Preparation method for improving acid resistance and temperature resistance of enzyme preparation

Abstract

The invention relates to the technical field of enzyme engineering, and discloses a preparation method for improving acid resistance and temperature resistance of an enzyme preparation, which comprises the following steps: step 1, selecting enzyme sources: selecting candida antarctica, serratia liquefaciens and pseudomonas pseudoalcaligenes as the enzyme sources; step 2, selecting an additive: adding the additive into the enzyme source; 3, culturing an enzyme source: selecting a culture medium for culturing, and setting the culture temperature and pH value range of the enzyme preparation; step 4, extracting an enzyme solution, namely breaking cells by using an enzymolysis method, and extracting the enzyme solution; 5, quality detection: detecting enzyme activity, acid resistance and temperature resistance; and step 6, packaging and storing: sealing and packaging the enzyme preparation which is qualified in quality detection by adopting a sterile container, regulating the temperature and Ph in a specified range according to a specified temperature condition and a specified Ph range, and preparing the enzyme preparation in three stages, so that the enzyme activity degree is still high after temperature gradient test and pH gradient test experiments.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to a preparation method for improving the acid and temperature resistance of an enzyme preparation. Background Art

[0002] The preparation method of traditional enzyme preparations mainly relies on microbial fermentation to produce enzymes. This method has the advantages of abundant resources, easy cloning of related genes, and easy mutagenesis breeding to increase yields. However, although these methods have advantages in some aspects, they still have obvious shortcomings in acid and temperature resistance. This causes traditional enzyme preparations to be easily inactivated in high temperature and acidic environments, limiting their application effect in feed processing and animal digestion. Summary of the invention

[0003] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides a preparation method for improving the acid and temperature resistance of an enzyme preparation, which has the advantages of high acid and temperature resistance of traditional enzyme preparations and solves the problem of poor acid and temperature resistance of traditional enzyme preparations.

[0004] (II) Technical solution To achieve the above object, the present invention provides the following technical solution: a preparation method for improving the acid and temperature resistance of an enzyme preparation, comprising the following steps: Step 1: Select enzyme source: select Antarctic Candida, Serratia liquefaciens and Pseudomonas pseudoalcaligenes as enzyme source; Step 2: Select additives: Add additives to the enzyme source; Step 3: Cultivate the enzyme source: select a culture medium for cultivation, and set the culture temperature and pH range of the enzyme preparation; Step 4: Extracting enzyme solution: using enzymatic method to break up cells and extract enzyme solution; Step 5: Quality testing: testing enzyme activity, acid and temperature resistance; Step 6. Packaging and storage: Enzyme preparations that pass quality inspection are sealed and packaged in sterile containers.

[0005] Preferably, in the step 1, Candida antarctica, Serratia liquefaciens and Pseudomonas pseudoalcaligenes are combined in a ratio of 1:3:2.

[0006] Preferably, in step 2, an additive is added to the enzyme source, and the additive consists of grape skin extract, lycopene, capsanthin and tea polyphenols in a ratio of 2:1:3:5.

[0007] Preferably, in step three, culture medium is selected for culture, and the weight ratio of the culture components includes: 25-28 parts of carbon source; 20-24 parts of nitrogen source; 13-16 parts of oil; 11-14 parts of minerals; 2-5 parts of vitamins; 8-11 parts of growth factors; and 0.5-1 parts of trace additives.

[0008] Preferably, the carbon source consists of glucose and glycerol in a ratio of 2:1; the nitrogen source consists of peptone and soybean cake powder in a ratio of 2:1; the oil is corn oil; the minerals consist of K2HPO4 (2 g / L), MgSO4·7H2O (1 g / L) and NaCl (5 g / L) in a ratio of 2:1:3; the vitamin is yeast extract; the growth factor is yeast extract; and the trace additives consist of octyl-agarose and antioxidants in a ratio of 4:3.

[0009] Preferably, in step 3, the culture temperature and pH range of the enzyme preparation are set, the culture temperature of the enzyme preparation is 15°C-30°C, the pH range is 4.5-9.0, and the culture is divided into three stages, and the process is as follows: Stage 1: Set the incubation temperature of the enzyme preparation to 18°C, use a plastic dropper to drip 5% hydrochloric acid, and adjust the pH value to 5.0; Stage 2: Set the incubation temperature of the enzyme preparation to 28°C and use a plastic dropper to add 10% sodium hydroxide to adjust the pH value to 7.3; Stage 3: Set the incubation temperature of the enzyme preparation to 30°C, use a plastic dropper to drip 10% sodium hydroxide to adjust the pH value to 9.0, and maintain this state for 10 hours.

[0010] Preferably, in step 4, the process of extracting the enzyme solution by enzymatic hydrolysis is as follows: S1. Pretreatment: Before enzymatic hydrolysis, the sample was defatted using ether; S2, preparation before enzymatic hydrolysis: prepare 25 mmol / L NH4HCO3, 10 mmol / LDTT, 55 mmol / LIAA, 100% acetonitrile, 50% acetonitrile, 10% acetonitrile, 25 mmol / L NH4HCO3 containing 55 mmol / L IAA, 1% stop solution, 2.5% trifluoroacetic acid, and 67% acetonitrile for MALDI; S3, enzymatic hydrolysis step: wash the sample three times with double distilled water, and then use 50% acetonitrile containing 25 mmol / L NH4HCO3 to keep warm at 37℃ for 30min. If the first decolorization is incomplete, decolorize again by adding 50% acetonitrile and then 100% acetonitrile for dehydration, add 25 mmol / L NH4HCO3 containing 10 mmol / L DTT, keep warm in a 57℃ water bath for 1h, cool to room temperature, add 25 mmol / L NH4HCO3 containing 55 mmol / L IAA, and place in the dark for 45min. Rinse the gel block with 25 mmol / L NH4HCO3 solution, 50% acetonitrile and 100% acetonitrile in turn. After each oscillation, place it for 10min and remove the liquid, seal it with sealing film, pierce holes, and freeze-dry for 30min. Make sure the sample is at the bottom of the tube, add appropriate amount of enzyme solution (25 uL / tube), place on ice for 30 minutes, then add covering solution (10% acetonitrile, 25 mmol / L NH4HCO3), keep warm at 37℃ for 16-18 hours, take out the sample from the 37℃ water bath, terminate the enzymatic reaction with 1% stop solution, transfer the supernatant to a clean EP tube, add 2.5% trifluoroacetic acid, 67% acetonitrile for MALDI, keep at 37℃ for 30 minutes, use an ultrasonic processor for ultrasonic treatment for 15 minutes, after 5 minutes, ultrasonicate again for 10 minutes, control the processor water temperature ≤40 degrees, and then use a centrifuge for centrifugation at 200 rpm. After standing for 2 hours, combine the supernatants and perform freeze-centrifugation to a volume that meets the requirements of mass spectrometry detection.

[0011] Preferably, in step five, the enzyme activity is detected by measuring the amount of enzyme product generated by colorimetry, fluorescence, titration, gasometrics, pH measurement or electrochemical methods.

[0012] Preferably, in step five, the acid and temperature resistance of the enzyme is detected: the acid resistance and temperature resistance of the enzyme are respectively measured on the amount of enzyme product generated by temperature gradient test and pH gradient test experiments.

[0013] Preferably, in step five, the packaging and storage are as follows: the enzyme preparation that has passed the quality inspection is added with a stabilizer, a protective agent or a carrier according to the stability of the enzyme and the application scenario, and is sealed and packaged in a sterile container and stored at a low temperature of 2-8 degrees Celsius.

[0014] Compared with the prior art, the present invention provides a preparation method for improving the acid and temperature resistance of enzyme preparations, which has the following beneficial effects: 1. The present invention uses temperature gradient test and pH gradient test experiments to help evaluate the stability and activity of the enzyme under different temperature and pH conditions. By specifying temperature conditions and Ph range, and adjusting the temperature and Ph within the specified range, the enzyme preparation is prepared in three stages, so that the enzyme activity level after the temperature gradient test and pH gradient test experiments is still high, indicating that the enzyme preparation of the present invention has acid and temperature resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram of the preparation steps of the present invention. DETAILED DESCRIPTION The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Example 1 See also Figure 1 , a preparation method for improving the acid and temperature resistance of an enzyme preparation, comprising the following steps: Step 1, select enzyme source: select Antarctic Candida, Serratia liquefaciens and Pseudomonas pseudoalcaligenes (Antarctic Candida, Serratia liquefaciens and Pseudomonas pseudoalcaligenes were purchased from China General Microbiological Culture Collection Management Center); Step 2: Select additives: Add additives to the enzyme source; Step 3: Cultivate the enzyme source: select a culture medium for cultivation, and set the cultivation temperature and pH range of the enzyme preparation. During the cultivation process, conditions such as temperature, pH value, and nutrient concentration have an important influence on the production and activity of the enzyme; Step 4: Extracting enzyme solution: using enzymatic hydrolysis to break up cells, release the enzymes therein, and extract the enzyme solution; Step 5: Quality testing: Detect enzyme activity, acid and temperature resistance, and measure the rate of enzyme-catalyzed specific reactions or the amount of product generated by the enzyme through colorimetry, fluorescence or titration experiments to evaluate the activity of the enzyme. Activity determination is not only used to test the purification effect, but also to provide a basis for dosage and applicable range for subsequent applications; Step 6. Packaging and storage: Enzyme preparations that pass quality inspection are sealed and packaged in sterile containers.

[0017] Specifically, in step 1, Candida antarctica, Serratia liquefaciens and Pseudomonas pseudoalcaligenes are combined in a ratio of 1:3:2.

[0018] Specifically, in step 2, an additive is added to the enzyme source, and the additive consists of grape skin extract, lycopene, capsanthin and tea polyphenols in a ratio of 2:1:3:5.

[0019] Specifically, in step three, a culture medium is selected for culturing, and the weight ratio of the culture components includes: 25-28 parts of carbon source; 20-24 parts of nitrogen source; 13-16 parts of oil; 11-14 parts of minerals; 2-5 parts of vitamins; 8-11 parts of growth factors; and 0.5-1 parts of trace additives.

[0020] Specifically, the carbon source consists of glucose and glycerol in a ratio of 2:1; the nitrogen source consists of peptone and soybean cake powder in a ratio of 2:1; the oil is corn oil; the minerals consist of K2HPO4 (2 g / L), MgSO4·7H2O (1 g / L) and NaCl (5 g / L) in a ratio of 2:1:3; the vitamin is yeast extract; the growth factor is yeast extract; and the trace additives consist of octyl-agarose and antioxidants in a ratio of 4:3.

[0021] Specifically, in step 3, the culture temperature and pH range of the enzyme preparation are set. The culture temperature of the enzyme preparation is 15°C-30°C, and the pH range is 4.5-9.0. The culture is divided into three stages, and the process is as follows: Phase 1: Set the culture temperature of the enzyme preparation to 18°C, use a plastic dropper to drip 5% hydrochloric acid, adjust the pH value to 5.0, and maintain this state for 36 hours. At a temperature of 18°C, the microorganisms can gradually adapt to the environment and begin to express acid-resistant enzymes to ensure sufficient growth and enzyme production; The second stage: set the culture temperature of the enzyme preparation to 28°C, use a plastic dropper to drip 10% sodium hydroxide to adjust the pH value to 7.3, and keep it in this state for 18 hours. This stage helps the liquefied Serratia, which is active in a neutral environment, to multiply rapidly and produce a large amount of enzymes; The third stage: Set the culture temperature of the enzyme preparation to 30°C, use a plastic dropper to add 10% sodium hydroxide to adjust the pH value to 9.0, and maintain this state for 10 hours to provide the best growth conditions for Pseudomonas pseudoalcaligenes. Under this temperature and alkaline environment, Pseudomonas pseudoalcaligenes can produce temperature-resistant enzymes and enzymes that are stable under alkaline conditions. Maintaining the culture time for 10 hours allows the microorganisms to adapt to environmental changes and produce the required enzymes.

[0022] Specifically, in step 4, the process of extracting the enzyme solution using enzymatic hydrolysis is as follows: S1. Pretreatment: Before enzymatic hydrolysis, the sample was defatted using ether; S2, preparation before enzymatic hydrolysis: prepare 25 mmol / L NH4HCO3, 10 mmol / LDTT, 55 mmol / LIAA, 100% acetonitrile, 50% acetonitrile, 10% acetonitrile, 25 mmol / L NH4HCO3 containing 55 mmol / L IAA, 1% stop solution, 2.5% trifluoroacetic acid, and 67% acetonitrile for MALDI; S3, enzymatic hydrolysis step: wash the sample three times with double distilled water to remove impurities, then use 50% acetonitrile containing 25 mmol / L NH4HCO3 to keep warm at 37℃ for 30min. If the first decolorization is incomplete, decolorize again by adding 50% acetonitrile and then 100% acetonitrile for dehydration, add 25 mmol / L NH4HCO3 containing 10 mmol / L DTT, keep warm in a 57℃ water bath for 1h to reduce the protein, cool to room temperature, add 25 mmol / L NH4HCO3 containing 55 mmol / L IAA, place in the dark for 45min, and use a concentration of 25 mmol / L NH4HCO3 solution, 50% acetonitrile and 100% acetonitrile were used to wash the gel block in turn. After each oscillation, the gel block was placed for 10 minutes and the liquid was removed. The tube was sealed with a sealing film, pierced, and freeze-dried for 30 minutes to ensure that the sample was at the bottom of the tube. An appropriate amount of enzyme solution (25uL / tube) was added, and the tube was placed on ice for 30 minutes. Then, a covering solution (10% acetonitrile, 25 mmol / L NH4HCO3) was added. The sample was kept warm at 37℃ for 16-18 hours. The sample was removed from the 37℃ water bath, and the enzymatic reaction was terminated with 1% stop solution. The supernatant was transferred to a clean EP tube, and 2.5% trifluoroacetic acid and 67% acetonitrile for MALDI, keep at 37℃ for 30min, use ultrasonic processor for ultrasonic treatment for 15min, after 5min, ultrasonicate again for 10min, control the water temperature of the processor ≤40℃, and then use centrifuge for centrifugation at 200 rpm. After standing for 2h, combine the supernatant and perform freeze centrifugation to dry it to a volume that meets the requirements of mass spectrometry detection.

[0023] Specifically, in step five, the enzyme activity is detected by measuring the amount of enzyme product generated by experiments using colorimetry, fluorescence, titration, gasometrics, pH measurement or electrochemical methods to evaluate the activity of the enzyme.

[0024] The results of the above determination methods were tabulated, and the enzyme activities were all excellent, as shown in Table 1: Table 1

[0025] From the above table 1, it can be seen that the finished enzyme obtained through the preparation steps of Example 1 has a high degree of activity.

[0026] Specifically, in step five, the acid and temperature resistance of the enzyme is detected: the acid and temperature resistance of the enzyme are measured by temperature gradient test and pH gradient test respectively on the amount of enzyme product generated, so as to evaluate the acid and temperature resistance of the enzyme.

[0027] The result data of the above determination method are tabulated, and the determination results are as follows in Table 2: Table 2

[0028] Information obtained from Table 2 above: the enzyme activity of the finished product prepared by the steps of Example 1 is still excellent under the temperature gradient test and the pH gradient test.

[0029] Specifically, in step five, packaging and storage: enzyme preparations that have passed quality inspection are added with stabilizers, protective agents or carriers according to the stability of the enzyme and the application scenario, sealed in sterile containers, and stored in a low temperature environment of 2-8 degrees Celsius to improve the long-term preservation effect of the enzyme preparations.

[0030] Comparative Example 1 On the basis of Example 1, only the culture temperature and pH value range of the enzyme preparation in step 3 are changed. The culture temperature of the enzyme preparation is 35° C., and the pH value is 11.

[0031] The activity of the enzyme is assessed by measuring the amount of enzyme product produced by colorimetry, fluorescence, titration, gasometrics, pH measurement or electrochemical methods.

[0032] The results of the above determination methods were tabulated, and the enzyme activities were all medium, as shown in Table 3: Table 3

[0033] From the above Table 3, it can be seen that the activity of the finished enzyme obtained through the preparation steps of Comparative Example 1 is moderate.

[0034] Specifically, the acid resistance and temperature resistance of the enzyme are measured by temperature gradient test and pH gradient test respectively, so as to evaluate the acid resistance and temperature resistance of the enzyme.

[0035] The result data of the above determination method are tabulated, and the determination results are shown in Table 4: Table 4

[0036] From the above table 4, it can be seen that the enzyme activity of the finished product prepared by the steps of comparative example 1 decreased from medium to general under the temperature gradient test and pH gradient test.

[0037] Example 2 On the basis of Example 1, only the culture temperature and pH range of the enzyme preparation in step 3 are changed. The culture temperature of the enzyme preparation is 15°C-30°C, the pH range is 4.5-9.0, and the culture is divided into three stages, and the process is as follows: Stage 1: Set the incubation temperature of the enzyme preparation to 26°C, use a plastic dropper to drip 5% hydrochloric acid, adjust the pH value to 7.0, and maintain it in this state for 28 hours; Phase II: Set the incubation temperature of the enzyme preparation to 28°C, use a plastic dropper to drip 10% sodium hydroxide to adjust the pH value to 7.5, and maintain it in this state for 20 hours.

[0038] Stage 3: Set the incubation temperature of the enzyme preparation to 29°C, use a plastic dropper to drip 10% sodium hydroxide to adjust the pH value to 8.0, and maintain this state for 12 hours.

[0039] The activity of the enzyme is assessed by measuring the amount of enzyme product produced by colorimetry, fluorescence, titration, gasometrics, pH measurement or electrochemical methods.

[0040] The results of the above determination methods were tabulated, and the enzyme activities were all excellent, as shown in Table 5: Table 5

[0041] From the above Table 5, it can be seen that the finished enzyme obtained through the preparation steps of Example 2 has a high degree of activity.

[0042] The acid and temperature resistance of the enzyme were measured by temperature gradient test and pH gradient test respectively, so as to evaluate the acid and temperature resistance of the enzyme.

[0043] The result data of the above determination method are tabulated, and the determination results are shown in Table 6: Table 6

[0044] Information obtained from Table 6 above: the enzyme activity of the finished product prepared by the steps of Example 2 is still excellent under the temperature gradient test and the pH gradient test.

[0045] Comparative Example 2 On the basis of Example 1, only the culture temperature and pH value range of the enzyme preparation in step 3 are changed. The culture temperature of the enzyme preparation is 10° C., and the pH value is 4.

[0046] Specifically, the amount of enzyme product generated is measured by colorimetry, fluorescence, titration, gasometrics, pH measurement or electrochemical methods to evaluate the activity of the enzyme.

[0047] The results of the above determination methods were tabulated, and the enzyme activities were all average, as shown in Table 7: Table 7

[0048] From the above Table 7, we can see that the activity of the finished enzyme obtained through the preparation steps of Comparative Example 2 is average.

[0049] Specifically, the acid resistance and temperature resistance of the enzyme are measured by temperature gradient test and pH gradient test respectively, so as to evaluate the acid resistance and temperature resistance of the enzyme.

[0050] The result data of the above determination method are tabulated, and the determination results are shown in Table 8: Table 8

[0051] From the above Table 8, it can be seen that the enzyme activity of the finished product prepared by the steps of Comparative Example 2 changes from fair to poor under the temperature gradient test and the pH gradient test.

[0052] Under the preparation conditions of Example 1-2 (optimal pH and temperature range), the enzyme exhibited excellent activity and high acid and high temperature resistance, while under the preparation conditions of Comparative Example 1-2 (non-ideal pH and temperature range), the activity and acid and high temperature resistance of the enzyme decreased significantly.

[0053] The temperature gradient test and the pH gradient test experiments are helpful to evaluate the stability and activity of the enzyme under different temperature and pH conditions. By specifying the temperature conditions and the Ph range, and adjusting the temperature and Ph within the specified range, the enzyme preparation is prepared in three stages, so that the enzyme activity level of the present invention is still high after passing the temperature gradient test and the pH gradient test experiments, indicating that the enzyme preparation of the present invention has the properties of acid and temperature resistance.

[0054] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the acid and temperature resistance of an enzyme preparation, characterized in that: The following steps are involved: Step 1: Select enzyme source: select Antarctic Candida, Serratia liquefaciens and Pseudomonas pseudoalcaligenes as enzyme source; Step 2: Select additives: Add additives to the enzyme source; Step 3: Cultivate the enzyme source: select a culture medium for cultivation, and set the culture temperature and pH range of the enzyme preparation; Step 4: Extracting enzyme solution: using enzymatic method to break up cells and extract enzyme solution; Step 5: Quality testing: testing enzyme activity, acid and temperature resistance; Step 6. Packaging and storage: Enzyme preparations that pass quality inspection are sealed and packaged in sterile containers.

2. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: In the step 1, the antarctic Candida, the liquefied Serratia and the pseudoalcaligenes are combined in a ratio of 1:3:

2.

3. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: In the step 2, an additive is added to the enzyme source, wherein the additive consists of grape skin extract, lycopene, capsanthin and tea polyphenols in a ratio of 2:1:3:

5.

4. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: In the step three, a culture medium is selected for culturing, and the weight ratio of the culture components includes: 25-28 parts of carbon source; 20-24 parts of nitrogen source; 13-16 parts of oil; 11-14 parts of minerals; 2-5 parts of vitamins; 8-11 parts of growth factors; and 0.5-1 parts of trace additives.

5. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 4, characterized in that: The carbon source consists of glucose and glycerol in a ratio of 2:1; the nitrogen source consists of peptone and soybean cake powder in a ratio of 2:1; the oil is corn oil; the minerals consist of K2HPO4 (2 g / L), MgSO4·7H2O (1 g / L) and NaCl (5 g / L) in a ratio of 2:1:3; the vitamin is yeast extract; the growth factor is yeast extract; and the trace additives consist of octyl-agarose and antioxidants in a ratio of 4:

3.

6. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: In the step 3, the culture temperature and pH range of the enzyme preparation are set. The culture temperature of the enzyme preparation is 15°C-30°C, and the pH range is 4.5-9.

0. The culture is divided into three stages, and the process is as follows: Stage 1: Set the incubation temperature of the enzyme preparation to 18°C, use a plastic dropper to drip 5% hydrochloric acid, and adjust the pH value to 5.0; Stage 2: Set the incubation temperature of the enzyme preparation to 28°C and use a plastic dropper to add 10% sodium hydroxide to adjust the pH value to 7.3; Stage 3: Set the incubation temperature of the enzyme preparation to 30°C, use a plastic dropper to drip 10% sodium hydroxide to adjust the pH value to 9.0, and maintain this state for 10 hours.

7. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: The process of extracting the enzyme solution by enzymatic hydrolysis in step 4: S1. Pretreatment: Before enzymatic hydrolysis, the sample was defatted using ether; S2. Preparation before enzymatic hydrolysis: prepare 25 mmol / L NH4HCO3, 10 mmol / LDTT, 55 mmol / LIAA, 100% acetonitrile, 50% acetonitrile, 10% acetonitrile, 25 mmol / L NH4HCO3 containing 55 mmol / L IAA, 1% stop solution, 2.5% trifluoroacetic acid, and 67% acetonitrile for MALDI; S3, enzymatic hydrolysis step: wash the sample three times with double distilled water, and then use 50% acetonitrile containing 25 mmol / L NH4HCO3 to keep warm at 37℃ for 30min. If the first decolorization is incomplete, decolorize again by adding 50% acetonitrile and then 100% acetonitrile for dehydration, add 25 mmol / L NH4HCO3 containing 10 mmol / L DTT, keep warm in a 57℃ water bath for 1h, cool to room temperature, add 25mmol / L NH4HCO3 containing 55 mmol / L IAA, and place in the dark for 45min. Rinse the gel block with 25 mmol / L NH4HCO3 solution, 50% acetonitrile and 100% acetonitrile in turn. After each oscillation, place it for 10min and remove the liquid, seal it with sealing film, pierce holes, and freeze-dry for 30min. Make sure the sample is at the bottom of the tube, add appropriate amount of enzyme solution (25 uL / tube), place on ice for 30 minutes, then add covering solution (10% acetonitrile, 25 mmol / L NH4HCO3), keep warm at 37℃ for 16-18 hours, take out the sample from the 37℃ water bath, terminate the enzymatic reaction with 1% stop solution, transfer the supernatant to a clean EP tube, add 2.5% trifluoroacetic acid, 67% acetonitrile for MALDI, keep at 37℃ for 30 minutes, use an ultrasonic processor for ultrasonic treatment for 15 minutes, after 5 minutes, ultrasonicate again for 10 minutes, control the processor water temperature ≤40 degrees, and then use a centrifuge for centrifugation at 200 rpm. After standing for 2 hours, combine the supernatants and perform freeze-centrifugation to a volume that meets the requirements of mass spectrometry detection.

8. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: In the step 5, the enzyme activity is detected by measuring the amount of enzyme product generated by colorimetry, fluorescence, titration, gas measurement, pH measurement or electrochemical method.

9. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: In the step 5, the acid and temperature resistance of the enzyme is detected: the acid resistance and temperature resistance of the enzyme are respectively measured by temperature gradient test and pH gradient test experiments on the amount of enzyme product generated.

10. A method for preparing an enzyme preparation for improving acid and temperature resistance according to claim 1, characterized in that: Packaging and storage in step 5: For enzyme preparations that have passed quality inspection, stabilizers, protective agents or carriers are added according to the stability of the enzyme and the application scenario, and they are sealed and packaged in sterile containers and stored at a low temperature of 2-8 degrees Celsius.

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