菌株；
    (4)对腈水解酶突变体表达菌株进行诱导表达，获得所述的腈水解酶突变体。
  6. 如权利要求1所述的腈水解酶突变体在催化外消旋异丁基丁二腈制备(S)-3-氰基-5-甲基己酸中的应用。
  7. 如权利要求6所述的应用，其特征在于，所述应用为以含腈水解酶突变体编码基因工程菌经发酵培养获得的湿菌体、湿菌体固定化细胞或者湿菌体超声破碎后提取的纯酶为催化剂，以外消旋异丁基丁二腈为底物，以pH 5.0～10.0的缓冲液为反应介质，在25～45℃、100～300rpm条件下进行水解反应，反应完全后，获得含(S)-3-氰基-5-甲基己酸的混合液，将混合液分离纯化，获得(S)-3-氰基-5-甲基己酸。
  8. 如权利要求7所述的应用，其特征在于，反应体系中，底物的终浓度为0.5～1.5M，催化剂的用量以湿菌体重量计为10～30g/L。
  9. 如权利要求7所述的应用，其特征在于，所述反应介质为pH 8.0的Tris-HCl缓冲液。
  10. 如权利要求7所述的应用，其特征在于，所述湿菌体为含腈水解酶突变体编码基因的重组大肠杆菌E.coli BL21(DE3)/pET28b-BrNIT 225-285或E.coli BL21(DE3)/pET28b-AtNIT 225-285，发酵培养的方法为：含腈水解酶突变体编码基因的重组大肠杆菌接种于含卡那霉素的LB培养基中，培养至OD 600＝0.6～0.8时，加入终浓度为0.1mM的异丙基-β-D-硫代吡喃半乳糖苷，28℃诱导培养10-12h，离心，收集菌体得到所述湿菌体。
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