WO2022095221A1 - 多肽标签、高度可溶性的重组腈水解酶及其在医药化学品合成中的应用 - Google Patents

多肽标签、高度可溶性的重组腈水解酶及其在医药化学品合成中的应用 Download PDF

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WO2022095221A1
WO2022095221A1 PCT/CN2020/135580 CN2020135580W WO2022095221A1 WO 2022095221 A1 WO2022095221 A1 WO 2022095221A1 CN 2020135580 W CN2020135580 W CN 2020135580W WO 2022095221 A1 WO2022095221 A1 WO 2022095221A1
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polypeptide tag
nitrilase
recombinant
enzyme
gkgkg
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薛亚平
谢冬
熊能
郑裕国
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浙江工业大学
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • 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)
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    • 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)
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
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    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to a polypeptide label, in particular to a general polypeptide label that can enhance the soluble expression of enzymes such as nitrilase, and its application in the synthesis of medicinal chemicals, belonging to the technical field of genetic engineering and protein engineering.
  • Nitrilohydrolase (EC 3.5.5.1) is an important class of hydrolases with a triplet of Glu-Lys-Cys catalytic active centers, which can selectively and efficiently catalyze the hydrolysis of cyano groups to carboxyl groups in one step. It has a wide range of applications in the synthesis of chemical products such as amino acids, vitamins and gabapentin, clopidogrel, baclofen, atorvastatin and other pharmaceutical chemicals.
  • fusion tags also known as fusion partners or soluble tags
  • these methods have some flaws more or less.
  • the selection of amino acids on the polypeptide tag and the length of the sequence may have great adverse effects on the enzymatic activity, stability, solubility or selectivity of the recombinase.
  • Some tags may even change the structure of the enzyme protein and cause the enzyme to lose its activity completely.
  • the object of the present invention is to provide a polypeptide tag, a recombinase containing a polypeptide tag and its application in the synthesis of pharmaceutical chemicals, the polypeptide tag can make the whole-cell catalyst activity and thermal stability of the recombinase containing the polypeptide tag obtained. Effective promotion. Solve the problems of poor thermal stability of the enzyme and insufficient enzyme activity to meet industrial large-scale applications.
  • the present invention provides a polypeptide tag.
  • the amino acids at both ends of the polypeptide tag are uncharged glycine (G), and the rest are glycine (G), histidine (H), glutamic acid (E) and aspartic acid.
  • G uncharged glycine
  • H histidine
  • E glutamic acid
  • S aspartic acid
  • D random combination of any one or more of lysine (K) and arginine (R); the length of the polypeptide tag is 5-11 amino acids.
  • amino acid sequence of the polypeptide tag is one of the following: GKGKG, GKGEG, GKGHG, GRGRG, GRGGG, GGHHG, GGDDG, GDGEG, GDGRG, GDGKG, GEGEG, GEGKG, GEGGG, GEGRG, GEGDG, GKGKG, GKGDG, GKGEG, GKGGG , GKGHG, GKGRG, GRGRG, GRGDG, GRGEG, GRGKG, GRGGG, GGGKG, GGGEG, GGHHG, GKGKG, GKGKGKG, GKGKGKGKG, GKGKGKGKG, GKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKG
  • both ends and the middle position of the polypeptide tag are uncharged glycine (G), and the rest are glycine (G), histidine (H), glutamic acid (E), aspartic acid (D)
  • G uncharged glycine
  • H histidine
  • E glutamic acid
  • D aspartic acid
  • K lysine
  • R arginine
  • polypeptide tag is further provided with a connecting peptide, and the amino acid sequence of the connecting peptide is one of the following: GS, GGS, GGGS, GGGGS.
  • the preferred polypeptide tag is GKGKG.
  • the present invention provides a recombinant enzyme containing the polypeptide tag, and the enzyme comprises nitrilase, lipase and deacylase.
  • the recombinant enzyme is preferably a recombinant nitrilase, which is obtained by linking the N-terminus of the amino acid sequence of the nitrilase with a polypeptide tag.
  • the ligation can adopt methods such as PCR amplification, one-step cloning, for example, a vector containing a nitrilase gene (preferably pET-28b(+)/AcN-M) is used as a template, a primer containing a polypeptide tag is designed, and PCR amplification is performed. , to obtain the nitrilase containing the peptide tag.
  • the manner in which the polypeptide tag is linked to the nitrilase through a linking peptide is one of the following: GKGKG-GS, GKGKG-GGS, GKGKG-GGGS, GKGKG-GGGGS.
  • the nitrilase gene of the present invention is cloned from Acidovorax facilis ZJB09122, the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.
  • the present invention also relates to a recombinant plasmid containing the recombinase encoding gene of the polypeptide tag (preferably a recombinant plasmid containing the recombinant nitrilase encoding gene), the recombinant plasmid is constructed by using pET-28b(+) as a vector, specifically: The plasmid of the recombinase gene is used as a template, and primers containing polypeptide tags are designed, whole plasmid PCR, nucleic acid gel electrophoresis and sequencing are performed for verification, and finally a recombinant plasmid is obtained.
  • the present invention also provides a recombinant genetically engineered bacterium constructed from the recombinase-encoding gene containing the polypeptide tag.
  • the host bacteria is constructed; the host bacteria is preferably Escherichia coli BL21 (DE3).
  • the present invention also provides an application of the polypeptide tag-containing recombinase in the preparation of gabapentin intermediate 1-cyanocyclohexylacetic acid, the application is: recombination genetic engineering with the polypeptide tag-containing recombinase encoding gene
  • the wet cells obtained by bacterial fermentation and culture or the purified enzyme after the purification of the wet cells are used as catalysts (preferably recombinant nitrilase), and 1-cyanocyclohexylacetonitrile (1-CN) is used as the substrate, and 0.2M, pH
  • wet cells are prepared as follows: the recombinant genetically engineered bacteria containing the polypeptide tag-containing recombinase (recombinant nitrilase) encoding gene are inoculated into LB (Luria-Bertani) medium, and after culturing at 37° C.
  • LB Lia-Bertani
  • the pure enzyme is prepared as follows: take the wet cells and evenly suspend them in 0.2M pH 7.0 Na 2 HPO 4 -NaH 2 PO 4 buffer solution, under ice bath conditions, carry out ultrasonic cell disruption, ultrasonic cell disruptor The power was set to 40W, the working time was 1s, the interval was 1s, and the total fragmentation time was 20min; then, the cell fragmentation solution was centrifuged at 12,000 ⁇ g, 4°C for 15min to remove cell debris and collect the crude enzyme solution; the volume of the buffer solution was Wet cell weight is 2mL/g;
  • the Ni column was equilibrated with loading buffer (Binding buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 50 mM imidazole, pH 8.0) at a flow rate of 2 mL/min; then, the crude enzyme solution was loaded at a flow rate of 2 mL/min , use the loading buffer to elute the impurity protein and weakly adsorbed protein; finally, use the elution buffer (Elution buffer: 50mM NaH 2 PO 4 , 300mM NaCl, 500mM imidazole, pH 8.0) for elution, the elution speed is 3 mL/min, based on the UV parameters of the protein purifier (Bio-Rad BioLogic LP chromatography system), when the UV ⁇ 2, the enzyme solution was collected, and when the UV ⁇ 2, the collection was ended; then a dialysis bag (Shanghai Rebs Biotechnology Co., Ltd. MD34-3500) was dialyzed in 50m
  • the method for preparing gabapentin from 1-cyanocyclohexylacetic acid according to the present invention is:
  • step (2) Dissolve the gabapent butyrolactam prepared in step (1) and dissolve it in 6M HCl and heat under reflux for 2.5h, after cooling to room temperature, add an equal volume of dichloromethane for extraction, and after standing for stratification, take the water phase at 0- Crystallization was carried out at 4°C, the white solid obtained by suction filtration was ground with acetone, acetone was removed by filtration and dried at 40°C to obtain gabapentin hydrochloride; all the obtained gabapentin hydrochloride was dissolved in water, heated to 40°C, 300rpm Fully dissolve under stirring, adjust pH to 7.0-7.5 with 6M NaOH; then add toluene, stir at 500rpm for 30min; finish stirring and crystallize at 0-4°C, filter to obtain a white solid which is the crude gabapentin, and use 60% methanol or isocyanide for the crude product Gabapentin is recrystallized from propanol and dried.
  • the volumetric consumption of HCl is 500mL/76.7g by weight of gabapentinolactam; the volumetric consumption of water for dissolving gabapentin hydrochloride is 500mL/76.7g by weight of gabapentinolactam; the volumetric consumption of toluene is by weight of gabapentinolactam Calculated as 125mL/76.7g.
  • the present invention also provides an application of the recombinase containing the polypeptide tag in the preparation of a clopidogrel intermediate (o-chloromandelic acid), the application is: using the recombinase (preferably recombinant nitrile) containing the polypeptide tag Hydrolase) of the genetically engineered bacteria obtained through fermentation and culture as catalyst, with o-chloromandelonitrile as substrate, and 0.2M, pH7.0 Na 2 HPO 4 -NaH 2 PO 4 buffer solution as reaction medium.
  • the reaction system was reacted in a constant temperature water bath at 35°C for 12 hours to obtain a reaction solution containing o-chloromandelic acid, which was separated and purified to obtain o-chloromandelic acid.
  • the amount of catalyst added is 50 g/L in terms of wet cell weight, and the final concentration of the substrate added is 1-2M.
  • the present invention also provides an application of the polypeptide-tagged recombinase in preparing ECBN (echinocandin B parent nucleus), the application is: the recombinase (preferably deacylase (NC_001136) containing the polypeptide-tagged recombinase (NC_001136 .10)) of the genetically engineered bacteria obtained by fermenting and culturing the wet cells as catalysts, using echinocandin B as a substrate, using 0.2M, pH7.0 Na 2 HPO 4 -NaH 2 PO 4 buffer and 1.5 % ⁇ -cyclodextrin (as a cosolvent) constitutes the reaction system, reacts in a constant temperature water bath at 35°C for 24 hours, and obtains a mixed reaction solution containing incompletely reacted substrates and partial products, which is separated and purified to obtain ECBN.
  • the amount of catalyst added is 50 g/L in terms of wet cell weight, and the final concentration of the substrate added is
  • the present invention provides a polypeptide label, and a recombinant nitrilase containing a polypeptide label and its application in the synthesis of medicinal chemicals such as gabapentin.
  • a recombinant nitrilase was constructed by linking a polypeptide tag to the N-terminus of the enzyme.
  • the polypeptide tag of the present invention is a positive charge tag.
  • the polypeptide tag is added to the N-terminus of the nitrilase gene for fusion expression, a recombinant strain is constructed, and the strain is induced to express for 12-14 hours, and the obtained whole cell catalyst is used in the preparation of gabapentin intermediates
  • the activity of 1-cyanocyclohexylacetic acid is as high as 3034.7U/g dcw, which significantly improves the soluble expression of nitrilase.
  • the same concentration of whole cell catalyst hydrolyzes 1M substrate, it is completed 30min faster than the parent enzyme, and the stability is better than that of the parent enzyme. this enzyme.
  • the method provided by the present invention can also be used for the biocatalytic reaction catalyzed by the nitrilase with other pharmaceutical intermediates as the substrate, and improve the activity of the whole-cell catalyst in the reaction; it can also be used to improve other different kinds of nitrilase or The solubility of other enzymes, and the activity of the corresponding whole-cell catalysts.
  • Figure 1 Map of recombinant plasmid pET-28b+/tag-AcN-M containing polypeptide tags.
  • Figure 2 Nucleic acid gel electrophoresis
  • lane M is Maker
  • lane 1 is the whole plasmid PCR product.
  • Figure 3 SDS-PAGE electropherogram of the disrupted supernatant and precipitated samples of recombinant strain E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M; where M is the molecular mass of standard protein, 1 , 2 are the cell disrupted supernatant and sediment of the original strain, respectively; 3, 4 are the cell disrupted supernatant and sediment of the recombinant strain, respectively.
  • Figure 4 Comparison of the relative cell viability of the original strain and the recombinant strain E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M at different temperatures; the original strain was tested under standard enzyme activity assay conditions The Vitality value is set to 100%.
  • Figure 5 Comparison of the relative viability of cells of the original strain and recombinant strain E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M under different pH; the original strain was tested under standard enzyme activity assay conditions The Vitality value is set to 100%.
  • Figure 6 Comparison of the accumulation concentration of the catalytic product in the cells of the original strain and the recombinant strain E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M at different substrate concentrations, A is the substrate concentration of 1M, B is the substrate concentration of 2M.
  • Specific enzymatic activity refers to the number of enzymatic activity units per unit weight (mg) of protein under specific conditions.
  • Resting cell viability assay method take 0.01g of resting cells and suspend them in 1mL, 0.2M, pH 7.0 Na 2 HPO 4 -NaH 2 PO 4 buffer, incubate at 35°C for 10min, add 0.03g (final concentration of 0.2M) to the bottom 1-cyanocyclohexylacetonitrile (1-CN), 200rpm, 35°C constant-temperature shaking reaction for 10min, centrifuged at 12,000rpm for 5min after the reaction, and the supernatant was collected to determine the product concentration.
  • 1-CN 1-cyanocyclohexylacetonitrile
  • the specific enzyme activity of the original strain was set as 100% under the standard enzyme activity assay conditions, and the ratio of the specific enzyme activity of the recombinant strain to that of the original strain was the relative cell viability (%).
  • Substrate 1-CN concentration detection method Gas chromatography: Agilent 7890A, chromatographic column: Agilent J&WHP-5 Column (30m ⁇ 0.32mm, film thickness 0.25 ⁇ m), the temperature of the inlet and detector is 320°C; the column temperature is 160°C , hold for 8min; carrier gas: high-purity helium; carrier gas flow: 1.0mL/min; injection volume: 1 ⁇ L; split ratio of 30:1.
  • Product 1-CA concentration detection method liquid chromatography: chromatographic column type is C18-H, 250mm ⁇ 4.6mm, J&K Scientific Ltd., China; chromatographic conditions are column temperature 40°C, UV detection wavelength 215nm, mobile phase 76 % buffer (0.58 g /L NH4H2PO4 and 1.83 g/L NaClO4 , pH 1.8) and 24 % acetonitrile.
  • the nitrilase gene is cloned from Acidovorax facilis ZJB09122, the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.
  • Acidovorax facilis (Acidovorax facilis ZJB09122) is deposited in the China Type Culture Collection with the deposit number CCTCC NO.M209044, which has been disclosed in the patent CN101629192B.
  • Example 1 Construction of recombinant plasmids containing polypeptide tags
  • the design principle is that the solubility of a protein is closely related to the hydrophobicity of the residues, and is also affected by the net charge of the protein or the ratio of helical residues.
  • Polar amino acids have an important effect on the solubility of proteins.
  • Palindromic element sequences typically consist of multiple repeating units containing one or two polar amino acids, are positively or negatively charged, have been reported to facilitate protein folding, and are typically less than 15 residues.
  • a pentapeptide tag in which both ends and the middle (ie, amino acids 1, 3, and 5) are uncharged glycine (G), and the rest (ie, amino acids 2 and 4) are glycine ( G), histidine (H), glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) any one or a random combination of two, specifically is one of the following: GDGDG, GDGEG, GDGRG, GDGKG, GDGGG, GEGEG, GEGKG, GEGGG, GEGRG, GEGDG, GKGKG, GKGHG, GKGRG, GRGRG, GRGDG, GRGEG, GRGKG, GRGGG, GGGKG, GGGEG , GGHHG, GGGKG.
  • linker peptide is designed, and the amino acid sequence is one of the following: GS, GGS, GGGS, GGGGS.
  • PCR system 25 ⁇ L 2 ⁇ Phanta Max Buffer (PCR system buffer), 1 ⁇ L dNTP Mix (dATP, dCTP, dGTP, dTTP), 1 ⁇ L template, 1 ⁇ L upstream primer, 1 ⁇ L downstream primer, 1 ⁇ L Phanta Max Super-Fidelity DNA Polymerase (High Fidelity DNA Polymerase), 20 ⁇ L of dd H2O in a total volume of 50 ⁇ L.
  • PCR reaction conditions were: pre-denaturation at 95°C for 5 min; 30 cycles of: denaturation at 95°C for 30 s, annealing at 55-65°C for 1 min, extension at 72°C for 5.5 min; 72°C for 10 min.
  • Axygen clean-up kit (purchased from Corning Life Sciences (Wujiang) Co., Ltd.) was used to purify (clean-up) the PCR product containing the recombinant plasmid pET-28b(+)/tag-AcN-M in Example 1,
  • the specific operations are as follows: add three times the volume of PCR-A buffer to the 5 ⁇ L PCR product of Example 1 and mix well, move it to a preparation tube, centrifuge at 12,000 rpm for 1 min, discard the filtrate, add 700 ⁇ L of W2 buffer to the preparation tube, and centrifuge at 12,000 rpm 1min, discard the filtrate, wash twice with W2 buffer; add 5 ⁇ L to the pre-thawed competent cells E.coli BL21 (DE3), ice bath for 30min, then heat shock at 42°C for 90s, ice bath again for 3-5min, add 700 ⁇ L LB Liquid medium, incubated at 37°C for 1h.
  • Example 3 Recombinant Escherichia coli expressing nitrilase
  • the Ni column was equilibrated with loading buffer (Binding buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 50 mM imidazole, pH 8.0) at a flow rate of 2 mL/min. Subsequently, the crude enzyme solution was loaded at a flow rate of 2 mL/min, and the impurity proteins and weakly adsorbed proteins were eluted with the loading buffer.
  • loading buffer 50 mM NaH 2 PO 4 , 300 mM NaCl, 50 mM imidazole, pH 8.0
  • MD34-3500 to dialyze in 50mM pH 7.0 Na 2 HPO 4 -NaH 2 PO 4 buffer overnight, take the retentate, which is the pure enzyme of nitrilase, ice bath Save it for later use, and use BCA kit to detect the protein content, which is the protein content of the supernatant.
  • Solubility (%) protein amount in supernatant/total protein amount ⁇ 100%.
  • the relative enzyme activity results show that the enzyme activity of recombinant E. coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M is 2.37 times that of the original strain, and the insertion of the tag hardly affects the large intestine normal growth of bacteria.
  • the protein electrophoresis experiment shown in Figure 3 shows that the soluble expression (protein amount in supernatant/total protein amount) of the recombinant strain is significantly enhanced. The solubility of the original strain is only 53.6%, while that of the recombinant strain reaches 87.9%.
  • Linker between the peptide tag and the target gene on the solubility of the original strain and recombinant strain E.coli BL21(DE3)/pET-28b(+)/tag-AcN-M
  • Example 4 The effect of temperature on the enzymatic activity of the original strain and recombinant strain E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M
  • Example 5 The effect of pH on the enzymatic activity of the original strain and recombinant strain E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M
  • 100 ⁇ L of resting cell suspension prepared by the method in Example 3 was added to 900 ⁇ L of buffers with different pH values (0.1M citric acid-sodium citrate buffer at pH 4.0-6.0; 0.2M Na 2 HPO at pH 6.0-8.0 4 -NaH 2 PO 4 buffer; 50mM Glycine-NaOH buffer with pH of 9.0-10.0) to form a 1mL reaction system, so that the resting cells in the reaction system were added at a concentration of 10g/L, and the reaction system was shaken at 35°C on the reactor. Preheat for 10 min, then add 1-CN with a final concentration of 0.2 M, react at 800 rpm and 35 °C for 10 min.
  • pH values 0.1M citric acid-sodium citrate buffer at pH 4.0-6.0; 0.2M Na 2 HPO at pH 6.0-8.0 4 -NaH 2 PO 4 buffer; 50mM Glycine-NaOH buffer with pH of 9.0-10.0
  • the samples were centrifuged at 12,000 rpm for 5 min, the supernatant was taken, and the 1-CA concentration in the supernatant was analyzed by HPLC. Under the same conditions, taking the original strain E. coli BL21 (DE3) as the control, the results are shown in Figure 5.
  • the optimum pH of the recombinant strain is 8.0, which has not changed compared with the original strain, and at the same pH, The cellular enzyme activities of the recombinant strains were higher than those of the original strains.
  • Example 6 Comparison of cell catalytic efficiency between original strain and recombinant strain E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M
  • Example 7 Preparation of gabapentbutyrolactam using 1-cyanocyclohexylacetic acid in the transformation solution produced by whole cell catalysis
  • the transformation solution obtained in Example 6 is centrifuged (8000rpm 10min) to remove the bacterial cells, and the collected filtrate is 1-cyanocyclohexylacetic acid, get 150mL of filtrate in a 500mL hydrogenation reactor, add 1.5g Raney nickel (type RTH-4110), 1 mL of triethylamine (analytical grade) and 500 ⁇ L of formic acid (analytical grade); feed nitrogen to replace air, repeat this 3 times to ensure that there is no air in the kettle; feed hydrogen again (keep the pressure during the reaction At 2Mpa), the 1000rpm reaction ended after 8h; after cooling, the Raney nickel was filtered and recovered, and the obtained filtrate was added with an equal volume of dichloromethane for extraction, and the organic phase was left to stand for stratification to get the organic phase at 40 ° C for rotary evaporation to obtain a solid, which is gabapentin.
  • Embodiment 8 Utilize hydrogenation to obtain gabapent butyrolactam to prepare medicinal chemicals-gabapentin
  • isopropanol recrystallization and drying is gabapentin.
  • the above experiments involve the extraction, suction filtration, and unused samples and used reagents that can be recycled.
  • the experimental results show that the yield of gabapentin hydrochloride reaches 81%, the yield of gabapentin obtained by recrystallization reaches 73.6%, and the yield of gabapentin after repeated recovery of mother liquor for 3-5 times reaches 93.2%.
  • the yields of intermediates and final gabapentin have reached a high level, and multiple sample and reagent recovery steps have reduced costs and waste water generation, meeting the concept of green chemistry, and realizing the efficient chemical-enzymatic method of pharmaceutical chemicals Production.
  • Example 9 Application effect of polypeptide tag on nitrilase from other sources
  • Example 1 The preferred polypeptide tag GKGKG of Example 1 was linked to nitrilase LNIT5 (Accession No.: AAR97494.1), nitrilase No. 385, 386 (Accession No.: AY487562) and derived from R. rhodochrous according to the method of Example 1 N-terminal of nitrilase (hereinafter referred to as RrNit) of K22 (Accession No.: Q02068.1). Solubility and relative cellular enzymatic activity were determined according to the method of Example 3.
  • the required reaction time of the recombinant strain was 3h, while the original strain
  • the reaction time required 4h, and the conversion rate of both reached more than 99%; however, in the case of 2M substrate, due to the high substrate concentration, after 12h of reaction, the conversion rate of the original strain to the substrate was 60.4%, while the recombinant Due to the increase of the effective protein content of the strain, the transformation rate reached 79.4% under the same conditions.
  • Embodiment 11 The application effect of preferred polypeptide tag in deacylase to convert echinocandin B to prepare echinocandin B parent nucleus
  • the preferred polypeptide tag GKGKG of Example 1 was connected to the deacylase (NC_001136.10) according to the method of Example 1, and the recombinant bacteria E.coli BL21(DE3)/pET-28b(+)/GKGKG-DEA (de-acylase) was constructed.
  • Acylase), its solubility and enzyme activity were determined, and the solubility of the deacylase containing the polypeptide tag was 2.8 times higher than that of the deacylase without the polypeptide tag, and the specific enzyme activity was increased by 358.5%.
  • Example 10 The catalyst in Example 10 was changed to 50g/L recombinant bacteria E.coli BL21(DE3)/pET-28b(+)/GKGKG-DEA (deacylase) resting cells, and the substrate was changed to a final concentration of 2g/L For echinocandin B, the reaction time was changed to 24h, and the others were the same as in Example 10.
  • the conversion rate of the substrate reached 60.6%, while the conversion rate of the deacylase without the polypeptide tag was only 35.7%. It shows that this peptide tag has a certain expansibility, but whether the solubilization effect on other enzymes is good or not needs to be discussed in depth.

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Abstract

提供一种重组腈水解酶及其在医药化学品合成中的应用，所述重组腈水解酶是在腈水解酶氨基酸序列的N端连接多肽标签；所述多肽标签两端位置均为不带电的甘氨酸G，其余为甘氨酸G、组氨酸H、谷氨酸E、天冬氨酸D、赖氨酸K、精氨酸R中任意一种或多种的随机组合。重组腈水解酶制备1-氰基环己基乙酸时活力高达3034.7U/g dcw，多肽标签的引入显著提高了腈水解酶的可溶性表达，相同浓度全细胞催化剂水解1M底物时较母本酶快30min完成，且稳定性优于母本酶。所述重组腈水解酶还能够催化合成其它医药中间体，并提高反应中全细胞催化剂的活力，提高其它不同种类腈水解酶的可溶性，以及相应的全细胞催化剂的活力。

Description

多肽标签、高度可溶性的重组腈水解酶及其在医药化学品合成中的应用 (一)技术领域
本发明涉及一种多肽标签，特别涉及一种可增强腈水解酶等酶可溶性表达的通用多肽标签，及在医药化学品合成中的应用，属于基因工程以及蛋白质工程技术领域。
(二)背景技术
腈水解酶(EC 3.5.5.1)是一类重要的具有Glu-Lys-Cys催化活性中心三联体的水解酶，可在一步反应中有选择地且有效地催化氰基水解为羧基，在有机酸、氨基酸、维生素等化工产品和如加巴喷丁、氯吡格雷、巴氯芬、阿托伐他汀等医药化学品的合成中具有广泛的应用。
迄今为止，已经报道了许多提高蛋白质可溶性表达水平的有效方法，一种是构建重组周质渗漏菌株或共表达周质伴侣蛋白，另一种是添加融合标签(也称为融合伴侣或溶解性标签)进行协同表达。但这些方法或多或少的存在一些缺陷。如多肽标签上氨基酸的选择、序列的长度可能对重组酶的酶活，稳定性，溶解度或选择性有很大副作用，某些标签甚至会改变酶蛋白的结构导致酶完全失去活性。对于不同的酶的结构和蛋白整体在催化体系中的带电属性，不同的多肽标签的引入会带来不同的效果，需要结合动力学模拟、同源建模等技术进行协助标签的设计。但是有效的多肽标签的优点之一是和目的基因连接后表达并不需要去除即可发挥酶蛋白的作用。其中，Sun-Ki Kim开发了一种新的聚阴离子多肽标签，显著提高了南极假丝酵母脂肪酶的表达水平和细胞外转运效率。此外，Han等人开发了一种新的融合标签[HE-MBP(Pyr)]以提高重组蛋白在大肠杆菌中的溶解度，研究表明目的蛋白(如单克隆抗体，抗原蛋白和聚合蛋白)溶解度有不同程度的改善。
在生物合成方法中，涉及到生物催化的过程通常都是使用全细胞催化剂(湿细胞)来进行的。一般认为产酶发酵中发酵液的体积酶活为比酶活和可溶性表达水平两者的结合。若能在不影响原始特性的前提下，提高酶的比酶活或有效蛋白质的数量将促进全细胞催化性能的进一步提高。所以，增加腈水解酶在大肠杆菌中的溶解度是有巨大意义的。然而增加大肠杆菌所产酶的溶解度是总是以牺牲活力为代价的。因此，在不降低催化水平的情况下合理地增加腈水解酶的溶解度是实现加巴喷丁中间体1-氰基环己基乙酸(1-CA)的高效生产仍需进一步研究。
(三)发明内容
本发明目的是提供一种多肽标签、含多肽标签的重组酶及其在医药化学品合成中的应用，所述多肽标签能够使含多肽标签的重组酶的全细胞催化剂活力和热稳定性均得到有效提升。解决酶热稳定性差，酶活不满足工业大规模应用等问题。
本发明采用的技术方案是：
本发明提供一种多肽标签，所述多肽标签两端的氨基酸均为不带电的甘氨酸(G)，其余为甘氨酸(G)、组氨酸(H)、谷氨酸(E)、天冬氨酸(D)、赖氨酸(K)、精氨酸(R)中任意一种或多种的随机组合；所述多肽标签的长度为5－11个氨基酸。
进一步，所述多肽标签氨基酸序列为下列之一：GKGKG、GKGEG、GKGHG、GRGRG、GRGGG、GHGHG、GDGDG、GDGEG、GDGRG、GDGKG、GEGEG、GEGKG、GEGGG、GEGRG、GEGDG、GKGKG、GKGDG、GKGEG、GKGGG、GKGHG、GKGRG、GRGRG、GRGDG、GRGEG、GRGKG、GRGGG、GGGKG、GGGEG、GHGHG、GKGKGKG、GKGKGKGKG、GKGKGKGKGKG。
进一步，优选所述多肽标签两端和中间位置均为不带电的甘氨酸(G)，其余为甘氨酸(G)、组氨酸(H)、谷氨酸(E)、天冬氨酸(D)、赖氨酸(K)、精氨酸(R)中任意一种或多种的随机组合，更优选构成具有回文元素的氨基酸序列。
进一步，所述多肽标签还设有连接肽，所述连接肽氨基酸序列为下列之一：GS、GGS、GGGS、GGGGS。
更进一步，优选多肽标签为GKGKG。
本发明提供一种含所述多肽标签的重组酶，所述酶包含腈水解酶，脂肪酶，脱酰基酶。
进一步，所述重组酶优选为重组腈水解酶，所述重组腈水解酶是将腈水解酶氨基酸序列的N端与多肽标签连接获得的。所述连接可以采用PCR扩增、一步克隆等方法，例如以含腈水解酶基因的载体(优选pET-28b(+)/AcN-M)为模板，设计含多肽标签的引物，经PCR扩增，获得含多肽标签的腈水解酶。
进一步，所述多肽标签通过连接肽与腈水解酶连接的方式为下列之一：GKGKG-GS、GKGKG-GGS、GKGKG-GGGS、GKGKG-GGGGS。
本发明所述腈水解酶基因克隆自敏捷食酸菌(Acidovorax facilis ZJB09122)，氨基酸序列为SEQ ID NO.1所示，核苷酸序列为SEQ ID NO.2所示。
本发明还涉及含有所述多肽标签的重组酶编码基因的重组质粒(优选重组腈水解 酶编码基因的重组质粒)，所述重组质粒以pET-28b(+)为载体构建，具体为：以含有所述重组酶基因的质粒作为模板，设计含多肽标签的引物，进行全质粒PCR，核酸凝胶电泳和测序进行验证，最终得到重组质粒。
本发明还提供一种由所述含多肽标签的重组酶编码基因构建的重组基因工程菌，所述重组基因工程菌是将含多肽标签的重组酶编码基因的载体pET-28b(+)，导入宿主菌构建而成；所述宿主菌优选为Escherichia coli BL21(DE3)。
本发明还提供一种所述含多肽标签的重组酶在制备加巴喷丁中间体1-氰基环已基乙酸中的应用，所述的应用为：以含多肽标签的重组酶编码基因的重组基因工程菌经发酵培养获得的湿菌体或湿菌体纯化后的纯酶为催化剂(优选重组腈水解酶)，以1-氰基环己基乙腈(1-CN)为底物，以0.2M、pH＝7.0的Na 2HPO 4-NaH 2PO 4缓冲液为反应介质构成转化体系，35℃、200rpm恒温水浴反应完全，获得含1-氰基环己基乙酸(1-CA)的转化液，转化液分离纯化，获得1-氰基环己基乙酸；所述1-氰基环己基乙酸经过后续加氢等步骤获得加巴喷丁；所述转化体系中，底物加入终浓度为1-2M，催化剂加入量以湿菌体重量计为50g/L。
进一步，所述湿菌体按如下方法制备：将含多肽标签的重组酶(重组腈水解酶)编码基因的重组基因工程菌接种至LB(Luria-Bertani)培养基，37℃培养12-14h后，获得单菌落；挑取单菌落接种到含0.5μg/mL卡那霉素的LB培养基中，37℃培养8h后，以体积浓度2％的接种量转接至含0.5μg/mL卡那霉素的发酵培养基中，37℃培养2h后，加入终浓度0.1mM的IPTG，28℃诱导产酶12-14h，12,000转离心10min，沉淀用0.9％的生理盐水清洗2次，收集湿菌体即为催化剂。所述发酵培养基组成：20g/L酵母粉，15g/L蔗糖，5g/L NaCl，0.9g/L三水合磷酸氢二钾，溶剂为水，pH＝6.8。
进一步，所述纯酶按如下方法制备：取湿菌体均匀悬浮于0.2M pH 7.0Na 2HPO 4-NaH 2PO 4缓冲液中，在冰浴条件下，进行超声波细胞破碎，超声波细胞破碎仪功率设定为40W，工作1s，间隔1s，总破碎时间为20min；随后，将细胞破碎液在12,000×g，4℃离心15min，去除细胞碎片，收集粗酶液；所述缓冲液体积用量以湿菌体重量计为2mL/g；
将Ni柱用上样缓冲液(Binding buffer：50mM NaH 2PO 4，300mM NaCl，50mM咪唑，pH 8.0)进行平衡，流速为2mL/min；随后，以2mL/min的流速将粗酶液上样，使用上样缓冲液洗脱杂蛋白以及弱吸附蛋白；最后，使用洗脱缓冲液(Elution buffer:50mM NaH 2PO 4，300mM NaCl，500mM咪唑，pH 8.0)进行洗脱，洗脱速度为3 mL/min，以蛋白纯化仪(Bio-Rad BioLogic LP层析***)UV参数为准，当UV≥2时，进行酶液的收集，当UV≤2时，结束收集；接着使用透析袋(上海雷布斯生物科技有限公司MD34-3500)在50mM pH 7.0Na 2HPO 4-NaH 2PO 4缓冲液中透析过夜，取截留液，即为腈水解酶纯酶。
本发明所述1-氰基环己基乙酸制备加巴喷丁的方法为：
(1)将含1-氰基环己基乙酸(1-CA)的转化液离心(8000rpm 10min)去除菌体细胞，收集的滤液即为1-氰基环己基乙酸，取滤液于加氢反应釜，加入雷尼镍(型号为RTH-4110)、三乙胺(分析纯)和甲酸(分析纯)；通入氮气置换空气，如此重复3次，保证釜内无空气存在；再次通入氢气(保持反应过程中压力在2Mpa)，1000rpm反应8h后结束；冷却之后过滤回收雷尼镍，得到的滤液加入等体积的二氯甲烷进行萃取，静置分层取有机相40℃下进行旋蒸得到固体即为加巴喷丁内酰胺，二氯甲烷可回收再利用；所述滤液体积用量以雷尼镍重量计为150mL/1.5g；所述三乙胺与滤液体积比为1:150；所述甲酸体积用量以滤液体积比为0.5:150；
(2)取步骤(1)中制备得到的加巴喷丁内酰胺溶解于6M HCl中加热回流2.5h，待冷却至室温后加入等体积的二氯甲烷进行萃取，静置分层后取水相在0-4℃下进行结晶，抽滤得到的白色固体用丙酮研磨，过滤除去丙酮并在40℃烘干得到加巴喷丁盐酸盐；将得到的所有的加巴喷丁盐酸盐溶于水中，加热至40℃，300rpm搅拌下充分溶解，使用6M NaOH调pH至7.0-7.5；后加入甲苯，500rpm搅拌30min；结束搅拌于0-4℃下进行结晶，过滤得到白色固体即为加巴喷丁粗品，粗品用60％甲醇或异丙醇重结晶并烘干即为加巴喷丁。所述HCl体积用量以加巴喷丁内酰胺重量计为500mL/76.7g；所述溶解加巴喷丁盐酸盐的水体积用量以加巴喷丁内酰胺重量计为500mL/76.7g；所述甲苯体积用量以加巴喷丁内酰胺重量计为125mL/76.7g。
本发明还提供一种所述含多肽标签的重组酶在制备氯吡格雷中间体(邻氯扁桃酸)中的应用，所述的应用为：将含所述多肽标签的重组酶(优选重组腈水解酶)的基因工程菌经发酵培养获得的湿菌体为催化剂，以邻氯扁桃腈为底物，以0.2M、pH7.0的Na 2HPO 4-NaH 2PO 4缓冲液为反应介质构成反应体系，35℃恒温水浴反应12h，获得含邻氯扁桃酸的反应液，分离纯化，得到邻氯扁桃酸。所述反应体系中，催化剂加入量以湿菌体重量计为50g/L，所述底物加入终浓度为1-2M。
本发明还提供一种所述多肽标签的重组酶在制备ECBN(棘白菌素B母核)中的应用，所述应用为：将含所述多肽标签的重组酶(优选脱酰基酶(NC_001136.10)) 的基因工程菌经发酵培养获得的湿菌体为催化剂，以棘白菌素B为底物，以0.2M、pH7.0的Na 2HPO 4-NaH 2PO 4缓冲液和1.5％β-环糊精(作为助溶剂)构成反应体系，35℃恒温水浴反应24h，获得含未完全反应的底物和部分产物的混合反应液，分离纯化，得到ECBN。所述反应体系中，催化剂加入量以湿菌体重量计为50g/L，所述底物加入终浓度为2g/L。
与现有技术相比，本发明有益效果主要体现在：本发明提供了一种多肽标签，以及含多肽标签的重组腈水解酶及其在加巴喷丁等医药化学品合成中的应用，通过在腈水解酶N端连接多肽标签构建重组腈水解酶。本发明多肽标签为正电荷标签，将多肽标签添加至腈水解酶基因的N端进行融合表达，构建重组菌株，并将此菌株诱导表达12-14h，得到的全细胞催化剂，在制备加巴喷丁中间体1-氰基环己基乙酸时活力高达3034.7U/g dcw，显著提高了腈水解酶的可溶性表达，相同浓度全细胞催化剂水解1M底物时较母本酶快30min完成，且稳定性优于母本酶。本发明提供的方法也可以用于该腈水解酶催化的以其它医药中间体为底物的生物催化反应，并提高反应中全细胞催化剂的活力；还可以用于提高其它不同种类腈水解酶或者其他酶的可溶性，以及相应的全细胞催化剂的活力。
(四)附图说明
图1：含多肽标签的重组质粒pET-28b+/tag-AcN-M图谱。
图2：核酸凝胶电泳图，泳道M为Maker，泳道1为全质粒PCR产物。
图3：重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M细胞破碎上清液和沉淀样品的SDS-PAGE电泳图；其中M为标准蛋白质的分子质量，1、2分别为原始菌株的细胞破碎上清液和沉淀；3、4分别为重组菌株的细胞破碎上清液和沉淀。
图4：原始菌株和重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞在不同温度作用下的相对活力比较；其中原始菌株在标准酶活测定条件下的活力值设置为100％。
图5：原始菌株和重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞在不同pH作用下的相对活力比较；其中原始菌株在标准酶活测定条件下的活力值设置为100％。
图6：原始菌株和重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞在不同底物浓度催化产物积累浓度的比较，A为底物浓度1M，B为底物浓度2M。
(五)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：
下列实施实例中所涉及的培养基如下：
LB固体培养基质量组成：5g/L酵母粉，10g/L蛋白胨，10g/L NaCl，2％琼脂粉，溶剂为水，pH＝7.0。
LB液体培养基：5g/L酵母粉，10g/L蛋白胨，10g/L NaCl，溶剂为水，pH＝7.0。
发酵培养基：20g/L酵母粉，15g/L蔗糖，5g/L NaCl，0.9g/L三水合磷酸氢二钾，溶剂为水，pH＝6.8。
下列实施实例中所涉及的检测方法如下：
酶活定义：在一定条件下，每分钟催化底物生成1μmol 1-氰基环己基乙酸(1-CA)所需的酶量定义为一个活力单位，记为U。
比酶活是指特定条件下，单位重量(mg)蛋白具有的酶活力单位数。
静息细胞活力测定方法：取0.01g静息细胞悬浮于1mL、0.2M、pH 7.0 Na 2HPO 4-NaH 2PO 4缓冲液，35℃保温10min，加入0.03g(终浓度为0.2M)底物1-氰基环己基乙腈(1-CN)，200rpm，35℃恒温振荡反应10min，反应结束后12,000rpm离心5min，取上清，测定产物浓度。
标准酶活测定条件下原始菌株的比酶活设为100％，重组菌株的比酶活与原始菌株的比酶活之比即为相对细胞活力(％)。
底物1-CN浓度检测方法：气相色谱：Agilent 7890A，色谱柱：Agilent J&WHP-5 Column(30m×0.32mm，膜厚0.25μm)，进样口及检测器温度为320℃；柱温160℃，保持8min；载气：高纯氦气；载气流量：1.0mL/min；进样量：1μL；分流比为30:1。
产物1-CA浓度检测方法：液相色谱：色谱柱类型为C18-H，250mm×4.6mm，J&K Scientific Ltd.，China；色谱条件为柱温40℃，紫外检测波长为215nm，流动相为76％缓冲液(0.58g/L NH 4H 2PO 4和1.83g/L NaClO 4，pH 1.8)和24％乙腈。
所述腈水解酶基因克隆自敏捷食酸菌(Acidovorax facilis ZJB09122)，氨基酸序列为SEQ ID NO.1所示，核苷酸序列为SEQ ID NO.2所示。敏捷食酸菌(Acidovorax facilis ZJB09122)保藏在中国典型培养物保藏中心，保藏编号为CCTCC NO.M209044，已在专利CN101629192B中披露。
实施例1：含多肽标签的重组质粒的构建
1、设计原则为：蛋白质的溶解度与残基的疏水性密切相关，同时也受到蛋白质净电荷或螺旋残基比例的影响。极性氨基酸对蛋白质的溶解度有重要影响。回文元素序列通常由含有一个或两个极性氨基酸的多个重复单元组成，具有正电荷或负电荷，有报道称其能够促进蛋白质折叠，且通常小于15个残基。
基于以上原则我们首先设计了一种五肽标签，其中两端和中间(即1、3、5位氨基酸)均为不带电的甘氨酸(G)，其余(即2、4位氨基酸)为甘氨酸(G)、组氨酸(H)、谷氨酸(E)、天冬氨酸(D)、赖氨酸(K)、精氨酸(R)中任意一种或两种的随机组合，具体为下列之一：GDGDG、GDGEG、GDGRG、GDGKG、GDGGG、GEGEG、GEGKG、GEGGG、GEGRG、GEGDG、GKGKG、GKGDG、GKGEG、GKGGG、GKGHG、GKGRG、GRGRG、GRGDG、GRGEG、GRGKG、GRGGG、GGGKG、GGGEG、GHGHG、GGGKG。
其次，设计连接肽，氨基酸序列为下列之一：GS、GGS、GGGS、GGGGS。
最后设计下列肽链延长的多肽标签及含有连接肽的多肽标签：GKGKGKG，GKGKGKGKG，GKGKGKGKGKG，GKGKG-GS，GKGKG-GGS，GKGKG-GGGS，GKGKG-GGGGS。
2、根据专利申请(CN104212784A)从敏捷食酸菌(Acidovorax facilis ZJB09122)中得到含表达载体pET-28b(+)的重组大肠杆菌BL21(DE3)/pET28b(+)-AcN2，再根据专利申请(CN107177576A)制备E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G。
提取E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G中的重组质粒pET28b(+)-AcN-T151V/C223A/C250G，即为重组质粒pET-28b(+)/AcN-M，其中AcN-M核苷酸序列为SEQ ID NO.2所示，氨基酸序列为SEQ ID NO.1所示。
以含有SEQ ID NO.1所示腈水解酶(AcN-M)的编码基因的重组质粒pET-28b(+)/AcN-M为模板，以含步骤1所述多肽标签的上下游引物(核苷酸序列如下表1所示)，进行PCR扩增反应，步骤1设计的多肽标签通过PCR扩增直接加到腈水解酶基因的N端，PCR扩增产物通过凝胶电泳验证PCR是否成功，随后以1μL/50μL扩增体系的量加入dpn I，37℃反应30min消化模板，取25μL测序验证(杭州擎科生物科技有限公司)，获得含重组质粒pET-28b(+)/tag-AcN-M的PCR产物，tag代表多肽标签，见表1。
PCR体系为：25μL 2×Phanta Max Buffer(PCR体系缓冲液)，1μL d NTP Mix (dATP,dCTP,dGTP,dTTP)，1μL模板，1μL上游引物，1μL下游引物，1μL Phanta Max Super-Fidelity DNA Polymerase(高保真DNA聚合酶)，20μL dd H 2O，总体积为50μL。
PCR反应条件为：预变性95℃，5min；30个循环：变性95℃，30s，退火55-65℃，1min，延伸72℃，5.5min；72℃10min。
表1含多肽标签的引物
Figure PCTCN2020135580-appb-000001
Figure PCTCN2020135580-appb-000002
实施例2：重组大肠杆菌的构建
采用Axygen clean-up试剂盒(购自康宁生命科学(吴江)有限公司)对实施例1中含重组质粒pET-28b(+)/tag-AcN-M的PCR产物进行纯化(clean-up)，具体操作为：向实施例1的5μL PCR产物中加入三倍体积的PCR-A buffer充分混匀，移到制备管中，12000rpm离心1min，弃滤液，向制备管中加入700μL W2 buffer，12000rpm离心1min，弃滤液，W2 buffer洗涤2次；取5μL加到预先解冻的感受态细胞E.coli BL21(DE3)，冰浴30min，之后42℃热击90s，再次冰浴3-5min，加入700μL LB液体培养基，37℃孵育1h。取500μL培养液接种到具有0.5μg/mL卡那抗性的LB固体培养基上，涂布均匀为止，37℃培养12-14h，挑菌测序验证，获得重组大肠杆菌E.coli BL21(DE3)/pET-28b(+)/tag-AcN-M。同样条件下，构建原始菌株E.coli BL21(DE3)/pET-28b(+)/AcN-M。
实施例3：重组大肠杆菌表达腈水解酶
1、静息细胞：将保藏在-80℃冰箱中由实施例2构建的重组大肠杆菌E.coli  BL21(DE3)/pET-28b(+)/tag-AcN-M菌株取出，接种至含0.5μg/mL卡那抗性的LB平板划线，37℃培养12-14h后，获得单菌落。挑取单菌落到10mL含0.5μg/mL卡那霉素的LB试管培养基中，37℃培养8h后，转接2mL至100mL含0.5μg/mL卡那霉素的发酵培养基中，37℃培养2h后，加入100μL IPTG(终浓度0.1mM)，28℃诱导产酶12-14h。12,000转离心10min，收集得到的菌体，用0.9％的生理盐水清洗2次后悬浮，获得静息细胞悬液，测定相对酶活。
2、纯酶：取步骤1方法制备的1g静息细胞均匀悬浮于10mL、0.2M pH 7.0 Na 2HPO 4-NaH 2PO 4缓冲液中，在冰浴条件下，进行超声波细胞破碎，超声波细胞破碎仪功率设定为40W，工作1s，间隔1s，总破碎时间为20min。随后，将细胞破碎液在12,000×g，4℃离心15min，去除细胞碎片，收集粗酶液，采用BCA试剂盒检测蛋白含量，即为总蛋白量。
将Ni柱用上样缓冲液(Binding buffer：50mM NaH 2PO 4，300mM NaCl，50mM咪唑，pH 8.0)进行平衡，流速为2mL/min。随后，以2mL/min的流速将粗酶液上样，使用上样缓冲液洗脱杂蛋白以及弱吸附蛋白。最后，使用洗脱缓冲液(Elution buffer:50mM NaH 2PO 4，300mM NaCl，500mM咪唑，pH 8.0)进行洗脱，洗脱速度为3mL/min，以蛋白纯化仪(Bio-Rad BioLogic LP层析***)UV参数为准，当UV≥2时，进行酶液的收集，当UV≤2时，结束收集。接着使用透析袋(上海雷布斯生物科技有限公司MD34-3500)在50mM pH 7.0Na 2HPO 4-NaH 2PO 4缓冲液中透析过夜，取截留液，即为腈水解酶纯酶，冰浴保存备用，采用BCA试剂盒检测蛋白含量，即为上清液蛋白量。
溶解度(％)＝上清液蛋白量/总蛋白量×100％。
表2含不同多肽标签的重组菌株的相对酶活及溶解度的对比
Figure PCTCN2020135580-appb-000003
Figure PCTCN2020135580-appb-000004
根据表2，相对酶活结果显示重组大肠杆菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的酶活是原始菌株的2.37倍，且标签的***几乎不影响大肠杆菌的正常生长。蛋白电泳实验如图3所示，重组菌株的可溶性表达(上清液蛋白量/总蛋白量)显著增强，原始菌株的溶解度仅为53.6％，而重组菌株溶解度达到87.9％。
4、多肽标签序列的长度对原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/tag-AcN-M溶解度的影响
表3含不同长度多肽标签重组菌株的相对酶活及溶解度的对比
Figure PCTCN2020135580-appb-000005
根据表3，多肽标签中的极性氨基酸对溶解度的影响改变较为明显，其中含有3个赖氨酸的多肽标签的腈水解酶溶解度达到了90.9％，而其他长度标签的腈水解酶溶解度变化不大。
5、多肽标签与目的基因之间的连接肽(Linker)对原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/tag-AcN-M溶解度的影响
表4含不同Linker的重组菌株的相对酶活及溶解度的对比
Figure PCTCN2020135580-appb-000006
Figure PCTCN2020135580-appb-000007
根据表4，Linker对多肽标签和目的基因的作用是很重要的，其中Linker的长度越长，腈水解酶的溶解度随之增加，但对其催化活力的抑制作用越来越强，且最长的Linker和多肽标签组合的重组菌株细胞的催化活力低至59.6％。
根据表2-表4，选取重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M进行后续试验。
实施例4：温度对原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M细胞酶活的影响
将200mM pH 7.0的Na 2HPO 4-NaH 2PO 4缓冲液900μL与100μL实施例3方法制备的静息细胞悬液混合后，构成1ml反应体系，使得反应体系中静息细胞加入量为10g/L，置于设定温度的振荡反应器上保温10min，温度分别为20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃和70℃，之后加入终浓度为0.2M的1-CN，在转速为800rpm的振荡反应器上反应10min后，取样离心，取上清，使用HPLC分析上清液中1-CA浓度。同样条件下，以原始菌株E.coli BL21(DE3)/pET-28b(+)/AcN-M为对照，结果如图4所示，重组菌株的最适温度为55℃，相比于原始菌株并没有发生改变，并且在相同温度下的，重组菌株的细胞酶活高于原始菌株。
实施例5：pH对原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M细胞酶活的影响
将实施例3方法制备的100μL静息细胞悬液加入900μL不同pH值的缓冲液(0.1M pH为4.0-6.0的柠檬酸-柠檬酸钠缓冲液；0.2M pH为6.0-8.0的Na 2HPO 4-NaH 2PO 4缓冲液；50mM pH为9.0-10.0的Glycine-NaOH缓冲液)中，构成1mL反应体系，使得反应体系中静息细胞加入浓度为10g/L，在35℃振荡反应器上预热10min，之后加入终浓度为0.2M的1-CN，800rpm，35℃反应10min。取样12,000rpm离心5min，取上清，使用HPLC分析上清液中1-CA浓度。同样条件下，以原始菌株E.coli BL21(DE3)为对照，结果如图5所示，重组菌株的最适pH为8.0，相比于原始菌株并没有发生改变，并且在相同pH下的，重组菌株的细胞酶活均高于 原始菌株。
实施例6：原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞催化效率比较
将实施例3方法制备的重组菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的湿菌体5g悬浮于100mL Na 2HPO 4-NaH 2PO 4缓冲液中(0.2M，pH＝7.0)，分别加入1.48g和2.2g 1-CN(终浓度分别1M和2M)，35℃恒温水浴反应12h。每隔30min取样，12,000rpm离心5min，取上清，使用HPLC分析上清液中1-CA浓度，并检测1-CN的浓度。同样条件下，以原始菌株E.coli BL21(DE3)-AcN-M为对照，结果如图6所示，在催化1M 1-CN时，重组菌株所需的反应时间为2h，而原始菌株的反应时间需2.5h，且转化率两者都达到了99％以上；然而在2M底物时，由于底物浓度过高，反应12h后，原始菌株对底物的转化率为77.5％，而重组菌株由于有效蛋白量的增加，相同条件下转化率达到了81.7％。
实施例7：利用全细胞催化生产的转化液中1-氰基环己基乙酸制备加巴喷丁内酰胺
将实施例6中获得转化液进行离心(8000rpm 10min)去除菌体细胞，收集的滤液即为1-氰基环己基乙酸，取150mL滤液于500mL加氢反应釜，加入1.5g雷尼镍(型号为RTH-4110)、1mL三乙胺(分析纯)和500μL甲酸(分析纯)；通入氮气置换空气，如此重复3次，保证釜内无空气存在；再次通入氢气(保持反应过程中压力在2Mpa)，1000rpm反应8h后结束；冷却之后过滤回收雷尼镍，得到的滤液加入等体积的二氯甲烷进行萃取，静置分层取有机相40℃下进行旋蒸得到固体即为加巴喷丁内酰胺，二氯甲烷可回收再利用。实验结果表明全细胞催化得到的转化液可直接用于下一步加氢反应，底物转化率达到了99.6％，加巴喷丁内酰胺得率得到95.8％，底物转化率和产物得率得到工业生产化学品要求。
实施例8：利用加氢得到加巴喷丁内酰胺制备医药化学品-加巴喷丁
取实施例7中制备得到的加巴喷丁内酰胺76.7g固体溶解于500mL 6M HCl中加热回流2.5h，待冷却至室温后加入等体积的二氯甲烷进行萃取，静置分层后取水相在0-4℃下进行结晶，抽滤得到的白色固体用丙酮研磨，过滤除去丙酮并在40℃烘干得到加巴喷丁盐酸盐，将得到的所有的加巴喷丁盐酸盐溶于500mL水中，加热至40℃，300rpm搅拌下充分溶解，使用6M NaOH调pH至7.0-7.5；后加入125mL甲苯，500rpm搅拌30min；结束搅拌于0-4℃下进行结晶，过滤得到白色固体即为加巴 喷丁粗品，粗品用60％甲醇或异丙醇重结晶并烘干即为加巴喷丁。以上实验涉及到萃取、抽滤、过滤操作中未用到的样品、使用后的试剂均可进行回收利用。实验结果表明加巴喷丁盐酸盐的收率达到了81％，重结晶得到的加巴喷丁收率达到73.6％，并且经过母液重复回收3-5次之后的加巴喷丁收率达到了93.2％。中间体及最终加巴喷丁的收率均达到了一个较高的水准，并且多次样品及试剂回收步骤降低了成本和废水的产生，满足绿色化学的理念，实现了医药化学品的化学-酶法高效生产。
实施例9：多肽标签在其他来源腈水解酶上的应用效果
将实施例1优选的多肽标签GKGKG按照实施例1的方法连接到腈水解酶LNIT5(Accession No.:AAR97494.1)，腈水解酶No.385,386(Accession No.:AY487562)及来源于R.rhodochrous K22(Accession No.:Q02068.1)的腈水解酶(以下简称RrNit)的N末端。按实施例3方法测定溶解度及相对细胞酶活。
实验结果如下表5，三种不同的腈水解酶的溶解度均有不同程度的提升，其中LNIT5提升最多，达到了1.9倍；并且三种腈水解酶以1-CN为底物时，细胞酶活提升水平均超过150％，充分说明该多肽标签对其他来源的腈水解酶的普适性。
表5不同来源腈水解酶相对酶活及溶解度的对比
Figure PCTCN2020135580-appb-000008
实施例10原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M催化合成氯吡格雷中间体(邻氯扁桃酸)的效率比较
将实施例3方法制备的重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的湿菌体5g悬浮于100mL Na 2HPO 4-NaH 2PO 4缓冲液中(0.2M，pH＝7.0)，分别加入终浓度分别1M和2M邻氯扁桃腈，35℃恒温水浴反应12h。每隔30min取样，12,000rpm离心5min，取上清，使用HPLC分析上清液中邻氯扁桃酸浓度，并检测邻氯扁桃腈的浓度。同样条件下，以原始菌株E.coli BL21(DE3)-AcN-M为对照，结果如表6所示，在催化1M邻氯扁桃腈时，重组菌株所需的反应时间为3h，而原始菌株的反应时间需4h，且转化率两者都达到了99％以上；然而在2M底物时，由于底物浓度过高，反应12h后，原始菌株对底物的转化率为60.4％，而重组菌株由于有效蛋白量的增加，相同条件下转化率达到了79.4％。
表6原始菌株及重组菌株催化合成氯吡格雷中间体的效率比较
Figure PCTCN2020135580-appb-000009
实施例11优选多肽标签在脱酰基酶中转化棘白菌素B制备棘白菌素B母核的应用效果
将实施例1优选的多肽标签GKGKG按照实施例1的方法连接到脱酰基酶(NC_001136.10)上，构建重组菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-DEA(脱酰基酶)，对其溶解度及酶活进行测定，含多肽标签的脱酰基酶较不含多肽标签的脱酰基酶溶解度提升2.8倍且比酶活提升358.5％。
将实施例10中催化剂改为50g/L的重组菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-DEA(脱酰基酶)静息细胞，底物改为终浓度2g/L棘白菌素B，反应时间改为24h，其他同实施例10，底物转化率达到60.6％，而不含多肽标签的脱酰基酶的转化率仅为35.7％。说明此多肽标签具有一定的扩展性，但在其他酶上增溶效果的优良与否需要进行深入的探讨。
虽然本发明已以较佳实例公开如上，但其并非用以限定本发明，任何熟悉此项技术的人，在不脱离本发明的精神和范围内，都可做各种的改动和修饰，因此本发明的保护范围应该以权力要求书所界定的为准。

Claims (10)

  1. 一种多肽标签，其特征在于所述多肽标签两端的氨基酸均为不带电的甘氨酸G，其余为甘氨酸G、组氨酸H、谷氨酸E、天冬氨酸D、赖氨酸K、精氨酸R中任意一种或多种的随机组合，所述的多肽标签的长度为5－11个氨基酸。
  2. 如权利要求1多肽标签，其特征在于所述多肽标签的氨基酸序列为下列之一：GKGKG、GKGEG、GKGHG、GRGRG、GRGGG、GHGHG、GDGDG、GDGEG、GDGRG、GDGKG、GEGEG、GEGKG、GEGGG、GEGRG、GEGDG、GKGKG、GKGDG、GKGEG、GKGGG、GKGHG、GKGRG、GRGRG、GRGDG、GRGEG、GRGKG、GRGGG、GGGKG、GGGEG、GHGHG、GKGKGKG、GKGKGKGKG、GKGKGKGKGKG。
  3. 如权利要求1所述多肽标签，其特征在于所述多肽标签含有连接肽，所述连接肽氨基酸序列为下列之一：GS、GGS、GGGS、GGGGS。
  4. 一种含权利要求1所述多肽标签的重组酶，其特征在于所述酶包括腈水解酶，脂肪酶，脱酰基酶。
  5. 如权利要求4所述含多肽标签的重组酶，其特征在于所述重组酶是将多肽标签连接至腈水解酶氨基酸序列的N端所得到的酶；所述腈水解酶的氨基酸序列为SEQ ID NO.1所示。
  6. 如权利要求5所述含多肽标签的重组酶，其特征在于所述多肽标签通过连接肽与腈水解酶N端连接的方式为下列之一：GKGKG-GS、GKGKG-GGS、GKGKG-GGGS、GKGKG-GGGGS。
  7. 一种含权利要求1所述多肽标签的重组酶的编码基因构建的重组基因工程菌。
  8. 一种含权利要求1所述多肽标签的重组酶在制备加巴喷丁中间体1-氰基环已基乙酸中的应用，其特征在于所述的应用为：以含多肽标签的重组腈水解酶的编码基因的重组基因工程菌经发酵培养获得的湿菌体或湿菌体纯化后的纯酶为催化剂，以1-氰基环己基乙腈为底物，以0.2M、pH＝7.0的Na 2HPO 4-NaH 2PO 4缓冲液为反应介质构成转化体系，35℃、200rpm恒温水浴反应完全，获得含1-氰基环己基乙酸的转化液，转化液分离纯化，获得1-氰基环己基乙酸；所述转化体系中，底物加入终浓度为1-2M，催化剂加入量以湿菌体重量计为50g/L。
  9. 如权利要求8所述的应用，其特征在于所述湿菌体按如下方法制备：将重组基因工程菌接种至LB培养基，37℃培养12-14h后，获得单菌落；挑取单菌落接种到含0.5μg/mL卡那霉素的LB试管培养基中，37℃培养8h后，以体积浓度2％的接种量转接至含0.5μg/mL卡那霉素的发酵培养基中，37℃培养2h后，加入终浓度0.1mM的IPTG，28 ℃诱导产酶12-14h，12,000转离心10min，沉淀用0.9％的生理盐水清洗2次，收集湿菌体。
  10. 一种含权利要求1所述多肽标签的重组酶在制备氯吡格雷中间体邻氯扁桃酸中的应用，其特征在于所述的应用为：将含多肽标签的重组腈水解酶的基因工程菌经发酵培养获得的湿菌体为催化剂，以邻氯扁桃腈为底物，以0.2M、pH7.0的Na 2HPO 4-NaH 2PO 4缓冲液为反应介质构成反应体系，35℃恒温水浴反应12h，获得含邻氯扁桃酸的反应液，分离纯化，得到邻氯扁桃酸；所述反应体系中，催化剂加入量以湿菌体重量计为50g/L，所述底物加入终浓度为1-2M。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104212784A (zh) * 2014-08-12 2014-12-17 浙江工业大学 重组腈水解酶、编码基因、突变体、工程菌及应用
CN104774825A (zh) * 2015-03-23 2015-07-15 浙江工业大学 腈水解酶突变体及其应用
CN107001409A (zh) * 2014-09-26 2017-08-01 株式会社钟化 疏水性肽的制造方法
CN107177576A (zh) * 2017-05-10 2017-09-19 浙江工业大学 腈水解酶突变体及其应用
CN111471668A (zh) * 2020-02-28 2020-07-31 浙江工业大学 一种腈水解酶突变体及其在制备1-氰基环己基乙酸中的应用

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE495244T1 (de) * 2000-03-31 2011-01-15 Du Pont Isolierung und expression eines nitrilase-gens von acidovorax facilis 72w
US7148051B2 (en) * 2004-08-16 2006-12-12 E. I. Du Pont De Nemours And Company Production of 3-hydroxycarboxylic acid using nitrilase
US7198927B2 (en) * 2004-12-22 2007-04-03 E. I. Du Pont De Nemours And Company Enzymatic production of glycolic acid
US8722354B2 (en) * 2012-04-03 2014-05-13 Empire Technology Development Llc Fluorescent labeling of living cells
CN105924532A (zh) * 2016-06-08 2016-09-07 盘古基因生物工程(南京)股份有限公司 一种可溶性碱性成纤维细胞生长因子融合蛋白表达载体及其应用
CN107445953B (zh) * 2017-07-25 2020-05-12 中国科学技术大学 可杀伤布氏锥虫的化合物及其在锥虫病治疗方面的应用
CN108486088B (zh) * 2018-02-14 2021-02-02 浙江工业大学 腈水解酶突变体及其应用
KR20210151945A (ko) * 2019-04-12 2021-12-14 립타이드 바이오사이언스, 인코포레이티드 대식세포 활성을 조정하기 위한 방법
CN110835366B (zh) * 2019-10-25 2021-08-27 中国科学院生物物理研究所 促进蛋白质可溶性表达的标签多肽及其用途
CN114164197B (zh) * 2020-01-13 2023-08-18 浙江工业大学 一种热稳定性和活力提高的腈水解酶突变体及其应用
CN111172140B (zh) * 2020-01-21 2022-04-19 浙江工业大学 一种腈水解酶突变体及其在制备抗癫痫药物中间体中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104212784A (zh) * 2014-08-12 2014-12-17 浙江工业大学 重组腈水解酶、编码基因、突变体、工程菌及应用
CN107001409A (zh) * 2014-09-26 2017-08-01 株式会社钟化 疏水性肽的制造方法
CN104774825A (zh) * 2015-03-23 2015-07-15 浙江工业大学 腈水解酶突变体及其应用
CN107177576A (zh) * 2017-05-10 2017-09-19 浙江工业大学 腈水解酶突变体及其应用
CN111471668A (zh) * 2020-02-28 2020-07-31 浙江工业大学 一种腈水解酶突变体及其在制备1-氰基环己基乙酸中的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KI MI-RAN; PACK SEUNG PIL: "Fusion tags to enhance heterologous protein expression", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 104, no. 6, 28 January 2020 (2020-01-28), Berlin/Heidelberg, pages 2411 - 2425, XP037044771, ISSN: 0175-7598, DOI: 10.1007/s00253-020-10402-8 *
VASILIKI PARASKEVOPOULOU, FRANCO FALCONE: "Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression", MICROORGANISMS, vol. 6, no. 2, pages 47, XP055635209, DOI: 10.3390/microorganisms6020047 *

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