CN110628744A - Method for separating and purifying esterifying enzyme from strong aromatic yeast - Google Patents
Method for separating and purifying esterifying enzyme from strong aromatic yeast Download PDFInfo
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- 235000021307 Triticum Nutrition 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
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- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- 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)
-
- 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)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- 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)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for separating and purifying esterifying enzyme from strong-flavor Daqu, which takes the strong-flavor Daqu as a test material and separates and purifies the esterifying enzyme in the Daqu by a method combining DEAE cellulose DE-52 ion exchange chromatography and glucan G-100 gel chromatography. The product of the invention is shown as two main bands when the purity of the product is checked by SDS-PAGE, the molecular weight is respectively 37.2kD and 30.8kD, after four-step purification, the esterifying enzyme is purified by 3.49 times, and the recovery rate of the enzyme activity is 28.01 percent.
Description
Technical Field
The invention belongs to the technical field of esterifying enzymes, and particularly relates to a method for separating and purifying esterifying enzymes from strong aromatic yeast.
Background
The yeast is a saccharification leaven which is prepared by taking wheat, barley, peas and the like as main raw materials, crushing, adding water, stirring, manually treading or mechanically pressing into brick-shaped yeast blanks and then culturing at a certain temperature and humidity. As a large amount of beneficial microorganisms in the natural environment are screened in the process of making the yeast, the finished product of the yeast contains rich wine-making microorganism strains, enzyme systems and material systems through fermentation and culture management. The Daqu has the functions of saccharification, fermentation, alcoholization and aroma generation, provides internal power for white spirit fermentation, and has a saying that the Daqu is the bone of the spirit.
The yeast for making hard liquor contains rich enzymes which can be divided into the following parts according to the action rule: amylase, protease, alcoholizing enzyme, esterifying enzyme, cellulase, xylanase and other enzymes. The esterifying enzyme is an important enzyme in the yeast for making hard liquor, and plays a key role in the aroma generation process in the later period of the fermentation of the white liquor. The esterifying enzyme is not a term in enzymology, and refers to a general name of lipase, esterase and phosphatase in the production of white spirit. The enzyme can catalyze both the synthesis and decomposition of esters. Thus, the liquor industry is accustomed to esterification and ester-decomposition enzymes, respectively.
The conventional method for separating and purifying the enzyme is to use ammonium sulfate precipitation, organic solvent precipitation, centrifugation (or filtration), ultrafiltration and other methods to carry out coarse separation on the enzyme, and then combine chromatography (ion exchange, gel filtration, hydrophobic chromatography, affinity chromatography and the like), electrophoresis and other methods to realize fine separation on the enzyme. With the development of downstream technologies in the biological industry, a new series of separation and purification technologies, such as membrane treatment technology, immune purification technology, aqueous two-phase system extraction, interfacial affinity chromatography technology, reverse micelle extraction, supercritical fluid extraction, high performance liquid chromatography and high performance liquid affinity chromatography technology, large-scale electrophoresis technology, countercurrent chromatography and displacement chromatography technology, also begin to be applied.
At present, good effects are obtained when the esterifying enzyme technology is applied to aroma enhancement of strong aromatic white spirit, an additional esterifying enzyme aroma enhancement method is used more frequently, and research reports on the esterifying enzyme existing in the yeast for making hard liquor are less. The current research mainly focuses on the separation and screening of monascus, rhizopus and aroma-producing yeast, optimization of enzyme (ester) producing conditions, and research and application of enzymology properties.
Disclosure of Invention
The invention aims to: the method for separating and purifying the esterifying enzyme from the strong aromatic Daqu is provided, the esterifying enzyme is directly separated and purified from the Daqu, the composition type and the molecular weight of the esterifying enzyme are preliminarily explored, a basis is provided for further researching the enzymology property of the esterifying enzyme, and a theoretical basis is provided for the application of the esterifying enzyme in the brewing process of the strong aromatic Chinese spirits.
The technical scheme adopted by the invention is as follows:
a method for separating and purifying esterifying enzyme from strong aromatic yeast comprises the following steps:
s1, smashing the strong-flavor Daqu, and then sieving with a 40-mesh sieve to obtain Daqu powder;
s2, placing the yeast powder obtained in the step S1 in a NaCl solution with the concentration of 0.2-0.4mol/L, pH6, soaking for 1-2h at the temperature of 3-5 ℃, and filtering to obtain a filtrate;
s3, stirring the filtrate obtained in the step S2 at 3-5 ℃, adding ammonium sulfate to ensure that the saturation of the solution reaches 25-35%, stirring for 15-30min, precipitating at 3-5 ℃ for 1.5-2.5h, then carrying out refrigerated centrifugation for 13-15min, taking the supernatant, adding ammonium sulfate to ensure that the saturation of the solution reaches 75-85%, stirring for 15min after the ammonium sulfate is completely dissolved, stirring for 15-30min, precipitating at 3-5 ℃ for 1.5-2.5h, carrying out refrigerated centrifugation for 16-25min, taking the precipitate, adding Tris-HCl buffer solution with the pH of 8.5 for redissolving, and then carrying out centrifugation at 3-5 ℃ to remove the precipitate to obtain a crude enzyme solution A;
s4, stirring the crude enzyme solution A obtained in the step S3 at 3-5 ℃, adding acetone with the volume being 3-4 times that of the crude enzyme solution A and precooled at-20 ℃, standing overnight at-20 ℃, then freezing and centrifuging for 13-15min, discarding supernatant, washing and removing impurities from precipitates, re-dissolving the precipitates by using Tris-HCl buffer solution with the pH value of 8.5, and centrifuging at 3-5 ℃ to remove the precipitates to obtain a crude enzyme solution B;
s5, using 0.02mol/L, pH-8.5 Tris-HCl buffer solution to balance DEAE cellulose DE-52 ion exchange chromatography column, filtering the crude enzyme solution B obtained in the step S4 by a 0.45 mu m microporous membrane, and then carrying out gradient elution to obtain concentrated solution;
s6, adding water into the glucan G-100 gel, boiling for 2 hours, standing and settling, removing supernatant, using the supernatant as a filler for column packing, using 0.02mol/L, pH ═ 8.5Tris-HCl buffer solution with the volume 2-3 times of that of a column bed as an eluent to stabilize the column bed, filtering the concentrated solution obtained in the step S5 through a 0.45-micron microporous filter membrane, and eluting to obtain the glucan G-100 gel.
Further, the volume ratio of the daqu powder to the NaCl solution in step S2 is 1: 5.
Further, centrifugation was performed at 14000rpm in a high-speed refrigerated centrifuge in step S3, and centrifugation was performed at 8000rpm at 4 ℃ in a high-speed refrigerated centrifuge in step S4.
Further, the specific manner of cleaning and removing impurities from the precipitate in step S4 is as follows: the precipitate was added with 80% acetone pre-cooled at-20 ℃ and centrifuged to remove the supernatant.
Further, the specific manner of performing gradient elution in step S5 is as follows: injecting the crude enzyme solution B into a sample injection ring of a protein purifier twice, wherein the volume ratio of the first injection to the second injection is 2:1, and after sample injection is finished, gradient elution is adopted during elution, and the flow rate is 1 mL/min.
Further, the specific manner of performing elution in step S6 is as follows: the concentrate was slowly added to the column and eluted with 0.1mol/L NaCl in 0.02mol/L, pH ═ 8.5Tris-HCl buffer at a flow rate of 0.5 mL/min.
The enzyme activity detection principle of the esterifying enzyme is as follows: the esterifying enzyme can be alpha-naphthyl acetate (C)12H10O2) Hydrolyzing alpha-naphthyl acetate into alpha-naphthol (C) under a proper temperature and acidic environment as a substrate10H8O) and acetic acid, alpha-naphthol and the color reagent fast blue B salt to form purple red azo compound, and the enzyme activity of the esterifying enzyme can be calculated by measuring the change of the light absorption value of the compound.
The method for measuring the content of the esterifying enzyme comprises the following steps: in the test, two methods, i.e., a Coomassie brilliant blue G-250 staining method and an ultraviolet absorption method, are used for measuring the content of the enzyme protein. When the ion exchange chromatography and the gel filtration chromatography need to roughly quantify the content of the enzyme protein in each tube of eluate collected in parts in the separation and purification process, an ultraviolet absorption method is used; the protein content is determined by Bradford staining when it is necessary to determine the rest of the assay. The protein content in the enzyme solution is calculated according to the following formula:
in the formula:
c is the content of the zymoprotein, mu g, obtained by checking a protein standard curve according to the absorbance value; n is the dilution multiple of the enzyme solution; 1 is the total volume of the added sample enzyme solution, mL; 103Is a conversion factor.
The method for measuring the activity of the esterifying enzyme comprises the following steps: adding pH 6.0 and 4.7mL of 0.02M phosphate buffer solution into an esterifying enzyme activity measuring system, transferring 0.1mL of diluted enzyme solution, and adding 10 times diluted alpha-naphthyl acetate ethanol solution (final concentration of 2.5 × 10)-3M)0.2mL, carrying out oscillation reaction at 35 ℃ for 15min, then adding 0.5mL of 0.02% fast blue B salt color development solution to terminate the reaction and develop color, mixing and developing color for 30s, adding 0.5mL of 6mol/L hydrochloric acid, shaking uniformly to stabilize the color development, carrying out color comparison at 537nm wavelength, and taking the inactivated enzyme solution as a blank sample.
Enzyme activity is defined as: under the above reaction conditions, the enzyme amount required for the esterifying enzyme to hydrolyze alpha-naphthyl acetate per minute to obtain 1 μ g of alpha-naphthol is 1 enzyme activity unit, i.e., 1U. The enzyme activity is calculated by the following formula:
in the formula: c. C1The concentration of alpha-naphthol in an enzyme activity detection system is calculated according to an alpha-naphthol standard curve and is mg/mL; n is the dilution multiple of the enzyme solution; v is the volume of enzyme solution added into the enzyme activity detection system, mL; c. C2The protein concentration of the enzyme solution is mg/mL; 5 is the reaction volume of the enzyme activity detection system, mL; 15 is reaction time, min; 103Is a conversion factor.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the method separates and purifies the esterifying enzyme in the yeast for the first time, and obtains the partially pure esterifying enzyme after four purification steps of ammonium sulfate precipitation, acetone precipitation, DEAE cellulose DE-52 ion exchange chromatography and glucan G-100 gel chromatography are carried out on crude enzyme liquid of the esterifying enzyme extracted from the yeast for the first time;
2. the product of the invention is shown as two main bands when the purity of the product is checked by SDS-PAGE, the molecular weight is respectively 37.2kD and 30.8kD, after four-step purification, the esterifying enzyme is purified by 3.49 times, and the recovery rate of the enzyme activity is 28.01 percent;
3. the present invention adopts (NH)4)2SO4Salting out in a segmented way at the saturation of 30-80%, the recovery rate of enzyme activity is higher, and enzyme protein precipitate in the precipitation range is collected;
4. in order to reduce the adverse effect of impurities such as pigment on the ion exchange chromatography efficiency, the invention adopts a method for removing pigment in the precipitate by an organic solvent acetone precipitation method, wherein the pigment is dissolved in an acetone solvent, the enzyme protein is precipitated, and simultaneously, a small amount of ammonium salt remained in the protein can be removed by the acetone precipitate;
5. the invention adopts DE-52 ion exchange chromatography and glucan G-100 gel chromatography to purify the esterifying enzyme in the yeast and obtains the partially pure esterifying enzyme.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a crude enzyme solution ammonium sulfate precipitation curve;
FIG. 2 is an ion exchange chromatogram of NGC esterases;
FIG. 3 is an elution profile of ion exchange chromatography for esterases DE-52;
FIG. 4 is a gel chromatography elution profile of esterifying enzyme dextran G-100;
FIG. 5 is a graph showing the results of the purity of the esterifying enzyme identified by SDS-PAGE.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
Examples
The method for separating and purifying the esterifying enzyme from the strong aromatic yeast provided by the preferred embodiment of the invention comprises the following specific steps:
(1) fully crushing the strong-flavor Daqu by using a crusher, sieving by using a 40-mesh sieve, filtering out larger particles, collecting Daqu powder, and storing the Daqu powder under the conditions of drying and avoiding light for later use.
(2) Taking NaCl solution as an extracting agent, dissolving Daqu powder in NaCl solution with concentration of 0.3mol/L and pH of 6, extracting at 4 deg.C for 1h with a solid-to-liquid ratio of 1:5, filtering, and storing the filtrate in a refrigerator at 4 deg.C.
(3) Adding ammonium sulfate into crude enzyme liquid of the esterifying enzyme in an ice bath at 4 ℃ under the condition of rotating a magnetic stirrer, leading the saturation degree to reach 30%, slowly stirring until the ammonium sulfate is completely dissolved, then stirring for 15min, putting the mixture into a refrigerator at 4 ℃ for precipitation for 2h, centrifuging the mixture in a high-speed refrigerated centrifuge at 14000rpm for 15min, taking supernatant fluid and discarding the precipitate, repeating the same operation method, adding the ammonium sulfate to lead the saturation degree to reach 80%, slowly stirring until the ammonium sulfate is completely dissolved, then stirring for 15min, putting the mixture into the refrigerator at 4 ℃ to lead target enzyme protein to be fully precipitated, centrifuging the mixture in the high-speed refrigerated centrifuge at 14000rpm for 20min after the precipitation is finished, collecting the precipitate, redissolving the precipitate by using Tris-HCl buffer solution with the pH being 8.5, and centrifuging the precipitate at 4 ℃ to remove insoluble precipitate, thus obtaining the crude enzyme liquid A for next acetone precipitation.
The recovery of enzyme activity was plotted against the saturation of ammonium sulfate, and the results are shown in FIG. 1. As can be seen from FIG. 1, when the crude enzyme solution is (NH)4)2SO4When the saturation is lower than 30%, the recovery rate of the activity of the precipitating enzyme is less than 5%, which indicates that only a very small amount of the target enzyme protein is precipitated, and most of the target enzyme protein is kept in the supernatant; with (NH)4)2SO4The saturation degree is increased, and the recovery rate of enzyme activity is increased along with the saturation degree, Namely (NH)4)2SO4When the saturation reaches 80%, the recovery rate of the enzyme activity reaches the maximum, namely 90.67%, which indicates that the target enzyme protein is mainly in the precipitate; thereafter (NH)4)2SO4Saturation degree is further increased, and the recovery rate of enzyme activity is reduced on the contrary, which is shown in (NH)4)2SO4Other hybrid proteins are co-precipitated with target enzyme proteins in the process of further increasing the saturation degree, so that the enzyme activity is recoveredThe rate decreased, indicating that the enzyme protein of interest is in (NH)4)2SO4At a saturation of 80%, the precipitation was essentially complete. So that (NH) is finally determined4)2SO4The fractional salting-out range is 30-80% of saturation, the zymoprotein precipitate in the precipitation range is collected, redissolved by Tris-HCl buffer solution with pH 8.5, and then the insoluble precipitate is removed by centrifugation at 4 ℃ to obtain crude zymofluid A.
(4) Slowly adding 3 times volume of pre-cooled acetone at-20 deg.C into the crude enzyme solution A in ice bath at 4 deg.C under the condition of rotation of magnetic stirrer, standing overnight at-20 deg.C, centrifuging at 8000rpm in a high-speed refrigerated centrifuge for 15min, and discarding supernatant; and adding a small amount of acetone pre-cooled to 80% at the temperature of-20 ℃ to re-suspend the zymoprotein precipitate, and centrifuging to wash out impurities remained on the zymoprotein, wherein the operation is carried out twice. The precipitate was collected and redissolved with Tris-HCl buffer solution at pH 8.5, and the insoluble precipitate was centrifuged off at 4 ℃ to give crude enzyme solution B for the next ion exchange chromatography.
During the preparation of the crude enzyme solution, the crude enzyme filtrate is found to be coffee, and pigments which enable the crude enzyme filtrate to be coffee are derived from the raw material of the yeast, and the pigments enter the solution in the process of extracting the esterifying enzyme; in the ammonium sulfate precipitation test process, the pigments are also found to be precipitated together with enzyme protein, the colors of the pigments still exist in enzyme liquid after redissolution, in order to reduce the adverse effect of impurities such as the pigments on the ion exchange chromatography efficiency, a method for removing the pigments in the precipitate by using an organic solvent acetone precipitation method is adopted, the pigments are dissolved in an acetone solvent, the enzyme protein is precipitated, and simultaneously, a small amount of ammonium salt remained in the protein is removed by using the acetone precipitate; after centrifugation, acetone precipitate is collected and dissolved again to be used as a sample loading liquid during DE-52 ion exchange chromatography, and the enzyme activity recovery rate of the step of acetone precipitation is 45.40 percent.
(5) The DEAE cellulose DE-52 filler was pretreated.
(6) Taking 9 small test tubes with scales, and numbering the test tubes to be 1-9; respectively filling the processed DEAE cellulose into 9 small test tubes, rinsing the test tube No. 1 with 10mL of citric acid-sodium citrate buffer solution (0.1mol/L) with pH5.0 for 10 times, adding 2mL of buffer solution with the same pH value, and slightly stirring the filler to fully balance the filler; sequentially increasing the pH value of the buffer solution used by each test tube by 0.5 unit, operating by the same method until the pH value of the buffer solution reaches No. 9 tube, and finishing the balance; adding 0.2mL of acetone into each test tube to precipitate the crude enzyme solution, standing for 2h, sucking the supernatant to measure the residual enzyme activity, and determining the pH value of the ion exchange chromatography buffer solution.
The results of the determination of the pH of the buffer for DE-52 ion exchange chromatography are given in Table 1 below. As can be seen from Table 1, the enzyme activity of the supernatant is decreased with the increase of pH value, when the pH value is 8.5, the enzyme activity is decreased to 0, which indicates that the esterifying enzyme in the crude enzyme solution is completely combined with the DE-52 ion exchanger, and then the pH value is increased, the enzyme activity is still 0, in order to ensure that the enzyme protein to be separated can be maximally combined with the ion exchanger filler under the proper pH environment and can be completely eluted under the proper salt ion concentration condition, the balance in the DE-52 ion exchange chromatography and the pH value of the buffer solution in the elution are selected to be 8.5 according to the test results.
TABLE 1 enzymatic Activity of supernatants at different pH values
(7) Closing a valve at the lower end of the Econo-Colum (20 multiplied by 1.8cm) ion exchange column, adding a small amount of deionized water, slightly stirring the processed DEAE-52 filler, conducting drainage by using a glass rod, filling the filler into the Econo-Colum (20 multiplied by 1.8cm) ion exchange column at one time, naturally settling the filler uniformly without cracks, opening the valve at the lower end of the column, and allowing water higher than the surface of the filler to flow out from the lower end until the liquid level is about 0.5cm higher than the filler; and starting the NGC Quest10 protein purification instrument, connecting the whole flow path, controlling the proper water flow speed of the whole flow path, observing whether the filler can descend, and if the filler can descend, continuing to follow the adapter until the filler can not descend any more, wherein the whole filler layer is uniform and consistent, has no crack and is completely sealed.
(8) Using 0.02mol/L Tris-HCl buffer solution with pH of 8.5 to balance DEAE cellulose DE-52 ion exchange chromatography column, switching the flow path to Inject mode, using a special injector for protein purification to Inject 6mL acetone precipitation crude enzyme liquid filtered by a 0.450m microporous membrane into the sample injection loop of the protein purification instrument in two times, wherein the first time is 4mL, and the second time is 2 mL. And after the sample introduction is finished, the flow path is switched to a Load mode, gradient elution is adopted during elution, the flow rate is lmL/min, and 2mL of solution is collected in each tube.
The results obtained from DE-52 ion exchange chromatography using an NGC Quest10 protein purifier are shown in FIG. 2, which is a map of the entire ion exchange chromatography process shown by ChromLab control software attached to an NGC Quest10 protein purifier: the method comprises the steps of flushing the whole flow path (an ion exchange column is connected in the flow path) by double distilled water, balancing the ion exchange column by using a pH 8.5Tris-HCl buffer solution, continuously passing the buffer solution through the column after crude enzyme liquid is loaded to wash off enzyme protein which is not hung on the column, and eluting target enzyme protein which is combined with DE-52 ionic agent by using a linear gradient of 0.02mol/L of 0-1mol/L NaCl and a pH 8.5Tris-HCl buffer solution. FIG. 3 is an elution curve obtained by detecting A280 and enzyme activity from the 1 st tube collected from the start of elution when the enzyme protein solution is eluted to the 30 th tube collected after the end of linear gradient elution (corresponding to the 21 st to 50 th tubes of NGC chromatogram) according to the chromatogram in FIG. 2. A total of 50 tubes (from flow-through to elution of the enzyme protein solution to the end of linear gradient elution) were collected by DE-52 ion exchange chromatography, with the first 20 tubes corresponding to flow-through peaks and the last 30 tubes corresponding to elution peaks. 20 tubes corresponding to flow-through peaks are combined, the enzyme activity of the detected esterifying enzyme is very low, and only 1.10 percent of the highest enzyme activity is obtained during elution, so that few target enzyme proteins without columns are obtained. When only one elution peak exists during the process of eluting target enzyme protein by using a 0.02mol/L and 8.5 pH Tris-HCl buffer solution containing 0-1mol/L NaCl in a linear gradient manner, through enzyme activity detection, the eluted enzyme solution with high enzyme activity of the esterifying enzyme is mainly concentrated between 27 th and 38 th tubes corresponding to the elution peak, the eluted solutions with high enzyme activity are combined, put into a dialysis bag to dialyze for 24 hours by using a vacuum freeze drying method, and then re-dissolved by using a small amount of 0.02mol/L and 8.5 pH Tris-HCl buffer solution for next step of gel chromatography of the glucan G-100.
(9) Weighing a proper amount of glucan G-100 gel, placing the glucan G-100 gel in a beaker, adding deionized water, boiling in a boiling water bath for 2 hours, standing until the glucan G-100 gel is completely settled, discarding the supernatant to remove suspended particles in the supernatant, and obtaining the glucan G-100 gel filler.
(10) Clamping a pipeline at the lower end of the gel filtration column by using a water stop clamp, adding a small amount of deionized water, gently stirring the treated glucan G-100 gel filler, draining by using a glass rod, filling the gel filler into the gel filtration column at one time, naturally and uniformly settling the filler, if the height of the filler is not enough, gently stirring the surface of the filler, continuously introducing the filler by using the glass rod until the filler is settled to the required height and has no cracks, and then finishing column filling.
(11) The gel packing is balanced by 0.02mol/L and pH 8.5Tris-HCl buffer solution for 2-3 times of the volume of the column bed, excessive balance buffer solution in the gel column is sucked by a suction pipe until the liquid level is about 1cm higher than the surface of the packing, DE-52 ion exchange is carried out to obtain 3mL of protein dialysis concentrated solution with enzyme activity (filtered by a 0.45 mu m microporous filter membrane), the protein dialysis concentrated solution is slowly added into the column by a dropper, 0.02mol/L NaCl containing 0.1mol/L and the Tris-HCl buffer solution with pH 8.5 mL/min are used for elution, and each tube contains 2 mL.
The eluate with esterase activity obtained by DE-52 ion exchange chromatography was dialyzed, concentrated and purified by Sephadex G-100 gel chromatography, and the result is shown in FIG. 4. As can be seen from FIG. 4, 54 tubes of eluates were collected by Sephadex G-100 gel chromatography, and there were two protein peaks in total, one larger protein peak and one smaller protein peak, and the enzyme activity was mainly concentrated in the 2 nd to 9 th tubes corresponding to the larger protein peak, and after combining, freeze-drying and concentrating, the purity was identified by SDS-PAGE.
Examples of the experiments
(1) Enzyme purity identification and enzyme molecular weight determination:
cleaning and airing the glass plate, and installing the glass plate according to the Mini-PROTECTAN 3 small vertical electrophoresis tank instruction; respectively filling 12% of separation glue and 5% of concentrated glue, and inserting into a sample application comb; after the zymoprotein sample liquid is processed, carefully pulling out the sample application comb, and respectively sucking 6 mu L of sample liquid to be analyzed by a microsyringe into sample application holes; after the sample application is finished, switching on a power supply, using 10mA direct current at the beginning, adding large current to 18mA after the sample is concentrated into a line in the concentrated glue part, and stopping electrophoresis when the bromophenol blue indicator reaches the bottom edge; after electrophoresis is finished, taking out the gel, putting the gel into a large culture dish, dyeing for 30min by using Coomassie Brilliant blue R-250 liquid, taking out the gel, repeatedly decoloring for a plurality of hours by using decoloring liquid until background color is transparent, and imaging the gel;
the molecular weight of the bands obtained by SDS-PAGE was analyzed by using quality one software from Bio-Rad.
After the active target enzyme solution collected by the dextran G-100 gel chromatography is frozen, dried and concentrated, the purity is identified, and the result is shown in figure 5, wherein M-marker is shown in the figure; 1-crude enzyme solution (reduction); 2-ammonium sulfate precipitation (reduction); 3-acetone precipitation (reduction); 4-anion exchange chromatography of the target enzyme concentrate (reduction); 5-gel chromatography of the enzyme concentrate of interest (non-reducing); 6-gel chromatography of the concentrated solution of the target enzyme (reduction).
As seen from FIG. 5, the esterases were electrophoresed after five steps of crude extraction, ammonium sulfate precipitation, acetone precipitation, DE-52 ion exchange chromatography, and dextran G-100 gel chromatography to finally show two main bands (shown by arrows in the figure), and no single band was obtained, i.e., the esterases present in the koji were not completely separated. From the results in the figure, the esterases isozymes which play the same catalytic function do exist in the yeast, which is similar to the distribution of esterases on the enzyme spectrum in the fen-flavor yeast. The samples collected by Sephadex G-100 gel chromatography in lanes 5 and 6 were subjected to SDS-PAGE, wherein the larger of the two major bands showed a difference in size from the results of the previous four steps, and the smaller of the two major bands showed the same results (at approximately the same electrophoretic position) as the results of the previous four steps, and the two major bands were analyzed by Quantitone software to have a size of 37.2kD and 30.8kD, respectively. The two main purification methods (DE-52 ion exchange chromatography and Sephadex G-100 gel chromatography) failed to separate the single component esterases, which may be related to the similar properties or molecular weights of some esterases in Daqu, as demonstrated by the elution profiles of DE-52 ion exchange chromatography and Sephadex G-100 gel chromatography: DE-52 ion exchange chromatography has only one elution peak, the enzyme activity is concentrated in the middle and rear sections of the elution peak, and the enzyme activity of the rest part is very low; the enzyme activity peak and the protein peak in the glucan G-100 gel chromatography elution curve are not completely coincident, other foreign proteins can be carried in target enzyme protein components, and the influence of the pH value and the ionic strength of a buffer solution during balance and elution on purification is also considered. By combining the above factor analysis, the test conditions need to be changed, and the method of performing hydrophobic chromatography or affinity chromatography by using different hydrophobic properties of the esterifying enzyme is considered to obtain single esterifying enzyme with different electrophoretic purities in the yeast.
In summary, the total protein, total enzyme activity, specific activity, purification fold and recovery rate of each of the five steps of crude esterification enzyme extraction, ammonium sulfate precipitation, acetone precipitation, DE-52 ion exchange chromatography and glucan G-100 gel chromatography were calculated, and the results are shown in Table 2 below:
TABLE 2 separation and purification results of esterases
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A method for separating and purifying esterifying enzyme from strong aromatic yeast is characterized by comprising the following steps:
s1, smashing the strong-flavor Daqu, and then sieving with a 40-mesh sieve to obtain Daqu powder;
s2, placing the yeast powder obtained in the step S1 in a NaCl solution with the concentration of 0.2-0.4mol/L, pH6, soaking for 1-2h at the temperature of 3-5 ℃, and filtering to obtain a filtrate;
s3, stirring the filtrate obtained in the step S2 at 3-5 ℃, adding ammonium sulfate to ensure that the saturation of the solution reaches 25-35%, stirring for 15-30min, precipitating at 3-5 ℃ for 1.5-2.5h, then carrying out refrigerated centrifugation for 13-15min, taking the supernatant, adding ammonium sulfate to ensure that the saturation of the solution reaches 75-85%, stirring for 15min after the ammonium sulfate is completely dissolved, stirring for 15-30min, precipitating at 3-5 ℃ for 1.5-2.5h, carrying out refrigerated centrifugation for 16-25min, taking the precipitate, adding Tris-HCl buffer solution with the pH of 8.5 for redissolving, and then carrying out centrifugation at 3-5 ℃ to remove the precipitate to obtain a crude enzyme solution A;
s4, stirring the crude enzyme solution A obtained in the step S3 at 3-5 ℃, adding acetone with the volume 3-4 times that of the crude enzyme solution A and precooled at-20 ℃, standing overnight at-20 ℃, then freezing and centrifuging for 13-15min, discarding supernatant, washing and removing impurities from the precipitate, re-dissolving the precipitate with Tris-HCl buffer solution with the pH value of 8.5, and centrifuging at 3-5 ℃ to remove the precipitate to obtain crude enzyme solution B;
s5, using 0.02mol/L, pH-8.5 Tris-HCl buffer solution to balance DEAE cellulose DE-52 ion exchange chromatography column, filtering the crude enzyme solution B obtained in the step S4 by a 0.45 mu m microporous membrane, and then carrying out gradient elution to obtain concentrated solution;
s6, adding water into the glucan G-100 gel, boiling for 2 hours, standing and settling, removing supernatant, using the supernatant as a filler for column packing, using 0.02mol/L, pH ═ 8.5Tris-HCl buffer solution with the volume 2-3 times of that of a column bed as an eluent to stabilize the column bed, filtering the concentrated solution obtained in the step S5 through a 0.45-micron microporous filter membrane, and eluting to obtain the glucan G-100 gel.
2. The method for separating and purifying esterases from Luzhou-flavor yeast as claimed in claim 1, wherein: the volume ratio of the Daqu powder to the NaCl solution in the step S2 is 1: 5.
3. The method of claim 1, wherein the step S3 is performed in a high-speed refrigerated centrifuge at 14000rpm, and the step S4 is performed in a high-speed refrigerated centrifuge at 8000rpm and 4 ℃.
4. The method for separating and purifying esterifying enzyme from Luzhou-flavor Daqu according to claim 1, wherein the specific manner of washing and removing impurities from the precipitate in the step S4 is as follows: the precipitate was added with 80% acetone pre-cooled at-20 ℃ and centrifuged to remove the supernatant.
5. The method for separating and purifying esterifying enzyme from Luzhou-flavor Daqu according to claim 1, wherein the gradient elution in step S5 is performed by: injecting the crude enzyme solution B into a sample injection ring of a protein purifier twice, wherein the volume ratio of the first injection to the second injection is 2:1, and after sample injection is finished, gradient elution is adopted during elution, and the flow rate is 1 mL/min.
6. The method for separating and purifying esterases from Luzhou-flavor Daqu according to claim 1, wherein the elution in step S6 is performed in a specific manner: the concentrate was slowly added to the column and eluted with 0.1mol/L NaCl in 0.02mol/L, pH ═ 8.5Tris-HCl buffer at a flow rate of 0.5 mL/min.
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