CN116656505B - High-yield alcohol dehydrogenase and acyltransferase strain, and culture method and application thereof - Google Patents
High-yield alcohol dehydrogenase and acyltransferase strain, and culture method and application thereof Download PDFInfo
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- 108010021809 Alcohol dehydrogenase Proteins 0.000 title claims abstract description 33
- 108010083294 ethanol acyltransferase Proteins 0.000 title claims abstract description 29
- 102000007698 Alcohol dehydrogenase Human genes 0.000 title claims abstract description 26
- 102000057234 Acyl transferases Human genes 0.000 title claims abstract description 14
- 238000012136 culture method Methods 0.000 title abstract description 3
- 241000228197 Aspergillus flavus Species 0.000 claims abstract description 18
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 40
- 108090000790 Enzymes Proteins 0.000 abstract description 40
- 150000002148 esters Chemical class 0.000 abstract description 26
- 230000000694 effects Effects 0.000 abstract description 22
- XKVUYEYANWFIJX-UHFFFAOYSA-N 5-methyl-1h-pyrazole Chemical compound CC1=CC=NN1 XKVUYEYANWFIJX-UHFFFAOYSA-N 0.000 abstract description 17
- 239000005969 1-Methyl-cyclopropene Substances 0.000 abstract description 14
- SHDPRTQPPWIEJG-UHFFFAOYSA-N 1-methylcyclopropene Chemical compound CC1=CC1 SHDPRTQPPWIEJG-UHFFFAOYSA-N 0.000 abstract description 14
- 244000005700 microbiome Species 0.000 abstract description 11
- 238000012216 screening Methods 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 5
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- 238000006243 chemical reaction Methods 0.000 description 13
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- 125000002252 acyl group Chemical group 0.000 description 6
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- 238000000034 method Methods 0.000 description 6
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- 102100034044 All-trans-retinol dehydrogenase [NAD(+)] ADH1B Human genes 0.000 description 3
- 101710193111 All-trans-retinol dehydrogenase [NAD(+)] ADH4 Proteins 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 108091023242 Internal transcribed spacer Proteins 0.000 description 3
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- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
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- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
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- 101710187573 Alcohol dehydrogenase 2 Proteins 0.000 description 2
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- 241000233866 Fungi Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
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- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 2
- 125000002777 acetyl group Chemical class [H]C([H])([H])C(*)=O 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
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- 238000000576 coating method Methods 0.000 description 2
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- LZCLXQDLBQLTDK-UHFFFAOYSA-N ethyl 2-hydroxypropanoate Chemical compound CCOC(=O)C(C)O LZCLXQDLBQLTDK-UHFFFAOYSA-N 0.000 description 2
- XIRNKXNNONJFQO-UHFFFAOYSA-N ethyl hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC XIRNKXNNONJFQO-UHFFFAOYSA-N 0.000 description 2
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 description 2
- 229940031154 kluyveromyces marxianus Drugs 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
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- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 1
- 102100034042 Alcohol dehydrogenase 1C Human genes 0.000 description 1
- 102100031794 Alcohol dehydrogenase 6 Human genes 0.000 description 1
- 102100039702 Alcohol dehydrogenase class-3 Human genes 0.000 description 1
- 102100031795 All-trans-retinol dehydrogenase [NAD(+)] ADH4 Human genes 0.000 description 1
- 102100026663 All-trans-retinol dehydrogenase [NAD(+)] ADH7 Human genes 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000013392 Carboxylesterase Human genes 0.000 description 1
- 108010051152 Carboxylesterase Proteins 0.000 description 1
- 101000796894 Coturnix japonica Alcohol dehydrogenase 1 Proteins 0.000 description 1
- 241000235646 Cyberlindnera jadinii Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 101710082056 Ethanol acetyltransferase 1 Proteins 0.000 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 description 1
- 101000780463 Homo sapiens Alcohol dehydrogenase 1C Proteins 0.000 description 1
- 101000775460 Homo sapiens Alcohol dehydrogenase 6 Proteins 0.000 description 1
- 101000959452 Homo sapiens Alcohol dehydrogenase class-3 Proteins 0.000 description 1
- 101000775437 Homo sapiens All-trans-retinol dehydrogenase [NAD(+)] ADH4 Proteins 0.000 description 1
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- 241000221961 Neurospora crassa Species 0.000 description 1
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- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 101000928111 Scheffersomyces stipitis (strain ATCC 58785 / CBS 6054 / NBRC 10063 / NRRL Y-11545) Alcohol dehydrogenase 1 Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 1
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- 229940116333 ethyl lactate Drugs 0.000 description 1
- FMMOOAYVCKXGMF-MURFETPASA-N ethyl linoleate Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OCC FMMOOAYVCKXGMF-MURFETPASA-N 0.000 description 1
- 229940031016 ethyl linoleate Drugs 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- 235000014655 lactic acid Nutrition 0.000 description 1
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- FMMOOAYVCKXGMF-UHFFFAOYSA-N linoleic acid ethyl ester Natural products CCCCCC=CCC=CCCCCCCCC(=O)OCC FMMOOAYVCKXGMF-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
-
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/67—Aspergillus flavus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention discloses a high-yield alcohol dehydrogenase and acyltransferase strain and a culture method thereof, wherein the strain is aspergillus flavus (Aspergillus flavus) strain T3 which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.40533. Through screening the methyl pyrazole and the 1-methylcyclopropene flat plate, the methyl pyrazole can inhibit the activity of alcohol dehydrogenase and the 1-methylcyclopropene can inhibit the activity of alcohol acyl transferase, and strains which can continuously grow on the methyl pyrazole and the 1-methylcyclopropene flat plate are screened, so that the enzyme activity of the target strain alcohol dehydrogenase and the enzyme activity of the alcohol acyl transferase are high. The strain is applied to white spirit production, the content of white spirit ester is improved by improving the content of specific esterifying enzyme of microorganisms in Daqu, the proportion of the strain is coordinated, the quality stability of white spirit is enhanced, and the quality of products is improved.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield alcohol dehydrogenase and acyltransferase strain.
Background
Esters are a very important class of flavors in white spirit. In white spirit, water and alcohol are main components, and besides, the white spirit also contains abundant and various trace components, and the trace components have a small content, but have a great effect on the flavor of the white spirit, and are responsible for determining the flavor of the white spirit and constituting l0% of the reasons for different styles of the white spirit. Among the various flavors, the esters are the largest in proportion, and the content and specific gravity of the flavors are important factors for determining the flavor and quality of the white spirit. In the process of brewing white spirit, ester substances are formed by esterification of acid and alcohol, and three formation routes are mainly adopted: the first is generated by the metabolic reaction of related microorganisms in the fermentation process of white wine, wherein the metabolic reaction comprises the interaction among different microorganisms; secondly, distiller's yeast (mainly distiller's yeast esterifying enzyme acts) and lipase are added in the white spirit fermentation process, and corresponding ester substances are synthesized under the catalysis of distiller's yeast or lipase; thirdly, the ester is synthesized by a simple organic chemical reaction, which is mainly reflected in the later fermentation period of white spirit and the storage process, and the free acid alcohol molecules synthesize the corresponding ester substances by the chemical reaction, but the chemical reaction has very low reaction rate and long reaction time under the condition of normal temperature, the esterification reaction can reach an equilibrium state only after a long time, and the esterification reaction rate is lower and is more difficult to reach equilibrium the more the carbon number of fatty acid is.
Esterifying enzyme is also called carboxylesterase, which is the general name of enzymes catalyzing synthesis of lower fatty acid ester, and in white spirit production, lipase, ester synthase and phosphatase are generally called, and the enzyme can catalyze both synthesis of ester and decomposition of ester. The esterifying enzyme can hydrolyze fat in water phase, can hydrolyze fat in fermented grains into glycerin and fatty acid, fatty acid can be esterified with ethanol to generate higher fatty acid such as ethyl oleate, ethyl linoleate, ethyl palmitate and the like, and simultaneously, under the condition of solid fermentation, the esterifying enzyme catalyzes small molecular acid such as caproic acid, lactic acid and the like to act with ethanol to generate flavor substances such as ethyl caproate, ethyl lactate and the like, so that the existence of the esterifying enzyme plays a role in the style of the wine.
The aldehyde and alcohol can undergo nucleophilic addition reaction to form hemiacetal. Typical hemiacetals are unstable and often act as intermediates in acetal formation reactions, but cyclic hemiacetals have some stability, and hemiacetals can continue to react with alcohols to give acetals. The hemiacetal reaction pathway refers to the oxidation of hemiacetal to ethyl acetate under the catalytic action of alcohol dehydrogenase ADH. Mainly in microorganisms such as heat-resistant Kluyveromyces marxianus, neurospora crassa, candida utilis and the like. Loser Christian et al have found that not only does esterification occur in Kluyveromyces marxianus to produce ethyl acetate, but that alcohol acyltransferase ATFl also plays an important role in the production of ethyl acetate, and more importantly have found that alcohol dehydrogenase gene ADH2 not only provides sufficient ethanol substrate for the formation of hemiacetal, but also can oxidize the hemiacetal to produce ethyl acetate. The total seven genes in yeast encode alcohol dehydrogenases (alcohol dehydrogenase, ADH), ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, respectively. During glucose fermentation, alcohol dehydrogenase 2 (ADH 2 p) catalyzes the dehydrogenation of ethanol to acetaldehyde, and the remaining six enzymes catalyze the conversion of acetaldehyde to ethanol. In the yeast alcohol dehydrogenase system, alcohol dehydrogenase 2 is the only enzyme in the cytoplasm of yeast that converts ethanol into acetaldehyde, and in the medium with ethanol as the only carbon source, yeast can only grow by the biochemical action of ADH2 p.
The alcohol acyl transferase is a double-substrate enzyme, can catalyze acyl CoA substrate and alcohol substrate to react to generate corresponding ester, is a key enzyme for synthesizing ester by biochemical reaction in organisms, and can generate corresponding ester by biocatalysis of alcohol and acyl CoA under the action of the alcohol acyl transferase, which is a main synthesis way of the ester in partial fungi. Studies show that the alcohol acetyl transferase coded by ATFl genes can catalyze the synthesis of acid esters such as ethyl acetate, isoamyl acetate and the like, and the enzyme is positioned on cell membranes of partial fungi and catalyzes different alcohols and acetyl CoA to form acetate.
The screening of the ester-producing microorganisms is mainly focused on the screening in the aspect of improving the esterification force, and specific enzymes are not determined, so that the synthetic reasons of the ester compounds are difficult to determine, and theoretical basis is difficult to provide for scientifically regulating and controlling the traditional white spirit brewing process. And the reports on the strains producing alcohol dehydrogenase and acyltransferase are less, most of the reports are research on modifying strains by utilizing a molecular method, but the ploidy of the strains applied to industrial production is often uncertain, and the genetic engineering strains are less applied to white wine brewing based on the food quality safety so as to avoid unpredictable changes caused by modifying the strains.
Disclosure of Invention
The object of the present invention is to provide a high-yield alcohol dehydrogenase and an acyltransferase strain capable of solving at least one of the above problems.
According to one aspect of the invention, a high-yield alcohol dehydrogenase and acyltransferase strain is provided, wherein the strain is Aspergillus flavus (Aspergillusflavus) strain T3 which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.40533.
According to another aspect of the present invention, there is disclosed a method for culturing a high-yield alcohol dehydrogenase and an acyltransferase strain, comprising the steps of:
S1, sampling: luzhou Laojiao Luzhou Daqu;
S2, primary screening, specifically: weighing 1g of Daqu powder, carrying out gradient dilution on the Daqu powder by using sterile physiological saline, respectively sucking 100 mu L of 10-4, 10-5 and 10-6 dilution suspensions, respectively coating the suspension on a carbon-free culture medium plate containing 100, 200, 300, 400, 500, 600, 700, 800, 900mg/L methylpyrazole and 5, 10, 15, 20, 30, 50, 100, 500 and 1000 mg/L1-methylcyclopropene, and culturing the culture medium plate at 30 for 4-5 d; then selecting strains on a plate with the least microorganism growth quantity, streaking, separating and purifying the strains on a PDA solid culture medium plate, and preserving the strains on a PDA inclined plane for later use;
s3, re-screening, specifically: streaking and activating the strain obtained by primary screening on a PDA solid culture medium plate, picking single bacterial colony, inoculating the bacterial colony into 5mL of PDW liquid culture medium, culturing the bacterial colony in a constant temperature shaking table at 30 and 220r/min overnight, inoculating the bacterial colony into 50mL of PDW liquid culture medium with the inoculum size of 1% of the volume fraction, culturing the bacterial colony in the constant temperature shaking table at 30 and 220r/min for 48 hours, and collecting bacterial colony;
S4, freezing thalli for 10min by using liquid nitrogen, adding an enzyme extracting solution and glass beads, then performing wall breaking treatment, oscillating for 30S, performing ice bath for 1min, circulating for multiple times, centrifuging for 2min at 12000r/min to obtain a supernatant to obtain a crude enzyme solution, and then performing enzyme activity measurement to obtain a target strain.
In some embodiments, the carbonless medium (g/L):(NH4)2SO4 5KH2PO4 1NaCl0.1MgSO47H2O 0.5CaCl2 0.1 and yeast powder are sterilized at 121 for 15min.
In some embodiments, the methylpyrazole and 1-methylcyclopropene plates are prepared by adding 20g/L agar, 20g/L absolute ethanol and a concentration of methylpyrazole and 1-methylcyclopropene on the basis of a carbon-free medium.
In some embodiments, the PDW broth formulation (g/L) is potato extract 6, glucose 20.
In some embodiments, the PDA solid medium formulation (g/L) is potato extract 6, glucose 20, agar 20.
An application of high-yield alcohol dehydrogenase and acyltransferase strains in the preparation process of white spirit.
The beneficial effects of the invention are as follows: through screening of the flat plates of the methylpyrazole and the 1-methylcyclopropene, the activity of the alcohol dehydrogenase is inhibited by the methylpyrazole, the activity of the alcohol acyltransferase is inhibited by the 1-methylcyclopropene, and thus the growth of mould is inhibited, however, the strains with relatively high alcohol dehydrogenase and alcohol acyltransferase activities can continue to grow on the flat plates of the methylpyrazole and the 1-methylcyclopropene due to the fact that the enzyme activities are only partially inhibited, and therefore the enzyme activities of the alcohol dehydrogenase and the alcohol acyltransferase of the target strain are high. The strain is applied to white wine production, and as aldehyde and alcohol can undergo nucleophilic addition reaction to generate hemiacetal, the hemiacetal is oxidized to generate ethyl acetate under the catalysis of alcohol dehydrogenase ADH; the alcohol acyl transferase is a double-substrate enzyme, can catalyze the reaction of acyl CoA substrate and alcohol substrate to generate corresponding ester, is a key enzyme for synthesizing ester by biochemical reaction in organisms, and can generate corresponding ester by biocatalysis of alcohol and acyl CoA under the action of the alcohol acyl transferase. Therefore, the content of the white spirit ester is improved by improving the content of the specific esterifying enzyme of the microorganism in the Daqu, the proportion of the white spirit ester is coordinated, the quality stability of the white spirit is enhanced, and the quality of a product is improved.
Drawings
FIG. 1 is a colony morphology of Aspergillus flavus of the present invention.
FIG. 2 is a further enlarged colony morphology of A.flavus of the present invention.
FIG. 3 is an enlarged view of Aspergillus flavus of the present invention under a microscope.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings.
Referring to fig. 1-3. A high-yield alcohol dehydrogenase and acyltransferase strain is Aspergillus flavus (Aspergillusflavus) strain T3 which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 3 months in 2023, and has an address of Xiyu No. 1, 3 in the Chaoyang region of Beijing city and a preservation number of CGMCC No.40533.
ITS nucleotide sequence of Aspergillus flavus strain T3
ITS1
TATTCGAGCTGAGGGTTCCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAAAGGCGGGGAAAGAAAAAGA;
ITS4
AGTGGGGATGCTACCTGATCCGAGGTCACCTGGAAAAAGATTGATTTGCGTTCGGCAAGCGCCGGCCGGGCCTACAGAGCGGGTGACAAAGCCCCATACGCTCGAGGATCGGACGCGGTGCCGCCGCTGCCTTTGGGGCCCGTCCCCCCCGGAGAGGGGACGACGACCCAACACACAAGCCGTGCTTGATGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGATGATTCACGGAATTCTGCAATTCACACTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGTTGAAAGTTTTAACTGATTGCGATACAATCAACTCAGACTTCACTAGATCAGACAGAGTTCGTGGTGTCTCCGGCGGGCGCGGGCCCGGGGCTGAGAGCCCCCGGCGGCCATGAATGGCGGGCCCGCCGAAGCAACTAAGGTACAGTAAACACGGGTGGGGAGGTTGGGCTCGCTAGGAACCCTACACTCGGTAATGATCCTTCCGA
The invention relates to a method for culturing high-yield alcohol dehydrogenase and acyltransferase strains, which comprises the following steps:
S1, sampling: luzhou Laojiao Luzhou Daqu;
s2, primary screening, specifically: weighing 1g of Daqu powder, carrying out gradient dilution on the Daqu powder by using sterile physiological saline, respectively sucking 100 mu L of 10 -410-5 and 10 -6 dilution suspensions, respectively coating the suspensions on a carbon-free culture medium flat plate containing 100, 200, 300, 400, 500, 600, 700, 800, 900mg/L methylpyrazole and 5, 10, 15, 20, 30, 50, 100, 500 and 1000 mg/L1-methylcyclopropene, and culturing the culture medium flat plate at 30 for 4-5 d; then selecting strains on a plate with the least microorganism growth quantity, streaking, separating and purifying on a PDA solid culture medium (Potato Dextrose Agar, potato dextrose agar culture medium) plate, and preserving on a PDA inclined plane for later use;
In this step, on a medium containing methylpyrazole and 1-methylcyclopropene and having ethanol as the sole carbon source, methylpyrazole inhibits the activity of alcohol acyltransferase by inhibiting the activity of alcohol dehydrogenase, and thus inhibits the growth of mold, whereas a strain having both alcohol dehydrogenase and alcohol acyltransferase activities relatively high can continue to grow on methylpyrazole and 1-methylcyclopropene plates because its enzyme activity is only partially inhibited. And culturing Daqu powder suspension with each concentration on each culture medium to obtain strains in different growth states, wherein practical culture results show that when the concentration of methylpyrazole is 800mg/L and the concentration of 1-methylcyclopropene is 20mg/L, the quantity of grown strains is minimum, mould on a flat plate containing 800mg/L of methylpyrazole and 20mg/L of 1-methylcyclopropene is selected, and the strains are separated and purified by streaking on a flat plate of a PDA solid culture medium and then stored on a PDA inclined surface for standby.
S3, re-screening, specifically: marking and activating the strain obtained by primary screening on a PDA solid culture medium plate, picking single bacterial colony, inoculating the single bacterial colony into 5mL of PDW liquid culture medium, culturing the single bacterial colony in a constant temperature shaking table at 30 and 220r/min overnight, inoculating the single bacterial colony into 50mL of PDW liquid culture medium with the inoculum size of 1 percent of volume fraction, culturing the single bacterial colony in the shaking table at 30 and 220r/min for 48 hours under shaking, ensuring the microbial quantity to be 1.25 multiplied by 10 10, taking 3mL of fermentation liquor, centrifuging the fermentation liquor at 15000r/min for 20min, and collecting all bacterial colonies;
S4, freezing the thalli for 10min by using liquid nitrogen, respectively adding an enzyme extract of alcohol dehydrogenase and acyltransferase and glass beads, wherein the adding amount of the glass beads is approximately the same as the volume amount of the thalli, finally performing wall breaking treatment by using a vortex oscillator, oscillating for 30S, ice-bathing for 1min, repeating 15 cycles, and centrifuging for 2min at 12000r/min to obtain a supernatant to obtain a crude enzyme solution, and then performing enzyme activity measurement to obtain the target strain.
Wherein, the carbon-free culture medium (g/L):(NH4)2SO4 5KH2PO4 1NaCl 0.1MgSO47H2O 0.5CaCl20.1 and yeast powder are sterilized at 121 for 15min.
The flat plate of the methylpyrazole and the 1-methylcyclopropene is prepared by adding 20g/L of agar, 20g/L of absolute ethyl alcohol and a certain concentration of methylpyrazole and 1-methylcyclopropene on the basis of a carbon-free culture medium.
PDW liquid medium formulation (g/L): potato extract 6 and glucose 20.
PDA solid medium formulation (g/L): potato extract 6, glucose 20 and agar 20.
AAT enzyme (i.e., alcohol acyltransferase) when performing an enzyme activity assay: using an AAT enzyme kit; wherein, the enzyme activity is defined as follows: the catalytic absorbance change per ml of fermentation broth per minute at 37was 0.001 unit to 1 enzyme activity unit).
ADH enzyme (i.e., alcohol dehydrogenase): NAD (nicotinamide adenine dinucleotide, abbreviated as coenzyme I) is added, and after shaking, the absorbance of the reaction system is measured at 340nm immediately, distilled water is zeroed, and the time is recorded. After the primary measurement is completed, the reaction tube is placed in a 30 incubator, the absorbance is measured at intervals of 0.5h, the change of the absorbance of the reaction system in 2.5h is measured, and the average value of three readings is obtained and the time is recorded. Wherein, the enzyme activity is defined as follows: the change of the catalytic absorbance value per milliliter of fermentation liquid per hour at the temperature of 30 in a reaction system is defined as 1 enzyme activity unit.
Table 1 shows ADH reaction system
Table 2 shows the corresponding conditions of the enzyme activities of the strains
As can be seen from the comparison of the above tables, the enzyme activities of both enzymes of T3 were high, wherein the enzyme activity of alcohol acyltransferase was 480U/mL and the enzyme activity of alcohol dehydrogenase was 411U/mL, so that T3 was used as the target strain.
The identification process for the ethanol dehydrogenase and alcohol acyltransferase producing mould strains is as follows:
(1) Morphological characteristics, culture characteristics and physiological and biochemical tests
And carrying out preliminary classification and identification according to the colony and the shape of the bacteria. As a result, as shown in FIG. 1, the colony structure was loose, the surface was yellowish-green and slightly brown, and the cell consisted of many complex branched hyphae.
(2) Sequence analysis and identification
1) PCR amplification was performed using fungal ITS amplification sequences, ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC).
Amplification system: 1TSE101 gold plate mix 45ul, ITS1 (10P) 2ul, ITS4 (10P) 2ul, DNA template 1ul.
PCR amplification procedure: pre-denaturation at 98for 3min, denaturation at 98for 10s, annealing at 53for 10s, extension at 72for 10s/kb,39 cycles, and finally extension at 72for 5min.
The PCR products were detected by 1% agarose gel electrophoresis. The PCR amplified product is sent to Beijing qingke biological company for sequencing after electrophoresis.
2) The measured ITS sequences were subjected to an on-line homology analysis with the sequences registered in the GenBank database by the BLAST program in the NCBI database. The results show that the ITS region sequence of the mould strain T3 has 100 percent of homology with the ITS region sequence of the aspergillus flavus (Aspergillus flavus) MT645322.1, and the strain is identified as the aspergillus flavus (Aspergillus flavus) by combining the conventional morphological characteristics, physiological characteristics and biochemical characteristics.
The application of the aspergillus flavus in the white spirit preparation process is that the strain is applied to white spirit production, as aldehyde and alcohol can generate nucleophilic addition reaction to generate hemiacetal, and then the hemiacetal is oxidized to generate ethyl acetate under the catalysis of alcohol dehydrogenase ADH; the alcohol acyl transferase is a double-substrate enzyme, can catalyze the reaction of acyl CoA substrate and alcohol substrate to generate corresponding ester, is a key enzyme for synthesizing ester by biochemical reaction in organisms, and can generate corresponding ester by biocatalysis of alcohol and acyl CoA under the action of the alcohol acyl transferase. Therefore, the content of the white spirit ester is improved by improving the content of the specific esterifying enzyme of the microorganism in the Daqu, the proportion of the white spirit ester is coordinated, the quality stability of the white spirit is enhanced, and the quality of a product is improved.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and improvements could be made by those skilled in the art without departing from the inventive concept, which falls within the scope of the present invention.
Claims (2)
1. A high-yield alcohol dehydrogenase and acyltransferase strain is characterized in that the strain is an aspergillus flavus (Aspergillus flavus) strain T3 which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.40533.
2. The use of a high yield alcohol dehydrogenase and acyltransferase strain of claim 1, wherein the use of aspergillus flavus in the preparation of white spirit.
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