CN112143681B - Bacillus belgii capable of producing feruloyl esterase and application thereof - Google Patents
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Abstract
The invention discloses a bacillus beiLeisi capable of producing feruloyl esterase and application thereof, belonging to the technical field of microorganisms. The invention provides a Bacillus velezensis SK52.002 strain, the Bacillus velezensis SK52.002 can produce ferulic acid esterase with high yield, which is specifically represented by the following steps: the Bacillus velezensis SK52.002 is inoculated into a fermentation medium for fermentation for 16 hours, so that the enzyme activity of the ferulic acid esterase in the fermentation liquid can reach 12mU/mL, and the Bacillus velezensis SK52.002 has great application prospect in the production of the ferulic acid esterase and ferulic acid.
Description
Technical Field
The invention relates to a bacillus beiLeisi capable of producing feruloyl esterase and application thereof, belonging to the technical field of microorganisms.
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, the low production of feruloyl esterase by the existing feruloyl esterase-producing strains greatly limits 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 Bacillus velezensis strain capable of producing ferulic acid esterase with high yield.
[ solution ]
In order to solve the problems, the invention provides a Bacillus subtilis SK52.002, wherein the Bacillus subtilis SK52.002 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020422 and the preservation date of 2020, 08 and 14 days.
The Bacillus belgii SK52.002 is derived from a soil sample in Wuxi city, Jiangsu province, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, the sequence obtained through sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is Bacillus belgii and is named as Bacillus belgii SK 52.002.
The invention also provides a method for producing feruloyl esterase, which comprises the steps of inoculating the Bacillus belgii SK52.002 into a fermentation medium for fermentation to obtain fermentation liquor, and then separating the feruloyl esterase from the fermentation liquor.
In one embodiment of the invention, the components of the fermentation medium comprise 5-50 g/L of wheat bran and 2-8 g/L, K g/L of tryptone2HPO4·3H2O 0～1.5g/L、KCl 0.2～0.5g/L、MgSO4·7H2O 0～0.6g/L、FeSO4·7H2O 0～0.1g/L。
In one embodiment of the invention, the components of the fermentation medium comprise wheat bran 45g/L, tryptone 5g/L, K2HPO4·3H2O 1.0g/L、KCl 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H2O 0.01g/L。
In one embodiment of the invention, the fermentation temperature is 28-37 ℃, the rotation speed is 150-200 r/min, and the time is 12-48 h.
The invention also provides the application of the Bacillus belgii SK52.002 or the method for producing the ferulic acid esterase in producing the ferulic acid esterase.
The invention also provides a method for producing ferulic acid, which comprises the steps of inoculating the Bacillus belgii SK52.002 into a fermentation culture medium for fermentation to obtain fermentation liquor, separating ferulic acid esterase from the fermentation liquor, adding ferulic acid esterase into a reaction system containing ferulic acid ester compounds for reaction to obtain reaction liquid, and finally separating ferulic acid from the reaction liquid;
or inoculating the Bacillus beiLeisi SK52.002 to a fermentation culture medium for fermentation to obtain a fermentation liquid, then inoculating the fermentation liquid to a reaction system containing ferulic acid ester compounds for reaction to obtain a reaction liquid, and finally separating 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 50 ℃ and the reaction time is 10 to 30 min.
The invention also provides application of the Bacillus belgii SK52.002 or the method for producing ferulic acid in production of ferulic acid, hydrolysis of lignocellulose, preparation of feed and papermaking.
Has the advantages that:
the invention provides a Bacillus velezensis SK52.002 strain, the Bacillus velezensis SK52.002 can produce ferulic acid esterase with high yield, which is specifically represented by the following steps: the Bacillus velezensis SK52.002 is inoculated into a fermentation medium for fermentation for 16 hours, so that the enzyme activity of the ferulic acid esterase in the fermentation liquid can reach 12mU/mL, and the Bacillus velezensis SK52.002 has great application prospect in the production of the ferulic acid esterase and ferulic acid.
Biological material preservation
A strain of Bacillus velezensis SK52.002, which is classified and named as Bacillus velezensis, is preserved in China center for type culture collection at 14.08.2020, with the preservation number of CCTCC NO: M2020422, and the preservation address of Wuhan university, Wuhan, China.
Drawings
FIG. 1: a cell morphology of Bacillus velezensis SK 52.002.
FIG. 2: high performance liquid chromatogram of ferulic acid standard.
FIG. 3: high performance liquid chromatogram of ferulic acid methyl ester standard.
FIG. 4: high performance liquid chromatogram of the reaction solution.
FIG. 5: LC-MS diagram of the reaction solution.
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 limited.
The media involved in the following examples are as follows:
strain isolation medium: 200g/L of potato, 20g/L of cane sugar and 15g/L of agar, and the pH is natural.
Screening a solid culture medium: filtering the N, N-dimethylformamide solution containing 100mg/L of ferulic acid ethyl ester by using a filter membrane of 0.22 mu m to obtain a ferulic acid ethyl ester solution; cooling the sterilized basal culture medium to 60 ℃, adding 10% (v/v) of ferulic acid ethyl ester solution into the basal culture medium, and shaking uniformly until the solution is uniform milky white;
wherein, the basic culture medium: NaNO3 2g/L、K2HPO4·3H2O 1g/L、KCl 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H20.01g/L of O and 15g/L of agar, and the pH is natural.
Seed culture medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride, and the pH is natural.
Fermentation medium: wheat bran 20g/L, NaNO3 2g/L、K2HPO4·3H2O 1g/L、KCl 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H2O0.01 g/L, pH is natural.
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.
Example 1: acquisition of Bacillus beilis SK52.002
The method comprises the following specific steps:
1. separating and purifying
Taking soil from Wuxi city of Jiangsu province as a sample, putting 1.0g of the sample into 10mL of sterile water filled with glass beads, and oscillating for 30min at 30 ℃ by a shaking table at 200r/min to fully separate thalli to obtain a mixed bacterial liquid; 0.5mL of the mixed bacterial liquid is sucked and added into a 10mL centrifuge tube filled with 4.5mL of normal saline under the aseptic environment to obtain 10-1Diluting the solution, repeating the dilutionStep (b) obtaining in sequence 10-2、10-3、10-4、10-5、10-6Diluting the solution; draw 100. mu.L of dilution gradient of 10-3、10-4、10-5、10-6Coating the diluent on a strain isolation culture medium, and performing inverted culture in a constant-temperature incubator at 30 ℃ for 24-48 h to obtain a diluted coating plate; selecting a single colony on the diluted coating plate, streaking the single colony on a screening solid culture medium, and performing inverted culture in a constant temperature incubator at 30 ℃ for 24 hours to obtain a single colony; observing whether a transparent ring appears around the single colony or not, and screening the single colony with the transparent ring around the single colony; a single colony was obtained by the co-screening, and the strain corresponding to the single colony was designated as SK 52.002.
2. Strain identification
The genome of the strain SK52.002 is extracted, the 16S rDNA of the strain SK52.002 is amplified and sequenced (finished by Shanghai Bioengineering Co., Ltd.), the 16S rDNA sequence of the strain SK52.002 obtained by sequencing analysis (the 16S rDNA sequence of the SK52.002 is shown as SEQ ID NO. 1) is compared in GenBank, and the result shows that the strain is confirmed to be Bacillus belgiensis and is named as Bacillus belgiensis (Bacillus velezensis) SK 52.002.
3. Observation of Bacillus beilesiensis SK52.002
Streaking a bacterial liquid dipped with Bacillus velezensis SK52.002 on an LB solid culture medium, and performing inverted culture in a constant-temperature incubator at 30 ℃ for 12h to obtain a single bacterial colony; selecting a single colony, inoculating the single colony to a seed culture medium, and culturing for 12h on a shaking table at 30 ℃ and 200r/min to obtain a bacterial liquid; the bacterial suspension was diluted 10-fold with sterile water, and the cell morphology was observed under an optical microscope (see FIG. 1).
As is clear from FIG. 1, the cell morphology of Bacillus velezensis SK52.002 is: rod-shaped, blunt-rounded at both ends, has the morphological characteristics of typical bacillus and belongs to gram-positive bacteria.
Example 2: production of feruloyl esterase
The method comprises the following specific steps:
the bacterial liquid of Bacillus velezensis SK52.002 obtained in example 1 is dipped and streaked on an LB solid culture medium, and is inversely cultured in a constant temperature and humidity incubator at 30 ℃ for 24-48 h to obtain a single bacterial colony; selecting single colony, inoculating to seed culture medium, and culturing in shaking table at 30 deg.C and 200r/min for 48 hr to obtain seed solution; inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 5% (v/v), and fermenting for 16h in a shaking table at 30 ℃ and 200r/min to obtain fermentation liquor.
Centrifuging the fermentation liquor at 4 deg.C and 10000rpm for 10min to obtain crude enzyme solution; and (3) measuring the enzyme activity of the ferulic acid esterase in the crude enzyme solution by the following method:
adding 250 μ L of the crude enzyme solution to 750 μ L of methyl ferulate solution with concentration of 0.003mol/L (the methyl ferulate solution is obtained by dissolving methyl ferulate in Tris-HCl buffer solution with pH of 8.0 and concentration of 0.05 mol/L) to obtain enzyme reaction system; reacting the enzyme reaction system in a water bath kettle at 50 ℃ for 30min, 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:
wherein the high performance liquid chromatogram of ferulic acid standard is shown in figure 2, the high performance liquid chromatogram of ferulic acid methyl ester standard is shown in figure 3, the high performance liquid chromatogram of reaction solution is shown in figure 4, and the linear relationship between ferulic acid concentration Y and ferulic acid concentration X in the high performance liquid chromatogram is 52514X-80.417(R is 52514X-80.417)20.9996) is calculated;
HPLC method adopts Agilent 1200 high performance liquid chromatograph; the column was ZORBAX Eclipse Plus C18(Agilent, 4.6 mm. times.150 mm, 3.5 μm); an ultraviolet detector; mobile phase A: 1% (v/v) acetic acid solution, mobile phase B: methanol; the flow rate is 1 mL/min; the column temperature is 30 ℃; the detection wavelength was 320nm and the gradient elution procedure is shown in Table 1.
The detection result is as follows: the enzyme activity of the ferulic acid esterase in the crude enzyme solution is 12 mU/mL.
To further prove that Bacillus velezensis SK52.002 can produce feruloyl esterase, the reaction solution was analyzed by LC-MS (see FIG. 5 for analysis results).
From fig. 3, it can be seen that the fragment m/z with the highest relative abundance of the product peak is 193, and the relative molecular mass of the target product is 194, which is consistent with the relative molecular mass of the ferulic acid monomer.
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 |
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 of south of the Yangtze river
<120> Bacillus belgii capable of producing feruloyl esterase and application thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1479
<212> DNA
<213> Bacillus belgii
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tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcggacag atgggagctt 60
gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg 120
ggataactcc gggaaaccgg ggctaatacc ggatggttgt ctgaaccgca tggttcagac 180
ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg 240
aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact 300
gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360
acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420
gttgttaggg aagaacaagt gccgttcaaa tagggcggca ccttgacggt acctaaccag 480
aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 540
ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg 600
gctcaaccgg ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa 660
ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact 720
ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc 780
ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg 840
ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900
ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 960
gaaccttacc aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg 1020
gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080
cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag 1260
ccaatcccac aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct 1320
ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380
caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta acctttatgg 1440
agccagccgc cgaaggtggg acagatgatt ggggtgaag 1479
Claims (9)
1. Bacillus belgiiBacillus velezensis) The bacillus beleisi is preserved in China collection center for culture collection of classical collection with the preservation number of CCTCC NO: M2020422 and the preservation date of 14 days 08 and 14 months 2020.
2. A method for producing feruloyl esterase, which comprises inoculating Bacillus belgii of claim 1 to a fermentation medium to ferment to obtain a fermentation broth, and separating feruloyl esterase from the fermentation broth.
3. The method of claim 2, wherein the fermentation medium comprises wheat bran 5-50 g/L, tryptone 2-8 g/L, K2HPO4·3H2O 0~1.5 g/L、KCl 0.2~0.5 g/L、MgSO4·7H2O 0~0.6 g/L、FeSO4·7H2O 0~0.1 g/L。
4. The method of claim 3, wherein the fermentation medium comprises wheat bran 45g/L, tryptone 5g/L, K2HPO4·3H2O 1.0 g/L、KCl 0.5 g/L、MgSO4·7H2O 0.5 g/L、FeSO4·7H2O 0.01 g/L。
5. The method for producing feruloyl esterase according to any one of claims 2 to 4, characterized in that the fermentation temperature is 28 to 37 ℃, the rotation speed is 150 to 200r/min, and the time is 12 to 48 hours.
6. Use of a Bacillus belgii according to claim 1 or a method of producing a feruloyl esterase according to any of claims 2 to 5 for producing a feruloyl esterase.
7. A method for producing ferulic acid, which is characterized in that the method comprises the steps of inoculating Bacillus belgii of claim 1 into a fermentation culture medium for fermentation to obtain a fermentation liquid, separating ferulic acid esterase from the fermentation liquid, adding ferulic acid esterase into a reaction system containing methyl ferulate for reaction to obtain a reaction liquid, and finally separating ferulic acid from the reaction liquid;
or, the method is that the Bacillus bleekii of claim 1 is inoculated into a fermentation culture medium for fermentation to obtain a fermentation liquid, then the fermentation liquid is inoculated into a reaction system containing methyl ferulate for reaction to obtain a reaction liquid, and finally ferulic acid is separated from the reaction liquid.
8. The method for producing ferulic acid according to claim 7, wherein the reaction temperature is 30-50 ℃ and the reaction time is 10-30 min.
9. Use of bacillus belgii according to claim 1 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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| CN104894017A (en) * | 2015-05-22 | 2015-09-09 | 徐州工程学院 | Feruloyl-esterase-producing Bacillus licheniformis strain and application thereof |
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| CN104894017A (en) * | 2015-05-22 | 2015-09-09 | 徐州工程学院 | Feruloyl-esterase-producing Bacillus licheniformis strain and application thereof |
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