Method for producing cellulase by Bacillus pumilus

Abstract

The present invention belongs to the field of biochemical production, relates to strain mutation and strain selection method of microbial fermentation, and is specifically related to a strain of Bacillus pumilus producing alkaline cellulase and its application. The strain is obtained after ultraviolet mutagenesis and diethyl sulfate mutagenesis breeding, specifically Bacillus pumilus WS-135, and the deposit number is CGMCC NO.18641. The alkaline cellulase produced by the strain is heat-resistant, alkali-resistant, high in enzyme activity, and stable in performance. At the same time, the present invention also optimizes the corresponding fermentation mechanism, and the fermentation mechanism has higher fermentation activity, higher extraction yield, lower manufacturing cost, and fermentation enzyme activity is on average more than 360U / mL; and a spray drying method for alkaline cellulase is developed, and a spraying method for an alkaline cellulase solid enzyme preparation is provided. The existing alkaline cellulase preservation and the inconvenient transportation are solved, and it has a wide range of application prospects.

Description

Technical field:

[0001] The invention belongs to the field of biochemical production, relates to strain mutation and strain breeding methods of microbial fermentation, and specifically relates to a strain of Bacillus pumilus producing alkaline cellulase and application thereof. Background technology:

[0002] In recent years, alkaline cellulase has played an important role in the research field at home and abroad. Alkaline cellulase belongs to endoglucanase and is also carboxymethyl cellulase. Its components do not contain β-glucosidase and exo-β-1,4-glucosidase of the cellulase system. Early research began in 1987. Ito added alkaline cellulase to detergent, which had a good decontamination effect on cotton fabrics. This alkaline cellulase was produced by alkaliphilic Bacillus KSM-635. The study of the decontamination mechanism showed that alkaline cellulase only affects the cellulose molecules in the non-crystalline region of natural cellulose, and has no obvious decomposition effect on cellulose in the crystalline region. The non-crystalline region only accounts for 10% of the total cotton fiber, so it will not significantly decompose cotton fabrics to reduce firmness and cause breakage. Using this characteristic to add enzymes to detergents can have a good effect, even if an excess of enzyme is added, the effect on the fabric is small. Alkaline Bacillus is the main strain that produces alkaline cellulase. In addition to its application in the washing industry, this enzyme has gradually been applied to chemical, food, papermaking, wastewater treatment and other industries in recent years. At present, the enzyme detergents in my country mainly contain lipase and alkaline protease. The research on alkaline cellulose is still in the initial stage.

[0003] At present, the main problems faced by alkaline cellulase are low enzyme activity, insufficient research on the properties of the enzyme, and enzyme production far from meeting the needs of industrial production. Therefore, the development and research of alkaline cellulase and its products have very important economic value and practical significance. Summary of the invention:

[0004] The present invention aims to provide a strain of Bacillus pumilus capable of producing alkaline cellulase, specifically Bacillus pumilus WS-135, which is obtained by ultraviolet mutagenesis and diethyl sulfate mutagenesis breeding, and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on October 9, 2019, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, zip code 100101, and the deposit number is CGMCC NO.18641. This strain greatly improves the fermentation enzyme production rate of alkaline cellulase and saves costs.

[0005] The second technical solution provided by the present invention is: application of the above-mentioned Bacillus pumilus WS-135 in the production of alkaline cellulase.

[0006] Furthermore, the present invention provides a method for producing alkaline cellulase by liquid microbial fermentation using Bacillus pumilus WS-135, which is as follows:

[0007] Fermentation tank culture: 5-15% of the seed solution is transferred into the fermentation medium. The culture conditions are: initial pH 8.0, temperature 28-35°C, rotation speed 100-600rpm, air ventilation volume 1-4vvm, tank pressure 0.05-0.08MPa, and ammonia is added during fermentation to control pH 8.0;

[0008] Furthermore, after 8 hours of fermentation, feed medium 1 was added to control the dissolved oxygen at 20-40% until the fermentation was completed;

[0009] Furthermore, after 24 h of fermentation, feed medium 2 was added at a fixed flow rate of 4 g per liter of fermentation liquid per hour until the fermentation was completed;

[0010] Furthermore, the fermentation cycle is 75-80h, during which time the enzyme activity in the fermentation tank increases slowly and the bacteria autolyze severely. At the end of fermentation, the alkaline cellulase activity in the fermentation broth can reach a maximum of 388U / mL.

[0011] The culture medium components and production and sterilization process used in the above culture process are as follows:

[0012] Fermentation enzyme production medium: glucose 1%-3%, corn flour 1%-3%, soybean cake powder 2%-4%, yeast extract powder 0.5%-1.5%, (NH4)2SO4 0.4%-1%, MgSO4 0.01%-0.04%, K2HPO4 0.2%-0.6%, FeSO40.05%-1%, and the rest is water, pH 8.0.

[0013] Fermentation tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilization for 30-35min.

[0014] Feed medium 1: glucose 40%-70%, natural pH.

[0015] Feed medium 2: 40% soybean meal hydrolysate.

[0016] Feed sterilization process: 121-123℃, 0.11-0.12MPa, sterilization for 30-35min.

[0017] Furthermore, the extraction and purification method of alkaline cellulase in the fermentation broth is as follows:

[0018] Perlite filter aid is added to the fermentation liquid for plate and frame filter pressing; diatomaceous earth is then added for fine filtration; the fine filtrate is ultrafiltered and concentrated using an ultrafiltration membrane; the concentrated liquid is filtered and sterilized using a sterile membrane (stabilizers and preservatives may be added) to obtain a liquid finished enzyme preparation.

[0019] Furthermore, the method for preparing a solid preparation of the above-mentioned liquid alkaline cellulase finished enzyme preparation by spray drying is as follows:

[0020] Take the liquid finished enzyme preparation, add the heat protectant according to the mass percentage of the enzyme liquid: 0.2%-0.8% MgSO4, 0.1%-0.5% ZnSO4, 0.5%-1% trehalose; add 6%-10% enzyme liquid drying agent maltodextrin according to the mass percentage of the enzyme liquid; adjust the inlet air temperature to 125-150°C and the outlet air temperature to 50-80°C, and spray dry to obtain a powdered enzyme preparation product.

[0021] The enzymatic properties of the cellulase produced by the present invention are as follows:

[0022] (1) Alkaline cellulase can maintain more than 80% activity at 45-70℃, has a wide range of action temperatures, and the optimum action temperature is 55℃.

[0023] (2) The activity of alkaline cellulase can be maintained above 80% at pH 8.0-11.0, with the optimum pH being 9.0.

[0024] (3) Alkaline cellulase still has more than 70% activity when kept at 80℃ for 3h, and has good heat stability.

[0025] (4) Alkaline cellulase still has more than 80% enzyme activity after 5 hours at pH 5.0-10.5, and has good alkali resistance and stability.

[0026] Beneficial effects:

[0027] The invention obtains a strain of Bacillus pumilus (WS-135) with high alkaline cellulase production through ultraviolet mutagenesis and diethyl sulfate mutagenesis. The strain adopts a new fermentation production process, so that the alkaline cellulase activity can reach 388U / mL, the enzyme production is high, the cycle is short, and energy consumption can be saved to a large extent, and the fermentation cost can be reduced. In addition, the alkaline cellulase produced by Bacillus pumilus (WS-135) has good alkali and high temperature resistance characteristics, a wide range of action temperature and action pH, and has broad application prospects. Description of the drawings:

[0028] Figure 1 . Optimal temperature range curve of alkaline cellulase;

[0029] Figure 2 .Optimal pH curve of alkaline cellulase;

[0030] Figure 3 . Temperature stability curve of alkaline cellulase;

[0031] Figure 4 . pH stability curve of alkaline cellulase. Specific implementation method:

[0032] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0033] The selection of mutant strains is based on the selective environment. Due to the differences in growth and metabolism between different microorganisms, strains that do not have the advantage of enzyme production are eliminated, and cells with enzyme production advantages are screened. Under different ultraviolet mutagenesis conditions, enzyme-producing bacteria are first irradiated with ultraviolet light for preliminary screening. After obtaining strains with gene mutations and large enzyme production, the mutated strains are cultured and chemically mutated with different doses of diethyl sulfate to obtain strains with higher enzyme production.

[0034] The alkaline cellulase activity assay and definition used in the present invention are:

[0035] Determination method: Use pH 9.0 glycine-NaOH solution as buffer (accurately measure 50 mL of 0.2 mol / L glycine and 8.8 mL of 0.2 mol / L sodium hydroxide, mix well, and make up to 200 mL), use 1% CMC-Na solution as substrate, and use DNS colorimetry to determine the amount of reducing sugar produced after enzymatic hydrolysis, which indicates the activity of the enzyme.

[0036] Take a 25mL graduated stoppered test tube, add 2mL of 1% CMC-Na solution prepared with pH 9.0 buffer solution, and place in a water bath (55±1℃) to preheat for 5min. Add 0.5mL of appropriately diluted enzyme solution to be tested, mix well with a vortex mixer, cover the stopper, place in a water bath (55±1℃), accurately time, and take out after 30min of reaction. Quickly add 3mL of DNS reagent and shake well. Color in a boiling water bath for 10 minutes, take out, quickly cool to room temperature, dilute to 25mL with water, and use a standard blank sample (2.5mL of glycine-NaOH solution, 3.0mL of DNS reagent, heated in a boiling water bath for 10min, cooled, and diluted to 25mL) as a control, and measure the absorbance A1 of the enzyme reaction solution at 540nm.

[0037] For the blank control, add DNS first and then enzyme solution, and the rest of the steps are the same. At a wavelength of 540nm on a spectrophotometer, use a 10mm colorimetric cup and a standard blank (2.5mL of glycine-NaOH solution, 3.0mL of DNS reagent, heated in a boiling water bath for 10min, cooled, and fixed to 25mL) as a control, and measure the absorbance A0 of the enzyme blank at 540nm.

[0038] Enzyme activity calculation formula:

[0039]

[0040] Wherein: X is the activity of cellulase in the diluted enzyme solution, U / ml; A1 is the absorbance of the enzyme reaction solution; A0 is the absorbance of the enzyme blank sample; K is the slope of the glucose standard curve; C0 is the intercept of the standard curve; M is the molar mass of glucose, g / mol; t is the enzymatic reaction time, min; n is the enzyme solution dilution multiple; 1000 is the conversion factor, 1mmol=1000μmol.

[0041] Under these conditions (temperature is 55°C and pH is 9.0 unless otherwise specified), the amount of enzyme that hydrolyzes the substrate to produce 1 μmol of glucose per minute is defined as one enzyme activity unit, expressed as U / mL.

[0042] Example 1 Mutagenesis Preparation

[0043] Seed culture medium: yeast extract 1.5%, peptone 1%, soluble starch 1%, NaCl 0.05%, KH2PO4 0.3%, Na2CO3 0.05%, and the rest is water, pH 8.0.

[0044] Preparation of bacterial suspension:

[0045] A strain of Bacillus pumilus preserved in the laboratory was taken as the starting strain and activated in the seed culture medium at 37°C for 24 hours. 10 mL of the bacterial solution was collected by centrifugation at 4°C and 5000 rpm / min for 10 minutes. The bacteria were washed and centrifuged twice with sterile saline. Sterile saline was added to the total volume of 10 mL. The bacteria were fully vortexed to suspend the bacteria and the bacterial suspension was prepared for use.

[0046] Example 2 UV mutagenesis

[0047] Mutagenesis of strains: Wrap the clean bench with paper, preheat the UV lamp for 40 minutes, and then irradiate with UV light. Place the collected bacterial suspension under the UV lamp at a distance of 25 cm for 100 seconds. Use Congo red staining to identify. The mutagenesis-treated bacterial suspension is stored in a dark refrigerator for 1-2 hours and diluted to 10 -4 , 10 -5 , 10 -60.2 mL of each was spread on a plate of separation medium, inverted and shaded at 37°C for 48 hours, and the unmutated bacterial solution was diluted and spread on the plate as a control. After the colonies grew, they were screened according to the size of the transparent circle around the colonies, and 7 mutants with large hydrolysis circles were obtained.

[0048] The seven mutants with large hydrolysis circles were selected and cultured in shake flasks for 48 h using fermentation enzyme production medium to measure the alkaline cellulase activity, with the original starting strain A as the control. The results are shown in Table 1. Strain A-5 had the highest enzyme activity, which could reach 36 U / mL.

[0049] Table 1 Results of positive mutant strain screening under UV mutagenesis

[0050]

[0051] Plate separation medium: CMC 1%, peptone 0.5%, yeast extract 0.5%, Na2CO3 0.05%, agar 2%, Congo red 0.03%, and the rest is water.

[0052] Fermentation enzyme production medium: glucose 2%, corn flour 2%, soybean cake powder 3%, yeast extract powder 1%, (NH4)2SO4 0.5%, MgSO4 0.03%, K2HPO4 0.5%, FeSO4 0.05%, and the rest is water, pH 8.0.

[0053] Example 3 Diethyl sulfate mutagenesis

[0054] The ultraviolet mutagenesis strain A-5 was used as the starting strain and cultured to prepare a bacterial suspension. 1 ml of diethyl sulfate was added to 9 ml of 95% ethanol to prepare a diethyl sulfate dilution. The diluted diethyl sulfate solution was mixed with the bacterial suspension at a ratio of 1:200 (diethyl sulfate dilution: bacterial suspension). The mixture was shaken for 25 min, and a 25% sodium thiosulfate solution was added to terminate the reaction. The bacterial suspension was diluted to 10 -4 , 10 -5 , 10 -6 0.2 mL of each was spread on the separation medium, inverted and shaded at 37°C for 48 hours, and the bacterial solution not treated with diethyl sulfate was diluted and spread on the plate as a control. After the colonies grew, they were screened according to the size of the transparent circle around the colonies, and 8 mutants with large hydrolysis circles were obtained.

[0055] The eight positive mutant strains were selected and cultured in shake flasks for 48 h using fermentation enzyme production medium. The alkaline cellulase activity was determined, and the strain that was not induced by diethyl sulfate was used as a control. The results are shown in Table 2.

[0056] Table 2 Results of positive mutant strain screening under diethyl sulfate

[0057]

[0058]

[0059] The enzyme activity of mutant B-7 reached 53.2U / mL. The strain was used as the research object to determine its genetic stability, and the enzyme activity experiment was carried out by shake flask fermentation. After 8 consecutive passages, the enzyme activity remained stable, and the results are shown in Table 3. The mutant B-7 was named Bacillus pumilus WS-135 and was preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with the preservation number CGMCC NO.18641.

[0060] Table 3 Genetic stability results of strain B-7

[0061]

[0062] Example 4 Production of alkaline cellulase by submerged fermentation of Bacillus pumilus WS-135 and product treatment

[0063] 1. Seed tank cultivation

[0064] Seed culture medium: yeast extract 1.5%, peptone 1%, soluble starch 1%, NaCl 0.05%, KH2PO4 0.3%, Na2CO3 0.05%, and the rest is water, pH 8.0.

[0065] Seed tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilize for 30 minutes.

[0066] Seed tank culture: The seed liquid after shake flask fermentation was inoculated into the seed tank at a ratio of 6% of the inoculation amount for culture. The culture conditions were temperature 34°C, tank pressure 0.06Mpa, air volume 3vvm, rotation speed 400rpm. Ammonia was added during the culture process to control the pH to 8.0. The culture time was 10h, and there were more bacteria under microscopic examination.

[0067] 2. Fermentation tank culture

[0068] Fermentation enzyme production medium: glucose 2%, corn flour 2%, soybean cake powder 3%, yeast extract powder 1%, (NH4)2SO4 0.5%, MgSO4 0.03%, K2HPO4 0.5%, FeSO4 0.05%, and the rest is water, pH 8.0.

[0069] Fermentation tank sterilization process: 121-123℃, 0.11-0.12MPa, sterilize for 30min.

[0070] Fermentation tank culture: transfer the bacterial liquid from the seed tank into the fermentation tank for culture at a ratio of 10% of the transplanting amount. The culture conditions are: temperature 34°C, tank pressure 0.06Mpa, rotation speed: 600rpm, air volume: 4vvm, and ammonia supplementation during the fermentation process to control pH 8.0.

[0071] 3. Fed-batch culture

[0072] Feed medium 1: glucose 60%, natural pH.

[0073] Feed medium 2: 40% soybean meal hydrolysate.

[0074] 40% soybean meal hydrolyzate production process:

[0075] Alkaline dissolution → alkaline protease hydrolysis → acidic protease hydrolysis → inactivation → filtration → concentration → spray drying → dissolution:

[0076] (1) Alkaline dissolution: add water to prepare 12.5% ​​soybean meal solution, stir at 400 rpm, adjust pH to 12.5 with 30% NaOH, and pre-dissolve at 50°C for 120 min;

[0077] (2) Alkaline protease hydrolysis: add 4000 U / g (calculated based on the mass of soybean meal) of alkaline protease to the above solution, add 30% NaOH to adjust the pH to 11.0, mix well, rotate at 400 rpm, and hydrolyze at 50°C for 120 min;

[0078] (3) Acidic protease hydrolysis: add 4000 U / g of acidic protease (calculated based on the mass of soybean meal) to the above enzymatic solution, rotate at 400 rpm, add HCl to adjust the pH to 4.0, and enzymolyze at 50°C for 60 min;

[0079] (4) Inactivation: Raise the temperature to 90°C for 20 min to inactivate the enzyme; obtain soybean meal liquid;

[0080] (5) Filtration: using plate and frame filter press to obtain filtrate;

[0081] (6) further spray drying the filtrate to obtain a powdered extract;

[0082] (7) Dissolve it into 40% concentration to obtain 40% soybean meal hydrolyzate.

[0083] Feed sterilization process: 121-123℃, 0.11-0.12MPa, sterilization for 30-35min.

[0084] Feed control: Feed was started after 8 hours of fermentation, and feed medium 1 was added in a flow manner. The feed amount was adjusted and gradually added, and the dissolved oxygen was controlled at 35% until the end of fermentation.

[0085] After 24 h of fermentation, feed medium 2 was added at a fixed flow rate of 4 g per liter of fermentation liquid per hour until the end of the fermentation.

[0086] 4. End of fermentation

[0087] The enzyme activity increased slowly and the fermentation was terminated when the bacterial autolysis was serious. The fermentation cycle was 75-80 hours. The fermentation broth was prepared for extraction and the alkaline cellulase activity in the fermentation broth was determined, as shown in Table 4.

[0088] Table 4 Fermentation test results in fermenter

[0089]

[0090] 5. Extraction and purification of alkaline cellulase

[0091] (1) Add 3% perlite filter aid and perform plate and frame filter pressing;

[0092] (2) adding 2% diatomaceous earth to the filtrate for fine filtration and clarification;

[0093] (3) ultrafiltration and concentration of the fine filtrate using a 20 KDa ultrafiltration membrane;

[0094] (4) Obtaining an ultrafiltration concentrate, and then filtering and sterilizing it with a sterile membrane to obtain a liquid alkaline cellulose finished enzyme preparation.

[0095] 6. Spray drying of alkaline cellulase

[0096] Take the liquid finished enzyme preparation, add 0.5% MgSO4, 0.3% ZnSO4, and 0.7% trehalose as heat protectants according to the mass percentage of the enzyme liquid; add 8% enzyme liquid drying agent maltodextrin according to the mass percentage of the enzyme liquid; adjust the inlet air temperature to 142°C and the outlet air temperature to 72°C, spray dry, and obtain a powdered enzyme preparation product.

[0097] Example 5 Enzymatic Properties 1

[0098] a. Optimal action temperature of alkaline cellulase

[0099] The liquid alkaline cellulose finished enzyme preparation prepared in batch 1 of Example 4 was taken, and the alkaline cellulose activity at 30-80°C was measured under pH 9.0 conditions, and the enzyme activity at 55°C was taken as 100%, and the relative enzyme activity was calculated respectively. The results are as follows Figure 1 As shown, the optimum reaction temperature is 55°C, and more than 80% of the activity can be maintained at 45-70°C. The experimental results show that the optimum reaction temperature range of the alkaline cellulase produced by the mutant strain is relatively wide, and it has great market application value and good application value in industrial production.

[0100] b. Optimal pH for alkaline cellulase

[0101] The liquid alkaline cellulose finished enzyme preparation prepared in batch 1 of Example 4 was taken, and the alkaline cellulose activity of the alkaline cellulose was measured at different pH values ​​(4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0) at a temperature of 55°C. The relative enzyme activity was calculated with the enzyme activity at pH 9.0 as 100%, and the relative enzyme activity change curve under different pH conditions was plotted. The measurement results are shown in FIG. Figure 2 As shown, the enzyme activity can be maintained above 80% when the pH is 8.0-11.0, and the optimum pH is 9.0, indicating that the alkaline cellulase produced by the bacteria can maintain its enzyme activity in a wider pH range.

[0102] Example 6 Enzymatic Properties 2

[0103] a. Temperature stability

[0104] The liquid alkaline cellulose finished enzyme preparation prepared in batch 1 of Example 4 was placed at 80°C for different time periods, and then the enzyme activity was measured at pH 9.0 and 55°C for stability test. The experimental results were expressed as relative enzyme activity, that is, the initial enzyme activity was set as 100% enzyme activity. The results are as follows: Figure 3 As shown, it was found that the enzyme activity gradually decreased with the extension of time, and there was still more than 70% enzyme activity after 3 hours of insulation time, indicating that alkaline cellulase has good storage stability at high temperature.

[0105] b. pH stability

[0106] The liquid alkaline cellulose finished enzyme preparation prepared in batch 1 of Example 4 was incubated at pH 4.0-12.0 for 5 h, and then the enzyme activity was measured at pH 9.0 and 55°C. The pH stability was studied as follows: Figure 4 As shown, the enzyme activity was above 80% under the condition of pH 5.0-10.5, indicating that the enzyme had good stability under this pH range. At pH 12, the enzyme activity was still 70%, indicating that the enzyme had strong alkali resistance.

[0107] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A method for producing cellulase by Bacillus pumilus, characterized in that: The details are as follows: Fermentation tank culture: 5-15% of the seed solution is transferred into the fermentation medium. The culture conditions are as follows: initial pH 8.0, temperature 28-35°C, rotation speed 100-600rpm, air ventilation volume 1-4vvm, tank pressure 0.05-0.08MPa, ammonia is added during fermentation to control pH 8.0, and the fermentation cycle is 75-80h. After 8 h of fermentation, feed medium 1 was added to control dissolved oxygen at 20-40% until the end of fermentation; After 24 h of fermentation, feed medium 2 was added at a fixed flow rate of 4 g per liter of fermentation liquid per hour until the fermentation was completed; The feed medium 1: 40%-70% glucose, natural pH; The feed medium 2: 40% soybean meal hydrolyzate; The Bacillus pumilus is specifically Bacillus pumilus ( Bacillus pumilus )WS-135, the deposit number is: CGMCC NO.18641.

2. The method according to claim 1, characterized in that The fermentation medium is: glucose 1%-3%, corn flour 1%-3%, soybean cake powder 2%-4%, yeast extract powder 0.5%-1.5%, (NH4)2SO4 0.4%-1%, MgSO4 0.01%-0.04%, K2HPO40.2%-0.6%, FeSO4 0.05%-1%, and the rest is water, pH 8.

0.

3. The method according to claim 1, characterized in that The extraction and purification method of alkaline cellulase in the fermentation broth is as follows: Perlite filter aid is added to the fermentation liquid for plate and frame filter pressing; diatomaceous earth is then added for fine filtration; the fine filtrate is ultrafiltered and concentrated using an ultrafiltration membrane; the concentrated liquid is filtered and sterilized using a sterile membrane to obtain a liquid finished enzyme preparation.

4. The method according to claim 3, characterized in that The method for preparing the solid preparation is as follows: Take the liquid finished enzyme preparation, add the heat protectant according to the mass percentage of the enzyme liquid: 0.2%-0.8% MgSO4, 0.1%-0.5% ZnSO4, 0.5%-1% trehalose; add 6%-10% enzyme liquid drying agent maltodextrin according to the mass percentage of the enzyme liquid; adjust the inlet air temperature to 125-150°C and the outlet air temperature to 50-80°C, spray dry, and obtain a powdered enzyme preparation product.

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