CN111718919A - Culture medium for producing esterifying enzyme and preparation method of crude esterifying enzyme preparation - Google Patents
Culture medium for producing esterifying enzyme and preparation method of crude esterifying enzyme preparation Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 115
- 239000001963 growth medium Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- 239000002609 medium Substances 0.000 claims abstract description 27
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 19
- 229930006000 Sucrose Natural products 0.000 claims abstract description 19
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01002—Arylesterase (3.1.1.2)
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Abstract
The invention discloses a culture medium for producing an esterifying enzyme and a preparation method of a crude esterifying enzyme preparation, wherein the culture medium comprises MgSO4、K2HPO4、FeSO4、KCl、MnSO4Sucrose and urea. When the crude enzyme preparation is prepared, an Aspergillus niger strain M is screened from the Daqu, then the Aspergillus niger strain M is inoculated into a culture medium for producing the esterifying enzyme, the mixture is subjected to constant-temperature shaking culture for 90-100 hours at the temperature of 28-35 ℃, a culture solution is separated and centrifuged, and a supernatant is taken to obtain the crude enzyme preparation of the esterifying enzyme. The medium for producing the esterifying enzyme comprises various inorganic salts, sucrose as a carbon source, urea as a nitrogen source and multi-component synergistic effect, can ensure the ion diversity of the medium while maintaining the pH stability of the medium, can ensure that the strain has sufficient nutrient supply in the fermentation process, and can provide the strain with the nutrient supplyProvides the optimal growth condition, can promote the strains to produce a large amount of esterifying enzymes, and ensures the activity of the esterifying enzymes.
Description
Technical Field
The invention belongs to the technical field of production of esterases, and particularly relates to a culture medium for producing esterases and a preparation method of a crude enzyme preparation of the esterases.
Background
Esterases (esterases e.c.3.1.1.2), also known as carboxylesterases, are enzymes that hydrolyze carboxylic ester bonds, but are also capable of catalyzing the synthesis of lower fatty acid esters. It has the ability to catalyze the synthesis and decomposition of esters, and therefore, the liquor industry is commonly called esterases or esterases.
The yeast for making strong aromatic Chinese spirits has a wide variety of microorganisms, wherein the mold is one of important ester-producing bacteria in brewing of strong aromatic Chinese spirits, and caproic acid (metabolism of pit mud functional bacteria) and ethanol (metabolism of a fermented grain system) are condensed to generate ethyl caproate with main fragrance by generating esterifying enzyme. By screening the esterifying enzyme strain with high metabolic activity, the generation of ethyl caproate is improved, and the aims of optimizing the quality of the wine and shortening the fermentation period are further fulfilled. The current research mainly focuses on the separation and screening of monascus, rhizopus and aroma-producing yeast, the optimization of enzyme (ester) producing conditions, and the research and application of enzymology properties. The study shows that the microorganisms capable of producing the esterifying enzyme exist in moulds, yeasts and bacteria. The esterases with different sources have different properties, and the catalytic reaction is greatly influenced by factors such as the concentration of reaction media, substrates and products. Therefore, the separation and purification of the purified esterifying enzyme and the enzymological properties of the esterifying enzyme are explored, so that the esterifying enzyme technology can be pertinently used for better serving for the white spirit production.
Disclosure of Invention
Aiming at the prior art, the invention provides a culture medium for producing an esterifying enzyme and a preparation method of a crude enzyme preparation of the esterifying enzyme, so as to achieve the aims of improving the yield of the esterifying enzyme and improving the activity of the esterifying enzyme.
In order to achieve the purpose, the invention adopts the technical scheme that: the medium for producing the esterifying enzyme comprises the following components in percentage by mass:
MgSO40.03～0.06wt％，K2HPO40.08～0.15wt％，FeSO40.001～0.002wt％，KCl0.03～0.06wt％，MnSO40.01-0.05 wt%, 1-3 wt% of cane sugar, 5-7 wt% of urea and the balance of deionized water.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the medium for producing the esterifying enzyme comprises the following components in percentage by mass:
MgSO40.05wt％，K2HPO40.1wt％，FeSO40.001wt％，KCl 0.05wt％，MnSO40.03 wt%, cane sugar 2 wt%, urea 6 wt%, and the balance of deionized water.
The medium for producing the esterifying enzyme of the present invention includes MgSO4、K2HPO4、FeSO4、KCl、MnSO4And the like. Wherein, MgSO4Providing Mg2+，FeSO4Providing Fe2+，MnSO4Providing Mn2+The three metal ions have synergistic effect, so that the metabolism, ion transport and the like of the esterifying enzyme can be promoted, and the activity of the esterifying enzyme is promoted. The culture medium takes sucrose as a carbon source, the sucrose can better adjust the osmotic pressure in the culture medium while providing carbon elements, so that the osmotic pressure in the culture medium can be kept stable for a long time, and the addition of the sucrose in the culture medium can reduce the pollution of microorganisms, reduce the competitive pressure of esterifying enzyme and enable the esterifying enzyme to survive more easily; the culture medium takes urea as a nitrogen source, and the urea can adjust the pH value in the culture medium while providing nitrogen elements, so that the pH value of the culture medium is kept stable for a long time, and formation and survival of the esterifying enzyme are facilitated.
Further, the medium for producing an esterifying enzyme of the present invention has a pH of 5.5 to 6.5.
The medium for producing the esterifying enzyme is prepared by the following steps:
(1) taking formula amount of MgSO4、K2HPO4KCl and MnSO4Dissolving them in water, and stirring them uniformly to obtain ionic solution;
(2) adding the sucrose and urea with the formula ratio into the ionic solution, and uniformly stirring to obtain a primary culture medium product;
(3) FeSO is added 5-10 min before use4Adding the mixture into the primary culture medium, uniformly stirring, and adjusting the pH value to 5.5-6.5 to obtain the culture medium for producing the esterifying enzyme.
The inventionWhen preparing the culture medium for producing the esterifying enzyme, FeSO is added finally4Can avoid Fe2+And the medium is oxidized to lose efficacy, so that the effect of the medium is effectively ensured, and a good environment is provided for the production of the esterifying enzyme.
In the invention, after the culture medium for producing the esterifying enzyme is prepared, the crude enzyme preparation of the esterifying enzyme is also prepared by using the culture medium, and the preparation of the crude enzyme preparation of the esterifying enzyme comprises the following steps:
s1: screening Aspergillus niger strain M from the Daqu (Query ID: lcl | Query _ 14659. Access: MN 190293.1);
s2: inoculating the Aspergillus niger strain into the prepared culture medium for producing the esterifying enzyme, carrying out constant-temperature shaking culture for 90-100 h at the temperature of 28-35 ℃, separating the culture solution and centrifuging, and taking the supernatant to obtain the crude esterifying enzyme preparation.
On the basis of the technical scheme, the preparation of the crude enzyme preparation of the esterifying enzyme can be improved as follows.
Further, the screening of Aspergillus niger strain M in S1 comprises the following steps:
SS 1: adding commercially available five-grain liquid strong flavor Daqu into sterile physiological saline, adding glass beads, scattering, standing for 30min, collecting supernatant, and making into 10% thick liquid-2g/mL bacterial suspension;
SS 2: uniformly coating the bacterial suspension on the surface of a mould culture medium, and culturing for 72h at 36 ℃; the mold culture medium comprises a substrate and emulsion, wherein the substrate and the emulsion are mixed according to a volume ratio of 3-5: 1; the matrix comprises the following components in parts by mass:
8-12 parts of peptone, 20-30 parts of agar, 4-6 parts of sodium chloride and 2-4 parts of beef extract;
the emulsion is formed by mixing tributyrin and polyvinyl alcohol according to the volume ratio of 1: 8-10;
SS 3: selecting the colony with the largest ratio of the diameter of the colony to the diameter of the transparent ring, and carrying out strain purification by using a plate marking method to obtain a strain M; the sequence of the strain M is shown in SEQ ID No. 1.
Further, mixing the substrate and the emulsion in the mold culture medium according to the volume ratio of 4: 1; wherein the matrix comprises the following components in parts by mass:
10 parts of peptone, 25 parts of agar, 5 parts of sodium chloride and 3 parts of beef extract;
the emulsion is formed by mixing tributyrin and polyvinyl alcohol according to the volume ratio of 1: 9.
Further, the culture temperature in S2 was 30 ℃ and the culture time was 96 hours.
The invention has the beneficial effects that: the medium for producing the esterifying enzyme comprises various inorganic salts, sucrose is used as a carbon source, urea is used as a nitrogen source, and multi-component synergistic effect is achieved, so that the ion diversity of the medium can be ensured while the pH of the medium is maintained to be stable, the sufficient nutrition supply of the strain in the fermentation process can be ensured, the optimal growth condition can be provided for the strain, the mass production of the esterifying enzyme by the strain can be promoted, and the activity of the esterifying enzyme can be ensured.
Drawings
FIG. 1 is an electrophoretogram of strain M;
FIG. 2 shows the effect of different carbon sources on enzyme production;
FIG. 3 is a graph showing the effect of different nitrogen sources on enzyme production;
FIG. 4 shows the effect of different fermentation temperatures on enzyme production;
FIG. 5 shows the effect of different fermentation times on enzyme production.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1: isolation of Strain M from Daqu
Adding 10g of commercial wuliangye aroma type Daqu ground into fine powder into 100mL of sterile physiological saline, adding glass beads, scattering, and standing for 30 min. 1mL of supernatant was taken and prepared into 10-2g/mL bacterial suspension. 0.2mL of the bacterial suspension was aspirated, uniformly applied to the surface of a mold medium, cultured at 36 ℃ for 72 hours, the size of a transparent circle around the colony was observed, and the ratio of the diameter (D) of the transparent circle to the diameter (D) of the colony was measured. And selecting the colony with the largest ratio of the colony diameter to the transparent ring diameter, and carrying out strain purification by using a plate marking method to obtain a strain M. The mold culture medium comprises a substrate and emulsion, wherein the substrate and the emulsion are mixed according to a volume ratio of 3-5: 1Combining; wherein the matrix comprises the following components in parts by mass:
8-12 parts of peptone, 20-30 parts of agar, 4-6 parts of sodium chloride and 2-4 parts of beef extract;
the emulsion is formed by mixing tributyrin and polyvinyl alcohol according to the volume ratio of 1: 8-10.
During the colony culture process, the growth, color, surface morphology, texture, edge shape, height, etc. of the colony are observed with a magnifying glass. The colony is white after 1d through culture observation; after 2d, the mixture turns to grey white and tends to be mature, and the bacterial colony is large and loose in texture when observed under a microscope; after 3 days, a large number of spores grow out and are gray; after 5 days, the bacterial colony becomes white, the reverse side of the bacterial colony is grey white, and hyphae are branched and have partitions; the strain has sufficient cells, and conidiophores are extremely coarse and long, and some cells are 1.5mm or so.
And then carrying out molecular biological identification on the strain M, wherein the specific operation is as follows: the inoculating loop is used for dipping the strain M, PCR amplification is carried out in a polymerase chain reaction system, and the amplification primer sequence is as follows:
the sequence of the upstream primer is as follows: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID No.2),
the sequence of the downstream primer is as follows: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID No. 3).
The PCR amplification conditions were: 98 deg.C, 3min, 98 deg.C, 10s, 53 deg.C, 10s, 72 deg.C, 10 s; 98. 35 cycles of 53 and 72 ℃; keeping at 72 deg.C for 5min and 4 deg.C. The PCR reaction system is as follows: 25 μ L T5Mix, 1 μ L16 Sr DNA-M, 1 μ L GenomicDNA, 22 μ L dH2And O. Carrying out 1% agarose gel electrophoresis detection and gel recovery on the PCR product, wherein the electrophoresis result is shown in figure 1; sequencing and identifying, wherein the sequence of the strain M is shown as SEQ ID No. 1.
Example 2: preparing culture medium for producing esterifying enzyme
The fermentation medium used in the production of the esterifying enzyme comprises the following components in percentage by mass:
MgSO40.03～0.06wt％，K2HPO40.08～0.15wt％，FeSO40.001～0.002wt％，KCl0.03～0.06wt％，MnSO40.01-0.05 wt%, 1-3 wt% of carbon source, 5-7 wt% of nitrogen source and the balance of deionized water.
The culture medium is prepared by the following steps:
(1) taking formula amount of MgSO4、K2HPO4KCl and MnSO4Dissolving them in water, and stirring them uniformly to obtain ionic solution;
(2) adding a carbon source and a nitrogen source with a formula amount into the ionic solution, and uniformly stirring to obtain a culture medium primary product;
(3) FeSO is added 5-10 min before use4Adding the mixture into the primary culture medium, uniformly stirring, and adjusting the pH value to 5.5-6.5 to obtain the culture medium for producing the esterifying enzyme.
1. Medium carbon source screening
Carbon is an important nutrient constituting the carbon skeleton in the cellular structure and metabolites of organisms, and is also a substance that provides energy to organisms. The microorganism has selectivity on carbon source utilization, different carbon sources are used in the fermentation medium, and the influence of the addition of different carbon source substances and the dosage thereof on the product result is examined by changing the content of the carbon sources. Sucrose (1.0%, 1.5%, 2.0%, 2.5%), glucose (1.0%, 1.5%, 2.0%, 2.5%), corn flour (1.0%, 1.5%, 2.0%, 2.5%), and soluble starch (1.0%, 1.5%, 2.0%, 2.5%) were used as carbon sources, and the results of enzyme activity measurement after cultivation are shown in FIG. 2. The method for measuring the enzyme activity comprises the following steps: 10mL of cyclohexane, 6.25mL of caproic acid, 3.65mL of ethanol and 0.2mL of enzyme solution (30 g of anhydrous sodium sulfate is added to each 500mL of all reagents) are weighed respectively and put into a 100mL conical flask, esterification reaction is carried out for 24h at 36 ℃ in a sealed mode, 0.5mL of supernatant is added with 5mL of water, two drops of phenolphthalein are added, and titration is carried out by using 0.05mol/L of NaOH until the end point is reached.
As can be seen from fig. 2, when sucrose, glucose, corn flour and soluble starch are used as carbon sources, the esterifying enzyme activity for sucrose production is the highest and is all higher than that of the glucose, the corn flour and the soluble starch, i.e. when sucrose is used as a carbon source, the strain is favorable for producing the esterifying enzyme, the enzyme activity value is 4.2111, the content of sucrose is changed, the enzyme activity is continuously improved when the addition amount is increased from 1.0% to 2.0%, the enzyme activity reaches a peak value at 2.0%, and then is slowly reduced.
2. Culture medium nitrogen source screening
The nitrogen source provides nitrogen-containing substances such as nucleic acid, protein and the like required by the microorganism for synthesizing cells, and enough nitrogen source is added in the process of making the yeast to be beneficial to the growth of thalli and the enzyme production. In the fermentation medium, different nitrogen sources were used and the content of the nitrogen source was changed, and soybean flour (4.0%, 5.0%, 6.0%, 7.0%), peptone (4.0%, 5.0%, 6.0%, 7.0%), yeast extract (4.0%, 5.0%, 6.0%, 7.0%), and urea (4.0%, 5.0%, 6.0%, 7.0%) were used as the nitrogen source, and the enzyme activity was measured after the cultivation, and the results are shown in FIG. 3.
As can be seen from FIG. 3, when soybean meal, peptone, yeast extract and urea are used as nitrogen sources, the esterifying enzyme produced by urea has the highest activity, and when yeast extract, peptone and soybean meal are used as secondary nitrogen sources, the esterifying enzyme produced by the strain is favorably produced by the strain. The content of sucrose is changed, the enzyme activity is in an increasing trend with the addition amount increased from 4.0% to 6.0%, the enzyme activity is rapidly increased from 5% to 6%, the enzyme activity reaches the highest value at 6.0% and is 3.9, and then the enzyme activity begins to decrease.
In summary, sucrose is selected as a carbon source and urea is selected as a nitrogen source in the medium for producing the esterifying enzyme, and the final composition of the medium is determined to comprise the following components in percentage by mass:
MgSO40.03～0.06wt％，K2HPO40.08～0.15wt％，FeSO40.001～0.002wt％，KCl0.03～0.06wt％，MnSO40.01-0.05 wt%, 1-3 wt% of cane sugar, 5-7 wt% of urea and the balance of deionized water.
Example 3: preparation of crude enzyme preparation of esterifying enzyme
After the strains are screened out and the fermentation culture medium is prepared, the preparation of the crude enzyme preparation of the esterifying enzyme can be started, the crude enzyme of the esterifying enzyme is prepared by the strain fermentation, and the fermentation is greatly influenced by the fermentation temperature and the fermentation time, so the fermentation temperature and the fermentation time are investigated, and the method is as follows.
1. Influence of different fermentation temperatures on enzyme production
Temperature is one of the important factors influencing enzyme production, cell metabolism and product synthesis need enzyme catalysis to complete, and the catalytic reaction can normally be carried out only when the temperature is suitable for the growth of thalli. The change of the fermentation temperature is controlled, and the influence of the change on the enzyme production is researched. The enzyme activity was measured after culturing the medium prepared in example 2 at 25 ℃ 28 ℃, 30 ℃, 32 ℃ and 35 ℃ for 96 hours using the medium as a fermentation medium, and the results are shown in FIG. 3.
As can be seen from FIG. 3, different fermentation temperatures differ in the esterases activity. With the continuous increase of the culture temperature, the thalli grow vigorously and the enzyme yield is increased gradually, and the enzyme activity reaches the maximum value at about 35 ℃ of culture, so that the temperature of 35 ℃ is the optimal fermentation temperature.
2. Effect of different fermentation times on enzyme production
In the liquid fermentation process, the fermentation time has obvious influence on the generation of thalli and enzyme production, the culture medium formula optimized in the embodiment 2 is used for fermentation culture, the enzyme activity is measured after the culture is respectively carried out for 3d, 5d, 7d and 9d at the temperature of 35 ℃, and the result is shown in figure 4.
As can be seen from FIG. 4, as the culture time was prolonged, the enzyme production amount increased gradually due to vigorous growth of the cells, and the enzyme activity reached the maximum value after 5 days of culture, and then decreased. Therefore, 96h was determined as the optimum incubation time.
In summary, the preparation of the crude enzyme preparation of the esterifying enzyme in the invention comprises the following specific operations:
inoculating the Aspergillus niger strain obtained by screening in the embodiment to the esterifying enzyme production culture medium prepared in the embodiment 2, carrying out constant temperature shaking culture at 28-35 ℃ for 90-100 h, separating the culture solution, centrifuging, and taking the supernatant to obtain the crude esterifying enzyme preparation.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Sequence listing
<110> Sichuan university of light chemical industry
<120> a culture medium for producing esterases and a method for preparing crude enzyme preparation of esterases
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>761
<212>DNA
<213> Aspergillus niger M (Aspergillus niger M)
<400>1
atttcagatc tagatgtgag tttgtttaag agaagaccca agttagatcc tctcgctagt 60
ccgtaaagat taagacctag ttcaagaaga gactgattga cttggaatta tgaactcact 120
aaccaagtcg ttgctcctca ggcactctag agtctacatc cggcaaatga ctaaagccaa 180
ttgcctagga ctaaatgtat ttaaggccat gacagcaact gaatgccatc aacacaagcc 240
catttccagc tcgcttaggt tcaaagaacc aagttgaact gattggtagt tgcagatact 300
gaaacaactg tgcctagtag attgactact aggcgcaaga tgcgttcgag aactcgatga 360
ttcgctatga atgcaagtcg caataattat cgcactttgc tacgctcttc atcgatgcga 420
gaaccaagag atccattgcc aagagttgtt tttaagttaa caactaactt tttcgtacat 480
catggtttac acgagaagaa taaacacctt tggggatagt tagtactaga gccccaaagg 540
cttgccttgc ttgctttgac tcgagacata ggtctcctga aagaagggtc catacgtctc 600
ttttcaagca gcctagatct tagtagtggc acaagtctcc taaaaggagg gtctctatgc 660
caacaaccag atctacagca caaggcagag ccaaccaagt tgacccgact ttgcacaaaa 720
ctgtgaggaa ctactagagg ggaagagacc cccaactagt g 761
<210>2
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tccgtaggtg aacctgcgg 19
<210>3
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tcctccgctt attgatatgc20
Claims (9)
1. The medium for producing the esterifying enzyme is characterized by comprising the following components in percentage by mass:
MgSO40.03～0.06wt％，K2HPO40.08～0.15wt％，FeSO40.001～0.002wt％，KCl 0.03～0.06wt％，MnSO40.01-0.05 wt%, 1-3 wt% of cane sugar, 5-7 wt% of urea and the balance of deionized water.
2. The medium for producing an esterifying enzyme according to claim 1, wherein: the composite material comprises the following components in percentage by mass:
MgSO40.05wt％，K2HPO40.1wt％，FeSO40.001wt％，KCl 0.05wt％，MnSO40.03 wt%, cane sugar 2 wt%, urea 6 wt%, and the balance of deionized water.
3. The medium for producing an esterifying enzyme according to claim 1 or 2, characterized in that: the pH value of the culture medium is 5.5-6.5.
4. The method for producing a medium for producing an esterifying enzyme according to claim 1 to 3, comprising the steps of:
(1) taking formula amount of MgSO4、K2HPO4KCl and MnSO4Dissolving them in water, and stirring them uniformly to obtain ionic solution;
(2) adding the sucrose and urea with the formula ratio into the ionic solution, and uniformly stirring to obtain a primary culture medium product;
(3) FeSO is added 5-10 min before use4Adding the mixture into the primary culture medium, uniformly stirring, and adjusting the pH value to 5.5-6.5 to obtain the culture medium for producing the esterifying enzyme.
5. A method for preparing crude enzyme preparation of esterifying enzyme by using yeast is characterized by comprising the following steps:
s1: screening aspergillus niger strains with a sequence shown as SEQ ID No.1 from the yeast for making hard liquor;
s2: inoculating the screened aspergillus niger strain into the esterifying enzyme production culture medium of any one of claims 1 to 3, carrying out constant temperature shaking culture for 90 to 100 hours at the temperature of 28 to 35 ℃, separating culture solution, centrifuging, and taking supernatant fluid to obtain the crude esterifying enzyme preparation.
6. The method for preparing crude enzyme preparation of esterifying enzyme by using Daqu according to claim 5, wherein the screening of Aspergillus niger strain M in S1 comprises the following steps:
SS 1: adding the fine powder of Daqu into sterile physiological saline, adding glass beads, scattering, standing for 30min, collecting supernatant, and making into 10% concentrate-2g/mL bacterial suspension;
SS 2: uniformly coating the bacterial suspension on the surface of a mould culture medium, and culturing for 72h at 36 ℃; the mold culture medium comprises a substrate and emulsion, wherein the substrate and the emulsion are mixed according to a volume ratio of 3-5: 1; the matrix comprises the following components in parts by mass:
8-12 parts of peptone, 20-30 parts of agar, 4-6 parts of sodium chloride and 2-4 parts of beef extract;
the emulsion is formed by mixing tributyrin and polyvinyl alcohol according to the volume ratio of 1: 8-10;
SS 3: and selecting the colony with the largest ratio of the colony diameter to the transparent ring diameter, and carrying out strain purification by using a plate marking method to obtain a strain M.
7. The method for preparing crude enzyme preparation of esterifying enzyme by using Daqu according to claim 6, wherein the substrate and the emulsion in the mold culture medium are mixed in a volume ratio of 4: 1; wherein the matrix comprises the following components in parts by mass:
10 parts of peptone, 25 parts of agar, 5 parts of sodium chloride and 3 parts of beef extract;
the emulsion is formed by mixing tributyrin and polyvinyl alcohol according to the volume ratio of 1: 9.
8. The method for preparing crude enzyme preparation of esterifying enzyme by using Daqu according to claim 5, wherein: the culture temperature in S2 was 35 ℃ and the culture time was 96 h.
9. The method for preparing crude enzyme preparation of esterifying enzyme by using Daqu according to claim 5, wherein: the Daqu is a five-grain liquor strong-flavor Daqu.
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Non-Patent Citations (5)
| Title |
|---|
| 吕梅: "Lichtheimia属HSM菌株酯酶发酵条件及酶学特性研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 黄丹等: "一株产己酸乙酯酯化酶霉菌的分离鉴定及产酶条件研究", 《酿酒科技》 * |
| 黄丹等: "一株产己酸乙酯酯化酶霉菌的分离鉴定及产酶条件研究", 《酿酒科技》, no. 02, 18 February 2008 (2008-02-18), pages 27 * |
| 黄丹等: "大曲酯化酶根霉菌的分离及产酶条件研究", 《食品与发酵科技》 * |
| 黄丹等: "大曲酯化酶根霉菌的分离及产酶条件研究", 《食品与发酵科技》, no. 03, 25 June 2010 (2010-06-25), pages 30 - 32 * |
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