CN104894017A - Feruloyl-esterase-producing Bacillus licheniformis strain and application thereof - Google Patents
Feruloyl-esterase-producing Bacillus licheniformis strain and application thereof Download PDFInfo
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Abstract
The invention discloses a feruloyl-esterase-producing Bacillus licheniformis strain and application thereof. The collection number of the Bacillus licheniformis DBM12 strain is CGMCC No.8672. The Bacillus licheniformis strain has the advantages of lower requirements for the carbon source and nitrogen source, short fermentation time, high safety and no toxicity, and can be used in food/feed industry. The Bacillus licheniformis strain has favorable hereditary stability, and is simple to operate. The feruloyl esterase synthesis capacity is basically unchanged after serial passage by more than 10 generations.
Description
Technical field
The invention belongs to technical field of microbial fermentation, be specifically related to a kind of Bacillus licheniformis and application thereof of producing feruloyl esterase.
Background technology
Forulic acid (ferulic acid, FA) is a kind of natural antioxidants be prevalent in plant, is a kind of phenolic acid in various plants.There is the function that calm, anticancer, anti-oxidant, atherosclerosis, platelet aggregation-against, reducing blood-fat etc. are unique.But forulic acid major part in plant exists with crosslinked form, and humans and animals can not directly absorb this kind of forulic acid, microorganism must be relied on to produce esterase forulic acid is dissociated out.On the other hand, in plant cell wall, forulic acid or dimer forulic acid are mainly connected on the arabinose residues of araboxylan with the form of ester bond, strengthen the intensity of araboxylan chain.But from spatially limiting the effective degraded to Mierocrystalline cellulose plant cell wall and hemicellulose of animal and microorganism.
Feruloyl esterase (EC 3.1.1.73, feruloyl esterase), also known as Ferulic acid esterase or cinnyl esterase, can be hydrolyzed the ester bond of forulic acid, the formation of dimerization forulic acid, forulic acid be discharged.It can combined action as mutual in zytase, cellulase and lignoenzyme other lytic enzymes, the polysaccharide comparatively up hill and dale in degradation of cell wall and xylogen.Therefore, feruloyl esterase has huge application potential at numerous areas such as food, medicine, papermaking, feed manufacturing, biosynthesizing and energy developments.
Since finding feruloyl esterase first in streptomycete, so far nearly 30 kinds of feruloyl esterases of separation and purification, but mainly concentrate on mould and aspergillus, minority is only had to be come from bacterium, such as Lactobacterium acidophilum, Bacillus subtillis etc., and the enzymatic property of their feruloyl esterase and aminoacid sequence all different.Because native fungal growth, production of enzyme and all restriction of fungi to growth conditions all govern the suitability for industrialized production that enzyme process prepares forulic acid.Therefore seek to be that fermentation strain production feruloyl esterase is significant with bacterium.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art and a kind of Bacillus licheniformis and application thereof of producing feruloyl esterase are provided, this bacterial strain not only to Carbon and nitrogen sources require lower, fermentation time is short, bacterial strain safety non-toxic, can use in food and feed industry; And genetic stability is good, simple to operate, more than continuous passage 10 generation, the synthesis capability of feruloyl esterase is substantially constant.
A kind of Bacillus licheniformis (Bacillus licheniformis) DBM12 producing feruloyl esterase, (address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC on January 2nd, 2014, postcode 100101), deposit number: CGMCC No.8672.
The screening method of Bacillus licheniformis DBM12 of the present invention, comprises the following steps:
Step 1, soil sample sampling and processing
Soil sample is picked up from rural area, Yunlong District, Xuzhou City of Jiangsu Province and is in animal waste compost in thermophilic fermentation.The soil sample gathered is put in room temperature preservation in the aseptic plastic bag of sealing, and carries out the separation of object bacterial strain in 1 week.
Get 5-10g soil sample to put into and 20-40mL sterilized water is housed and the triangular flask being placed with little granulated glass sphere, be placed on shaking table, 120-160r/min rotates concussion 20-30min, and then triangular flask is placed in 70-80 DEG C of heating in water bath 15-30min, period shakes once in 5min.
Step 2, enrichment culture
Get the soil sample after 1-2mL thermal treatment in the triangular flask that 30-50mL enrichment medium one is housed, at 160-180r/min rotating speed 50-60 DEG C, 20-24h is cultivated in concussion.Then get 1-2mL nutrient solution to be inoculated into and to be equipped with in the enrichment medium two of 50mL concentration 5-10%, continue concussion at 160-180r/min rotating speed 50-60 DEG C and cultivate 18-20h.
Enrichment medium one: yeast extract 0.5g, KH
2pO
40.1g, NH
4nO
30.1g, NaCl 0.1g, MgSO
47H
2o 0.25g, micro-1mL, Ferulic acid ethylester 1g, deionized water 100mL, regulate pH4.0.
Enrichment medium two: NH
4nO
30.5g, urea 0.1g, KH
2pO
40.1g, NaCl 0.1g, Ferulic acid ethylester 1g, deionized water 100mL, regulate pH3.0.
Trace element: EDTA 5g, CaCl
22H
2o 6g, FeSO
47H
2o 6g, MnCl
24H
2o 1.15g, CoCl
26H
2o 0.8g, ZnSO
47H
2o 0.7g, CuCl
22H
2o 0.3g, H
3bO
30.3g, (NH
4)
6mo
7o
24H
2o 0.25g, deionized water 1000mL.
Step 3, primary dcreening operation
The nutrient solution sterilized water of second time enrichment is diluted, gets respectively and be diluted to 10
-2, 10
-3, 10
-4, 10
-5, 10
-6diluent 150-250 μ L coat on primary dcreening operation substratum one, after the bacterium liquid of media surface is completely dry, culture dish seals with sealed membrane, culture dish is placed in 40-45 DEG C of incubator and cultivates 24-48h, then culture dish is placed in 10-20 DEG C of incubator and continues to cultivate 6-12h.
Single bacterium colony point of picking different shape is received on primary dcreening operation substratum two, 45-60 DEG C of constant temperature culture 24-48h.To pick out on primary dcreening operation substratum two growth better and the transparent circle diameter bacterial strain larger with colony diameter ratio, streak inoculation in pure medium, in 45-50 DEG C of cultivation 1-2d.Then picking single bacterium colony is wherein rule separation again, repetitive operation 3 times.Examine under a microscope the single thalline of form as continuous several times and then think bacterial strain purifying.Finally by the 4 DEG C of preservations in slant preservation substratum of the inoculation of purifying, then sieve again further.
Primary dcreening operation substratum one: peptone 0.5g, yeast extract 0.1g, NaNO
30.3g, K
2hPO
40.1g, KCl 0.5g, MgSO
47H
2o 0.025g, FeSO
47H
2o 0.001g, agar 2.5, deionized water 100mL, regulates pH4.5.
Primary dcreening operation substratum two: KH
2pO
40.2g, (NH
4)
2sO
40.4g, MgSO
47H
2o 0.03g, FeSO
47H
2o 0.001g, MnSO
40.001g, ZnCl
20.001g, agar 2.5g, deionized water 100mL, pH4.0.After substratum 121 DEG C, 15min sterilizing, when being cooled to 60-70 DEG C, add Ferulic acid ethylester solution 1mL, after fully shaking up, in the culture dish on average down to 3 diameter 90mm.
Ferulic acid ethylester solution: 0.2g Ferulic acid ethylester is added in 1.5mL sterile centrifugation tube, then add 500 μ LN, dinethylformamide dissolving, and then add 500 μ L sterilized waters, fully shake up.
Step 4, multiple sieve
The bacterial strain streak inoculation obtained by primary dcreening operation is in activating solid substratum, and 40-45 DEG C of constant incubator cultivates 16-20h.Being inoculated by the bacterial strain activated in the triangular flask that 30-50mL seed culture fluid is housed, is shake constant temperature culture 16-20h under 40-45 DEG C, shaking speed 160-200r/min frequency with culture temperature.Be inoculated in multiple sieve fermention medium according to the inoculum size of 4-10%, shaking table concussion is cultivated.Fermentation condition is: 250mL triangular flask, and liquid amount is 30-60mL, and shaking table concussion frequency is 160-200r/min, leavening temperature 40-50 DEG C, fermentation time 24-48h.After fermentation ends, get fermented liquid with the centrifugal 5-10min of 6000-1000r/min rotating speed, get supernatant liquor and detect enzymic activity.
Sieve seed culture medium again: peptone 1g, extractum carnis 0.5g, yeast powder 0.5g, NaCl 0.5g, K
2hPO
40.02g, add deionized water to 100mL, pH4.5,121 DEG C, sterilizing 15min.
Fermentative medium formula: Zulkovsky starch 0.5g, peptone 1g, extractum carnis 0.5g, NaCl 0.5g, KH
2pO
40.1g, MgSO
47H
2o 0.04g, deionized water 100mL, pH4.5,121 DEG C, sterilizing 15min.
Described Bacillus licheniformis (Baclicus lincheniformis) DBM12 produces feruloyl esterase and the application prepared in forulic acid in fermentation, comprises the following steps:
Step 1, ferments Bacillus licheniformis DBM12 bacterial strain, fermentation period 2-3d, leavening temperature 35-40 DEG C, inoculum size 4-10%, shaking speed 160-200r/min;
Step 2, by the centrifugal 20min of fermented liquid 8000r/min of step 1, supernatant 0.22 μm of filtration, collects filtrate;
Step 3, be 75% by ammonium sulfate saturation ratio, in step 2 gained filtrate, add ammonium sulfate, 4 DEG C of standing 12h, the centrifugal 20min of 8000r/min, collecting precipitation, dissolves with 20mmol/L, pH6.0 phosphate buffer solution, the desalination of loading Sephedex G25 molecular sieve gel post, carry out wash-out with the phosphoric acid buffer of pH6.5, elution rate is 15 ~ 20ml/h, obtains crude enzyme liquid;
Step 4, is added to step 3 gained crude enzyme liquid in bran powder, then adds water, and regulates pH6.5, shaking table shakes frequency 120r/min, 50 DEG C of enzymolysis 12h, and boiling water goes out enzyme, and centrifuging and taking supernatant, is forulic acid.
As the further improvement of foregoing invention, the formula of the fermention medium described in step 1 is: the bran powder 2g of peptone 2g, glucose 0.5g, excessively 60 mesh sieves, excessively 60 mesh sieve husk powder 1g, yeast extract 0.5g, NaCl 0.4g, KH
2pO
40.1g, MgSO
40.05g, distilled water 100mL, pH7.0-7.5.
As the further improvement of foregoing invention, in step 4, crude enzyme liquid consumption is 5mL, and bran powder consumption is 10g, and water consumption is 150mL.
Bacillus licheniformis of the present invention (Baclicus lincheniformis) DBM12 not only to Carbon and nitrogen sources require lower, fermentation time is short, bacterial strain safety non-toxic, can use in food and feed industry; And genetic stability is good, simple to operate, more than continuous passage 10 generation, the synthesis capability of feruloyl esterase is substantially constant; Feruloyl esterase that this bacterial strain produces is heat-resisting acidproof, in feed manufacturing, have important application.
Accompanying drawing explanation
Fig. 1 is the multiple sieve lithograph in embodiment 1;
Fig. 2 is the suitableeest action pH and the ph stability graphic representation of feruloyl esterase in embodiment 3;
Fig. 3 is feruloyl esterase optimum temperature graphic representation in embodiment 3;
Fig. 4 is the thermostability graphic representation of feruloyl esterase in embodiment 3.
Embodiment
Embodiment 1
The screening method of Bacillus licheniformis (Baclicus lincheniformis) DBM12, comprises the following steps:
Step 1, soil sample sampling and processing
Soil sample is picked up from rural area, Yunlong District, Xuzhou City of Jiangsu Province and is in animal waste compost in thermophilic fermentation.The soil sample gathered is put in room temperature preservation in the aseptic plastic bag of sealing, and carries out the separation of object bacterial strain in 1 week.
Get 5-10g soil sample to put into and 20-40mL sterilized water is housed and the triangular flask being placed with little granulated glass sphere, be placed on shaking table, 120-160r/min rotates concussion 20-30min, and then triangular flask is placed in 70-80 DEG C of heating in water bath 15-30min, period shakes once in 5min.
Step 2, enrichment culture
Get the soil sample after 1-2mL thermal treatment in the triangular flask that 30-50mL enrichment medium one is housed, at 160-180r/min rotating speed 50-60 DEG C, 20-24h is cultivated in concussion.Then get 1-2mL nutrient solution to be inoculated into and to be equipped with in the enrichment medium two of 50mL concentration 5-10%, continue concussion at 160-180r/min rotating speed 50-60 DEG C and cultivate 18-20h.
Enrichment medium one: yeast extract 0.5g, KH
2pO
40.1g, NH
4nO
30.1g, NaCl 0.1g, MgSO
47H
2o 0.25g, micro-1mL, Ferulic acid ethylester 1g, deionized water 100mL, regulate pH4.0.
Enrichment medium two: NH
4nO
30.5g, urea 0.1g, KH
2pO
40.1g, NaCl 0.1g, Ferulic acid ethylester 1g, deionized water 100mL, regulate pH3.0.
Trace element: EDTA 5g, CaCl
22H
2o 6g, FeSO
47H
2o 6g, MnCl
24H
2o 1.15g, CoCl
26H
2o 0.8g, ZnSO
47H
2o 0.7g, CuCl
22H
2o 0.3g, H
3bO
30.3g, (NH
4)
6mo
7o
24H
2o 0.25g, deionized water 1000mL.
Step 3, primary dcreening operation
The nutrient solution sterilized water of second time enrichment is diluted, gets respectively and be diluted to 10
-2, 10
-3, 10
-4, 10
-5, 10
-6diluent 150-250 μ L coat on primary dcreening operation substratum one, after the bacterium liquid of media surface is completely dry, culture dish seals with sealed membrane, culture dish is placed in 40-45 DEG C of incubator and cultivates 24-48h, then culture dish is placed in 10-20 DEG C of incubator and continues to cultivate 6-12h.
Single bacterium colony point of picking different shape is received on primary dcreening operation substratum two, 45-60 DEG C of constant temperature culture 24-48h.To pick out on primary dcreening operation substratum two growth better and the transparent circle diameter bacterial strain larger with colony diameter ratio, streak inoculation in pure medium, in 45-50 DEG C of cultivation 1-2d.Then picking single bacterium colony is wherein rule separation again, repetitive operation 3 times.Examine under a microscope the single thalline of form as continuous several times and then think bacterial strain purifying.Finally by the 4 DEG C of preservations in slant preservation substratum of the inoculation of purifying, then sieve again further.Primary dcreening operation result is as shown in table 1.
Primary dcreening operation substratum one: peptone 0.5g, yeast extract 0.1g, NaNO
30.3g, K
2hPO
40.1g, KCl 0.5g, MgSO
47H
2o 0.025g, FeSO
47H
2o 0.001g, agar 2.5, deionized water 100mL, regulates pH4.5.
Primary dcreening operation substratum two: KH
2pO
40.2g, (NH
4)
2sO
40.4g, MgSO
47H
2o 0.03g, FeSO
47H
2o 0.001g, MnSO
40.001g, ZnCl
20.001g, agar 2.5g, deionized water 100mL, pH4.0.After substratum 121 DEG C, 15min sterilizing, when being cooled to 60-70 DEG C, add Ferulic acid ethylester solution 1mL, after fully shaking up, in the culture dish on average down to 3 diameter 90mm.
Ferulic acid ethylester solution: 0.2g Ferulic acid ethylester is added in 1.5mL sterile centrifugation tube, then add 500 μ LN, dinethylformamide dissolving, and then add 500 μ L sterilized waters, fully shake up.
Step 4, multiple sieve
The bacterial strain streak inoculation obtained by primary dcreening operation is in activating solid substratum, and 40-45 DEG C of constant incubator cultivates 16-20h.Being inoculated by the bacterial strain activated in the triangular flask that 30-50mL seed culture fluid is housed, is shake constant temperature culture 16-20h under 40-45 DEG C, shaking speed 160-200r/min frequency with culture temperature.Be inoculated in multiple sieve fermention medium according to the inoculum size of 4-10%, shaking table concussion is cultivated.Fermentation condition is: 250mL triangular flask, and liquid amount is 30-60mL, and shaking table concussion frequency is 160-200r/min, leavening temperature 40-50 DEG C, fermentation time 24-48h.After fermentation ends, get fermented liquid with the centrifugal 5-10min of 6000-1000r/min rotating speed, get supernatant liquor and detect enzymic activity.
Sieve seed culture medium again: peptone 1g, extractum carnis 0.5g, yeast powder 0.5g, NaCl 0.5g, K
2hPO
40.02g, add deionized water to 100mL, pH4.5,121 DEG C of sterilizing 15min.
Fermentative medium formula: Zulkovsky starch 0.5g, peptone 1g, extractum carnis 0.5g, NaCl 0.5g, KH
2pO
40.1g, MgSO
47H
2o 0.04g, deionized water 100mL, pH4.5,121 DEG C of sterilizing 15min.
Sodium phosphate dibasic-citric acid solution (pH4.5) 100mL of enzyme dull and stereotyped detection method: 0.02mol/L alive, agar powder 2.5g, 121 DEG C, after 15min sterilizing, when being cooled to 60-70 DEG C, add Ferulic acid ethylester solution 1mL, after fully shaking up, in the culture dish on average down to 3 diameter 90mm.The punch tool that agar fully solidifies rear diameter 6mm evenly punches about 3-10.Getting supernatant 100 μ L after fermented liquid is centrifugal is added in hole, and 45 DEG C are spent the night, and check transparent circle situation.Result as shown in Figure 1.
Step 5, culture presevation
Just the bacterial classification that obtains of screening ground is rule on beef extract-peptone, is separated to single bacterium and falls behind on LB slant medium and rule, cultivate 1-2 days for 40 DEG C, be placed in preservation in 4 DEG C of refrigerators.
LB slant culture based formulas is: peptone 1g, NaCl 0.5g, yeast powder 0.5g, agar 1.5g, pH value 5.0,121 DEG C of sterilizing 15min.
Table 1 primary dcreening operation plate screening result
| Bacterium | Colony diameter D/cm | Transparent circle diameter d/cm | d/D |
| DBM3 | 0.3 | 2.2 | 7.33 |
| DBM4 | 0.4 | 1.5 | 3.75 |
| DBM6 | 0.15 | 1.1 | 3.67 |
| DBM9 | 0.52 | 1.5 | 2.88 |
| DBM10 | 0.15 | 2.6 | 3.71 |
| DBM16 | 0.4 | 2.3 | 5.75 |
| DBM18 | 0.3 | 1.1 | 3.67 |
| DBM24 | 0.4 | 2.1 | 5.25 |
| DBM26 | 0.3 | 2.9 | 9.67 |
| DBM12 | 0.3 | 3.1 | 10.33 |
| DBM28 | 0.4 | 2.2 | 5.50 |
| DBM29 | 0.6 | 2.3 | 3.83 |
| DBM35 | 0.4 | 3.2 | 8.00 |
| DBM42 | 0.6 | 2.1 | 3.50 |
| DBM47 | 0.2 | 1.1 | 5.50 |
| DBM49 | 0.3 | 2.4 | 8.00 |
| DBM51 | 0.4 | 2.4 | 6.00 |
| DBM58 | 0.5 | 2.1 | 4.20 |
Obtained strains cultivates 24h on beef extract-peptone solid medium, and bacterium colony is circular, diameter 2.4-3.2mm; Bacterium colony is white, opaque, and surface is comparatively flat and dry, and firm attachment substratum, is difficult to picking.Static gas wave refrigerator in beef extract-peptone liquid nutrient medium, without precipitation, liquid is limpid, and media surface forms white elephant skin film, and there is particle on surface, and film is thinner.Observe thalli morphology, Gram-positive, process of growth produces gemma, and sporangiocyst is without expanding, and without parasporal crystal, thalline is shaft-like, has mobility.The morphology that bacterial strain DBM12 is concrete and physiological and biochemical property see the following form 2.Bacillus licheniformis in bacterium colony, dyeing characteristic and physiological and biochemical property and " uncle outstanding bacterium reflect handbook " (the 8th edition) relatively, tentatively determines that bacterial strain DBM12 is Bacillus licheniformis (Bacillus licheniformis).
Table 2 bacterial strain DBM12 morphology and physio-biochemical characteristics
| Characteristic | Result | Characteristic | Result |
| Anaerobic growth | + | Gelatin hydrolysate | + |
| Sporocyst | + | Hydrolyzed starch | + |
| Catalase test | + | Urea utilizes | - |
| V.P tests | + | Nitrate utilizes | + |
| Yelkin TTS is tested | - | Indole test | - |
| Methyl red test | + | PEARLITOL 25C | + |
| Catalase | + | D-wood sugar | + |
| Citrate trianion utilizes | + | L-arabinose | + |
| 2％NaCl | + | D-Glucose | + |
| 5％NaCl | + | Ferment is hydrolyzed | + |
| 7％NaCl | + | Growth temperature 10 DEG C | + |
| pH5.7 | + | Growth temperature 50 DEG C | + |
Extract the application's bacterial strain DBM12 genome, according to primers the most conservative in bacterial 16 S rDNA, wherein forward primer is for F:5'-AGAGTTTGATCCTGGCTCAG-3'(is as shown in SEQ ID NO.2); Reverse primer is for R:5'-AAGGAGGTGATCCAGCCGCA-3'(is as shown in SEQ ID NO.3).PCR reaction system is (20 μ L): ddH
2o 12.7 μ L, 10 × PCR buffer 2.0 μ L, dNTP mix (2.5mmol/L) 2.0 μ L, forward primer (10 μm of ol/L) 1.0 μ L, reverse primer (10 μm of ol/L) 1.0 μ L, Taq FlexiPolymerase (5U/ μ L) 0.3 μ L, template 1.0 μ L.Thermal circulation parameters 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 55 DEG C of annealing 45s, 72 DEG C extend 2min, circulate 30 times, and 72 DEG C extend 10min.Check order with pGM-T carrier cloning.Record sequence as shown in SEQ ID NO.1, the 16S rDNA sequence of the close bacterium obtained with comparison from NCBI adopts ClustalX1.83 to carry out Multiple Sequence Alignment, the 16S rDNA nucleotide sequence of display bacterial strain DBM12 and the 16S rDNA nucleotide sequence homology of Bacillus licheniformis (EU162839) the highest, maximum comparability (maxident) reaches 99.2%.The morphological feature of comprehensive DBM12 bacterial strain, physiological and biochemical property and 16S rDNA nucleotide sequence homology analytical results, be accredited as Bacillus licheniformis Bacillus licheniformis by bacterial strain DBM12.
Embodiment 2 is applied Bacillus licheniformis DBM12 fermentative production feruloyl esterase and is prepared forulic acid
Ferment screening the Bacillus licheniformis DBM12 bacterial strain obtained, fermentative medium formula is: peptone 2g, the bran powder 2g of glucose 0.5g, excessively 60 mesh sieves, excessively 60 mesh sieve husk powder 1g, yeast extract 0.5g, NaCl 0.4g, KH
2pO
40.1g, MgSO
40.05g, distilled water 100ml, pH7.0-7.5.Fermentation condition is: fermentation period 2-3d, leavening temperature 35-40 DEG C, inoculum size 4-10%, shaking speed 160-200r/min.
After fermentation ends, centrifugal for fermented liquid 8000r/min 20min is removed thalline, and filters remaining thalline and insoluble substance with 0.22 μm, collect filtrate.According to ammonium sulfate saturation ratio 75%, to to add ammonium sulfate in top fermentation filtrate, after fully dissolving, 4 DEG C of hold over night, the centrifugal 20min of 8000r/min, collecting precipitation, dissolves with 20mmol/L, pH6.0 phosphate buffer solution, the desalination of loading Sephedex G25 molecular sieve gel post, chromatography column is to carry out wash-out with identical buffered soln after the phosphoric acid buffer of pH6.5 balance, and elution rate is 15 ~ 20ml/h, obtains crude enzyme liquid.Its rate of recovery of recovery method of the present invention reaches more than 95%, by the thick enzyme cryopreservation obtained.
In 500mL triangular flask, add destarching bran powder 10g, add the feruloyl esterase 5mL that Bacillus licheniformis DBM12 fermentation of the present invention obtains, water 150mL, regulate pH6.5, temperature is 50 DEG C, and shaking table shakes frequency 120r/min, enzymolysis time 12h.Boiling water goes out enzyme, and centrifuging and taking supernatant measures forulic acid concentration.Forulic acid burst size is up to 8.24mg/g wheat bran with this understanding, and forulic acid release rate reaches more than 80%.Utilize strain fermentation of the present invention to produce ferulic acid ester production forulic acid efficiency high, transform fast, pollution-free, be applicable to industrial production forulic acid.
The enzyme of embodiment 3 feruloyl esterase is lived and enzymatic property measures
In the present embodiment with embodiment 2 gained fermented liquid for sample measures.
1. the ferulic acid ester enzyme activity determination of fermented liquid
In 2mL centrifuge tube, add 0.5mL enzyme liquid, then add 0.5mL Ferulic acid methylester solution, fully mix, 50 DEG C of insulation 20min, then adding 1mL volume fraction is that the glacial acetic acid of 10% is with termination reaction.Sample, in the centrifugal 10min of 12000r/min, retains supernatant liquor, suitably after dilution, with the ferulaic acid content in high performance liquid chromatography (HPLC) method working sample.Blank sample is the enzyme liquid boiling deactivation, and treatment process is the same.The enzyme of feruloyl esterase is lived and is defined: at 50 DEG C, pH value is under the condition of 6.5, per minute esterlysis Ferulic acid methylester, generates the enzyme amount needed for 1 μm of ol forulic acid.Forulic acid adopts high effective liquid chromatography for measuring.
2. the feruloyl esterase enzymatic property of fermented liquid
1) Optimun pH
The pH scope selected and system following (50mmol/L): (citric acid solution 2.0 ~ 4.5; Hac buffer 4 ~ 5.5; Phosphate buffer solution 6 ~ 8; Tris-HCl buffered soln 7 ~ 9; Glycine buffer 8 ~ 12).By comparing enzyme activity difference under buffer system, draw the optimal pH of enzyme reaction.With most high enzymatic activity for 100%, other enzyme activity is enzyme activity with the ratio of most high enzymatic activity, and take shelf time as X-coordinate, enzyme activity is ordinate zou mapping, the results are shown in Figure 2.The optimum pH of this enzyme is pH5.0-8.0, and when pH is between 4.0 ~ 9.0, this enzyme all has higher relative enzyme to live.Therefore feruloyl esterase soda acid adaptability is better, within the scope of wider pH, can play high catalysis activity.
2) pH tolerance
Damping fluid (the citric acid solution 2.0 ~ 4.5 of enzyme liquid and different pH value; Hac buffer 4 ~ 5.5; Phosphate buffer solution 6 ~ 8; Tris-HCl buffered soln 7 ~ 9; Glycine buffer 8 ~ 12) after 37 DEG C of insulation 12h, then diluted with damping fluid, make pH value all reach pH6.5, measure enzyme activity.With most high enzymatic activity for 100%, other enzyme activity is enzyme activity with the ratio of most high enzymatic activity, and take shelf time as X-coordinate, enzyme activity is ordinate zou mapping, the results are shown in Figure 2.Result shows that enzyme all has stronger stability within the scope of detected pH, and relative enzyme is lived all more than 90%.Illustrate that this enzyme has good ph stability in wider pH scope.
3) optimum temperature
After suitably being diluted by the phosphoric acid buffer of enzyme liquid pH6.5, at 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C and 80 DEG C, measure feruloyl esterase enzyme activity respectively.With most high enzymatic activity for 100%, other enzyme activity is enzyme activity with the ratio of most high enzymatic activity, with enzyme activity determination temperature for X-coordinate, enzyme activity is ordinate zou mapping, and result as shown in Figure 3, the optimal reactive temperature of enzyme is 50 DEG C, between 35 ~ 65 DEG C, all have higher vigor, residual enzyme vigor, more than 85%, declines significantly higher than 65 DEG C of enzyme activities, 80 DEG C time, residual enzyme vigor is 40%.Therefore enzyme temperature when catalyzed reaction be set between 35 ~ 65 DEG C comparatively suitable.
4) temperature tolerance
After suitably being diluted by the phosphoric acid buffer of enzyme liquid pH6.5, in 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C and 80 DEG C, be incubated different time respectively, then at 50 DEG C, measure its remaining enzyme activity.As seen from Figure 4, this enzyme has stronger thermostability, 40 DEG C ~ 70 DEG C insulation 100min, and enzyme activity does not almost lose, and 80 DEG C are incubated the enzyme activity that 60min still retains 80%.With most high enzymatic activity for 100%, other enzyme activity is enzyme activity with the ratio of most high enzymatic activity, and take shelf time as X-coordinate, enzyme activity is ordinate zou mapping, and result as shown in Figure 3.This feruloyl esterase is better at 40 ~ 70 DEG C of scope internal stabilities, 70 DEG C, 60min time enzyme activity influenced hardly, therefore this enzyme belongs to heat-resisting feruloyl esterase.This enzyme is placed and namely within 10 days, still can be retained higher enzyme activity in 240 hours under pH4.0,30 DEG C of conditions.
Sequence table
<110> Xuzhou Engineering Institute
<120> mono-kind produces Bacillus licheniformis and the application thereof of feruloyl esterase
<130> 001
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1505
<212> DNA
<213> Bacillus licheniformis (Baclicus lincheniformis)
<400> 1
gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggaccgacgg gagcttgctc 60
ccttaggtca gcggcggacg agtgagtaac acgtgggtaa cctgcctgta agactgggat 120
aactccagga aaccggggct aataccggat gcttgattga acagcatggt tcaatcataa 180
aaggtggctt tcagctacca cttacagatg gacccgcggc gcattagcta gttggtgagg 240
taacggctca ctaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg 300
actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg 360
aaagtctgac ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aaactctgtt 420
gttagagaag aacaagtacc gttcgaatag ggcggtacct tgacggtacc taaccagaaa 480
gccacggcta actacgtgcc agcagccgcg gtaatacgta gatggcaagc gttgtccgga 540
attattgggc gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct 600
caaccgggga gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc 660
cacgtgtagc ggcgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc 720
tggtctgtaa ctgacgctga ggcgcgaaag cgtggggagc gaacaggatc agataccctg 780
gtagtccacg ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg 840
cagcaaacgc attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga 900
attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960
ccttaccagg tcttgacatc ctcttacaac cctagagata gggcttcccc ttcgggggca 1020
gagtgacagg tggtgactgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080
gcaacgagcg caacccttga tcttagttgc cagcattcag ttgggcactc taaggagact 1140
gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1200
gggctacaca cgtgctacaa tgggcavaac aaagggcagc gaagccgcga ggctaagcca 1260
atcccacaaa tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga 1320
atcgctcgta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac 1380
cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttggagcc 1440
agccgccgaa ggtgggacag atgattgggg tgaagtcgta acaaggtagc cgtatcggaa 1500
ggtgc 1505
<210> 2
<211> 20
<212> DNA
<213> forward primer
<400> 2
agagtttgatcctggctcag 20
<210> 3
<211> 20
<212> DNA
<213> reverse primer
<400> 3
aaggaggtgatccagccgca 20
Claims (5)
1. the Bacillus licheniformis producing feruloyl esterase (
bacillus licheniformis) DBM12, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number on January 2nd, 2014: CGMCC No.8672.
2. product feruloyl esterase according to claim 1 Bacillus licheniformis (
bacillus licheniformis) DBM12 produces feruloyl esterase the application prepared in forulic acid by fermentation.
3. product feruloyl esterase according to claim 2 Bacillus licheniformis (
bacillus licheniformis) DBM12 produces feruloyl esterase the application prepared in forulic acid by fermentation, it is characterized in that: comprise the following steps:
Step 1, ferments Bacillus licheniformis DBM12 inoculation to fermention medium, fermentation period 2-3d, leavening temperature 35-40 DEG C, inoculum size 4-10%, shaking speed 160-200r/min;
Step 2, by the centrifugal 20min of fermented liquid 8000r/min of step 1, supernatant 0.22 μm of filtration, collects filtrate;
Step 3, be 75% by ammonium sulfate saturation ratio, in step 2 gained filtrate, add ammonium sulfate, 4 DEG C of standing 12h, the centrifugal 20min of 8000r/min, collecting precipitation, dissolves with 20mmol/L, pH6.0 phosphate buffer solution, the desalination of loading Sephedex G25 molecular sieve gel post, carry out wash-out with the phosphoric acid buffer of pH6.5, elution rate is 15 ~ 20ml/h, obtains crude enzyme liquid;
Step 4, is added to step 3 gained crude enzyme liquid in bran powder, then adds water, and regulates pH6.5, shaking table shakes frequency 120r/min, 50 DEG C of enzymolysis 12h, and boiling water goes out enzyme, and centrifuging and taking supernatant, is forulic acid.
4. product feruloyl esterase according to claim 3 Bacillus licheniformis (
bacillus licheniformis) DBM12 produces feruloyl esterase the application prepared in forulic acid by fermentation, it is characterized in that: the formula of the fermention medium described in step 1 is: peptone 2g, glucose 0.5g, cross 60 mesh sieves bran powder 2g, cross 60 mesh sieve husk powder 1g, yeast extract 0.5g, NaCl 0.4g, KH
2pO
40.1g, MgSO
40.05g, distilled water 100mL, pH7.0-7.5.
5. product feruloyl esterase according to claim 3 Bacillus licheniformis (
baclicus lincheniformis) DBM12 produces feruloyl esterase the application prepared in forulic acid in fermentation, it is characterized in that: in step 4, crude enzyme liquid consumption is 5mL, bran powder consumption is 10g, and water consumption is 150mL.
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Cited By (10)
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| CN105154371A (en) * | 2015-09-30 | 2015-12-16 | 山东大学 | Lactobacillus amylovorus for producing feruloyl esterase and application of lactobacillus amylovorus |
| CN105695431A (en) * | 2016-03-17 | 2016-06-22 | 江南大学 | Esterase and application thereof |
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| CN106167795A (en) * | 2016-09-07 | 2016-11-30 | 江南大学 | A kind of esterase and application thereof |
| CN106167794A (en) * | 2016-09-07 | 2016-11-30 | 江南大学 | A kind of esterase and application thereof |
| CN108823131A (en) * | 2018-07-02 | 2018-11-16 | 中国科学院微生物研究所 | Lactobacillus fermentum for high yield of feruloyl esterase and application thereof |
| CN110372495A (en) * | 2019-08-28 | 2019-10-25 | 河南工业大学 | A kind of method of extraction purification ferulic acid from green wheat kernel wheat bran of simplicity |
| CN112143681A (en) * | 2020-09-28 | 2020-12-29 | 江南大学 | Bacillus belgii capable of producing feruloyl esterase and application thereof |
| CN113308424A (en) * | 2021-04-25 | 2021-08-27 | 天津科技大学 | Bacillus pumilus for producing feruloyl esterase and application thereof |
| WO2022207015A3 (en) * | 2022-05-09 | 2023-03-23 | 江苏省农业科学院 | Lactobacillus farciminis sr2 and use thereof |
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| CN105154371B (en) * | 2015-09-30 | 2018-02-23 | 山东大学 | One plant of Lactobacillus amylovorus for producing feruloyl esterase and its application |
| CN105154371A (en) * | 2015-09-30 | 2015-12-16 | 山东大学 | Lactobacillus amylovorus for producing feruloyl esterase and application of lactobacillus amylovorus |
| CN105695431A (en) * | 2016-03-17 | 2016-06-22 | 江南大学 | Esterase and application thereof |
| CN105695431B (en) * | 2016-03-17 | 2020-05-08 | 江南大学 | Esterase and application thereof |
| CN106167795B (en) * | 2016-09-07 | 2019-12-24 | 江南大学 | Esterase and application thereof |
| CN106167794A (en) * | 2016-09-07 | 2016-11-30 | 江南大学 | A kind of esterase and application thereof |
| CN106167794B (en) * | 2016-09-07 | 2019-11-26 | 江南大学 | A kind of esterase and its application |
| CN106167795A (en) * | 2016-09-07 | 2016-11-30 | 江南大学 | A kind of esterase and application thereof |
| CN106148301A (en) * | 2016-09-07 | 2016-11-23 | 江南大学 | A kind of esterase and application thereof |
| CN108823131A (en) * | 2018-07-02 | 2018-11-16 | 中国科学院微生物研究所 | Lactobacillus fermentum for high yield of feruloyl esterase and application thereof |
| CN110372495A (en) * | 2019-08-28 | 2019-10-25 | 河南工业大学 | A kind of method of extraction purification ferulic acid from green wheat kernel wheat bran of simplicity |
| CN112143681A (en) * | 2020-09-28 | 2020-12-29 | 江南大学 | Bacillus belgii capable of producing feruloyl esterase and application thereof |
| CN112143681B (en) * | 2020-09-28 | 2021-10-19 | 江南大学 | Bacillus belgii capable of producing feruloyl esterase and application thereof |
| CN113308424A (en) * | 2021-04-25 | 2021-08-27 | 天津科技大学 | Bacillus pumilus for producing feruloyl esterase and application thereof |
| WO2022207015A3 (en) * | 2022-05-09 | 2023-03-23 | 江苏省农业科学院 | Lactobacillus farciminis sr2 and use thereof |
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