阅读说明：本技术 基于宏蛋白质组学技术对浓香大曲进行酶系结构解析的方法 (Method for carrying out enzyme system structure analysis on aroma Daqu based on macroproteomics technology ) 是由 赵鑫锐 李江华 范伟业 施思 赵东 乔宗伟 郑佳 于 2021-01-21 设计创作，主要内容包括：本发明属于宏蛋白质组学技术领域,具体涉及一种基于宏蛋白质组学技术对浓香大曲进行酶系结构解析的方法。针对目前还未见有对浓香大曲中酶种类和酶成分的具体解析的方法的问题,本发明提供了一种基于宏蛋白质组学技术对浓香大曲进行酶系结构解析的方法,通过建立浓香大曲粗酶液和宏蛋白质组样品的提取方法以及浓香大曲酶系结构的分析方法,实现了应用宏蛋白质组学对浓香大曲酶系组成的解析,解决了之前的研究缺少对浓香大曲未知酶系的深入解析的问题。本发明为白酒中风味物质的解析提供了理论基础,具有重要的意义。(The invention belongs to the technical field of macroproteomics, and particularly relates to a method for carrying out enzyme system structure analysis on aroma yeast based on the macroproteomics technology. Aiming at the problem that a method for specifically analyzing the enzyme types and the enzyme components in the aroma Daqu is not available at present, the invention provides a method for analyzing the enzyme system structure of the aroma Daqu based on a macroproteomics technology, and the analysis of the aroma Daqu enzyme system composition by applying the macroproteomics is realized by establishing an extraction method of crude enzyme liquid and a macroproteinaceous sample of the aroma Daqu and an analysis method of the aroma Daqu enzyme system structure, so that the problem that the prior research lacks deep analysis of the unknown enzyme system of the aroma Daqu is solved. The method provides a theoretical basis for the analysis of flavor substances in the white spirit, and has important significance.)

1. The method for carrying out enzyme system structure analysis on the aroma Daqu based on the macro-proteomics technology is characterized by comprising the following steps of:

a. extracting a thick fragrant yeast crude enzyme solution and measuring various enzyme activities in the crude enzyme solution; the specific operation of extracting the crude enzyme solution is as follows: crushing the aromatic Daqu, and extracting with Tris-HCl as a solvent, wherein the adding amount of the Daqu and the Tris-HCl solvent is 1: 5-10, the pH value in the extraction process is 5.0-7.0, and the extraction time is 0.5-2 h;

b. extracting a macro protein sample and analyzing by adopting a macro proteomics technology;

the extraction method comprises the following specific operation steps: adding a BPP extracting solution into a Daqu sample, shaking, centrifuging, taking supernatant, adding a Tris-saturated phenol solution with the same volume, shaking again and centrifuging to obtain a phenol layer containing protein, adding the BPP extracting solution with the same volume, shaking, centrifuging to take supernatant, adding an ammonium acetate methanol solution with the same volume, precipitating the protein overnight at the temperature of-20 ℃, adding acetone into the precipitate, centrifuging to remove supernatant, and dissolving the protein by adopting a lysate to obtain a sample to be detected;

enzymolysis of the extracted sample: adding dithiothreitol into the extracted protein, and incubating for 1h at 37 ℃; cooling to room temperature, adding iodoacetamide, and standing at room temperature for 30 min; adding pancreatin at 37 deg.C overnight, adding formic acid to stop enzyme digestion, collecting digested fraction, and storing at-80 deg.C for MS analysis;

c. and comparing the macro-proteomics technology analysis result with the enzyme activity determination result.

2. The method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology according to claim 1, wherein the method comprises the following steps: the enzyme in step a comprises: at least one of an alpha-amylase, a protease, a cellulase or a pectinase.

3. The method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology according to claim 1, wherein the method comprises the following steps: the ratio of the Daqu sample to the BPP extract added in the step b is 0.4 g: 1 mL.

4. The method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology according to claim 1, wherein the method comprises the following steps: and c, the lysis solution in the step b is lysis solution containing 8M urea.

5. The method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology according to claim 1, wherein the method comprises the following steps: the final concentration of dithiothreitol in step b is 10 mM.

6. The method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology according to claim 1, wherein the method comprises the following steps: the final concentration of iodoacetamide in step b is 30 mM.

7. The method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology according to claim 1, wherein the method comprises the following steps: the adding amount of the pancreatin in the step b is as follows: the mass ratio of pancreatin to protein is 1: 25.

8. The method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology according to claim 1, wherein the method comprises the following steps: the concentration of formic acid in step b is 0.1%.

Technical Field

The invention belongs to the technical field of macroproteomics, and particularly relates to a method for carrying out enzyme system structure analysis on aroma yeast based on the macroproteomics technology.

Background

White spirit is the most globally consumed strong spirit, and it and vodka, brandy, whisky are called world's four major distilled spirits. White spirit can be divided into three main types according to the difference of flavor characteristics: strong white spirit, sauce-flavor white spirit and delicate fragrance white spirit. The fermentation of white spirit adopts solid saccharification fermentation process for thousands of years, and the core of the process lies in its leaven-distiller's yeast, which is used to provide microorganisms and enzymes for the whole brewing process, and the microorganisms and enzymes have important effect on the formation of various flavor substances in white spirit. The yeast used for brewing the aroma-enriched white spirit is aroma-enriched yeast, and many researches are carried out on the aroma-enriched yeast in the industry, but the composition of an enzyme system in the aroma-enriched yeast is not substantially disclosed. The current research on the aroma Daqu is mainly focused on two aspects. Firstly, the method focuses on the detection of enzyme activity of the strong aromatic yeast and generally detects enzymes commonly seen in the yeast. Secondly, the dynamic analysis of the microbial community in the aroma Daqu is to analyze the microbial composition and change of the aroma Daqu from the microbial level. However, further studies on the types of enzymes in the koji and the enzyme activities of various enzymes are lacking, and development is yet to be made.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: at present, no specific analysis method for enzyme types and enzyme components in the aroma Daqu exists, and the effect of the aroma Daqu in liquor brewing cannot be determined from the level of the enzyme.

The technical scheme for solving the technical problems comprises the following steps: provides a method for carrying out enzyme system structure analysis on aroma Daqu based on a macroproteomics technology. The method comprises the following steps:

a. extracting a thick fragrant yeast crude enzyme solution and measuring various enzyme activities in the crude enzyme solution; the specific operation of extracting the crude enzyme solution is as follows: crushing the aromatic Daqu, and extracting with Tris-HCl as a solvent, wherein the adding amount of the Daqu and the Tris-HCl solvent is 1: 5-10, the pH value in the extraction process is 5.0-7.0, and the extraction time is 0.5-2 h;

b. extracting a macro protein sample and analyzing by adopting a macro proteomics technology;

the extraction method comprises the following specific operation steps: adding a BPP extracting solution into a Daqu sample, shaking, centrifuging, taking supernatant, adding a Tris-saturated phenol solution with the same volume, shaking again and centrifuging to obtain a phenol layer containing protein, adding the BPP extracting solution with the same volume, shaking, centrifuging to take supernatant, adding an ammonium acetate methanol solution with the same volume, precipitating the protein overnight at the temperature of-20 ℃, adding acetone into the precipitate, centrifuging to remove supernatant, and dissolving the protein by adopting a lysate to obtain a sample to be detected;

enzymolysis of the extracted sample: adding dithiothreitol into the extracted protein, and incubating for 1h at 37 ℃; cooling to room temperature, adding iodoacetamide, and standing at room temperature for 30 min; adding pancreatin at 37 deg.C overnight, adding formic acid to stop enzyme digestion, collecting digested fraction, and storing at-80 deg.C for MS analysis;

c. and comparing the macro-proteomics technology analysis result with the enzyme activity determination result.

Wherein, in the method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology, the enzyme in the step a comprises the following steps: at least one of an alpha-amylase, a protease, a cellulase or a pectinase.

Wherein, in the method for carrying out enzyme system structure analysis on the aroma Daqu based on the macro-proteomics technology, the ratio of the Daqu sample added in the step b to the BPP extracting solution is 0.4 g: 1 mL.

In the method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology, the lysis solution in the step b is lysis solution containing 8M urea.

Wherein, in the method for carrying out enzyme system structure analysis on the aroma Daqu based on the macro-proteomics technology, the final concentration of the dithiothreitol in the step b is 10 mM.

Wherein, in the method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology, the final concentration of the iodoacetamide in the step b is 30 mM.

In the method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology, the adding amount of pancreatin in the step b is as follows: the mass ratio of pancreatin to protein is 1: 25.

Wherein, in the method for analyzing the enzyme system structure of the aroma Daqu based on the macro-proteomics technology, the concentration of the formic acid in the step b is 0.1%.

The invention has the beneficial effects that:

the invention successfully establishes the extraction method of the aroma Daqu crude enzyme solution and the macro-proteome sample and the analysis method of the aroma Daqu enzyme system structure, realizes the analysis of the aroma Daqu enzyme system composition by applying macro-proteomics, and solves the problem that the previous research lacks deep analysis of the aroma Daqu unknown enzyme system. Through GO and KEGG biological function analysis, the prediction of the function of enzyme in the strong aromatic Daqu and the participation of metabolic pathway is realized, the result provides clues for further understanding the irreplaceable function of the Daqu in liquor brewing, and lays a foundation for analyzing the mechanism of the formation of the characteristic flavor substances of the strong aromatic liquor.

Drawings

FIG. 1 shows the measurement results of the enzyme activities of common enzymes in the aroma yeast;

FIG. 2 shows the effect of protein extraction in different combinations;

FIG. 3 shows GO analysis results of the aroma Daqu enzyme system;

FIG. 4 shows the results of KEGG pathway analysis of the aroma Daqu enzyme system.

Detailed Description

The invention provides a method capable of analyzing the types of proteins in aromatic Daqu and tracing the species sources of the proteins, which analyzes the enzyme system structure of the aromatic Daqu through a macro-proteomics technology. All proteins in the strong aromatic Daqu are classified into three types of plants, animals and microorganisms. Microbial proteins are further divided into eukaryotic and prokaryotic microbial proteins. According to the invention, eukaryotic microorganisms and prokaryotic microorganisms with the highest protein content and corresponding protein abundance in the aroma yeast are analyzed through species composition analysis of proteins (through SWISS and Uniprot database annotation on mass spectrum data, the ID, name and source of the proteins can be searched, then the sources of all the proteins are further counted, and organisms from the same level are classified into one class), and the protein abundance is 36.81%.

The method of the invention not only can singly analyze the species composition of the protein in the strong aromatic Daqu, but also can be applied to the difference research of the species compositions of different types of Daqu proteins.

Specifically, the method for carrying out enzyme system structure analysis on the aroma Daqu based on the macro-proteomics technology comprises the following steps:

a. extracting a thick fragrant yeast crude enzyme solution and measuring various enzyme activities in the crude enzyme solution; the specific operation of extracting the crude enzyme solution is as follows: crushing the aromatic Daqu, and extracting with Tris-HCl as a solvent, wherein the adding amount of the Daqu and the Tris-HCl solvent is 1: 5-10, the pH value in the extraction process is 5.0-7.0, and the extraction time is 0.5-2 h; the enzyme comprises at least one of alpha-amylase, protease, cellulase or pectinase;

b. extracting a macro protein sample and analyzing by adopting a macro proteomics technology;

the extraction method comprises the following specific operation steps: adding a BPP extracting solution (the proportion of the two is 0.4 g: 1mL) into a Daqu sample, shaking, centrifuging, taking supernatant, adding a Tris-saturated phenol solution with the same volume, shaking and centrifuging again to obtain a phenol layer containing protein, adding a BPP extracting solution with the same volume, shaking and centrifuging to take supernatant, adding an ammonium acetate methanol solution with the same volume, precipitating the protein overnight at the temperature of-20 ℃, adding acetone into the precipitate, centrifuging to remove the supernatant, and dissolving the protein by using a lysis solution containing 8M urea to obtain a sample to be detected;

enzymolysis of the extracted sample: directly taking the extracted protein, and putting 50 mu g of protein per sample solution into an EP (ethylene propylene glycol) tube; dithiothreitol (10mM IAA (which is iodoacetamide) in H) was added to a final concentration of 10mM₂O), incubating for 1h at 37 ℃. Cooled to room temperature and added with IAA (50mM IAA in H) to a final concentration of 30mM₂O), standing at room temperature for 30 min. Pancreatin (pancreatin: protein 1:25) was added at 37 ℃ overnight. The next day, 50. mu.L of 0.1% formic acid was added to stop the digestion, and the digested fractions were collected and stored at-80 ℃ for MS analysis.

c. And comparing the macro-proteomics technology analysis result with the enzyme activity determination result.

The invention can also analyze all enzymes in the strong aromatic yeast and analyze the action of the enzymes through biological function. All enzymes in the strong aromatic yeast are also divided into plant enzymes, animal enzymes, eukaryotic microbial enzymes and prokaryotic microbial enzymes, namely, each enzyme can be traced back to a source organism. All enzymes in the strong aromatic Daqu are systematically divided into seven major classes according to an EC number classification system: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases and translocation enzymes. The measurement results of the enzyme activities of common enzymes in the aroma yeast are shown in figure 1, and the protein extraction effects of different combination modes are shown in figure 2.

Further, the biological functions of the enzymes in the aroma Daqu are analyzed through GO database analysis. The GO analysis comprises three categories of biological process, cellular component and molecular function, and all enzymes enriched in each large category can be respectively found out. The GO analysis results of the aroma Daqu enzyme system are shown in fig. 3.

In addition, the invention analyzes the metabolic pathway of the aromatic Daqu in which the enzyme participates through analysis of the KEGG database, and can find out the most main metabolic process in which the enzyme in the aromatic Daqu participates synergistically. The results of the KEGG pathway analysis of the aroma Daqu enzyme system are shown in fig. 4.

The invention utilizes macro-proteomics to prove that the strong aromatic Daqu has the capability of synthesizing certain typical flavor substances of the strong aromatic white spirit from the perspective of enzyme. The key enzyme of valine, which is an important prerequisite for synthesizing isobutanol, is found in the results of macro proteomics, and key enzymes related to valine synthesis comprise ketol-acid reductoisomerase (EC:1.1.1.86), dihydroxy-acid dehydratase (EC:4.2.1.9) and bridged-chain amino aminotransferase (EC: 2.6.1.42). The present inventors have found that lactate dehydrogenases include L-lactate dehydrogenases (EC:1.1.1.27) and D-lactate dehydrogenases (EC: 1.1.2.4). In addition, the present inventors have found a key enzyme 3-hydroxy butyryl-CoA dehydrogenase (EC:1.1.1.157) in the pathway of synthesizing butyric acid, an important precursor for synthesizing ethyl hexanoate. One of the purposes of analyzing the structure of the strong aromatic Daqu enzyme system is to establish a relationship with the flavor substances of the strong aromatic white spirit, find an enzyme related to the synthesis of important flavor substances in a macro-proteomics result, and prove that the enzyme system in the strong aromatic Daqu has an important role in forming the flavor substances of the strong aromatic white spirit.

The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.

The reagents used in the examples are all common commercial products.

EXAMPLE 1 optimization of crude enzyme solution extraction method

Breaking Daqu, extracting with Tris-HCL as solvent, centrifuging the extract twice to obtain crude enzyme solution, and detecting enzyme activity. When the pH value of Tris-HCL is 7, the leaching time is 2 hours, the leaching effect is best when the feed-liquid ratio is 1: 5, and the total protein concentration of the leaching liquor is 285.68 mu g/mL at most, which is obviously higher than that of other optimized combination modes.

The extracted crude enzyme liquid is used for detecting various enzyme activities, the alpha-amylase is determined by adopting a reducing sugar determination method-DNS determination method, and the measurement result shows that the alpha-amylase activity of superior aroma Daqu (PBBQ) is 86.98 +/-1.14U/g, and the enzyme activity of common aroma Daqu (NBBQ) is 83.43 +/-2.61U/g;

the protease activity is determined according to SB _ T10317-1999, the protease activity of PBBQ is 360.45 + -2.62U/g, and the protease activity of NBBQ is 213.92 + -10.49U/g;

cellulase activity was determined according to QB 2583-2003; the cellulase activity of PBBQ is 187.95 plus or minus 2.74U/g, and the cellulase activity of NBBQ is 56.19 plus or minus 0.78U/g;

the pectase activity is determined by adopting a reducing sugar determination method-DNS determination method, the pectase activity of PBBQ is 17.41 +/-0.27U/g, and the pectase activity of NBBQ is 10.73 +/-0.38U/g.

Example 2 establishment of a Macroproteome sample extraction method

Taking out the Daqu sample stored at a proper temperature, adding a proper amount of Daqu sample into an MP shaking tube, then adding a proper amount of BPP extracting solution, and adding a proper amount of grinding beads. Shaking by using a high-throughput tissue grinder, centrifuging under corresponding conditions after shaking to obtain supernatant, transferring the supernatant in 3 MP shaking tubes to the same centrifugal tube, adding a Tris-saturated phenol solution with the same volume, shaking and centrifuging again to obtain a phenol layer containing protein, adding an extracting solution with the same volume, then shaking and centrifuging to obtain supernatant containing protein, adding a precooled ammonium acetate methanol solution with the same volume, and precipitating the protein overnight at-20 ℃; and centrifuging to remove the supernatant in a shaking way on the second day, adding precooled acetone into the precipitate, uniformly mixing, centrifuging to remove the supernatant, and repeating the operation twice. After the lysate is used for dissolving the protein sample, protein concentration determination and SDS-PAGE operation show that the macro-proteome sample extracted by the method meets the requirement of mass spectrum analysis.

Example 3 enzymatic digestion and Mass Spectrometry of Macroproteome samples

Enzymolysis of the macroprotein sample: after protein quantification, 50. mu.g of protein per sample solution was placed in an EP tube; DTT (dithiothreitol) (10mM IAA (iodoacetamide) in H) was added to a final concentration of 10mM₂O), incubating for 1h at 37 ℃. Cooled to room temperature and added with IAA (50mM IAA in H) to a final concentration of 30mM₂O), standing at room temperature for 30 min. Pancreatin (pancreatin: protein 1:25) was added at 37 ℃ overnight. The next dayThe digestion was stopped by adding 50. mu.L of 0.1% FA formic acid, and the digested fractions were collected and stored at-80 ℃ for MS analysis.

LC-MS/MS analysis: each fraction was resuspended in buffer a (0.1% formic acid, FA). Separation was performed using an EASY-nLC 1000 system (Thermo Fisher Scientific, USA) and Orbitrap Fusion (Thermo Fisher Scientific, USA) mass spectrometer. The peptide was passed through a C18 trap column (3um, 0.1X 20mm) at a flow rate of 0.3. mu.L/min. Peptides were desalted on-line and loaded onto a C18 column (1.9um, 150um x 120mm) for 120 min using a gradient of 1% -96% buffer B (0.08% FA and 80% ACN). The mass spectrometer is operated in positive mode using a data-dependent acquisition method. A full MS scan (250-. All MS/MS spectral data (RAW files) were searched in the Unit-gallon FASTA database using Maxquant and Proteome scanner software (Thermo Fisher Scientific). The mass tolerance of the precursor for the search was 20ppm and the mass tolerance of the fragment ions was + -0.5 Da. The largest missing cleavage sites were two, the enzyme specificity was trypsin, and the search was performed under variable modifications of oxidation (M) and acetyl (protein N-terminal) to set the cysteine carbamoylmethylation as a fixed modification.

Example 4 species composition of koji and analysis of biological function of enzymes

After the species composition analysis of the source of the aromatic Daqu protein by eliminating plants (all from wheat), various common yeasts and molds were found in the aromatic Daqu, and the proportion of the protein they produce was high. Metabolites produced by the metabolism of the bacteria play different roles in the process of yeast fermentation and the subsequent process of white spirit brewing. For example, monascus can produce various enzyme substances such as protease, amylase, glucoamylase, pectinase, maltase and the like in the growth metabolic process. Analysis of KEGG metabolic pathway shows that most of enzymes in the aroma Daqu are related to carbohydrate metabolism and amino acid biosynthesis, and the two pathways enrich a large amount of enzymes in the aroma Daqu. And the further metabolism of amino acid can generate a plurality of precursors of typical flavor substances of the strong aromatic white spirit, and researches have shown that the amino acid in the white spirit has important influence on the synthesis of higher alcohol. Including isoamyl alcohol derived from leucine, 1-propanol derived from threonine, isobutyl alcohol derived from valine, 2-phenylethyl alcohol derived from phenylalanine, and the sour, sweet, bitter and astringent tastes of white spirits. The results are shown in fig. 4, and it can be seen that species sources of proteins in the aroma Daqu and biological processes mainly involved in aroma Daqu enzyme systems are analyzed by a macro-proteomics technology.

Example 5 validation of enzyme Activity measurements on Macro-proteomics results

Through the detection of the enzyme activity of the top-grade yeast and the common yeast of the aromatic Daqu, all proteins related to corresponding enzymes are searched in a macro-proteomics result, and whether the enzyme activity reflected by the content of the proteins is consistent with the enzyme activity detection result or not is calculated. The macro-protein result shows that the total content of all the proteins related to the alpha-amylase in the aroma superior Daqu is higher than that of the aroma common Daqu, and the result shows that the alpha-amylase activity of the former is higher than that of the latter and is consistent with the enzyme activity detection result.