A method for producing alkaline cellulase

Abstract

The present invention belongs to the field of microbial technology, and more particularly to a kind of high-yield strain and production method for producing alkaline cellulase. The strain is obtained after NTG mutagenesis, specifically Bacillus amyloliquefaciens MN101202, and the deposit number is CGMCC No.26079. The carboxymethyl cellulase activity level of the strain for industrial fermentation production can reach more than 1100-1200U / mL, which greatly reduces the production cost, is conducive to the large-scale application of alkaline cellulase, and saves energy and reduces emissions. The produced cellulase has the characteristics of having higher enzyme activity under low temperature conditions, maintaining the stability of enzyme activity within the alkaline pH range, being stable under the action of various surfactants and filter aids in washing powder, and being able to resist the degradation of alkaline protease, the optimum reaction temperature is 30 DEG C, and it is kept warm for 2h under 60 DEG C, and still can maintain more than 85% enzyme activity, and the thermal stability is good; the optimum reaction pH is 10.0, and it is treated for 2h under pH12 conditions, and the enzyme can maintain more than 85% of its activity, and has good alkali resistance.

Description

Technical Field:

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a method for producing cold-resistant and alkali-resistant cellulase. Background Art:

[0002] Cellulose is the most abundant renewable resource in the world. Using microbial cellulase to degrade cellulose has the characteristics of being more economical, effective, and resource-saving compared with traditional physical and chemical methods. Therefore, cellulase has good application prospects in industries such as biofuels, food, feed, medicine, textiles, detergents, and papermaking.

[0003] Cellulase refers to a multi-component complex enzyme system that can degrade cellulose to produce small molecule substances such as cellobiose and glucose. According to the application conditions of cellulose, it can be divided into acidic cellulase, neutral cellulase, and alkaline cellulase. At present, the industrial application of cellulase mainly focuses on fields such as the detergent industry, bioenergy, denim washing, and textile finishing. Among them, acidic cellulase is mainly applied in the bioenergy field, where cellulose substances are completely degraded through the degradation action of cellulase, and its hydrolysis products can be further fermented to produce hydrogen, ethanol, biodiesel, etc.; in the washing industry, adding alkaline cellulase to detergents can improve the washing effect; in the textile finishing and denim washing industries, neutral cellulase has great advantages due to its mild reaction conditions, low reaction temperature, and good fabric finishing effect.

[0004] Adding lipase, protease, or amylase to detergents can make dirt easier to wash off from fabrics. However, dirt not only adheres to the fiber surface but also easily enters the internal tissue of the fiber and is enclosed. After repeated washing of the fabric, yellowing, fading, and hardening will occur at the most severely worn parts. Alkaline cellulase is a new type of enzyme used in detergents. It not only has good dirt-removing ability but also can still make cotton fabrics soft, bright-colored, with clear fabric texture, and as good as new after multiple uses.

[0005] Natural cellulose is composed of two parts: crystalline and non-crystalline. The fiber structure of the crystalline part is tight, and it is difficult for dirt to penetrate. The fiber structure of the non-crystalline part is loose, resulting in easy penetration of dirt. Alkaline cellulase can act on the non-crystalline region inside the fabric fiber, making the cellulose structure of the cotton fabric expand and effectively softening the gel structure formed by fiber molecules and water. This makes the dirt enclosed in it easily dissolve out from the fiber gaps, thus improving the detergency, and can also make the washed fabric softer and more brightly colored.

[0006] Cellulase can generally be divided into three types of enzyme components, namely exoglucanase, endoglucanase, and cellobiase. The alkaline cellulase added to detergents is endoglucanase, that is, carboxymethyl cellulase (CMCase), which has a relatively high hydrolysis activity only for the cellulose in the amorphous region and a very low hydrolysis activity for the cellulose in the crystalline region. Therefore, it will not cause a significant decrease in the strength of the fabric or damage to the fabric. So, alkaline cellulase is an ideal additive for detergents. Alkaline cellulase is a special functional washing enzyme with a decontamination mechanism and performance different from other enzyme species. The application of cellulase has changed the decontamination mechanism of traditional detergents and has important application prospects in the detergent industry.

[0007] At present, there are many strains that can produce cellulase, including actinomycetes, bacteria, fungi, etc. However, the cellulase they produce is usually acidic or slightly acidic. And detergents are strongly alkaline, usually with a pH value above 9.0. Therefore, the cellulase used for washing must have a relatively high enzyme activity under the condition of pH above 9.0. In addition, from the international situation, washing is moving towards low temperature, and the washing characteristics in China are medium and normal temperature or even cold water washing. Therefore, it is of great significance to develop a cellulase with high activity, low temperature resistance, and alkalinity resistance. Summary of the Invention:

[0008] To solve the above technical problems, one of the technical solutions provided by the present invention is a high-yield strain of low-temperature and alkali-resistant cellulase. This strain is obtained by NTG mutagenesis of the Bacillus amyloliquefaciens strain DXH20 preserved in the laboratory, specifically Bacillus amyloliquefaciens MN101202. This strain was deposited on November 8, 2022, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101), and the deposit number is CGMCC No. 26079.

[0009] Another technical solution provided by the present invention is the application of Bacillus amyloliquefaciens MN101202, especially its application in the production of cellulase.

[0010] Another technical solution provided by the present invention is a liquid fermentation production method of cellulase with high fermentation enzyme activity and low manufacturing cost, which can be achieved through the following measures:

[0011] Fermentation process conditions in the fermenter: inoculum size 1-2%, tank pressure 0.05-0.08 MPa, culture temperature 36-38 °C, rotation speed 200-800 rpm. When the pH rises to 7.6, feeding starts, controlling the pH at 7.1-7.2. Culture for about 70-80 h. When the enzyme activity growth slows down and partial autolysis of the bacteria starts, discharge the fermenter.

[0012] At the end of fermentation, the enzyme activity in the fermentation broth is 1100-1200 U / mL.

[0013] Furthermore, the composition of the fermenter medium is as follows: CMC-Na 1-1.2%, glucose 1-1.5%, wheat bran 2-2.5%, corn steep liquor 2-3%, potassium dihydrogen phosphate 0.5-0.8%, magnesium sulfate 0.15-0.2%, ammonium sulfate 1.5-2.0%, and the rest is water, pH 7.2-7.5 ("%" represents mass-volume percentage).

[0014] Furthermore, the sterilization process conditions in the fermenter: at 121 °C - 124 °C, 0.11-0.12 MPa, sterilize for 30 min.

[0015] Furthermore, the composition of the feeding medium is as follows: CMC-Na 5-6%, glucose 40-60%, corn steep liquor 3.5-4.5%, ammonium sulfate 3-4%, potassium dihydrogen phosphate 3-4%, and the rest is water, pH 7.2-7.5 ("%" represents mass-volume percentage).

[0016] The cellulase prepared by the present invention has the following enzymatic characteristics:

[0017] (1) The optimal reaction temperature is 30 °C. At 20 °C, more than 85% of the enzyme activity remains, which is beneficial for washing under natural low-temperature conditions. When incubated at 50 °C for 2 h, the enzyme activity basically does not decrease. When incubated at 60 °C for 2 h, more than 85% of the enzyme activity can still be maintained, showing good thermal stability and being suitable for high-temperature treatment during the formation of solid detergents.

[0018] (2) The optimal reaction pH is 10.0. When treated at pH 12 for 2 h, the relative enzyme activity still remains above 85%, showing good alkali resistance.

[0019] Beneficial effects:

[0020] The Bacillus amyloliquefaciens provided by the present invention has outstanding substantial features and significant progress compared with the prior art, and can produce the following positive effects:

[0021] 1. The invention first provides a mutant strain Bacillus amyloliquefaciens MN101202 with high-yield, low-temperature and alkali-tolerant cellulase. The carboxymethyl cellulase activity level in industrial fermentation using this strain can reach over 1100 - 1200 U / mL, greatly reducing the production cost, facilitating the large-scale application of alkaline cellulase, and saving energy and reducing emissions.

[0022] 2. This cellulase also has the characteristics of having high enzyme activity under low-temperature conditions, maintaining the stability of enzyme activity within the alkaline pH range, being stable under the action of various surfactants and filtering aids in laundry detergents, and being able to resist degradation by alkaline proteases. The optimal reaction temperature is 30 °C, and after incubation at 60 °C for 2 h, it can still maintain more than 85% of its enzyme activity, showing good thermal stability; the optimal reaction pH is 10.0, and after treatment at pH 12 for 2 h, the enzyme can maintain more than 85% of its activity, showing good alkali tolerance.

[0023] 3. This cellulase is mainly an endo-type cellulase, which has a relatively high hydrolysis activity only for the cellulose in the amorphous region, while having a very low hydrolysis activity for the cellulose in the crystalline region, and will not cause a significant decrease in the fabric strength or damage to the fabric, and can be widely applied to the detergent industry. Description of the Drawings:

[0024] Figure 1 Optimal reaction temperature curve graph.

[0025] Figure 2 Thermal stability curve graph.

[0026] Figure 3 Optimal reaction pH curve graph.

[0027] Figure 4 pH stability curve graph.

[0028] Figure 5 Substrate specificity of alkaline cellulase.

[0029] Figure 6 Curve graph of the performance of resisting degradation by alkaline protease.

[0030] Figure 7 Curve graph of the influence of common surfactants and additives in detergents on cellulase activity.

[0031] Figure 8 Determination of the detergency efficiency of alkaline cellulase. Detailed Description of the Invention:

[0032] In order to make the objectives, technical solutions and advantages of this patent clearer, the following further details this patent in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not used to limit the present invention.

[0033] Mutation Breeding of the Strain in Example 1

[0034] 1. NTG Mutation

[0035] Inoculate the original strain DXH20 into the seed medium and culture it at 37°C until the logarithmic phase. Then collect the bacterial cells, centrifuge at 8000 rpm for 5 min, wash the bacterial cell precipitate twice with physiological saline, and finally resuspend the bacterial cells with Tris buffer at pH 7.0. Add the NTG mother liquor to make the final concentration 0.375 g / L. Then react at 37°C for 30 min. After appropriate dilution, take 100 μL and spread it on the screening plate, and culture it at 37°C for about 24 h to calculate the lethality rate. Add an appropriate amount of 1% congo red solution to the cultured petri dish, stain for 20 min, discard the staining solution, add an appropriate amount of 1 mol / L NaCl solution, pour out the NaCl solution after 20 min, select the mutant strain according to the ratio of the diameter of the transparent circle around the colony to the diameter of the colony, pick the colony with a larger ratio and further streak isolate it. Transfer the isolated single bacterium to the seed medium, and transfer it to a shake flask for re-screening after the seeds grow well. Finally, a mutant strain MN101202 with an initial screening shake flask enzyme activity of 155 U / mL is screened through NTG mutation. The shake flask enzyme activity of the original strain DXH20 is 48 U / mL, which is 3.2 times higher than that of the original strain.

[0036] (1) Plate Screening Medium

[0037] CMC-Na 2%, glucose 0.5%, yeast powder 1%, potassium dihydrogen phosphate 0.2%, ammonium sulfate 0.6%, magnesium sulfate 0.2%, agar 2%, the rest is water, pH 7.2.

[0038] (2) Seed Medium

[0039] Glucose 2%, beef extract 1%, peptone 2%, potassium dihydrogen phosphate 0.5%, ammonium sulfate 1.2%, the rest is water, pH 7.2.

[0040] (3) Fermentation Shake Flask Medium

[0041] CMC-Na 2%, glucose 1%, yeast powder 2%, corn steep liquor 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1%, ammonium sulfate 0.5%, sodium carbonate 0.3%, the rest is water, pH 7.2.

[0042] (4) Culture Conditions

[0043] Isolation Plate: Culture at 37°C for 24 h;

[0044] Liquid Seeds: Culture at 37°C for 8 h, shaker speed 220 rpm;

[0045] Fermentation shake flask: Incubate at 37°C for 72 h, with a shaker speed of 220 rpm and an inoculum size of 2%.

[0046] 2. Stability subculture experiment of the high-yield cellulase strain MN101202

[0047] Pick a single colony of MN101202 with good growth on the screening plate and inoculate it into a seed flask. Incubate at 37°C and 220 rpm for about 8 h, then transfer it to a fermentation shake flask at an inoculum size of 2%. Incubate at 37°C and 220 rpm for about 72 h, and measure the enzyme activity. The results of the shake flask for subculturing this strain 10 times continuously are shown in Table 1:

[0048] Table 1. Results of stability detection of strain MN101202

[0049] Flask enzyme activity (U / mL) Relative enzyme activity (%) F1 156 100 F2 153 98.1 F3 165 105.8 F4 159 101.9 F5 150 96.2 F6 162 103.8 F7 165 105.8 F8 159 101.9 F9 153 98.1 F10 153 98.1

[0050] The mutant strain was subcultured for 10 generations. As can be seen from Table 1 of the experimental results, the heredity of this mutant strain is stable.

[0051] Example 2 Method for measuring alkaline cellulase activity

[0052] (1) Definition of cellulase activity used in the present invention

[0053] Under the conditions of 40°C and pH 9.0, the amount of enzyme required to hydrolyze CMC-Na to produce 1 μmol of glucose per minute is defined as 1 enzyme activity unit.

[0054] (2) Method for measuring enzyme activity

[0055] The DNS method was used to detect the activity of the alkaline cellulase of the present invention. The cellulase hydrolyzes the cellulose substrate (CMC-Na) to release reducing sugar under certain temperature and pH conditions (if not otherwise specified, the detection conditions are 40°C and pH 9.0). After the 3,5-dinitrosalicylic acid solution and the reducing sugar solution are heated together, they are reduced to a brownish-red amino compound. The amount of reducing sugar and the color depth of the brownish-red substance are in a proportional relationship within a certain range, so it can be used for colorimetric determination.

[0056] The specific method is as follows:

[0057] ① Take four 25-mL stoppered test tubes (one blank tube and three sample tubes).

[0058] ② Accurately add 1.5 mL of 1% CMC-Na solution prepared with pH 9.0 sodium carbonate-sodium bicarbonate buffer to each of the four test tubes.

[0059] ③ Accurately add 0.5 mL of the diluted enzyme solution to be measured to each of the three sample tubes (the blank tube is not added), mix well, and cover with a stopper.

[0060] ④Place the four test tubes simultaneously in a water bath at (40 ± 0.1) °C, accurately time the reaction for 30 min, and then take them out.

[0061] ⑤Immediately and accurately add 3.0 mL of DNS reagent to each test tube. Then, accurately add 0.5 mL of the diluted enzyme solution to be measured to the blank tube and shake well. Place the four test tubes simultaneously in a boiling water bath, heat for 10 min, take them out, quickly cool to room temperature, add water to make up the volume to 25 mL, and shake well.

[0062] ⑥Adjust the instrument zero with the blank tube (control solution). Measure the absorbance of the sample solutions in the three parallel test tubes at a wavelength of 540 nm on a spectrophotometer and take the average value. Determine the content of reducing sugar by referring to the standard curve.

[0063] Calculation formula for enzyme activity: X = A × n × 1000 / (0.5 × 180 × 30)

[0064] X—Carboxymethyl cellulase activity (U / mL);

[0065] A—Content of reducing sugar (mg) obtained from the standard curve based on the absorbance;

[0066] n—Dilution factor of the enzyme sample;

[0067] 180—Molecular weight of glucose;

[0068] 30—Reaction time (min);

[0069] 0.5—Converted to 1 mL of the enzyme solution;

[0070] 1000—Conversion of millimoles to micromoles.

[0071] Example 3 Liquid fermentation of strain MN101202 to produce cellulase

[0072] 1. Seed tank scale-up culture

[0073] (1) Seed tank medium: Glucose 2%, soybean cake powder 1%, peptone 2%, corn steep liquor 2%, potassium dihydrogen phosphate 0.5%, ammonium sulfate 1.2%, the rest is water, pH 7.2.

[0074] (2) Sterilization process conditions for the seed tank: Sterilize at 121 °C and 0.12 MPa for 30 min;

[0075] (3) Culture process conditions for the seed tank: Tank pressure 0.05 MPa, culture temperature 37 °C, stirring speed 400 rpm, inoculation amount 2%, pH controlled at 7.2;

[0076] (4) Conditions for transferring the seed tank: The bacterial cells are deeply stained, thick and strong, and there are no contaminants.

[0077] 3. Cellulase Production by Liquid Fermentation

[0078] (1) Fermentation Tank Medium: 1% CMC-Na, 1% glucose, 2% wheat bran, 3% corn steep liquor, 0.5% potassium dihydrogen phosphate, 0.2% magnesium sulfate, 2.0% ammonium sulfate, with the rest being water, pH 7.2 (“%” represents mass-volume percentage).

[0079] (2) Fermentation Tank Sterilization Process Conditions: Sterilize for 30 min at 121°C and 0.12 MPa.

[0080] (3) Fermentation Tank Process Conditions: Tank pressure 0.05 MPa, culture temperature 37°C, rotation speed 200 - 800 rpm (control dissolved oxygen at 30 - 35% by adjusting the rotation speed), inoculation amount 2%. Start feeding when the pH rises to 7.6, control the pH at 7.1 - 7.2 (initial feeding amount is 10 g / L / h). Reduce the feeding amount when the pH is lower than 7.2, increase the feeding amount when the pH is higher than 7.4, and adjust the feeding amount appropriately according to the pH situation. Stop fermentation when the cell autolysis is severe and the enzyme activity does not increase significantly.

[0081] 4. Feeding

[0082] (1) Feeding Medium: 5% CMC-Na, 50% glucose, 3.5% corn steep liquor, 3% ammonium sulfate, 4% dipotassium hydrogen phosphate, with the rest being water, pH 7.2 (“%” represents mass-volume percentage).

[0083] (2) Feeding Method: Start feeding when the pH rises to 7.6, control the pH at 7.1 - 7.2 (initial feeding amount is 10 g / L / h). Reduce the feeding amount when the pH is lower than 7.2, increase the feeding amount when the pH is higher than 7.4, and adjust the feeding amount appropriately according to the pH situation.

[0084] 5. Stop Fermentation

[0085] Cultivate for 70 - 80 h. When the enzyme activity growth is slow and the cells start to autolyze, stop fermentation.

[0086] 6. Extraction and Refinement of Alkaline Cellulase

[0087] After fermentation, add 40% water and 0.3% calcium chloride based on the volume of the fermentation broth, add 3% perlite filter aid, and perform plate and frame pressure filtration. Ultrafilter and concentrate the clarified filtered enzyme solution with an ultrafiltration membrane. Add 20% glycerol as a stabilizer, 10% sodium chloride, and 0.15% potassium sorbate as a preservative to the concentrated solution, and then filter and sterilize with diatomaceous earth to obtain the finished enzyme preparation of alkaline cellulase.

[0088] Fermentation was carried out using the above-mentioned Bacillus amyloliquefaciens mutant strain CGMCC No. 26079 (MN101202) and the culture medium. Table 2 shows the fermentation cycle and the enzyme activity of the fermentation broth for 6 batches of fermentation. The average enzyme activity of the fermentation broth measured by the method described in Example 2 was 1163 U / mL.

[0089] Table 2. Results of 50L small tank fermentation experiment

[0090] Batch Fermentation cycle (h) Fermentation enzyme activity (U / mL) 1 75 1122 2 76 1182 3 76 1158 4 78 1176 5 76 1183 6 79 1157

[0091] As can be seen from Table 2, the fermentation level of the mutagenized strain MN101202 was relatively stable, and the alkaline cellulase activity reached over 1100 U / mL.

[0092] At the same time, using the same method as above, the wild-type Bacillus amyloliquefaciens strain DXH20 was used as the production strain to ferment and produce cellulase and prepare the finished enzyme preparation for subsequent determination of enzyme properties.

[0093] Example 4 Enzyme property analysis

[0094] (1) Optimal reaction temperature

[0095] Take the finished cellulase prepared in Example 3, and using the method described in Example 2, at normal condition pH 9.0, measure the cellulase activity at 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 °C respectively, and calculate the relative enzyme activity with the highest enzyme activity as 100%. The results are as Figure 1 shown. The optimal reaction temperature of the cellulase produced by the mutagenized strain MN101202 is 30 °C, and there is still more than 85% of the enzyme activity at 20 °C, while the wild type only has about 50% of the enzyme activity at 20 °C, indicating that the cellulase produced by this mutant strain is more suitable for washing under natural low temperature conditions and more meets the requirements of normal temperature washing.

[0096] (2) Thermal stability

[0097] Take the finished cellulase prepared in Example 3, place the enzyme solution in a water bath at 50, 55, 60, 65, 70, 75, 80 °C for heat preservation treatment for 2 h respectively, and measure the enzyme activity using the method described in Example 2, and calculate the relative enzyme activity with the original enzyme activity without treatment as 100%. The experimental results are as Figure 2 shown. The enzyme activity of the cellulase produced by the mutagenized strain MN101202 basically does not lose at 50 °C for 2 h of heat preservation, and still can maintain more than 85% of the enzyme activity at 60 °C for 2 h of heat preservation, while the relative enzyme activity of the wild type is only 65%. Compared with the wild type, it has better thermal stability and is more suitable for high-temperature treatment during the forming process of solid detergents.

[0098] (3) Optimal reaction pH

[0099] Take the finished cellulose enzyme prepared in Example 3, and according to the enzyme activity determination method described in Example 2, at a temperature of 40 °C, measure the cellulose enzyme activity under the conditions of pH values of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, and 13.0 respectively. Calculate the relative enzyme activity with the highest enzyme activity as 100%. The measurement results are as Figure 3 shown. The cellulose enzyme activity produced by the mutant strain MN101202 is the highest when the pH is around 10.0, which is basically the same as that of the wild type. Under the condition of pH 12.0, the relative enzyme activity can still remain above 80%, while that of the wild type is only 63%. Since the pH value in the washing liquid is basically in the alkaline region of 10 - 11, it is required that the enzyme used as a detergent ingredient needs to adapt to the alkaline environment, and the cellulose enzyme produced by this strain just meets this requirement.

[0100] (4) Acid and alkali resistance

[0101] Take the finished cellulose enzyme prepared in Example 3, and place it in sodium carbonate - sodium bicarbonate buffer solutions with pH values of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0 respectively. After standing at room temperature for 2 h, measure the enzyme activity, and calculate the relative enzyme activity with the original enzyme activity without treatment as 100%. The measurement results are as Figure 4 shown. After the cellulose enzyme produced by the mutant strain MN101202 is treated for 2 h under the condition of pH 12, the relative enzyme activity still remains above 85%. Compared with the wild type, it has better alkali resistance.

[0102] Determination of the substrate specificity of the enzyme in Example 5

[0103] Take the finished cellulose enzyme prepared in Example 3, and according to the enzyme activity determination method described in Example 2, measure the enzyme activities with 1% CMC - Na, 1% microcrystalline cellulose (Avicel), 1% cellulose powder, and filter paper strips (0.5 × 0.5 cm) as substrates respectively (replace the "1% CMC - Na solution" in step ② of the enzyme activity determination method in Example 2), and calculate the relative enzyme activity with the highest enzyme activity as 100%. The measurement results are as Figure 5As shown in the figure, the cellulase produced by the mutant strain has stronger substrate specificity than the wild type. The cellulase CMCase activity of the mutagenized strain MN101202 is relatively strong, and it has basically no hydrolysis ability for cellulose with a relatively high crystallinity such as Avicel, cellulose powder, and filter paper strips. This indicates that the cellulase produced by this mutant strain is mainly endo-cellulase, which only has a relatively high hydrolysis activity for cellulose in the amorphous region, while having a very low hydrolysis activity for cellulose in the crystalline region. Therefore, it will not cause a significant decrease in the fabric strength or damage to the fabric. So this cellulase is an ideal additive for detergents.

[0104] Example 6 Determination of the resistance of cellulase to alkaline protease degradation

[0105] Currently, alkaline protease is generally added to detergents, which may have a certain hydrolysis effect on other types of enzymes added to the detergents, causing them to become inactivated. Therefore, it is necessary to determine the resistance of this cellulase to alkaline protease. Take the cellulase finished product prepared in Example 3, add alkaline protease at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 U / mL, and react under oscillation at 40°C for 1 h. According to the enzyme activity determination method described in Example 2, determine the residual enzyme activity of cellulose, and calculate the relative enzyme activity with the original enzyme activity of the untreated sample as 100%. The determination results are as Figure 6 shown. For the cellulase produced by the mutagenized strain MN101202, adding a low concentration of alkaline protease has basically no effect on its enzyme activity. When the concentration increases to 6 U / mL, the enzyme activity begins to decline. When the concentration increases to 10 U / mL, the relative enzyme activity of the mutant can still remain above 70%, while the relative enzyme activity of the wild type drops to 52%. Compared with the wild type, the alkaline cellulase of the present invention has better resistance to alkaline protease degradation and is more suitable for use in detergents.

[0106] Example 7 Effects of common surfactants and auxiliaries in detergents on cellulase activity

[0107] Currently, the main surfactants added to detergents are LAS and AEO-9, and the main auxiliaries used are 4A zeolite, sodium polyacrylate, sodium silicate, sodium carbonate, EDTA, sodium sulfate. In addition, perfume is also added. Take the cellulase finished product produced by the mutagenized strain MN101202 prepared in Example 3, and add 0.2, 0.4, 0.6, 0.8, 1 g / L of the above surfactants and auxiliaries respectively, and react at 40°C for 1 h, and determine the residual enzyme activity, and calculate the residual enzyme activity with the original enzyme activity of the untreated sample as 100%. The determination results are as Figure 7 shown. The above-mentioned surfactants and auxiliaries have basically no effect on the enzyme activity of the cellulase produced by MN101202 within a relatively wide range of usage concentrations and can be widely used in detergent products.

[0108] Determination of the Detergent Efficacy of Cellulase in Example 8

[0109] Take the finished cellulase product produced by the mutagenized strain MN101202 prepared in Example 3 and add it to the corresponding test solution at 30 U / L. The method for determining detergency is carried out according to the regulations of GB / T 13174-2021. Three kinds of stained cloth test pieces, JB-01, JB-02, and JB-03, are used, and the whiteness F1 of each test piece before washing is read with a whiteness meter at 457 nm. The detergent efficacy of cellulase on these three kinds of stained cloths is determined. The washing test is carried out in a vertical detergent machine. During the test, 250 mg / kg of hard water is used to prepare 1 L of test solution with a concentration of 0.2% for the sample and the standard detergent respectively and pour it into the corresponding detergent bathtub. Place the bathtub in the corresponding position and install the stirring impeller. Adjust the instrument to keep the washing test temperature at 30°C. Put the test pieces with measured whiteness into each bathtub one by one, start stirring, and keep the rotation speed at 120 rpm. Stop after the washing process lasts for 20 min. Take out the test pieces from each detergent bathtub, put them into a rinser for rinsing and dehydration, repeat rinsing four times. After the last rinsing and dehydration, take out the test pieces and place them in an enamel tray to dry at room temperature, and measure the whiteness F2. Calculate the detergency R of each detergent on various stained cloths. The formula for detergency is as follows:

[0110] R = (F2 - F1) / (f2 - f1)

[0111] In the formula: R—detergency;

[0112] F1—whiteness of the test piece before washing with the test solution, %;

[0113] F2—whiteness of the test piece after washing with the test solution, %;

[0114] f1—whiteness of the test piece before washing with the standard solution, %;

[0115] f2—whiteness of the test piece after washing with the standard solution, %.

[0116] The measurement results are as Figure 8 shown. Under the same detergent and the same washing conditions, adding a small amount of cellulase produced by MN101202 to the detergent can greatly improve the detergency. In addition, the detergency of the enzyme-added detergent varies with the type of stained cloth, but its detergency is greatly improved compared with the non-enzyme-added detergent, indicating that this mutant alkaline cellulase has good detergency and is suitable for the detergent industry.

[0117] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this patent, several variations, combinations, and improvements can be made to the above-mentioned embodiments, and these all fall within the protection scope of this patent. Therefore, the protection scope of this patent shall be subject to the claims.

Claims

1. A liquid fermentation production method for producing cellulase, characterized in that: The fermentation process conditions of the fermentation tank are as follows: inoculation amount 1-2%, tank pressure 0.05-0.08 MPa, culture temperature 36-38°C, rotation speed 200-800 rpm, feed when pH rises to 7.6, control pH 7.1-7.2, culture to 70-80h, release the tank when enzyme activity increases slowly and the bacteria begin to partially autolyze; The production strain is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MN101202, the deposit number is CGMCC No. 26079.

2. The liquid fermentation production method for producing cellulase according to claim 1, characterized in that: The fermentation tank culture medium composition is as follows: CMC-Na 1-1.2%, glucose 1-1.5%, bran 2-2.5%, corn steep liquor 2-3%, potassium dihydrogen phosphate 0.5-0.8%, magnesium sulfate 0.15-0.2%, ammonium sulfate 1.5-2.0%, and the rest is water, pH 7.2-7.

5.

3. The liquid fermentation production method for producing cellulase according to claim 1, characterized in that: The composition of the feed medium is as follows: CMC-Na 5-6%, glucose 40-60%, corn steep liquor 3.5-4.5%, ammonium sulfate 3-4%, potassium dihydrogen phosphate 3-4%, and the rest is water, pH 7.2-7.5.

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