WO2022041397A1 - 一种阿魏酸酯酶及其应用 - Google Patents

一种阿魏酸酯酶及其应用 Download PDF

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WO2022041397A1
WO2022041397A1 PCT/CN2020/119269 CN2020119269W WO2022041397A1 WO 2022041397 A1 WO2022041397 A1 WO 2022041397A1 CN 2020119269 W CN2020119269 W CN 2020119269W WO 2022041397 A1 WO2022041397 A1 WO 2022041397A1
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ferulic acid
fermentation
esterase
acid esterase
bacillus subtilis
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张涛
江波
段晓莉
缪铭
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Jiangnan University
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Jiangnan University
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Priority claimed from CN202010898408.0A external-priority patent/CN111944782B/zh
Priority claimed from CN202010900348.1A external-priority patent/CN111909881B/zh
Priority claimed from CN202011048393.5A external-priority patent/CN112210523B/zh
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to JP2021560888A priority Critical patent/JP7489119B2/ja
Priority to US17/485,682 priority patent/US12270058B2/en
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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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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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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    • 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
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01073—Feruloyl esterase (3.1.1.73)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • C—CHEMISTRY; METALLURGY
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the invention relates to a ferulic acid esterase and an application thereof, belonging to the technical field of microorganisms.
  • Ferulic acid is widely present in plant cell walls, and is connected with polysaccharides, cellulose, and lignin in the form of ester bonds or ether bonds to form a complex network skeleton structure in the cell wall to maintain the integrity of the cell wall. At the same time reduce the biodegradation rate.
  • Ferulic acid is recognized as a safe antioxidant in the world, and is listed as a food additive in Japan, the United States and other countries. In addition, ferulic acid also has the functions of ultraviolet absorption, antibacterial and anti-inflammatory, anti-cancer and lowering blood lipids, etc., and has wide application prospects in the fields of medicine, cosmetics, and paper making.
  • the methods of producing ferulic acid mainly include plant extraction method, chemical synthesis method and biological enzyme method.
  • the plant extraction method mainly extracts and separates natural ferulic acid from plants such as angelica, coptis, rice bran, wheat bran and other plants through acid-base hydrolysis.
  • using this method to produce ferulic acid will destroy other high-value chemical components in the plant, and at the same time, due to the production of ferulic acid by the acid-base hydrolysis method, there are many by-products, and the product separation is difficult, and the use of this method to produce ferulic acid consumes a lot of energy ,polluted environment.
  • the chemical synthesis method mainly uses vanillin as the raw material to produce ferulic acid through a series of organic reactions.
  • vanillin as the raw material to produce ferulic acid through a series of organic reactions.
  • the production of ferulic acid using this method will increase the cost of separation, and the use of The ferulic acid produced by this method cannot be used directly as a pharmaceutical raw material.
  • using this method to produce ferulic acid also has defects such as long reaction time and serious environmental pollution.
  • the biological enzymatic method is mainly to produce ferulic acid by adding ferulic acid esterase (Ferruloyl esterase, Ferulic acid esterase, FAE, EC3.1.1.73) to the reaction system containing ferulic acid ester compounds to produce ferulic acid.
  • ferulic acid esterase Feruloyl esterase, Ferulic acid esterase, FAE, EC3.1.1.73
  • the use of biological enzyme method to produce ferulic acid has the advantages of mild reaction conditions, high specificity, and environmental friendliness. hot spot.
  • the literature due to the low specific enzyme activity of the existing ferulic acid esterase, for example, the literature "Cao Yan, Xia Qile, Chen Jianbing, et al.
  • the technical problem to be solved by the present invention is to provide a ferulic acid esterase (Feruloyl esterase, Ferulic acid esterase, FAE, E.C. 3.1.1.73) with a higher specific enzyme activity.
  • a ferulic acid esterase Feruloyl esterase, Ferulic acid esterase, FAE, E.C. 3.1.1.73
  • the present invention provides a ferulic acid esterase, and the amino acid sequence of the ferulic acid esterase is shown in SEQ ID NO.2.
  • the present invention also provides a gene encoding the above-mentioned ferulic acid esterase.
  • nucleotide sequence of the gene is shown in SEQ ID NO.3.
  • the present invention also provides a recombinant plasmid carrying the above gene.
  • the expression vector of the recombinant plasmid is pMA5 plasmid or pUB plasmid.
  • the present invention also provides a host cell, which carries the above-mentioned gene or the above-mentioned recombinant plasmid.
  • the host cell is Bacillus pumilus or Bacillus subtilis.
  • Bacillus subtilis is Bacillus subtilis 168, Bacillus subtilis WB800, Bacillus subtilis WB600 or Bacillus subtilis 1A751.
  • the present invention also provides a method for producing ferulic acid esterase.
  • the method includes firstly inoculating the above-mentioned host cells into a fermentation medium for fermentation to obtain a fermentation liquid, and then separating and obtaining ferulic acid esterase from the fermentation liquid. .
  • the present invention also provides the application of the above-mentioned host cell or the above-mentioned method for producing ferulic acid esterase in the production of ferulic acid esterase.
  • the present invention also provides a method for producing ferulic acid.
  • the method comprises the following steps: firstly adding the above ferulic acid esterase to a reaction system containing ferulic acid ester compounds for reaction to obtain a reaction solution; ferulic acid is obtained by separation;
  • the method is to first inoculate the above-mentioned host cells into a fermentation medium for fermentation to obtain a fermentation broth, then separate and obtain ferulic acid esterase from the fermentation broth, and then add the ferulic acid esterase to a ferulic acid-containing solution
  • the reaction is carried out in the reaction system of the ester compound to obtain a reaction solution, and finally the ferulic acid is separated from the reaction solution;
  • the method is to first inoculate the above-mentioned host cells into a fermentation medium for fermentation to obtain a fermentation liquid, then add the fermentation liquid to a reaction system containing ferulate compounds for reaction to obtain a reaction liquid, and finally obtain a reaction liquid from Ferulic acid was obtained by separation from the reaction solution.
  • the ferulate ester compound is methyl ferulate or ethyl ferulate.
  • the temperature of the reaction is 30-90° C. and the time is 10-30 min.
  • the present invention also provides the application of the above ferulic acid esterase or the above gene or the above recombinant plasmid or the above host cell or the above method in producing ferulic acid, hydrolyzing lignocellulose, preparing feed and making paper.
  • the present invention provides a ferulic acid esterase with amino acid sequence as shown in SEQ ID NO.2, the specific enzyme activity of this ferulic acid esterase is as high as 519U/g, therefore, this ferulic acid esterase is in There is a huge application prospect in the production of ferulic acid.
  • the present invention provides a strain of recombinant Bacillus subtilis, this recombinant Bacillus subtilis uses Bacillus subtilis as a host, and expresses the gene encoding the ferulic acid esterase shown in SEQ ID NO.2, the recombinant Bacillus subtilis Bacillus can produce high ferulic acid esterase, which is embodied in: the recombinant Bacillus subtilis is inoculated into the fermentation medium for fermentation, and fermented for 14 hours, the enzyme activity of ferulic acid esterase in the cell crushing supernatant can be as high as Therefore, this recombinant Bacillus subtilis has great application prospects in the production of ferulic acid esterase and ferulic acid.
  • Figure 1 Colony morphology of Bacillus pumilus SK52.001.
  • Figure 2 Morphology of Bacillus pumilus SK52.001.
  • Figure 3 High performance liquid chromatogram of ferulic acid standard.
  • Figure 4 High performance liquid chromatogram of methyl ferulate standard.
  • Figure 5 High performance liquid chromatogram of the reaction solution.
  • Figure 8 The yield of ferulic acid in the reaction solution.
  • the ferulic acid standard product involved in the following examples was purchased from Beijing Bailingwei Technology Co., Ltd.; the methyl ferulate involved in the following examples was purchased from Alfa Aesar (China) Chemical Co., Ltd.; in the following examples
  • the bovine albumin involved and Coomassie brilliant blue G-250 were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.
  • the pMA5 plasmid involved in the following examples was purchased from Youbao Bio
  • the Escherichia coli (Escherichia coli) involved in the following examples DH5 ⁇ was purchased from General Biotechnology Co., Ltd.
  • Bacillus subtilis WB800 involved in the following examples was purchased from NTCC Type Culture Collection Center
  • lysozyme involved in the following examples was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • Tris (hydroxymethyl) aminomethane (Tris) and EDTA disodium dihydrate involved in the following examples were purchased from Shanghai McLean Bio
  • Strain isolation medium potato 200g/L, sucrose 20g/L, agar 15g/L, natural pH.
  • Screening of solid medium use 0.22 ⁇ m filter membrane to filter N,N-dimethylformamide solution containing 100 mg/L ethyl ferulate to obtain ethyl ferulate solution; cool the sterilized basal medium After reaching 60°C, add 10% (v/v) ethyl ferulate solution to the basal medium, and shake until it is a uniform milky white solution;
  • basal medium NaNO 3 2g/L, K 2 HPO 4 ⁇ 3H 2 O 1g/L, KCl 0.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, FeSO 4 ⁇ 7H 2 O 0.01g/ L, agar 15g/L, pH natural.
  • Seed medium potato 200g/L, sucrose 20g/L, natural pH.
  • Basic fermentation medium wheat bran 20g/L, NaNO 3 2g/L, K 2 HPO 4 3H 2 O 1g/L, KCl 0.5g/L, MgSO 4 7H 2 O 0.5g/L, FeSO 4 7H 2 O 0.01g/L, pH is natural.
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, natural pH.
  • LB solid medium tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar powder 15g/L.
  • Super Rich medium tryptone 25g/L, yeast extract 20g/L, dipotassium hydrogen phosphate 3.0g/L, glucose 30g/L, natural pH.
  • Lysis solution Tris 6.057g/L, NaCl 5.844g/L, adjust pH to 8.0 with hydrochloric acid, and dilute to 1L.
  • Dialysate Tris 6.057g/L, adjust pH to 8.0 with hydrochloric acid, and dilute to 1L.
  • Example 1 Acquisition of Bacillus pumilus SK52.001
  • strain SK52.001 The genome of strain SK52.001 was extracted, the 16S rDNA of strain SK52.001 was amplified and sequenced (completed by Tianyi Huiyuan Biotechnology Co., Ltd.), and the 16S rDNA sequence of strain SK52.001 obtained by sequencing analysis (SK52.
  • the 16S rDNA sequence of 001 (shown in SEQ ID NO.1) was compared in GenBank, and the results showed that this strain was indeed Bacillus pumilus and was named Bacillus pumilus SK52.001.
  • Bacillus pumilus SK52.001 is: short rod-shaped, with blunt rounded ends, with typical morphological characteristics of Bacillus, belonging to Gram-positive bacteria.
  • the single colony of Bacillus pumilus SK52.001 obtained in Example 1 was picked and inoculated into the seed medium, and cultivated for 18h in a shaking table at 30°C and 200r/min to obtain seed liquid;
  • the % (v/v) inoculum was inoculated into the basal fermentation medium, and fermented for 26 hours in a shaker at 30° C. and 200 r/min to obtain a fermentation broth.
  • the fermentation broth was centrifuged at 4°C and 10,000 rpm for 10 min to obtain a crude enzyme solution; the enzyme activity of ferulic acid esterase in the crude enzyme solution was determined, and the assay method was as follows:
  • methyl ferulate solution is obtained by dissolving methyl ferulate in Tris with a pH of 8.0 and a concentration of 0.05 mol/L).
  • the enzyme activity of ferulic acid esterase is defined as: at 50°C, the amount of enzyme required to decompose methyl ferulate to generate 1 ⁇ mol of ferulic acid per minute is 1 unit of enzyme activity (1U);
  • the high performance liquid chromatogram of the ferulic acid standard is shown in Figure 3
  • the high performance liquid chromatogram of the methyl ferulate standard product is shown in Figure 4
  • the high performance liquid chromatogram of the reaction solution is shown in Figure 5.
  • HPLC method adopts Agilent 1200 high performance liquid chromatograph; chromatographic column is ZORBAX Eclipse Plus C18 (Agilent, 4.6mm ⁇ 150mm, 3.5 ⁇ m); UV detector; mobile phase A: 1% (v/v) acetic acid solution , mobile phase B: methanol; flow rate 1 mL/min; column temperature 30 ° C; detection wavelength 320 nm, the gradient elution procedure is shown in Table 1.
  • the detection result is: the enzyme activity of ferulic acid esterase in the crude enzyme solution is 195U/L.
  • Example 1 On the basis of Example 1, the basal fermentation medium was replaced with fermentation medium A to E, respectively, to obtain fermentation broth A to E;
  • fermentation medium A wheat bran 45g/L, tryptone 5g/L, K 2 HPO 4 ⁇ 3H 2 O 1.0g/L, KCl 0.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, FeSO 4 ⁇ 7H 2 O 0.01g/L, pH 6.0;
  • Fermentation medium B de-starch wheat bran 10g/L, NaNO 3 2g/L, K 2 HPO 4 ⁇ 3H 2 O 1.0g/L, KCl 0.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, FeSO 4 ⁇ 7H 2 O 0.01g/L, pH is natural;
  • the treatment method of de-starch wheat bran is: soak the wheat bran with potassium acetate solution with a concentration of 0.3% (w/w) to obtain a mixture; put the mixture at 95 After 1 h in a water bath at °C and stirring constantly, rinse with deionized water repeatedly until the starch is completely removed, and dry at 105 °C to constant weight for use;
  • Fermentation broth C wheat bran 10g/L, NaNO 3 2g/L, K 2 HPO 4 ⁇ 3H 2 O 1.0g/L, KCl 0.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, FeSO 4 7H 2 O 0.01g/L, natural pH;
  • Fermentation broth D wheat bran 45g/L, NaNO 3 2g/L, K 2 HPO 4 ⁇ 3H 2 O 1.0g/L, KCl 0.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, FeSO 4 ⁇ 7H 2 O 0.01g/L, pH is natural.
  • Fermentation broth E wheat bran 45g/L, yeast extract 5g/L, K 2 HPO 4 ⁇ 3H 2 O 1.0g/L, KCl 0.5g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, FeSO 4 ⁇ 7H 2 O 0.01g/L, pH is natural.
  • the fermentation broths A ⁇ E were centrifuged at 4°C and 10000rpm for 10min respectively to obtain crude enzyme solutions A ⁇ E; the enzymatic activity of ferulic acid esterase in crude enzyme solutions A ⁇ E was measured, and the determination results were: crude enzyme solutions A ⁇ E
  • the enzymatic activities of ferulic acid esterase in E were 421U/L, 73U/L, 102U/L, 241U/L and 344U/L, respectively. It can be seen that when fermentation medium A is used, Bacillus pumilus SK52.001 produces the highest yield of ferulic acid esterase.
  • Coomassie brilliant blue G-250 Weigh 100 mg of Coomassie brilliant blue G-250 and dissolve it in 50 mL of 90% (v/v) ethanol, add 100 mL of 85% (v/v) phosphoric acid, and dilute to 1 L with distilled water to obtain Coomassie brilliant blue G-250 dye solution ; Weigh 100 mg of bovine albumin to dissolve, and dilute to 100 mL with distilled water to obtain a standard protein solution; draw 0, 0.02, 0.04, 0.06, 0.08, 0.10 mL of standard protein solution and add 1.0, 0.98, 0.96, 0.94, 0.92, After 0.90 mL of distilled water, 5 mL of Coomassie brilliant blue G-250 dye solution was added and mixed evenly to obtain a mixed solution; the mixed solution was reacted at 25°C for 2 min to obtain a reaction solution; the absorbance of the reaction solution was measured at a wavelength of 595 nm, and plotted Protein concentration standard curve (see Figure 7 for the standard curve).
  • the detection results were as follows: the protein concentration in crude enzyme solution A was 0.81 mg/mL, and the specific enzyme activity of ferulic acid esterase in crude enzyme solution A was 519 U/g.
  • the gene of ferulic acid esterase was amplified from the genome of Bacillus pumilus SK52.001 by PCR and sequenced.
  • the sequencing result is: the amino acid sequence of ferulic acid esterase in crude enzyme solution A is as SEQ ID As shown in NO.2, the nucleotide sequence of the gene encoding the ferulic acid esterase in the crude enzyme solution A is as shown in SEQ ID NO.3.
  • the single colony of Bacillus pumilus SK52.001 obtained in Example 1 was picked and inoculated into the seed medium, and cultivated for 18h in a shaking table at 30°C and 200r/min to obtain seed liquid; % (v/v) of the inoculum amount is inoculated into the basic fermentation medium, and fermented in a shaker at 30 ° C and 200 r/min for 40 hours to obtain a fermentation broth; during the fermentation process, the fermentation broth is continuously sampled; the sampled fermentation broth Centrifuge at 4°C and 10000rpm for 10min to obtain crude enzyme solution; add 250 ⁇ L of crude enzyme solution to 750 ⁇ L of methyl ferulate solution with a concentration of 0.003mol/L (methyl ferulate solution is obtained by mixing methyl ferulate Dissolved in Tris-HCl buffer with pH 8.0 and concentration 0.05mol/L) to obtain an enzyme reaction system; react the enzyme reaction system in a 50°C water bath for 30 min to obtain a reaction solution.
  • the detection method of the output of ferulic acid in the reaction solution is:
  • reaction solution was first inactivated by boiling water for 10 min, then filtered through a 0.22 ⁇ m membrane, and finally the crude enzyme solution after inactivation of the enzyme was used as a blank control, and the concentration of ferulic acid in the reaction solution was determined by HPLC (high performance liquid chromatography);
  • HPLC method adopts Agilent 1200 high performance liquid chromatograph; chromatographic column is ZORBAX Eclipse Plus C18 (Agilent, 4.6mm ⁇ 150mm, 3.5 ⁇ m); UV detector; mobile phase A: 1% (v/v) acetic acid solution , mobile phase B: methanol; flow rate 1 mL/min; column temperature 30 ° C; detection wavelength 320 nm, the gradient elution procedure is shown in Table 1.
  • the ferulic acid constant was 280 mg/L after fermenting the crude enzyme solution for 26 h and reacting for 30 min.
  • the gene encoding ferulic acid esterase (SEQ ID NO. 3); the gene encoding ferulic acid esterase and the pMA5 plasmid are connected with homologous recombinase Exnase II to obtain a ligated product; the ligated product is transformed into Escherichia coli DH5 ⁇ competent cells; the transformed Escherichia coli DH5 ⁇ competent cells are The cells were coated with LB solid medium (containing 100 ⁇ g ⁇ mL -1 ampicillin), and cultured upside down at 37°C for 24 h; positive transformants were picked, plasmids were extracted, and the recombinant plasmid PMA5-fae was obtained after verification was correct; The recombinant plasmid PMA5-fae was transformed into Bacillus subtilis WB800 to
  • the primers are as follows:
  • fae-F aaaaggagcgatttacatatgATGAACTTACAAGAGCAAATCAAAATCGCTGC (SEQ ID NO. 4);
  • gagctcgactctagaggatccTTAATGGTGATGGTGATGATGTTCAAATGCCTTT (SEQ ID NO. 5).
  • the transformants of the recombinant Bacillus subtilis WB800/PMA5-fae obtained in Example 1 were streaked on LB solid medium (containing 100 ⁇ g ⁇ mL -1 kanamycin), and cultured upside down in a 30°C constant temperature incubator 12h to obtain a single colony; pick a single colony and inoculate it into LB liquid medium (containing 100 ⁇ g ⁇ mL -1 kanamycin), cultivate at 37 ° C and 200r/min for 12h to obtain a seed liquid; the seed liquid is 3% ( The inoculum of v/v) was transferred to Super Rich medium, and cultured at 37° C. and 200 r/min for 60 h to obtain a fermentation broth.
  • the fermentation broth was sampled at regular intervals with a sampling volume of 1 mL; the fermentation broth was centrifuged at 4°C and 10,000 rpm for 15 min to obtain fermentation supernatant and precipitate; 1 mL of lysate was added to the precipitate to resuspend the bacteria. A resuspension (whole cells) was obtained.
  • the enzyme activity of ferulic acid esterase is defined as: at 50°C, the amount of enzyme required to decompose methyl ferulate to generate 1 ⁇ mol of ferulic acid per minute is 1 unit of enzyme activity (1U);
  • the high performance liquid chromatogram of the ferulic acid standard is shown in Figure 3
  • the high performance liquid chromatogram of the methyl ferulate standard product is shown in Figure 4
  • the high performance liquid chromatogram of the reaction solution is shown in Figure 5.
  • HPLC method adopts Agilent 1200 high performance liquid chromatograph; chromatographic column is ZORBAX Eclipse Plus C18 (Agilent, 4.6mm ⁇ 150mm, 3.5 ⁇ m); UV detector; mobile phase A: 1% (v/v) acetic acid solution , mobile phase B: methanol; flow rate 1 mL/min; column temperature 30 ° C; detection wavelength 320 nm, the gradient elution procedure is shown in Table 1.
  • Bacillus subtilis WB800 as a control, the transformants of recombinant Bacillus subtilis WB800/PMA5-fae obtained in Example 7 were streaked on LB solid medium (containing 100 ⁇ g ⁇ mL -1 kanamycin), Inverted culture in a constant temperature incubator at 30°C for 12h to obtain a single colony; pick a single colony and inoculate it into LB liquid medium (containing 100 ⁇ g ⁇ mL -1 kanamycin), incubate at 37°C, 200r/min for 12h to obtain seed liquid ; The seed liquid was transferred to Super Rich medium with 3% (v/v) inoculum, and cultured at 37°C and 200r/min for 14h to obtain fermentation broth.
  • LB solid medium containing 100 ⁇ g ⁇ mL -1 kanamycin
  • LB liquid medium containing 100 ⁇ g ⁇ mL -1 kanamycin
  • the intracellular ferulic acid esterase obtained by the fermentation of recombinant Bacillus subtilis WB800/PMA5-fae was 82.53U/mL, which was 82.53U/mL than that of the extracellular fermentation carried out to 60h.
  • the ferulic acid esterase activity was 4 times higher at 20.80U/mL; at the same time, the intracellular ferulic acid esterase activity of recombinant Bacillus subtilis WB800/pMA5-fae was significantly higher than that of Bacillus subtilis WB800 Enzyme activity. Therefore, the intracellular ferulic acid esterase fermented for 14 h was selected as the basis for subsequent enzyme purification.
  • Example 3 After the cell crushed supernatant obtained by fermentation of the recombinant Bacillus subtilis WB800/PMA5-fae obtained in Example 3 was purified with a nickel column, the dialysate containing 0.01mol/L EDTA was first dialyzed three times, and then the EDTA-free dialysate was used. The dialysate was dialyzed three times to obtain pure enzyme; the pure enzyme was placed in a 4°C refrigerator for later use.
  • Coomassie brilliant blue G-250 Weigh 100 mg of Coomassie brilliant blue G-250 and dissolve it in 50 mL of 90% (v/v) ethanol, add 100 mL of 85% (v/v) phosphoric acid, and dilute to 1 L with distilled water to obtain Coomassie brilliant blue G-250 dye solution ; Weigh 100 mg of bovine albumin to dissolve, and dilute to 100 mL with distilled water to obtain a standard protein solution; draw 0, 0.02, 0.04, 0.06, 0.08, 0.10 mL of standard protein solution and add 1.0, 0.98, 0.96, 0.94, 0.92, After 0.90 mL of distilled water, add 5 mL of Coomassie brilliant blue G-250 dye solution and mix evenly to obtain a mixed solution; react the mixed solution at room temperature (25°C) for 5 min to obtain a reaction solution; measure the absorbance of the reaction solution at a wavelength of 560 nm , and draw a standard curve of protein concentration (see Figure 7 for the standard curve).
  • the detection results were: the protein concentration of the pure enzyme diluted 20 times was 0.15 mg/mL, and the specific enzyme activity of ferulic acid esterase of the pure enzyme diluted 20 times was 2230 U/mg.

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Abstract

一种阿魏酸酯酶及其应用，该阿魏酸酯酶的氨基酸序列如SEQ ID NO.2所示，此阿魏酸酯酶的比酶活高达519U/g。一株重组枯草芽孢杆菌，以枯草芽孢杆菌为宿主，表达编码氨基酸序列如SEQ ID NO.2所示的阿魏酸酯酶的基因，将此重组枯草芽孢杆菌接种至发酵培养基中进行发酵，发酵14h，细胞破碎上清液中阿魏酸酯酶的酶活高达82.53U/mL。

Description

一种阿魏酸酯酶及其应用 技术领域
本发明涉及一种阿魏酸酯酶及其应用，属于微生物技术领域。
背景技术
阿魏酸(Ferulic acid，FA)广泛存在于植物细胞壁中，与多糖、纤维素、木质素通过酯键或醚键的形式连接构成细胞壁中复杂的网状骨架结构，以维持细胞壁的完整性，同时降低生物分解率。
阿魏酸是世界上公认的安全抗氧化剂，在日本、美国等国家被列入食品添加剂中。此外，阿魏酸还具有紫外吸收、抗菌消炎、防癌降血脂等作用，在医药、化妆品、造纸等领域均有广泛的应用前景。
目前，生产阿魏酸的方法主要有植物提取法、化学合成法以及生物酶法。其中，植物提取法主要是通过酸碱水解的方式从当归、黄连、米糠、麦麸等植物中提取分离天然阿魏酸，但是，由于酸碱水解法会引起细胞壁中其他化学成分的改变，因此，使用此方法生产阿魏酸会破坏植物内其他高价值化学成分，同时，由于酸碱水解法生产阿魏酸的副产物多，产物分离困难，并且，使用此方法生产阿魏酸能耗大、污染环境。化学合成法主要是以香草醛为基原料通过一系列有机反应生产阿魏酸，但是，由于产物中掺杂顺式阿魏酸，使用此方法生产阿魏酸会使分离成本增加，并且，使用此方法生产得到的阿魏酸无法直接作为医药原料使用，另外，使用此方法生产阿魏酸还具有反应时间长、对环境污染严重等缺陷。
生物酶法主要是通过将阿魏酸酯酶(Feruloyl esterase，Ferulic acid esterase，FAE，E.C.3.1.1.73)添加至含有阿魏酸酯类化合物的反应体系中进行反应以生产阿魏酸，与生物提取法、酸碱水解法以及化学合成法相比，使用生物酶法生产阿魏酸具有反应条件温和、专一性高、对环境友好等优点，这些优点使得生物酶法成为生产阿魏酸的研究热点。但是，由于现有的阿魏酸酯酶比酶活较低，例如，文献“曹艳,夏其乐,陈剑兵,等.1株阿魏酸酯酶产生菌的筛选鉴定及发酵条件优化[J].微生物学杂志,2019，(5)：8-15.”中，来源于溜曲霉的阿魏酸酯酶的比酶活仅有231.34U/g，这大大限制了使用生物酶法生产阿魏酸的工业化进程。
因此，急需找到一种比酶活高的阿魏酸酯酶。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种比酶活高的阿魏酸酯酶(Feruloyl esterase，Ferulic  acid esterase，FAE，E.C.3.1.1.73)。
[技术方案]
为解决上述问题，本发明提供了一种阿魏酸酯酶，所述阿魏酸酯酶的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种基因，所述基因编码上述阿魏酸酯酶。
在本发明的一种实施方式中，所述基因的核苷酸序列如SEQ ID NO.3所示。
本发明还提供了一种重组质粒，所述重组质粒携带上述基因。
在本发明的一种实施方式中，所述重组质粒的表达载体为pMA5质粒或pUB质粒。
本发明还提供了一种宿主细胞，所述宿主细胞携带上述基因或上述重组质粒。
在本发明的一种实施方式中，所述宿主细胞为短小芽孢杆菌(Bacillus pumilus)或枯草芽孢杆菌(Bacillus subtilis)。
在本发明的一种实施方式中，所述枯草芽孢杆菌为枯草芽孢杆菌168、枯草芽孢杆菌WB800、枯草芽孢杆菌WB600或枯草芽孢杆菌1A751。
本发明还提供了一种生产阿魏酸酯酶的方法，所述方法为先将上述宿主细胞接种至发酵培养基中进行发酵，得到发酵液，然后从发酵液中分离得到阿魏酸酯酶。
本发明还提供了上述宿主细胞或上述生产阿魏酸酯酶的方法在生产阿魏酸酯酶中的应用。
本发明还提供了一种生产阿魏酸的方法，所述方法为先将上述阿魏酸酯酶添加至含有阿魏酸酯类化合物的反应体系中进行反应，得到反应液，然后从反应液中分离得到阿魏酸；
或者，所述方法为先将上述宿主细胞接种至发酵培养基中进行发酵，得到发酵液，然后从发酵液中分离得到阿魏酸酯酶，再将阿魏酸酯酶添加至含有阿魏酸酯类化合物的反应体系中进行反应，得到反应液，最后从反应液中分离得到阿魏酸；
或者，所述方法为先将上述宿主细胞接种至发酵培养基中进行发酵，得到发酵液，然后将发酵液添加至含有阿魏酸酯类化合物的反应体系中进行反应，得到反应液，最后从反应液中分离得到阿魏酸。
在本发明的一种实施方式中，所述阿魏酸酯类化合物为阿魏酸甲酯或阿魏酸乙酯。
在本发明的一种实施方式中，所述反应的温度为30～90℃、时间为10～30min。
本发明还提供了上述阿魏酸酯酶或上述基因或上述重组质粒或上述宿主细胞或上述方法在生产阿魏酸、水解木质纤维素、制备饲料以及造纸中的应用。
有益效果：
(1)本发明提供了一种氨基酸序列如SEQ ID NO.2所示的阿魏酸酯酶，此阿魏酸酯酶的比酶活高达519U/g，因此，此阿魏酸酯酶在生产阿魏酸中具有巨大的应用前景。
(2)本发明提供了一株重组枯草芽孢杆菌，此重组枯草芽孢杆菌以枯草芽孢杆菌为宿主，表达编码氨基酸序列如SEQ ID NO.2所示的阿魏酸酯酶的基因，此重组枯草芽孢杆菌可高产阿魏酸酯酶，具体体现在：将此重组枯草芽孢杆菌接种至发酵培养基中进行发酵，发酵14h，即可使细胞破碎上清液中阿魏酸酯酶的酶活高达82.53U/mL，因此，此重组枯草芽孢杆菌在生产阿魏酸酯酶以及阿魏酸中具有巨大的应用前景。
生物材料保藏
一株短小芽孢杆菌(Bacillus pumilus)SK52.001，分类学命名为Bacillus pumilus，已于2020年8月14日保藏于中国典藏培养物保藏中心，保藏编号为CCTCC NO:M 2020421，保藏地址为中国武汉，武汉大学。
附图说明
图1：短小芽孢杆菌(Bacillus pumilus)SK52.001的菌落形态。
图2：短小芽孢杆菌(Bacillus pumilus)SK52.001的菌体形态。
图3：阿魏酸标准品的高效液相色谱图。
图4：阿魏酸甲酯标准品的高效液相色谱图。
图5：反应液的高效液相色谱图。
图6：反应液的LC-MS图。
图7：蛋白浓度标准曲线。
图8：反应液中阿魏酸的产量。
具体实施方式
下述实施例中涉及的阿魏酸标准品购自北京百灵威科技有限公司；下述实施例中涉及的阿魏酸甲酯购自阿法埃莎(中国)化学有限公司；下述实施例中涉及的牛白蛋白和考马斯亮蓝G-250购自上海国药集团化学试剂有限公司；下述实施例中涉及的pMA5质粒购自优宝生物；下述实施例中涉及的大肠杆菌(Escherichia coli)DH5α购自通用生物技术有限公司；下述实施例中涉及的枯草芽孢杆菌WB800购自NTCC典型培养物保藏中心；下述实施例中涉及的溶菌酶购自生工生物工程(上海)股份有限公司；下述实施例中涉及的三(羟甲基)氨基甲烷(Tris)和乙二胺四乙酸二钠二水合物购自上海麦克林生化科技有限公司
下述实施例中涉及的培养基如下：
菌株分离培养基：马铃薯200g/L、蔗糖20g/L、琼脂15g/L，pH自然。
筛选固体培养基：使用0.22μm的滤膜过滤含有100mg/L阿魏酸乙酯的N,N-二甲基甲酰胺溶液，得到阿魏酸乙酯溶液；将经灭菌的基础培养基冷却至60℃后，向基础培养基中添加10％(v/v)的阿魏酸乙酯溶液，摇匀至呈均匀的乳白色溶液；
其中，基础培养基：NaNO 32g/L、K 2HPO 4·3H 2O 1g/L、KCl 0.5g/L、MgSO 4·7H 2O 0.5g/L、FeSO 4·7H 2O 0.01g/L、琼脂15g/L，pH自然。
种子培养基：马铃薯200g/L、蔗糖20g/L，pH自然。
基础发酵培养基：麦麸20g/L、NaNO 32g/L、K 2HPO 4·3H 2O 1g/L、KCl 0.5g/L、MgSO 4·7H 2O 0.5g/L、FeSO 4·7H 2O 0.01g/L，pH自然。
LB液体培养基：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L，pH自然。
LB固体培养基：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L、琼脂粉15g/L。
Super Rich培养基：胰蛋白胨25g/L、酵母提取物20g/L、磷酸氢二钾3.0g/L、葡萄糖30g/L，pH自然。
下述实施例中涉及的溶液如下：
裂解液：Tris 6.057g/L，NaCl 5.844g/L，盐酸调pH 8.0，定容至1L。
透析液：Tris 6.057g/L，盐酸调pH 8.0，定容至1L。
实施例1：短小芽孢杆菌SK52.001的获取
具体步骤如下：
1、短小芽孢杆菌SK52.001的分离纯化
以来源于江苏省无锡市贡湖湾湿地地区的土壤为样本，取1.0g样本放入10mL装入玻璃珠的无菌水中，于30℃，200r/min摇床振荡30min，使菌体充分分离，得到混合菌液；吸取0.5mL混合菌液在无菌环境下加入装有4.5mL生理盐水的10mL离心管中，得到10 -1稀释液，重复上述稀释步骤，依次得到10 -2、10 -3、10 -4、10 -5、10 -6稀释液；分别吸取100μL稀释梯度为10 -3、10 -4、10 -5、10 -6稀释液涂布于菌株分离培养基上，于30℃恒温培养箱中倒置培养24～48h，得到稀释涂布平板；挑取稀释涂布平板上的单菌落在筛选固体培养基上划线，于30℃恒温培养箱中倒置培养24h，得到单菌落；观察单菌落周围是否有透明圈出现，筛选周围有透明圈的单菌落；此次共筛得一个单菌落，将与此单菌落对应的菌株命名为SK52.001。
2、短小芽孢杆菌SK52.001的鉴定
提取菌株SK52.001的基因组，将菌株SK52.001的16S rDNA进行扩增和测序(由天一辉远生物科技有限公司完成)，将测序分析得到的菌株SK52.001的16S rDNA序列(SK52.001的16S rDNA序列如SEQ ID NO.1所示)在GenBank中进行比对，结果显示此菌株确为短小 芽孢杆菌，命名为短小芽孢杆菌(Bacillus pumilus)SK52.001。
3、短小芽孢杆菌SK52.001的观察
蘸取短小芽孢杆菌(Bacillus pumilus)SK52.001的菌液在LB固体培养基上划线，于30℃恒温培养箱倒置培养12h，得到单菌落；挑取单菌落接种至LB液体培养基中于30℃、200r/min的摇床上培养12h，得到菌液；将菌液用无菌水稀释100倍后涂布于LB固体培养基上，于30℃培养箱中培养18h后观察到菌落形态(菌落形态见图1)；将菌液用无菌水稀释20倍后在光学显微镜下观察到菌体形态(菌体形态见图2)。
由图1可知，短小芽孢杆菌(Bacillus pumilus)SK52.001的菌落形态为：圆形，较薄，中间呈浅黄色，边缘微白，表面黏稠湿润，边缘凹凸不整齐。
由图2可知，短小芽孢杆菌(Bacillus pumilus)SK52.001的菌体形态为：短杆状，两端钝圆，具有典型的芽孢杆菌的形态特征，属于革兰氏阳性菌。
实施例2：阿魏酸酯酶的生产
具体步骤如下：
挑取实施例1获得的短小芽孢杆菌(Bacillus pumilus)SK52.001的单菌落接种至种子培养基中，于30℃、200r/min的摇床中培养18h，得到种子液；将种子液按照5％(v/v)的接种量接种至基础发酵培养基中，于30℃、200r/min的摇床中发酵26h，得到发酵液。
将发酵液于4℃、10000rpm下离心10min，获得粗酶液；测定粗酶液中阿魏酸酯酶的酶活，测定方法如下：
将250μL粗酶液添加至750μL浓度为0.003mol/L的阿魏酸甲酯溶液(阿魏酸甲酯溶液是通过将阿魏酸甲酯溶于pH为8.0、浓度为0.05mol/L的Tris-HCl缓冲液中获得的)，得到酶反应体系；将酶反应体系于50℃水浴锅中反应30min后，先用沸水灭酶10min，然后经0.22μm膜过滤，得到反应液；以灭酶后的粗酶液为空白对照，采用HPLC法(高效液相色谱法)测定反应液中阿魏酸浓度；将阿魏酸浓度代入阿魏酸酯酶活力计算公式中，得到粗酶液中阿魏酸酯酶的酶活；
其中，阿魏酸酯酶酶活的定义为：在50℃下，每分钟分解阿魏酸甲酯生成1μmol阿魏酸所需要的酶量为1个酶活力单位(1U)；
阿魏酸酯酶活力计算公式如下：
Figure PCTCN2020119269-appb-000001
阿魏酸标准品的高效液相色谱图见图3，阿魏酸甲酯标准品的高效液相色谱图见图4，反 应液的高效液相色谱图见图5，阿魏酸浓度根据高效液相色谱图中的峰面积Y与阿魏酸浓度X之间的线性关系Y＝52514X-80.417(R 2＝0.9996)计算而得；
HPLC法采用Agilent 1 200型高效液相色谱仪；色谱柱为ZORBAX Eclipse Plus C18(Agilent，4.6mm×150mm，3.5μm)；紫外检测器；流动相A：1％(v/v)的乙酸溶液，流动相B：甲醇；流速1mL/min；柱温30℃；检测波长320nm，梯度洗脱程序如表1所示。
检测结果为：粗酶液中阿魏酸酯酶的酶活为195U/L。
为进一步证明短小芽孢杆菌(Bacillus pumilus)SK52.001可产阿魏酸酯酶，将反应液LC-MS分析(分析结果见图6)。
由图6可知，产物峰相对丰度最高的碎片m/z＝193，而目标产物的相对分子质量为194，与阿魏酸单体的相对分子量一致。
表1洗脱程序
时间/min A相/％ B相/％
0 90 10
0.23 70 30
1.66 50 50
4.97 0 100
5.57 85 15
7.52 90 10
7.60 90 10
实施例3：阿魏酸酯酶的生产
具体步骤如下：
在实施例1的基础上，将基础发酵培养基分别替换为发酵培养基A～E，得到发酵液A～E；
其中，发酵培养基A：小麦麸皮45g/L、胰蛋白胨5g/L、K 2HPO 4·3H 2O 1.0g/L、KCl 0.5g/L、MgSO 4·7H 2O 0.5g/L、FeSO 4·7H 2O 0.01g/L，pH 6.0；
发酵培养基B：去淀粉小麦麸皮10g/L、NaNO 32g/L、K 2HPO 4·3H 2O 1.0g/L、KCl 0.5g/L、MgSO 4·7H 2O 0.5g/L、FeSO 4·7H 2O 0.01g/L，pH自然；去淀粉小麦麸皮的处理方法为：用浓度为0.3％(w/w)的醋酸钾溶液浸泡小麦麸皮，得到混合物；将混合物在95℃水浴1h并不断搅拌后，使用去离子水反复冲洗至淀粉完全去除，于105℃下烘干至恒重备用；
发酵培养液C：小麦麸皮10g/L、NaNO 32g/L、K 2HPO 4·3H 2O 1.0g/L、KCl 0.5g/L、MgSO 4·7H 2O 0.5g/L、FeSO 4·7H 2O 0.01g/L，pH自然；
发酵培养液D：小麦麸皮45g/L、NaNO 32g/L、K 2HPO 4·3H 2O 1.0g/L、KCl 0.5g/L、 MgSO 4·7H 2O 0.5g/L、FeSO 4·7H 2O 0.01g/L，pH自然。
发酵培养液E：小麦麸皮45g/L、酵母提取物5g/L、K 2HPO 4·3H 2O 1.0g/L、KCl 0.5g/L、MgSO 4·7H 2O 0.5g/L、FeSO 4·7H 2O 0.01g/L，pH自然。
将发酵液A～E分别于4℃、10000rpm下离心10min，获得粗酶液A～E；测定粗酶液A～E中阿魏酸酯酶的酶活，测定结果为：粗酶液A～E中阿魏酸酯酶的酶活分别为421U/L、73U/L、102U/L、241U/L、344U/L。可见，使用发酵培养基A时，短小芽孢杆菌(Bacillus pumilus)SK52.001产阿魏酸酯酶的产量最高。
实施例4：阿魏酸酯酶的性能
具体步骤如下：
测定粗酶液A中阿魏酸酯酶的比酶活，测定方法如下：
称取100mg考马斯亮蓝G-250溶于50mL 90％(v/v)乙醇中后，加入100mL 85％(v/v)磷酸，并用蒸馏水定容至1L，得到考马斯亮蓝G-250染液；称取100mg牛白蛋白溶解，并用蒸馏水定容至100mL，得到标准蛋白溶液；分别吸取0、0.02、0.04、0.06、0.08、0.10mL标准蛋白溶液分别加入1.0、0.98、0.96、0.94、0.92、0.90mL蒸馏水中后，分别加入5mL考马斯亮蓝G-250染液混合均匀，得到混合液；将混合液于25℃下反应2min，得到反应液；将反应液于595nm波长下测定吸光度，并绘制蛋白浓度标准曲线(标准曲线见图7)。
取100μL实施例2获得的粗酶液A，用蒸馏水定容至1mL，得到稀释液；在稀释液中加入5mL考马斯亮蓝G-250染液混合均匀，得到混合液；将混合液于25℃下反应2min，得到反应液；将反应液于595nm波长下测定吸光度；根据测得的吸光度和蛋白浓度标准曲线，计算得到粗酶液A中的蛋白浓度，并根据粗酶液A中的蛋白浓度，计算得到粗酶液A中阿魏酸酯酶的比酶活；
其中，阿魏酸酯酶的比酶活的计算公式如下：
Figure PCTCN2020119269-appb-000002
检测结果为：粗酶液A中的蛋白浓度为0.81mg/mL、粗酶液A中阿魏酸酯酶的比酶活519U/g。
通过PCR从短小芽孢杆菌(Bacillus pumilus)SK52.001基因组中扩增出阿魏酸酯酶的基因并进行测序，测序结果为：粗酶液A中的阿魏酸酯酶的氨基酸序列如SEQ ID NO.2所示，编码粗酶液A中的阿魏酸酯酶的基因的核苷酸序列如SEQ ID NO.3所示。
实施例5：阿魏酸的生产
具体步骤如下：
挑取实施例1获得的短小芽孢杆菌(Bacillus pumilus)SK52.001的单菌落接种至种子培养基中，于30℃、200r/min的摇床中培养18h，得到种子液；将种子液按照5％(v/v)的接种量接种至基础发酵培养基中，于30℃、200r/min的摇床中发酵40h，得到发酵液；发酵过程中，不断对发酵液取样；将取样的发酵液于4℃、10000rpm下离心10min，获得粗酶液；将250μL粗酶液添加至750μL浓度为0.003mol/L的阿魏酸甲酯溶液(阿魏酸甲酯溶液是通过将阿魏酸甲酯溶于pH为8.0、浓度为0.05mol/L的Tris-HCl缓冲液中获得的)，得到酶反应体系；将酶反应体系于50℃水浴锅中反应30min，得到反应液。
测定反应液中阿魏酸的产量(检测结果见图8)；
其中，反应液中阿魏酸的产量的检测方法为：
将反应液先用沸水灭酶10min，然后经0.22μm膜过滤，最后以灭酶后的粗酶液为空白对照，采用HPLC法(高效液相色谱法)测定反应液中阿魏酸浓度；
HPLC法采用Agilent 1 200型高效液相色谱仪；色谱柱为ZORBAX Eclipse Plus C18(Agilent，4.6mm×150mm，3.5μm)；紫外检测器；流动相A：1％(v/v)的乙酸溶液，流动相B：甲醇；流速1mL/min；柱温30℃；检测波长320nm，梯度洗脱程序如表1所示。
如图8所示，发酵26h粗酶液，反应30min后，阿魏酸常量为280mg/L。
实施例6：重组枯草芽孢杆菌的构建
具体步骤如下：
以实施例1获得的短小芽孢杆菌(Bacillus pumilus)SK52.001的基因组为模板，以fae-F/fae-R为引物，通过PCR扩增获得编码阿魏酸酯酶的基因(SEQ ID NO.3)；将编码阿魏酸酯酶的基因和pMA5质粒用同源重组酶Exnase II连接，得到连接产物；将连接产物转化至大肠杆菌DH5α感受态细胞中；将转化后的大肠杆菌DH5α感受态细胞涂布LB固体培养基(含有100μg·mL -1氨苄霉素)，37℃倒置培养24h；挑取阳性转化子，提取质粒，测序验证，验证正确即获得重组质粒PMA5-fae；将获得的重组质粒PMA5-fae转化枯草芽孢杆菌WB800，得到转化产物；将转化产物涂布在LB固体培养基(含有100μg·mL -1卡那霉素)上，于37℃恒温培养箱中倒置培养8～12h，得到转化子；将转化子进行PCR验证，验证正确即获得重组枯草芽孢杆菌Bacillus subtilisWB800/PMA5-fae；
其中，引物如下：
fae-F：aaaaggagcgatttacatatgATGAACTTACAAGAGCAAATCAAAATCGCTGC(SEQ ID NO.4)；
fae-R：gagctcgactctagaggatccTTAATGGTGATGGTGATGATGTTCAAATGCCTTT(SEQ ID NO.5)。
实施例7：阿魏酸酯酶的生产
具体步骤如下：
将实施例1获得的重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae的转化子在LB固体培养基(含有100μg·mL -1卡那霉素)上划线，于30℃恒温培养箱中倒置培养12h，得到单菌落；挑取单菌落接种至LB液体培养基(含有100μg·mL -1卡那霉素)，于37℃、200r/min培养12h，得到种子液；将种子液以3％(v/v)的接种量转接至Super Rich培养基中，于37℃、200r/min培养60h，得到发酵液。
发酵过程中，每隔一段时间对发酵液进行取样，取样量1mL；将发酵液于4℃、10000rpm下离心15min，获得发酵上清液和沉淀；在沉淀中加入1mL裂解液将菌体重悬，得到重悬液(全细胞)。
检测发酵不同时间获得的发酵上清液和重悬液中阿魏酸酯酶的酶活(检测结果见表2)，测定方法如下：
将10μL发酵上清液或重悬液添加至990μL浓度为0.003mol/L的阿魏酸甲酯溶液(阿魏酸甲酯溶液是通过将阿魏酸甲酯溶于pH为8.0、浓度为0.05mol/L的Tris-HCl缓冲液中获得的)，得到酶反应体系；将酶反应体系于50℃水浴锅中反应10min后，先用沸水灭酶10min，然后经0.22μm膜过滤，得到反应液；以灭酶后的粗酶液为空白对照，采用HPLC法(高效液相色谱法)测定反应液中阿魏酸浓度；将阿魏酸浓度代入阿魏酸酯酶活力计算公式中，得到粗酶液中阿魏酸酯酶的酶活；
其中，阿魏酸酯酶酶活的定义为：在50℃下，每分钟分解阿魏酸甲酯生成1μmol阿魏酸所需要的酶量为1个酶活力单位(1U)；
阿魏酸酯酶活力计算公式如下：
Figure PCTCN2020119269-appb-000003
阿魏酸标准品的高效液相色谱图见图3，阿魏酸甲酯标准品的高效液相色谱图见图4，反应液的高效液相色谱图见图5，阿魏酸浓度根据高效液相色谱图中的峰面积Y与阿魏酸浓度X之间的线性关系Y＝52514X-80.417(R 2＝0.9996)计算而得；
HPLC法采用Agilent 1 200型高效液相色谱仪；色谱柱为ZORBAX Eclipse Plus C18(Agilent，4.6mm×150mm，3.5μm)；紫外检测器；流动相A：1％(v/v)的乙酸溶液，流 动相B：甲醇；流速1mL/min；柱温30℃；检测波长320nm，梯度洗脱程序如表1所示。
由表2可知，当发酵进行到14h时，重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae发酵获得的重悬液中阿魏酸酯酶的酶活达到3.20U/mL；当发酵进行到60h时，重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae发酵获得的发酵上清液中阿魏酸酯酶的酶活达20.80U/mL；在发酵到14h之后，重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae发酵获得的重悬液中阿魏酸酯酶的酶活先降低后升高，这是由于菌体达到稳定期后不断死亡使菌体数量减少，胞内酶活力下降，菌体死亡自溶后营养物质增多，使菌体数量缓慢上升，进而阿魏酸酯酶活力缓慢增加；在发酵到40h之后，重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae发酵获得的发酵上清液中阿魏酸酯酶的酶活持续不断增长，这可能是由于菌体不断死亡自溶破碎后，胞内的阿魏酸酯酶释放出来。
表2发酵不同时间获得的发酵上清液和重悬液中阿魏酸酯酶的酶活
组别 发酵上清液(U/mL) 重悬液(U/mL)
发酵6h 0.20 0.17
发酵14h 2.13 3.20
发酵20h 1.24 1.01
发酵25h 10.01 1.51
发酵37h 17.92 1.94
发酵42h 15.10 2.27
发酵48h 16.15 1.86
发酵54h 18.81 1.45
发酵60h 20.80 1.60
实施例8：阿魏酸酯酶的生产
具体步骤如下：
以枯草芽孢杆菌WB800为对照，将实施例7获得的重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae的转化子在LB固体培养基(含有100μg·mL -1卡那霉素)上划线，于30℃恒温培养箱中倒置培养12h，得到单菌落；挑取单菌落接种至LB液体培养基(含有100μg·mL -1卡那霉素)，于37℃、200r/min培养12h，得到种子液；将种子液以3％(v/v)的接种量转接至Super Rich培养基中，于37℃、200r/min培养14h，得到发酵液。
发酵结束后，取5mL发酵液，将发酵液于4℃、8000rpm下离心15min，获得发酵上清液和沉淀；在沉淀中加入5mL裂解液将菌体重悬，得到重悬液(全细胞)；将重悬液超声破 碎15min，得到细胞破碎液；将细胞破碎液于4℃、8000rpm下离心15min，得到细胞破碎上清液。
检测枯草芽孢杆菌WB800和重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae发酵获得的发酵上清液和细胞破碎上清液中阿魏酸酯酶的酶活(检测结果见表3)。
由表3可知，当发酵进行到14h时，重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae发酵获得的胞内阿魏酸酯酶的酶活为82.53U/mL，比发酵进行到60h的胞外阿魏酸酯酶活力20.80U/mL高4倍；同时，重组枯草芽孢杆菌Bacillus subtilis WB800/pMA5-fae的胞内阿魏酸酯酶活力明显高于枯草芽孢杆菌WB800的胞内阿魏酸酯酶活力。因此，选用发酵14h的胞内阿魏酸酯酶作为后续酶纯化的基础。
表3不同枯草芽孢杆菌发酵获得的发酵上清液和细胞破碎上清液中阿魏酸酯酶的酶活
Figure PCTCN2020119269-appb-000004
实施例9：阿魏酸酯酶的性能
具体步骤如下：
将实施例3获得的重组枯草芽孢杆菌Bacillus subtilis WB800/PMA5-fae发酵获得的细胞破碎上清液用镍柱纯化后，先使用含有0.01mol/L EDTA的透析液透析三次，再使用不含EDTA的透析液透析三次，得到纯酶；将纯酶放置在4℃冰箱中备用。
将纯酶稀释0～50倍后，参照实施例5中的方法检测纯酶的阿魏酸酯酶酶活，检测结果见表4。
由表4可知，选择20倍的稀释倍数最佳，测定稀释20倍的纯酶的阿魏酸酯酶酶活，测定方法如下：
称取100mg考马斯亮蓝G-250溶于50mL 90％(v/v)乙醇中后，加入100mL 85％(v/v)磷酸，并用蒸馏水定容至1L，得到考马斯亮蓝G-250染液；称取100mg牛白蛋白溶解，并用蒸馏水定容至100mL，得到标准蛋白溶液；分别吸取0、0.02、0.04、0.06、0.08、0.10mL标准蛋白溶液分别加入1.0、0.98、0.96、0.94、0.92、0.90mL蒸馏水中后，分别加入5mL考马斯亮蓝G-250染液混合均匀，得到混合液；将混合液于室温(25℃)下反应5min，得到反应液；将反应液于560nm波长下测定吸光度，并绘制蛋白浓度标准曲线(标准曲线见图7)。
取500μL纯酶，在纯酶中加入500μL蒸馏水后，再加入5mL考马斯亮蓝G-250染液混合均匀，得到混合液；将混合液于室温(25℃)下反应5min，得到反应液；将反应液于595 nm波长下测定吸光度；根据测得的吸光度和蛋白浓度标准曲线，计算得到纯酶的蛋白浓度，并根据稀释20倍的纯酶中阿魏酸酯酶活力，计算得到阿魏酸酯酶比酶活；
其中，阿魏酸酯酶比酶活的计算公式如下：
Figure PCTCN2020119269-appb-000005
检测结果为：稀释20倍的纯酶的蛋白浓度为0.15mg/mL、稀释20倍的纯酶的阿魏酸酯酶比酶活为2230U/mg。
表4稀释不同倍数的纯酶的阿魏酸酯酶酶活
纯酶稀释倍数 加酶体积(稀释后)/μL 酶活力(U/mL) 备注
0 10 47.85 底物完全反应
2 10 60.53 底物几乎反应完全
5 10 142.25 底物几乎反应完全
20 10 334.50 峰高度比为4：3
50 10 210.89 峰高度比为1：4
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (14)

  1. 一种阿魏酸酯酶，其特征在于，所述阿魏酸酯酶的氨基酸序列如SEQ ID NO.2所示。
  2. 一种基因，其特征在于，所述基因编码权利要求1所述的阿魏酸酯酶。
  3. 如权利要求2所述的一种基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.3所示。
  4. 一种重组质粒，其特征在于，所述重组质粒携带权利要求2或3所述的基因。
  5. 如权利要求4所述的一种重组质粒，其特征在于，所述重组质粒的表达载体为pMA5质粒或pUB质粒。
  6. 一种宿主细胞，其特征在于，所述宿主细胞携带权利要求2或3所述的基因或权利要求4或5所述的重组质粒。
  7. 如权利要求6所述的一种宿主细胞，其特征在于，所述宿主细胞为短小芽孢杆菌(Bacillus pumilus)或枯草芽孢杆菌(Bacillus subtilis)。
  8. 如权利要求7所述的一种宿主细胞，其特征在于，所述枯草芽孢杆菌为枯草芽孢杆菌168、枯草芽孢杆菌WB800、枯草芽孢杆菌WB600或枯草芽孢杆菌1A751。
  9. 一种生产阿魏酸酯酶的方法，其特征在于，所述方法为先将权利要求6-8任一项所述的宿主细胞接种至发酵培养基中进行发酵，得到发酵液，然后从发酵液中分离得到阿魏酸酯酶。
  10. 权利要求6-8任一项所述的宿主细胞或权利要求9所述的生产阿魏酸酯酶的方法在生产阿魏酸酯酶中的应用。
  11. 一种生产阿魏酸的方法，其特征在于，所述方法为先将权利要求1所述的阿魏酸酯酶添加至含有阿魏酸酯类化合物的反应体系中进行反应，得到反应液，然后从反应液中分离得到阿魏酸；
    或者，所述方法为先将权利要求6-8任一项所述的宿主细胞接种至发酵培养基中进行发酵，得到发酵液，然后从发酵液中分离得到阿魏酸酯酶，再将阿魏酸酯酶添加至含有阿魏酸酯类化合物的反应体系中进行反应，得到反应液，最后从反应液中分离得到阿魏酸；
    或者，所述方法为先将权利要求6-8任一项所述的宿主细胞接种至发酵培养基中进行发酵，得到发酵液，然后将发酵液添加至含有阿魏酸酯类化合物的反应体系中进行反应，得到反应液，最后从反应液中分离得到阿魏酸。
  12. 如权利要求11所述的一种生产阿魏酸的方法，其特征在于，所述阿魏酸酯类化合物为阿魏酸甲酯或阿魏酸乙酯。
  13. 如权利要求11或12所述的一种生产阿魏酸的方法，其特征在于，所述反应的温度为30～90℃、时间为10～30min。
  14. 权利要求1所述的阿魏酸酯酶或权利要求2或3所述的基因或权利要求4或5所述的 重组质粒或权利要求6-8任一项所述的宿主细胞或权利要求9所述的生产阿魏酸酯酶的方法或权利要求11-13任一项所述的生产阿魏酸的方法在生产阿魏酸、水解木质纤维素、制备饲料以及造纸中的应用。
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