CN104894017B - A kind of bacillus licheniformis for producing feruloyl esterase and its application - Google Patents
A kind of bacillus licheniformis for producing feruloyl esterase and its application Download PDFInfo
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Abstract
The invention discloses a kind of bacillus licheniformis for producing feruloyl esterase and its application, the bacillus licheniformis (Bacillus licheniformis) DBM12, deposit number：CGMCC No.8672.The bacillus licheniformis of the present invention to carbon source and nitrogen source require relatively low, fermentation time is short, bacterial strain is safe and non-toxic, can not only use in food and feed industry；Simple to operate and genetic stability is good, more than generation, the synthesis capability of feruloyl esterase is basically unchanged for continuous passage 10.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to it is a kind of produce feruloyl esterase bacillus licheniformis and
It is applied.
Background technology
Forulic acid (ferulic acid, FA) is a kind of natural being prevalent in plant, is plant more
A kind of phenolic acid in thing.With the uniqueness such as calmness, anticancer, anti-oxidant, antiatherosclerosis, platelet aggregation-against, reducing blood lipid
Function.But forulic acid largely exists in plant in the form of crosslinking, humans and animals can not directly absorb this kind of forulic acid,
Microorganism must be relied on to produce esterase by forulic acid separate out.On the other hand, in plant cell wall, forulic acid or dimer
Forulic acid is mainly connected on the arabinose residues of araboxylan in the form of ester bond, enhancing araboxylan chain
Intensity.But from spatially limiting the effective degraded of animal and microorganism to cellulose in plant cell wall and hemicellulose.
Feruloyl esterase (EC 3.1.1.73, feruloyl esterase) is also known as Ferulic acid esterase or cinnamoyl esterase,
Forulic acid can be hydrolyzed, the ester bond that dimerization forulic acid is formed, forulic acid is discharged.It can be with other hydrolases such as xylan
Enzyme, cellulase and the mutual synergy of lignoenzyme, the more polysaccharide and lignin thoroughly in degradation of cell wall.Therefore,
Feruloyl esterase suffers from huge in numerous areas such as food, medicine, papermaking, feed manufacturing, biosynthesis and energy developments
Application potential.
Since feruloyl esterase is found in streptomycete first, nearly 30 kinds of feruloyl esterases of separated purifying so far, but
Mould and aspergillus are concentrated mainly on, only minority comes from bacterium, such as lactobacillus acidophilus, Bacillus subtillis etc., and
The enzymatic property and amino acid sequence of their feruloyl esterase are all different.Due to native fungal growth, production of enzyme with
And a variety of limitations of the fungi to growth conditions all govern the industrialized production that enzyme process prepares forulic acid.Therefore seek using bacterium as hair
Yeast-like fungi strain production feruloyl esterase is significant.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art and provides a kind of bacillus licheniformis for producing feruloyl esterase
And its application, the bacterial strain to carbon source and nitrogen source require relatively low, fermentation time is short, bacterial strain is safe and non-toxic, can not only raise in food
Used in material industry；Simple to operate and genetic stability is good, continuous passage 10 is more than generation, the synthesis capability of feruloyl esterase
It is basically unchanged.
A kind of bacillus licheniformis (Bacillus licheniformis) DBM12 for producing feruloyl esterase, in 2014
On January 2, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, and (address is：BeiJing, China
The institute 3 of city Chaoyang District North Star West Road 1, postcode 100101), deposit number：CGMCC No.8672.
Bacillus licheniformis DBM12 of the present invention screening technique, comprises the following steps：
Step 1, soil sample collection and processing
Soil sample picks up from the animal waste compost that Xuzhou City of Jiangsu Province Yunlong District rural area is in hot fermentation.The soil of collection
Sample is put in the aseptic plastic bag of sealing in room temperature preservation, and carries out the separation of purpose bacterial strain within 1 week.
Take 5-10g soil samples to be put into equipped with 20-40mL sterilized waters and be placed with the triangular flask of small bead, be placed on shaking table,
120-160r/min rotation concussion 20-30min, triangular flask is then placed in 70-80 DEG C of heating water bath 15-30min, during which every
Shaken once in 5min.
Step 2, enrichment culture
The soil sample after 1-2mL heat treatments is taken in the triangular flask equipped with 30-50mL enriched mediums one, in 160-180r/
Concussion and cultivate 20-24h at 50-60 DEG C of min rotating speeds.Then 1-2mL nutrient solutions are taken to be inoculated into the richness equipped with 50mL concentration 5-10%
Collect in culture medium two, continue the concussion and cultivate 18-20h at 50-60 DEG C of 160-180r/min rotating speeds.
Enriched medium one：Yeast extract 0.5g, KH2PO40.1g, NH4NO30.1g, NaCl 0.1g, MgSO4·
7H2O 0.25g, micro- 1mL, ferulic acid ethyl ester 1g, deionized water 100mL, adjust pH4.0.
Enriched medium two：NH4NO30.5g, urea 0.1g, KH2PO40.1g, NaCl 0.1g, ferulic acid ethyl ester 1g, go
Ionized water 100mL, adjust pH3.0.
Trace element：EDTA 5g, CaCl2·2H2O 6g, FeSO4·7H2O 6g, MnCl2·4H2O 1.15g,
CoCl2·6H2O 0.8g, ZnSO4·7H2O 0.7g, CuCl2·2H2O 0.3g, H3BO30.3g, (NH4)6Mo7O2·4H2O
0.25g, deionized water 1000mL.
Step 3, primary dcreening operation
The nutrient solution sterilized water of second of enrichment is diluted, takes be diluted to 10 respectively-2、10-3、10-4、10-5、10-6It is dilute
Release liquid 150-250 μ L to be coated on primary dcreening operation culture medium one, after the bacterium solution of media surface is completely dry, culture dish is sealed with sealed membrane
Mouthful, 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 continued
Cultivate 6-12h.
The single bacterium drop point of picking different shape is connected on primary dcreening operation culture medium two, 45-60 DEG C of incubated 24-48h.Pick out
Preferable and transparent loop diameter and the larger bacterial strain of colony diameter ratio are grown on primary dcreening operation culture medium two, streak inoculation is in purifying
Culture medium, 1-2d is cultivated in 45-50 DEG C.Then picking single bacterium colony therein is rule separation again, is repeated 3 times.As continuously
Repeatedly observe that the single thalline of form then thinks that bacterial strain has purified under the microscope.Finally by the inoculation of purifying in inclined-plane
4 DEG C preserve in storage medium, then further secondary screening.
Primary dcreening operation culture medium one：Peptone 0.5g, yeast extract 0.1g, NaNO30.3g, K2HPO40.1g, KCl
0.5g, MgSO4·7H2O 0.025g, FeSO4·7H2O 0.001g, agar 2.5, deionized water 100mL, adjust pH4.5.
Primary dcreening operation culture medium two：KH2PO40.2g, (NH4)2SO40.4g, MgSO4·7H2O 0.03g, FeSO4·7H2O
0.001g, MnSO40.001g, ZnCl20.001g, agar 2.5g, deionized water 100mL, pH4.0.121 DEG C of culture medium,
After 15min sterilizings, when being cooled to 60-70 DEG C, ferulic acid ethyl ester solution 1mL is added, it is average down to 3 diameters after fully shaking up
In 90mm culture dish.
Ferulic acid ethyl ester solution：0.2g ferulic acid ethyl esters are added in 1.5mL sterile centrifugation tubes, add 500 μ LN, N- bis-
NMF dissolves, and then adds 500 μ L sterilized waters, fully shakes up.
Step 4, secondary screening
The bacterial strain streak inoculation that primary dcreening operation is obtained is into activating solid culture medium, 40-45 DEG C of constant incubator culture 16-
20h.The bacterial strain activated is inoculated in the triangular flask equipped with 30-50mL seed culture fluids, using cultivation temperature as 40-45
DEG C, shake incubated 16-20h under shaking speed 160-200r/min frequencies.Secondary screening is inoculated into according to 4-10% inoculum concentration
In fermentation medium, shaking table concussion and cultivate.Fermentation condition is：250mL triangular flasks, liquid amount 30-60mL, shaking table concussion frequency
For 160-200r/min, 40-50 DEG C of fermentation temperature, fermentation time 24-48h.After fermentation ends, zymotic fluid is taken with 6000-
1000r/min rotating speeds centrifuge 5-10min, take supernatant to detect enzymatic activity.
Secondary screening seed culture medium：Peptone 1g, beef extract 0.5g, dusty yeast 0.5g, NaCl 0.5g, K2HPO4 0.02g、
Adding deionized water, sterilize 15min to 100mL, pH4.5,121 DEG C.
Fermentative medium formula：Soluble starch 0.5g, peptone 1g, beef extract 0.5g, NaCl 0.5g, KH2PO4
0.1g、MgSO4·7H2O 0.04g, deionized water 100mL, pH4.5,121 DEG C, sterilize 15min.
Described bacillus licheniformis (Baclicus lincheniformis) DBM12 produces feruloyl esterase simultaneously in fermentation
The application in forulic acid is prepared, is comprised the following steps：
Step 1, bacillus licheniformis DBM12 bacterial strains are fermented, fermentation period 2-3d, 35-40 DEG C of fermentation temperature, connect
Kind amount 4-10%, shaking speed 160-200r/min；
Step 2, the zymotic fluid 8000r/min of step 1 is centrifuged into 20min, 0.22 μm of filtering of supernatant, collects filtrate；
Step 3, it is 75% by ammonium sulfate saturation degree, ammonium sulfate is added into step 2 gained filtrate, 4 DEG C stand 12h,
8000r/min centrifuges 20min, collects precipitation, with 20mmol/L, the dissolving of pH6.0 phosphate buffer solutions, loading Sephedex G25
Molecular sieve gel post desalination, is eluted with pH6.5 phosphate buffer, and elution rate is 15~20ml/h, obtains crude enzyme liquid；
Step 4, step 3 gained crude enzyme liquid is added in wheatfeed, added water, adjusted pH6.5, shake frequency on shaking table
120r/min, 50 DEG C of enzymolysis 12h, boiling water enzyme deactivation, as centrifuging and taking supernatant, forulic acid.
As the further improvement of foregoing invention, the formula of the fermentation medium described in step 1 is：Peptone 2g, grape
Sugared 0.5g, cross 60 mesh sieves wheatfeed 2g, cross 60 mesh sieve husk powder 1g, yeast extract 0.5g, NaCl 0.4g,
KH2PO40.1g、MgSO40.05g, distilled water 100mL, pH7.0-7.5.
As the further improvement of foregoing invention, crude enzyme liquid dosage is 5mL in step 4, and wheatfeed dosage is 10g, and water is used
Measure as 150mL.
Bacillus licheniformis (Baclicus lincheniformis) DBM12 of the present invention not only will to carbon source and nitrogen source
Ask relatively low, fermentation time is short, bacterial strain is safe and non-toxic, can be used in food and feed industry；And genetic stability is good, operation
Simply, more than generation, the synthesis capability of feruloyl esterase is basically unchanged for continuous passage 10；It is heat-resisting resistance to that the bacterial strain produces feruloyl esterase
Acid, there is important application in feed manufacturing.
Brief description of the drawings
Fig. 1 is the secondary screening lithograph in embodiment 1；
Fig. 2 is the most suitable action pH and ph stability curve map of feruloyl esterase in embodiment 3；
Fig. 3 is feruloyl esterase optimum temperature curve map in embodiment 3；
Fig. 4 is the heat endurance curve map of feruloyl esterase in embodiment 3.
Embodiment
Embodiment 1
Bacillus licheniformis (Baclicus lincheniformis) DBM12 screening technique, comprises the following steps：
Step 1, soil sample collection and processing
Soil sample picks up from the animal waste compost that Xuzhou City of Jiangsu Province Yunlong District rural area is in hot fermentation.The soil of collection
Sample is put in the aseptic plastic bag of sealing in room temperature preservation, and carries out the separation of purpose bacterial strain within 1 week.
Take 5-10g soil samples to be put into equipped with 20-40mL sterilized waters and be placed with the triangular flask of small bead, be placed on shaking table,
120-160r/min rotation concussion 20-30min, triangular flask is then placed in 70-80 DEG C of heating water bath 15-30min, during which every
Shaken once in 5min.
Step 2, enrichment culture
The soil sample after 1-2mL heat treatments is taken in the triangular flask equipped with 30-50mL enriched mediums one, in 160-180r/
Concussion and cultivate 20-24h at 50-60 DEG C of min rotating speeds.Then 1-2mL nutrient solutions are taken to be inoculated into the richness equipped with 50mL concentration 5-10%
Collect in culture medium two, continue the concussion and cultivate 18-20h at 50-60 DEG C of 160-180r/min rotating speeds.
Enriched medium one：Yeast extract 0.5g, KH2PO40.1g, NH4NO30.1g, NaCl 0.1g, MgSO4·
7H2O 0.25g, micro- 1mL, ferulic acid ethyl ester 1g, deionized water 100mL, adjust pH4.0.
Enriched medium two：NH4NO30.5g, urea 0.1g, KH2PO40.1g, NaCl 0.1g, ferulic acid ethyl ester 1g, go
Ionized water 100mL, adjust pH3.0.
Trace element：EDTA 5g, CaCl2·2H2O 6g, FeSO4·7H2O 6g, MnCl2·4H2O 1.15g,
CoCl2·6H2O 0.8g, ZnSO4·7H2O 0.7g, CuCl2·2H2O 0.3g, H3BO30.3g, (NH4)6Mo7O2·4H2O
0.25g, deionized water 1000mL.
Step 3, primary dcreening operation
The nutrient solution sterilized water of second of enrichment is diluted, takes be diluted to 10 respectively-2、10-3、10-4、10-5、10-6It is dilute
Release liquid 150-250 μ L to be coated on primary dcreening operation culture medium one, after the bacterium solution of media surface is completely dry, culture dish is sealed with sealed membrane
Mouthful, 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 continued
Cultivate 6-12h.
The single bacterium drop point of picking different shape is connected on primary dcreening operation culture medium two, 45-60 DEG C of incubated 24-48h.Pick out
Preferable and transparent loop diameter and the larger bacterial strain of colony diameter ratio are grown on primary dcreening operation culture medium two, streak inoculation is in purifying
Culture medium, 1-2d is cultivated in 45-50 DEG C.Then picking single bacterium colony therein is rule separation again, is repeated 3 times.As continuously
Repeatedly observe that the single thalline of form then thinks that bacterial strain has purified under the microscope.Finally by the inoculation of purifying in inclined-plane
4 DEG C preserve in storage medium, then further secondary screening.Primary dcreening operation result is as shown in table 1.
Primary dcreening operation culture medium one：Peptone 0.5g, yeast extract 0.1g, NaNO30.3g, K2HPO40.1g, KCl
0.5g, MgSO4·7H2O 0.025g, FeSO4·7H2O 0.001g, agar 2.5, deionized water 100mL, adjust pH4.5.
Primary dcreening operation culture medium two：KH2PO40.2g, (NH4)2SO40.4g, MgSO4·7H2O 0.03g, FeSO4·7H2O
0.001g, MnSO40.001g, ZnCl20.001g, agar 2.5g, deionized water 100mL, pH4.0.121 DEG C of culture medium,
After 15min sterilizings, when being cooled to 60-70 DEG C, ferulic acid ethyl ester solution 1mL is added, it is average down to 3 diameters after fully shaking up
In 90mm culture dish.
Ferulic acid ethyl ester solution：0.2g ferulic acid ethyl esters are added in 1.5mL sterile centrifugation tubes, add 500 μ LN, N- bis-
NMF dissolves, and then adds 500 μ L sterilized waters, fully shakes up.
Step 4, secondary screening
The bacterial strain streak inoculation that primary dcreening operation is obtained is into activating solid culture medium, 40-45 DEG C of constant incubator culture 16-
20h.The bacterial strain activated is inoculated in the triangular flask equipped with 30-50mL seed culture fluids, using cultivation temperature as 40-45
DEG C, shake incubated 16-20h under shaking speed 160-200r/min frequencies.Secondary screening is inoculated into according to 4-10% inoculum concentration
In fermentation medium, shaking table concussion and cultivate.Fermentation condition is：250mL triangular flasks, liquid amount 30-60mL, shaking table concussion frequency
For 160-200r/min, 40-50 DEG C of fermentation temperature, fermentation time 24-48h.After fermentation ends, zymotic fluid is taken with 6000-
1000r/min rotating speeds centrifuge 5-10min, take supernatant to detect enzymatic activity.
Secondary screening seed culture medium：Peptone 1g, beef extract 0.5g, dusty yeast 0.5g, NaCl 0.5g, K2HPO4 0.02g、
Add deionized water to 100mL, pH4.5,121 DEG C of sterilizing 15min.
Fermentative medium formula：Soluble starch 0.5g, peptone 1g, beef extract 0.5g, NaCl 0.5g, KH2PO4
0.1g、MgSO4·7H2100mL, pH4.5,121 DEG C of O 0.04g, deionized water sterilizing 15min.
Enzyme activity flat board detection method：0.02mol/L disodium hydrogen phosphate-citric acid solution (pH4.5) 100mL, fine jade
Cosmetics 2.5g, 121 DEG C, after 15min sterilizings, when being cooled to 60-70 DEG C, add ferulic acid ethyl ester solution 1mL, after fully shaking up,
It is averaged down in 3 diameter 90mm culture dish.Agar uses diameter 6mm card punch uniformly to punch about 3-10 after fully solidifying
It is individual.The μ L of supernatant 100 are taken to be added in hole after zymotic fluid is centrifuged, 45 DEG C overnight, are checked transparent circle situation.As a result it is as shown in Figure 1.
Step 5, culture presevation
The strain by screening obtained is rule on beef extract-peptone, single bacterium is separated to and falls behind in LB slant mediums
Upper line, 40 DEG C are cultivated 1-2 days, are placed in 4 DEG C of refrigerators and are preserved.
LB inclined-plane culture based formulas is：Peptone 1g, NaCl 0.5g, dusty yeast 0.5g, agar 1.5g, pH value 5.0,121
DEG C sterilizing 15min.
The primary dcreening operation plate screening result of table 1
| Bacterium | Colony diameter D/cm | Transparent loop 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 cultivate 24h on beef extract-peptone solid medium, and bacterium colony is circular, diameter 2.4-3.2mm；Bacterium colony
Opaque for white, surface is relatively flat and dries, firm attachment culture medium, it is difficult to picking.In beef extract-peptone fluid nutrient medium
Middle static gas wave refrigerator, no precipitation, liquid is limpid, and media surface forms white elephant skin film, and there is a particle on surface, and film is relatively thin.Observe bacterium
Volume morphing, Gram-positive, growth course produce gemma, and sporangiocyst is without expanding, and no parasporal crystal, thalline is shaft-like, have fortune
Dynamic property.The specific morphology of bacterial strain DBM12 and physiological and biochemical property see the table below 2.Bacterium colony, dyeing characteristic and physiological and biochemical property with
《The outstanding bacterium mirror handbook of uncle》Relatively, it is lichens gemma to primarily determine that bacterial strain DBM12 to bacillus licheniformis in (the 8th edition)
Bacillus (Bacillus licheniformis).
The bacterial strain DBM12 morphology of table 2 and physio-biochemical characteristics
| Characteristic | As a result | Characteristic | As a result |
| Anaerobic growth | + | Gelatin hydrolysate | + |
| Sporangium | + | Hydrolysis starch | + |
| Catalase test | + | Urea utilizes | - |
| V.P is tested | + | Nitrate utilizes | + |
| Lecithin is tested | - | Indole test | - |
| Methyl red test | + | PEARLITOL 25C | + |
| Catalase | + | D- xyloses | + |
| Citrate utilizes | + | L-arabinose | + |
| 2%NaCl | + | D-Glucose | + |
| 5%NaCl | + | Ferment hydrolyzes | + |
| 7%NaCl | + | 10 DEG C of growth temperature | + |
| pH5.7 | + | 50 DEG C of growth temperature | + |
The application bacterial strain DBM12 genomes are extracted, according to primers most conservative in bacterial 16 S rDNA, wherein
Forward primer is F：5'-AGAGTTTGATCCTGGCTCAG-3'(is as shown in SEQ ID NO.2)；Reverse primer is R：5'-
AAGGAGGTGATCCAGCCGCA-3'(is as shown in SEQ ID NO.3).PCR reaction systems are (20 μ L):ddH2The μ L of O 12.7,
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, reversely draw
Thing (10 μm of ol/L) 1.0 μ L, Taq FlexiPolymerase (5U/ μ L) 0.3 μ L, the μ L of template 1.0.94 DEG C of thermal circulation parameters are pre-
5min is denatured, 94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 2min, is circulated 30 times, 72 DEG C of extension 10min.Use pGM-T
Carrier cloning is sequenced.Sequence is measured as shown in SEQ ID NO.1, the 16S rDNA with comparing the close bacterium obtained from NCBI
Sequence carries out Multiple Sequence Alignment using ClustalX1.83, display bacterial strain DBM12 16S rDNA nucleotide sequences with
Bacillus licheniformis (EU162839) 16S rDNA nucleotide sequence homology highests, maximum comparability
(maxident) 99.2% is reached.Morphological feature, physiological and biochemical property and the 16S rDNA nucleotides sequences of comprehensive DBM12 bacterial strains
Row homology analysis result, bacterial strain DBM12 is accredited as bacillus licheniformis Bacillus licheniformis.
The application bacillus licheniformis DBM12 fermenting and producings feruloyl esterase of embodiment 2 simultaneously prepares forulic acid
The bacillus licheniformis DBM12 bacterial strains that screening obtains are fermented, fermentative medium formula is：Peptone 2g,
Glucose 0.5g, the wheatfeed 2g for crossing 60 mesh sieves, cross 60 mesh sieve husk powder 1g, yeast extract 0.5g, NaCl 0.4g, KH2PO4
0.1g、MgSO40.05g, distilled water 100ml, pH7.0-7.5.Fermentation condition is：Fermentation period 2-3d, fermentation temperature 35-40
DEG C, inoculum concentration 4-10%, shaking speed 160-200r/min.
After fermentation ends, zymotic fluid 8000r/min centrifugations 20min is removed into thalline, and remaining bacterium is filtered out with 0.22 μm
Body and insoluble matter, collect filter liquor.According to ammonium sulfate saturation degree 75%, ammonium sulfate is added in top fermentation filter liquor, is filled
After dividing dissolving, 4 DEG C stand overnight, and 8000r/min centrifugation 20min, precipitation are collected, with 20mmol/L, pH6.0 phosphate buffer solutions
Dissolving, loading Sephedex G25 molecular sieve gel post desalinations, chromatographic column are delayed after being balanced with pH6.5 phosphate buffer with identical
Rush solution to be eluted, 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 Cord blood of acquisition.
Desizing wheatfeed 10g is added in 500mL triangular flasks, adds bacillus licheniformis DBM12 fermentations system of the present invention
Feruloyl esterase 5mL, water 150mL, adjust pH6.5, temperature is 50 DEG C, frequency 120r/min is shaken on shaking table, during enzymolysis
Between 12h.Boiling water enzyme deactivation, centrifuging and taking supernatant measure asafoetide acid concentration.Forulic acid burst size is up to 8.24mg/g on this condition
Wheat bran, forulic acid release rate reach more than 80%.Ferulic acid ester production forulic acid efficiency high is produced using strain fermentation of the present invention,
Conversion is fast, pollution-free, suitable for industrial production forulic acid.
Enzyme activity and the enzymatic property measure of the feruloyl esterase of embodiment 3
It is measured in the present embodiment using the gained zymotic fluid of embodiment 2 as sample.
1. the ferulic acid ester enzyme activity determination of zymotic fluid
0.5mL enzyme liquids are added into 2mL centrifuge tubes, add 0.5mL Ferulic acid methylester solution, are fully mixed, 50 DEG C of guarantors
Warm 20min, the glacial acetic acid that then addition 1mL volume fractions are 10% is with terminating reaction.Sample centrifuges in 12000r/min
10min, retain supernatant, after appropriate dilution, with the ferulaic acid content in high performance liquid chromatography (HPLC) method determination sample.Blank
Sample is the enzyme liquid for boiling inactivation, and processing method is same as above.The enzyme activity definition of feruloyl esterase：At 50 DEG C, pH value is 6.5 condition
Under, esterlysis Ferulic acid methylester per minute, generate the enzyme amount needed for 1 μm of ol forulic acid.Forulic acid is surveyed using high performance liquid chromatography
It is fixed.
2. the feruloyl esterase enzymatic property of zymotic fluid
1) Optimun pH
The pH scopes and system of selection are following (50mmol/L)：(citric acid solution 2.0~4.5；Hac buffer
4~5.5；Phosphate buffer solution 6~8；Tris-HCl cushioning liquid 7~9；Glycine buffer 8~12).By relatively more slow
Enzyme activity difference under system is rushed, draws the optimal pH of enzyme reaction.Using highest enzyme activity as 100%, other enzyme activities and highest enzyme
The ratio of vigor is enzyme activity, and using the holding time as abscissa, enzyme activity is mapped for ordinate, as a result sees Fig. 2.Should
The optimum pH of enzyme is pH5.0-8.0, and when pH is between 4.0~9.0, the enzyme has higher relative enzyme activity.Therefore asafoetide
Acid esters enzyme soda acid adaptability is preferable, in the range of wider pH, can play high catalysis activity.
2) pH tolerances
Buffer solution (the citric acid solution 2.0~4.5 of enzyme liquid and different pH value；Hac buffer 4~5.5；Phosphoric acid
Cushioning liquid 6~8；Tris-HCl cushioning liquid 7~9；Glycine buffer 8~12) in 37 DEG C be incubated 12h after, then with delay
Fliud flushing is diluted, and pH value is reached pH6.5, determines enzyme activity.Using highest enzyme activity as 100%, other enzyme activities with most
The ratio of high enzymatic activity is enzyme activity, and using the holding time as abscissa, enzyme activity is mapped for ordinate, as a result sees figure
2.As a result show that enzyme is respectively provided with stronger stability in the range of the pH detected, with respect to enzyme activity more than 90%.Illustrate this
Enzyme has good ph stability in wider pH scopes.
3) optimum temperature
After enzyme liquid is suitably diluted with pH6.5 phosphate buffer, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C,
Feruloyl esterase enzyme activity is determined respectively at 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C and 80 DEG C.Using highest enzyme activity as
100%, the ratio of other enzyme activities and highest enzyme activity is enzyme activity, using enzyme activity determination temperature as abscissa, with respect to enzyme
Vigor be ordinate mapping, as a result as shown in figure 3, the optimal reactive temperature of enzyme be 50 DEG C, be respectively provided between 35~65 DEG C compared with
High vigor, remaining enzyme activity is more than 85%, and higher than 65 DEG C enzyme activities decline significantly, and at 80 DEG C, remaining enzyme activity is
40%.Therefore enzyme temperature in catalytic reaction be set in it is more suitable between 35~65 DEG C.
4) temperature tolerance
After enzyme liquid is suitably diluted with pH6.5 phosphate buffer, respectively in 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C and 80 DEG C
Different time is incubated, then its remaining enzyme activity is determined at 50 DEG C.From fig. 4, it can be seen that the enzyme has a stronger heat endurance, 40 DEG C
~70 DEG C of insulation 100min, enzyme activity almost do not lose, and 80 DEG C of insulation 60min still retain 80% enzyme activity.With highest enzyme
Vigor is 100%, and the ratio of other enzyme activities and highest enzyme activity is enzyme activity, using the holding time as abscissa, relatively
Enzyme activity is mapped for ordinate, as a result as shown in Figure 3.The feruloyl esterase is preferable in 40~70 DEG C of scope internal stabilities, 70
DEG C, 60min when enzyme activity be barely affected, therefore the enzyme belongs to heat-resisting feruloyl esterase.The enzyme is in pH4.0,30 DEG C of conditions
Lower place i.e. 10 days 240 hours can still retain higher enzyme activity.
Sequence table
<110>Xuzhou Engineering Institute
<120>A kind of bacillus licheniformis for producing feruloyl esterase and its application
<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 (3)
1. it is a kind of produce feruloyl esterase bacillus licheniformis (Bacillus licheniformis) DBM12, in 2014 1
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number on 2nd：CGMCC
No.8672；
It is described production feruloyl esterase bacillus licheniformis (Bacillus licheniformis) DBM12 can pass through ferment production
Feruloyl esterase simultaneously prepares forulic acid, specifically includes following steps：
Step 1, bacillus licheniformis DBM12 inoculations are fermented to fermentation medium, fermentation period 2-3d, fermentation temperature
35-40 DEG C, inoculum concentration 4-10%, shaking speed 160-200r/min of degree；
Step 2, the zymotic fluid 8000r/min of step 1 is centrifuged into 20min, 0.22 μm of filtering of supernatant, collects filtrate；
Step 3, it is 75% by ammonium sulfate saturation degree, ammonium sulfate is added into step 2 gained filtrate, 4 DEG C stands 12h, 8000r/
Min centrifuges 20min, collects precipitation, with 20mmol/L, the dissolving of pH6.0 phosphate buffer solutions, loading Sephedex G25 molecular sieves
Gel column desalination, eluted with pH6.5 phosphate buffer, elution rate 15-20mL/h, obtain crude enzyme liquid；
Step 4, step 3 gained crude enzyme liquid is added in wheatfeed, added water, adjusted pH6.5, shake frequency 120r/ on shaking table
Min, 50 DEG C of enzymolysis 12h, boiling water enzyme deactivation, as centrifuging and taking supernatant, forulic acid；
The thick enzyme that the crude enzyme liquid reclaims to obtain enzyme activity after 40 DEG C of -70 DEG C of insulation 100min does not change.
2. it is according to claim 1 production feruloyl esterase bacillus licheniformis (Bacillus licheniformis)
DBM12, it is characterised in that：The formula of fermentation medium described in step 1 is：Peptone 2g, glucose 0.5g, cross 60 mesh sieves
Wheatfeed 2g, cross 60 mesh sieve husk powder 1g, yeast extract 0.5g, NaCl 0.4g, KH2PO4 0.1g、MgSO40.05g, steam
Distilled water 100mL, pH7.0-7.5.
3. it is according to claim 1 production feruloyl esterase bacillus licheniformis (Baclicus lincheniformis)
DBM12, it is characterised in that：Crude enzyme liquid dosage is 5mL in step 4, and wheatfeed dosage is 10g, water consumption 150mL.
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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 |
| 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 |
| CN106167794B (en) * | 2016-09-07 | 2019-11-26 | 江南大学 | A kind of esterase and its application |
| CN106148301A (en) * | 2016-09-07 | 2016-11-23 | 江南大学 | A kind of esterase and application thereof |
| CN108823131B (en) * | 2018-07-02 | 2020-07-28 | 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 |
| CN112143681B (en) * | 2020-09-28 | 2021-10-19 | 江南大学 | Bacillus belgii capable of producing feruloyl esterase and application thereof |
| CN113308424B (en) * | 2021-04-25 | 2022-12-06 | 天津科技大学 | Bacillus pumilus for producing feruloyl esterase and application thereof |
| CN114908011B (en) * | 2022-05-09 | 2023-09-05 | 江苏省农业科学院 | Lactobacillus sausage and application thereof |
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