CN112210523B - Recombinant bacillus subtilis for producing feruloyl esterase and application thereof - Google Patents
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Abstract
The invention discloses a recombinant bacillus subtilis for producing feruloyl esterase and application thereof, belonging to the technical field of biology. The invention provides a recombinant bacillus subtilis which can produce feruloyl esterase with high yield, and the recombinant bacillus subtilis is specifically represented by the following steps: the recombinant bacillus subtilis is inoculated into a fermentation culture medium for fermentation for 14 hours, so that the enzyme activity of the ferulic acid esterase in cell disruption supernatant can reach 82.53U/mL, and the recombinant bacillus subtilis has huge application prospect in the production of the ferulic acid esterase and ferulic acid.
Description
Technical Field
The invention relates to a recombinant bacillus subtilis for producing feruloyl esterase and application thereof, belonging to the technical field of biology.
Background
Ferulic Acid (FA) is widely present in plant cell walls, and is connected with polysaccharide, cellulose and lignin in the form of ester bonds or ether bonds to form a complex reticular framework structure in the cell walls, so that the integrity of the cell walls is maintained, and the biological decomposition rate is reduced.
Ferulic acid is a world-recognized safe antioxidant and is included in food additives in japan, the united states, and other countries. In addition, ferulic acid also has the effects of ultraviolet absorption, antibiosis and antiphlogosis, cancer prevention and blood fat reduction and the like, and has wide application prospects in the fields of medicines, cosmetics, paper making and the like.
At present, methods for producing ferulic acid mainly include plant extraction methods, chemical synthesis methods and biological enzyme methods. The plant extraction method mainly extracts and separates natural ferulic acid from angelica sinensis, coptis chinensis, rice bran, wheat bran and other plants in an acid-base hydrolysis mode, but the acid-base hydrolysis method can cause changes of other chemical components in cell walls, so that the method for producing ferulic acid can damage other high-value chemical components in plants, meanwhile, the method for producing ferulic acid by the acid-base hydrolysis method has a lot of byproducts, the separation of products is difficult, and the method for producing ferulic acid has high energy consumption and pollutes the environment. The chemical synthesis method mainly uses vanillin as a base raw material to produce ferulic acid through a series of organic reactions, however, the cis-ferulic acid is doped in a product, so that the separation cost is increased when the method is used for producing the ferulic acid, the ferulic acid produced by the method cannot be directly used as a medicinal raw material, and in addition, the method for producing the ferulic acid also has the defects of long reaction time, serious environmental pollution and the like.
The biological enzyme method is mainly characterized in that Feruloyl esterase (Feruloyl esterase, ferulic acid esterase, FAE, E.C. 3.1.1.73) is added into a reaction system containing a ferulate compound to react to produce Ferulic acid. However, since the existing strains capable of producing feruloyl esterase have low feruloyl esterase production, for example, li Cuicui et al, inoculated cladosporium cladosporioides into a fermentation medium for fermentation for 3d, can only make the enzyme activity of the feruloyl esterase in the fermentation broth reach 175.4U/L (see, in particular, references: li Cuicui, mao Jian, liu Shuangping, etc.. Cladosporium screening, fermentation characteristics and application research in yellow wine [ J ]. Food and biotechnology reports, 2018,37 (08): 793-801.); hu Bohan, et al, inoculated Aspergillus fumigatus into a fermentation medium for fermentation for 4 days, only allowed the enzyme activity of feruloyl esterase in the fermentation broth to reach 32.7U/L (see, in particular, references: hu Bohan, wu Hui, lifuxiang, et al, screening of feruloyl esterase-producing strains and phenolic acid release research [ J ]. Modern food technology, 2015 (07): 99-105.), which greatly limited the industrial process of producing ferulic acid by using a biological enzyme method.
Therefore, it is urgently needed to find a strain capable of producing ferulic acid esterase with high yield.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide a recombinant bacillus subtilis capable of highly producing feruloyl esterase.
[ solution ]
In order to solve the problems, the invention provides a recombinant bacillus subtilis which takes bacillus subtilis as a host to express a gene for coding feruloyl esterase; the amino acid sequence of the ferulic acid esterase is shown in SEQ ID NO. 1.
In one embodiment of the invention, the nucleotide sequence of the gene encoding feruloyl esterase is shown in SEQ ID NO. 2.
In one embodiment of the present invention, the recombinant bacillus subtilis expresses a gene encoding feruloyl esterase using bacillus subtilis as a host and a pMA5 plasmid, a pUB110 plasmid or a pTSC plasmid as an expression vector.
In one embodiment of the present invention, the bacillus subtilis is bacillus subtilis 168, bacillus subtilis WB800, bacillus subtilis WB600 or bacillus subtilis 1a751.
The invention also provides a method for producing feruloyl esterase, which comprises the steps of inoculating the recombinant bacillus subtilis to a fermentation culture medium for fermentation to obtain fermentation liquor, and then separating the feruloyl esterase from the fermentation liquor.
The invention also provides the application of the recombinant bacillus subtilis or the method in the production of feruloyl esterase.
The invention also provides a method for producing ferulic acid, which comprises the steps of inoculating the recombinant bacillus subtilis to a fermentation culture medium for fermentation to obtain fermentation liquor, separating ferulic acid esterase from the fermentation liquor, adding the ferulic acid esterase to a reaction system containing a ferulic acid ester compound for reaction to obtain reaction liquid, and finally separating ferulic acid from the reaction liquid;
or, the method comprises the steps of firstly inoculating the recombinant bacillus subtilis to a fermentation culture medium for fermentation to obtain a fermentation liquid, then adding the fermentation liquid into a reaction system containing the ferulic acid ester compound for reaction to obtain a reaction liquid, and finally separating the ferulic acid from the reaction liquid.
In one embodiment of the present invention, the ferulic acid ester compound is ferulic acid methyl ester or ferulic acid ethyl ester.
In one embodiment of the present invention, the reaction temperature is 30 to 90 ℃ and the reaction time is 10 to 30min.
The invention also provides the application of the recombinant bacillus subtilis or the method in the production of ferulic acid, the hydrolysis of lignocellulose, the preparation of feed and papermaking.
Has the advantages that:
the invention provides a recombinant bacillus subtilis which can produce feruloyl esterase with high yield, and the recombinant bacillus subtilis is specifically represented by the following steps: the recombinant bacillus subtilis is inoculated into a fermentation culture medium for fermentation for 14 hours, so that the enzyme activity of the ferulic acid esterase in cell disruption supernatant can reach 82.53U/mL, and the recombinant bacillus subtilis has huge application prospect in the production of the ferulic acid esterase and ferulic acid.
Biological material preservation
A strain of Bacillus pumilus (Bacillus pumilus) SK52.001 is taxonomically named as Bacillus pumilus and is preserved in China collection culture collection at 8 months and 14 days in 2020, with the preservation number of CCTCC NO: M2020421 and the preservation address of Wuhan university in Wuhan, china.
Drawings
FIG. 1: high performance liquid chromatogram of ferulic acid standard.
FIG. 2: high performance liquid chromatogram of ferulic acid methyl ester standard.
FIG. 3: high performance liquid chromatogram of the reaction solution.
FIG. 4: protein concentration standard curve.
Detailed Description
The ferulic acid standard referred to in the following examples was purchased from Bailingwei technologies, beijing; the methyl ferulate referred to in the examples below was purchased from alfa aesar (china) chemical ltd; bovine albumin and Coomassie Brilliant blue G-250 referred to in the examples below were purchased from Shanghai pharmaceutical group, chemicals, inc.; the pMA5 plasmids referred to in the examples below were purchased from Youbao organisms; coli (Escherichia coli) DH 5. Alpha. Referred to in the examples below was purchased from general Biotechnology Ltd; bacillus subtilis WB800 referred to in the following examples was purchased from NTCC type culture Collection; the lysozyme referred to in the following examples was purchased from Biotechnology engineering (Shanghai) GmbH; tris (hydroxymethyl) aminomethane (Tris) and disodium ethylenediaminetetraacetate dihydrate referred to in the examples below were purchased from Shanghai Michelin Biotech, inc.
The media involved in the following examples are as follows:
LB liquid medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride, and the pH is natural.
LB solid Medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 15g/L of agar powder.
Super Rich medium: 25g/L of tryptone, 20g/L of yeast extract, 3.0g/L of dipotassium phosphate and 30g/L of glucose, and the pH is natural.
The solutions referred to in the following examples are as follows:
lysis solution: tris 6.057g/L, naCl 5.844g/L, hydrochloric acid to adjust pH to 8.0, and constant volume to 1L.
Dialyzate: tris 6.057g/L, hydrochloric acid to adjust pH to 8.0, and constant volume to 1L.
Example 1: construction of recombinant Bacillus subtilis
The method comprises the following specific steps:
obtaining a gene (SEQ ID NO. 1) encoding feruloyl esterase by PCR amplification using the genome of Bacillus pumilus (Bacillus pumilus) SK52.001 obtained in example 1 as a template and fae-F/fae-R as a primer; encoding the feruloyl esteraseConnecting the plasmid and pMA5 by homologous recombinase Exnase II to obtain a connection product; transforming the ligation product into E.coli DH5 alpha competent cells; the transformed E.coli DH 5. Alpha. Competent cells were spread on LB solid medium (containing 100. Mu.g. ML) -1 Ampicillin) and is subjected to inverted culture at 37 ℃ for 24h; selecting positive transformants, extracting plasmids, verifying sequencing, and obtaining a recombinant plasmid PMA5-fae after verifying correctness; transforming the obtained recombinant plasmid PMA5-fae into Bacillus subtilis WB800 to obtain a transformation product; the transformed product was spread on LB solid medium (containing 100. Mu.g.mL) -1 Kanamycin), and performing inverted culture in a constant-temperature incubator at 37 ℃ for 8-12 h to obtain a transformant; carrying out PCR verification on the transformant, and obtaining the recombinant Bacillus subtilis WB800/PMA5-fae after the verification is correct;
wherein, the primers are as follows:
fae-F：aaaaggagcgatttacatatgATGAACTTACAAGAGCAAATCAAAATCGCTGC(SEQ ID NO.3)；
fae-R：gagctcgactctagaggatccTTAATGGTGATGGTGATGATGTTCAAATGCCTTT(SEQ ID NO.4)。
example 2: production of feruloyl esterase
The method comprises the following specific steps:
the transformant of the recombinant Bacillus subtilis WB800/PMA5-fae obtained in example 1 was cultured in LB solid medium (containing 100. Mu.g. ML) -1 Kanamycin) is marked out, and inverted culture is carried out in a constant-temperature incubator at 30 ℃ for 12 hours to obtain a single bacterial colony; single colonies were picked and inoculated into LB liquid medium (containing 100. Mu.g. ML) -1 Kanamycin) is cultured for 12 hours at 37 ℃ and 200r/min to obtain seed liquid; transferring the seed solution into a SuperRich culture medium at an inoculum size of 3% (v/v), and culturing at 37 ℃ and 200r/min for 60h to obtain a fermentation solution.
Sampling the fermentation liquor at intervals during the fermentation process, wherein the sampling amount is 1mL; centrifuging the fermentation liquid at 4 deg.C and 10000rpm for 15min to obtain fermentation supernatant and precipitate; the pellet was resuspended in 1mL of lysate to obtain a resuspension (whole cells).
The enzyme activities of the ferulic acid esterase in the fermentation supernatant and the suspension obtained at different times of fermentation (the detection results are shown in table 2) are detected by the following detection method:
adding 10 μ L of the fermentation supernatant or the resuspension solution to 990 μ L of a methyl ferulate solution of 0.003mol/L concentration (the methyl ferulate solution is obtained by dissolving methyl ferulate in a Tris-HCl buffer solution of pH 8.0 of concentration 0.05 mol/L) to obtain an enzyme reaction system; reacting the enzyme reaction system in a water bath kettle at 50 ℃ for 10min, inactivating the enzyme with boiling water for 10min, and filtering by a 0.22 mu m membrane to obtain a reaction solution; taking the enzyme-inactivated crude enzyme solution as a blank control, and determining the concentration of ferulic acid in the reaction solution by HPLC (high performance liquid chromatography); substituting the ferulic acid concentration into a ferulic acid esterase activity calculation formula to obtain the enzyme activity of the ferulic acid esterase in the crude enzyme solution;
wherein the enzyme activity of the ferulic acid esterase is defined as: at 50 ℃, the enzyme amount required for decomposing the ferulic acid methyl ester to generate 1 mu mol of ferulic acid per minute is 1 enzyme activity unit (1U);
the calculation formula of the activity of the ferulic acid ester enzyme is as follows:
the high performance liquid chromatogram of ferulic acid standard is shown in figure 1, the high performance liquid chromatogram of ferulic acid methyl ester standard is shown in figure 2, the high performance liquid chromatogram of reaction solution is shown in figure 3, and the concentration of ferulic acid is Y =52514X-80.417 (R is based on the linear relationship between peak area Y and concentration X of ferulic acid in the high performance liquid chromatogram 2 = 0.9996);
HPLC method adopts Agilent 1 200 high performance liquid chromatograph; the column was ZORBAX Eclipse Plus C18 (Agilent, 4.6 mm. Times.150mm, 3.5 μm); an ultraviolet detector; mobile phase A:1% (v/v) acetic acid solution, mobile phase B: methanol; the flow rate is 1mL/min; the column temperature is 30 ℃; the detection wavelength was 320nm and the gradient elution procedure is shown in Table 1.
As can be seen from Table 2, when the fermentation is carried out for 14 hours, the enzyme activity of the ferulic acid esterase in the resuspension obtained by fermenting the recombinant Bacillus subtilis WB800/PMA5-fae reaches 3.20U/mL; when the fermentation is carried out for 60 hours, the enzyme activity of the ferulic acid esterase in the fermentation supernatant obtained by fermenting the recombinant Bacillus subtilis WB800/PMA5-fae reaches 20.80U/mL; after fermenting for 14 hours, the enzyme activity of the ferulic acid esterase in the resuspension obtained by fermenting the recombinant Bacillus subtilis WB800/PMA5-fae is firstly reduced and then increased, because the thalli are continuously dead after reaching the stable period to reduce the number of the thalli, the activity of intracellular enzyme is reduced, nutrient substances are increased after the thalli are dead and autolyzed, the number of the thalli is slowly increased, and then the activity of the ferulic acid esterase is slowly increased; after fermentation for 40 hours, the enzyme activity of the ferulic acid esterase in the fermentation supernatant obtained by fermenting the recombinant Bacillus subtilis WB800/PMA5-fae is continuously increased, which is probably because the intracellular ferulic acid esterase is released after the bacteria are continuously dead and crushed by autolysis.
TABLE 1 elution procedure
| Time/min | A phase/% | B phase/%) |
| 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 |
TABLE 2 enzyme Activity of Feruloyl esterase in fermentation supernatants and resuspensions obtained at different fermentation times
| Group of | Fermentation supernatant (U/mL) | Resuspension (U/mL) |
| Fermenting for 6h | 0.20 | 0.17 |
| Fermenting for 14h | 2.13 | 3.20 |
| Fermenting for 20h | 1.24 | 1.01 |
| Fermenting for 25h | 10.01 | 1.51 |
| Fermenting for 37h | 17.92 | 1.94 |
| Fermenting for 42h | 15.10 | 2.27 |
| Fermenting for 48h | 16.15 | 1.86 |
| Fermenting for 54h | 18.81 | 1.45 |
| Fermenting for 60h | 20.80 | 1.60 |
Example 3: production of feruloyl esterase
The method comprises the following specific steps:
the transformant of the recombinant Bacillus subtilis WB800/PMA5-fae obtained in example 1 was cultured in LB solid medium (containing 100. Mu.g. ML) with Bacillus subtilis WB800 as a control -1 Kanamycin) is marked, and inverted culture is carried out in a constant temperature incubator at 30 ℃ for 12h to obtain a single colony; single colonies were picked and inoculated into LB liquid medium (containing 100. Mu.g. ML) -1 Kanamycin) is cultured for 12 hours at 37 ℃ and 200r/min to obtain seed liquid; seed liquid is expressed in 3% (v-v) transferring the inoculum size of the strain into a SuperRich culture medium, and culturing at 37 ℃ and 200r/min for 14h to obtain a fermentation liquid.
After the fermentation is finished, taking 5mL of fermentation liquor, centrifuging the fermentation liquor for 15min at 4 ℃ and 8000rpm, and obtaining fermentation supernatant and precipitate; adding 5mL of lysate into the precipitate to resuspend the thalli to obtain a resuspension solution (whole cells); ultrasonically crushing the heavy suspension for 15min to obtain a cell crushing liquid; the cell disruption solution was centrifuged at 8000rpm for 15min at 4 ℃ to obtain a cell disruption supernatant.
The enzyme activities of ferulic acid esterase in fermentation supernatant and cell disruption supernatant obtained by fermenting the Bacillus subtilis WB800 and the recombinant Bacillus subtilis WB800/PMA5-fae are detected (the detection results are shown in Table 3).
As can be seen from Table 3, when the fermentation is carried out for 14h, the enzyme activity of the intracellular feruloyl esterase obtained by fermenting the recombinant Bacillus subtilis WB800/PMA5-fae is 82.53U/mL, which is 4 times higher than the enzyme activity of the extracellular feruloyl esterase obtained by fermenting for 60h, which is 20.80U/mL; meanwhile, the intracellular ferulic acid esterase activity of the recombinant Bacillus subtilis WB800/pMA5-fae is obviously higher than that of the Bacillus subtilis WB 800. Therefore, intracellular feruloyl esterase fermented for 14h was selected as the basis for subsequent enzyme purification.
TABLE 3 enzyme Activity of Feruloyl esterase in fermentation supernatants and cell disruption supernatants obtained by fermentation of different Bacillus subtilis
Example 4: feruloyl esterase Performance
The method comprises the following specific steps:
purifying cell disruption supernatant obtained by fermenting the recombinant Bacillus subtilis WB800/PMA5-fae obtained in the embodiment 3 by using a nickel column, dialyzing by using dialysate containing 0.01mol/L EDTA for three times, and dialyzing by using dialysate not containing EDTA for three times to obtain pure enzyme; the pure enzyme was placed in a refrigerator at 4 ℃ until use.
After diluting the pure enzyme by 0 to 50 times, the method of example 5 was referenced to detect the ferulic acid esterase enzyme activity of the pure enzyme, and the detection results are shown in Table 4.
As can be seen from Table 4, the ferulic acid esterase enzyme activity of the pure enzyme diluted 20 times was determined by selecting the optimum dilution factor of 20 times, and the determination method was as follows:
weighing 100mg of Coomassie brilliant blue G-250, dissolving in 50mL of 90% (v/v) ethanol, adding 100mL of 85% (v/v) phosphoric acid, and diluting to 1L with distilled water to obtain a Coomassie brilliant blue G-250 dye solution; weighing 100mg of bovine albumin, dissolving, and fixing the volume to 100mL by using distilled water to obtain a standard protein solution; respectively sucking 0mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL and 0.10mL of standard protein solution, respectively adding 1.0 mL, 0.98 mL, 0.96 mL, 0.94 mL, 0.92 mL and 0.90mL of distilled water, respectively adding 5mL of Coomassie brilliant blue G-250 dye solution, and uniformly mixing to obtain a mixed solution; reacting the mixed solution at room temperature (25 ℃) for 5min to obtain reaction solution; the reaction solution was measured for absorbance at a wavelength of 560nm, and a standard curve for protein concentration was plotted (see FIG. 4 for the standard curve).
Taking 500 mu L of pure enzyme, adding 500 mu L of distilled water into the pure enzyme, then adding 5mL of Coomassie brilliant blue G-250 dye solution, and uniformly mixing to obtain mixed solution; reacting the mixed solution at room temperature (25 ℃) for 5min to obtain reaction solution; measuring the absorbance of the reaction solution at the wavelength of 595 nm; calculating to obtain the protein concentration of the pure enzyme according to the measured absorbance and the protein concentration standard curve, and calculating to obtain the specific enzyme activity of the ferulic acid esterase according to the activity of the ferulic acid ester enzyme in the pure enzyme diluted by 20 times;
wherein, the calculation formula of the specific enzyme activity of the ferulic acid esterase is as follows:
the detection result is as follows: the protein concentration of the pure enzyme diluted by 20 times is 0.15mg/mL, and the specific enzyme activity of the ferulic acid esterase of the pure enzyme diluted by 20 times is 2230U/mg.
TABLE 4 Feruloyl esterase enzyme Activity of pure enzymes diluted by different fold
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university in south of the Yangtze river
<120> recombinant bacillus subtilis for producing feruloyl esterase and application thereof
<160> 4
<170> PatentIn version 3.3
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Glu Met Asp Asn His Trp Cys Pro Val Ile Ala Asp Arg Ala Gln Cys
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Ile Val Val Asn Val Glu Tyr Gln Leu Ala Pro Glu His Pro Phe Pro
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Ala Ala Leu His Glu Cys Tyr Asp Val Leu Lys Trp Leu Tyr Glu His
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Pro Asp Glu Leu Gln Ile Asp Pro Asn Arg Val Ala Ile Gly Gly His
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Ser Ala Gly Gly Asn Leu Ala Thr Ala Ala Cys Leu Leu Asn Ile Gln
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Lys Gly Asn Pro Val Pro Ile Val Tyr Gln Val Leu Asp Tyr Pro Pro
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Leu Asp Leu Ala Thr Asp Pro Ala Glu Lys Pro Ala Phe Glu Glu Ala
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aagcagccgc ttcccgtttt tgtgaattta catggcggag gatttatcct aggcagtgct 240
gaaatggata accactggtg tccggtcatt gcagaccgag cgcaatgtat cgtcgtcaat 300
gtcgagtatc agcttgcccc agagcaccct tttccagcag ctcttcatga atgctacgat 360
gtgctgaagt ggctgtatga acaccctgat gagcttcaaa tagatcctaa tagagtagcc 420
attggcggac atagtgcagg aggaaacttg gcaacggctg cttgtctctt aaatattcaa 480
aaagggaacc cagtcccgat tgtctatcaa gtgcttgatt atccgccgct tgatttagcc 540
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tttaatgcct tctatctgca aggccaagat ccgcacaatc cgctcgtttc tccaatcttt 660
gccgatcgtt catccttggc tcaactgcca ccagctctcg ttatcacagc tgaaagagat 720
tcgctagctc aagaagccga acaatatgcg gagaagttaa aagaagcagg ggtagatgtc 780
acgtacagac agtttaaagg agtccctcac gccttcacgc atgctggaga tttagaaata 840
gctgaagaag cttggcatct gatgagtgat caattgaaaa aggcatttga ataa 894
<210> 3
<211> 53
<212> DNA
<213> Artificial sequence
<400> 3
aaaaggagcg atttacatat gatgaactta caagagcaaa tcaaaatcgc tgc 53
<210> 4
<211> 55
<212> DNA
<213> Artificial sequence
<400> 4
gagctcgact ctagaggatc cttaatggtg atggtgatga tgttcaaatg ccttt 55
Claims (10)
1. The recombinant bacillus subtilis is characterized in that the recombinant bacillus subtilis uses bacillus subtilis as a host to express a gene for coding feruloyl esterase; the amino acid sequence of the ferulic acid esterase is shown in SEQ ID NO. 1.
2. The recombinant Bacillus subtilis of claim 1, wherein the nucleotide sequence of the gene encoding feruloyl esterase is shown in SEQ ID No. 2.
3. The recombinant Bacillus subtilis of claim 1 or 2 wherein the recombinant Bacillus subtilis is a Bacillus subtilis-based host and the pMA5 plasmid, pUB110 plasmid or pTSC plasmid is an expression vector for expressing a gene encoding feruloyl esterase.
4. The recombinant bacillus subtilis according to claim 1 or 2, wherein the bacillus subtilis is bacillus subtilis 168, bacillus subtilis WB800, bacillus subtilis WB600 or bacillus subtilis 1a751.
5. The recombinant Bacillus subtilis strain of claim 3, wherein the Bacillus subtilis strain is Bacillus subtilis 168, bacillus subtilis WB800, bacillus subtilis WB600 or Bacillus subtilis 1A751.
6. A method for producing feruloyl esterase, which comprises the steps of inoculating the recombinant Bacillus subtilis of any one of claims 1 to 5 into a fermentation medium for fermentation to obtain a fermentation broth, and separating the feruloyl esterase from the fermentation broth.
7. Use of a recombinant bacillus subtilis according to any one of claims 1-5 or a method according to claim 6 for the production of a feruloyl esterase.
8. A method for producing ferulic acid, which is characterized in that the method comprises the steps of inoculating the recombinant Bacillus subtilis of any one of claims 1 to 5 into a fermentation culture medium for fermentation to obtain a fermentation broth, separating ferulic acid esterase from the fermentation broth, adding ferulic acid esterase into a reaction system containing ferulic acid ester compounds for reaction to obtain a reaction solution, and finally separating ferulic acid from the reaction solution;
or, the method is that the recombinant bacillus subtilis of any one of claims 1 to 5 is inoculated into a fermentation culture medium for fermentation to obtain a fermentation liquid, then the fermentation liquid is added into a reaction system containing ferulic acid ester compounds for reaction to obtain a reaction liquid, and finally ferulic acid is separated from the reaction liquid; the ferulic acid ester compound is ferulic acid methyl ester or ferulic acid ethyl ester.
9. The method for producing ferulic acid of claim 8, wherein the temperature of the reaction is 50 ℃ and the time is 10-30 min.
10. Use of the recombinant Bacillus subtilis of any one of claims 1-5 for the production of ferulic acid, for the hydrolysis of lignocellulose, for the preparation of feed and for the production of paper.
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| CN202011048393.5A CN112210523B (en) | 2020-09-29 | 2020-09-29 | Recombinant bacillus subtilis for producing feruloyl esterase and application thereof |
| PCT/CN2020/119269 WO2022041397A1 (en) | 2020-08-31 | 2020-09-30 | Feruloyl esterase and application thereof |
| JP2021560888A JP7489119B2 (en) | 2020-08-31 | 2020-09-30 | Ferulic acid esterase and its applications |
| US17/485,682 US20220002689A1 (en) | 2020-08-31 | 2021-09-27 | Feruloyl Esterase and Application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008116319A1 (en) * | 2007-03-27 | 2008-10-02 | The Royal Institution For The Advancement Of Learning/Mcgill University | Bioproduction of ferulic acid and uses thereof |
| CN102066409A (en) * | 2008-04-17 | 2011-05-18 | 诺维信公司 | Polypeptides having ferulic acid esterase activity and polynucleotides encoding same |
| CN106434598A (en) * | 2016-09-07 | 2017-02-22 | 江南大学 | Esterase and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008116319A1 (en) * | 2007-03-27 | 2008-10-02 | The Royal Institution For The Advancement Of Learning/Mcgill University | Bioproduction of ferulic acid and uses thereof |
| CN102066409A (en) * | 2008-04-17 | 2011-05-18 | 诺维信公司 | Polypeptides having ferulic acid esterase activity and polynucleotides encoding same |
| CN106434598A (en) * | 2016-09-07 | 2017-02-22 | 江南大学 | Esterase and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 阿魏酸酯酶的研究与应用进展;王丽等;《山东农业大学学报(自然科学版)》;20161231;第47卷(第4期);第628-635页 * |
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