A strain with high production of alkaline cellulase and its application

Abstract

The present invention belongs to the field of microbial technology, and particularly relates to a low-temperature alkaline-resistant cellulase high-yield strain and its application. The strain is obtained after NTG mutagenesis, specifically Bacillus amyloliquefaciens MN101201, 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, etc., the optimum reaction temperature is 30 DEG C, and it is kept warm for 2h under 60 DEG C, and the enzyme activity of more than 85% can still be maintained, 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 invention belongs to the technical field of microorganisms, and particularly relates to a low-temperature alkali-resistant cellulase high-yield strain and application thereof. Background technology:

[0002] Cellulose is the most abundant renewable resource in the world. Using microbial cellulase to degrade cellulose is more economical, effective and energy-saving than 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 generate small molecules such as cellobiose and glucose. According to the application conditions of cellulose, it can be divided into acid cellulase, neutral cellulase and alkaline cellulase. At present, the industrial application of cellulase is mainly concentrated in the fields of detergent industry, bioenergy, denim washing and textile finishing. Among them, acid cellulase is mainly used in the field of bioenergy. Cellulose is completely degraded through the degradation of cellulase, and its hydrolysis products can be further fermented to produce hydrogen, ethanol and biodiesel; in the washing industry, adding alkaline cellulase to detergent can improve the washing effect; in the textile finishing and denim washing industry, 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 detergent can make it easier to clean dirt from fabrics. However, dirt not only adheres to the surface of the fiber, but also easily enters the internal tissue of the fiber and is sealed. After repeated washing, the fabric will turn yellow, become old and hardened in the parts with more severe wear. Alkaline cellulase is a new type of enzyme for detergents. It not only has a good ability to remove dirt, but also can make cotton fabrics soft, brighter, and clearer after repeated use.

[0005] Natural cellulose is composed of two parts: crystalline and non-crystalline. The fiber structure of the crystalline part is tight, making it difficult for dirt to penetrate, while the fiber structure of the non-crystalline part is loose, making it easy for dirt to penetrate. Alkaline cellulase can act on the non-crystalline area inside the fabric fiber, making the cellulose structure of the cotton fabric bulky and the gel structure formed by the fiber molecules and water effectively softened, which makes it easy for the dirt trapped in it to dissolve from the gaps between the fibers, thereby improving the decontamination ability and making the washed fabric softer and brighter in color.

[0006] Cellulase can generally be divided into three types of enzyme components, namely exoglucanase, endo-glucanase and cellobiase. The alkaline cellulase added to detergent is endo-glucanase, namely carboxymethyl cellulase (CMCase), which has high hydrolysis activity only for cellulose in the non-crystalline area, but very low hydrolysis activity for cellulose in the crystalline area. Therefore, it will not cause a significant decrease in fabric strength 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 that are different from other enzymes. 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., but the cellulase they produce is usually acidic or neutral to acidic. Detergents are highly alkaline, and the pH value is usually above 9.0. Therefore, cellulase for washing must have a high enzyme activity under conditions above pH 9.0. In addition, from the international situation, washing is in the process of low temperature, and the washing characteristics of China are washing at normal temperature or even cold water. Therefore, it is of great significance to develop low-temperature alkaline-resistant cellulase with high activity. Summary of the invention:

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

[0009] The second technical solution provided by the present invention is that Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )Application of MN101202, especially in the production of cellulase.

[0010] The third 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 by the following measures:

[0011] Fermentation tank fermentation process conditions: inoculation amount 1-2%, tank pressure 0.05-0.08 MPa, culture temperature 36-38℃, rotation speed 200-800 rpm, start feeding when pH rises to 7.6, control pH 7.1-7.2, culture for about 70-80h, release the tank when enzyme activity increases slowly and the bacteria begin to partially autolyze;

[0012] At the end of fermentation, the enzyme activity of the fermentation broth was 1100-1200U / mL;

[0013] Furthermore, 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 ("%" indicates mass volume percentage).

[0014] Furthermore, the fermentation tank sterilization process conditions are: 121°C-124°C, 0.11-0.12 MPa, sterilization for 30 minutes.

[0015] Furthermore, the feed medium composition 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 ("%" indicates mass volume percentage).

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

[0017] (1) The optimum reaction temperature is 30°C. At 20°C, the enzyme activity is still above 85%, which is conducive to washing under natural low temperature conditions. The enzyme activity is basically not lost when kept at 50°C for 2 hours, and it can still maintain above 85% of the enzyme activity when kept at 60°C for 2 hours. The thermal stability is good, which is suitable for high temperature treatment in the solid detergent molding process.

[0018] (2) The optimum reaction pH is 10.0. After treatment at pH 12 for 2 h, the relative enzyme activity still remained above 85%, indicating good alkali resistance.

[0019] Beneficial effects:

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

[0021] 1. The invention first provides a mutant strain of Bacillus amyloliquefaciens MN101202 that produces high-yield low-temperature alkaline-resistant cellulase. The activity level of carboxymethyl cellulase produced by industrial fermentation using this strain can reach above 1100-1200 U / mL, which greatly reduces the production cost, is conducive to the large-scale application of alkaline cellulase, and saves energy and reduces emissions.

[0022] 2. The cellulase has the characteristics of high enzyme activity under low temperature conditions, stability of enzyme activity within the alkaline pH range, stability under the action of various surfactants and filter aids in washing powder, and resistance to degradation by alkaline protease. The optimum reaction temperature is 30℃. After being kept at 60℃ for 2h, the enzyme activity can still be maintained at more than 85%, and the thermal stability is good; the optimum reaction pH is 10.0. After being treated at pH12 for 2h, the enzyme can maintain more than 85% of its activity and has good alkali resistance.

[0023] 3. The cellulase is mainly an endo-type cellulase, which has a high hydrolysis activity only on cellulose in the non-crystalline area, but a very low hydrolysis activity on cellulose in the crystalline area. It will not cause a significant decrease in fabric strength or damage to the fabric, and can be widely used in the detergent industry. Description of the drawings:

[0024] Figure 1 Optimum reaction temperature curve.

[0025] Figure 2 Thermal stability curve.

[0026] Figure 3 Optimal reaction pH curve.

[0027] Figure 4 pH stability graph.

[0028] Figure 5 Substrate specificity of alkaline cellulase.

[0029] Figure 6 The performance curve of resistance to alkaline protease degradation.

[0030] Figure 7 Curve chart showing the effects of commonly used surfactants and additives in detergents on cellulase activity.

[0031] Figure 8 Determination of the detergent efficiency 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] Example 1 Mutation breeding of strains

[0034] 1. NTG mutagenesis

[0035] The starting strain DXH20 was inoculated into the seed culture medium and cultured at 37°C until the logarithmic phase. Then the bacteria were collected, centrifuged at 8000rpm for 5min, and the bacterial precipitate was washed twice with physiological saline. Finally, the bacteria were resuspended with Tris buffer at pH 7.0, and NTG mother solution was added to make the final concentration 0.375g / L. Then reacted at 37°C for 30min, and after appropriate dilution, 100μL was applied to the screening plate, cultured at 37°C for about 24h, and the lethality was calculated. Add an appropriate amount of 1% Congo red solution to the cultured culture dish, stain for 20min, discard the dye solution, add an appropriate amount of 1mol / L NaCl solution, and pour out the NaCl solution after 20min. Select the mutant strain according to the diameter of the transparent circle around the colony and the diameter of the colony, pick the colony with a large ratio for further streaking separation, and transfer the separated single bacteria to the seed culture medium. After the seeds grow well, enter the shaking bottle for rescreening. Through NTG mutagenesis, a mutant strain MN101202 was finally screened out with an initial shake flask enzyme activity of 155U / mL. The shake flask enzyme activity of the starting strain DXH20 was 48U / mL, which was 3.2 times higher than that of the starting strain.

[0036] (1) Plate screening culture 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%, and the rest is water, pH 7.2.

[0038] (2) Seed culture 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%, and the rest is water, pH 7.2.

[0042] (4) Culture conditions

[0043] Separation plate: culture at 37°C for 24 h;

[0044] Liquid seeds: culture at 37°C for 8 h, shaker at 220 rpm;

[0045] Fermentation shake flask: culture at 37°C for 72 h, shaker speed of 220 rpm, inoculation amount of 2%.

[0046] 2. Stability test of cellulase high-yielding strain MN101202

[0047] A single colony of MN101202 with good growth on the screening plate was selected and inoculated into a seed bottle, cultured at 37°C, 220rpm for about 8h, transferred to a fermentation shake flask at a 2% inoculation rate, cultured at 37°C, 220rpm for about 72h, and the enzyme activity was measured. The results of the shake flask after 10 consecutive passages of the strain are shown in Table 1:

[0048] Table 1. Stability test results of strain MN101202

[0049]

[0050] The mutant strain was subcultured for 10 generations. The experimental results shown in Table 1 show that the mutant strain has good genetic stability.

[0051] Example 2 Alkaline Cellulase Activity Assay Method

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

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

[0054] (2) Enzyme activity determination method

[0055] The activity of the alkaline cellulase of the present invention is detected by the DNS method. The cellulase hydrolyzes the cellulose substrate (CMC-Na) under certain temperature and pH conditions (if not specifically stated, the detection conditions are 40°C and pH 9.0) to release reducing sugars. The 3,5-dinitrosalicylic acid solution is reduced to a brown-red amino compound after being heated with the reducing sugar solution. Within a certain range, the amount of reducing sugar is proportional to the depth of the color of the brown-red substance, so it can be used for colorimetric determination.

[0056] The specific method is as follows:

[0057] ① Take four 25mL 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 into each of the four test tubes.

[0059] ③ Accurately add 0.5 mL of the diluted enzyme solution to be tested into three sample tubes (no addition to the blank tube), mix well, and cover.

[0060] ④ Place the four test tubes in a (40±0.1)℃ water bath at the same time, accurately time them, react for 30 minutes, and then take them out.

[0061] ⑤ Immediately 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 tested to the blank tube and shake well. Put the four test tubes into a boiling water bath at the same time, heat for 10 minutes, take them out, quickly cool to room temperature, add water to make up to 25 mL, and shake well.

[0062] ⑥ Use a blank tube (control solution) to adjust the instrument zero point, and measure the absorbance of the sample solution in three parallel test tubes at a wavelength of 540nm on the spectrophotometer, and take the average value. Calculate the reducing sugar content by checking the standard curve.

[0063] Enzyme activity calculation formula: X = A × n × 1000 / (0.5 × 180 × 30)

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

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

[0066] n—dilution multiple of enzyme sample;

[0067] 180—glucose molecular weight;

[0068] 30—reaction time (min);

[0069] 0.5—converted into 1mL of enzyme solution;

[0070] 1000—millimoles converted to micromoles.

[0071] Example 3 Production of cellulase by liquid fermentation of MN101202 strain

[0072] 1. Seed tank expansion culture

[0073] (1) Seed tank culture medium: 2% glucose, 1% soybean meal, 2% peptone, 2% corn steep liquor, 0.5% potassium dihydrogen phosphate, 1.2% ammonium sulfate, and the rest water, pH 7.2.

[0074] (2) Seed tank sterilization process conditions: 121°C, 0.12MPa, sterilization for 30min;

[0075] (3) Seed tank culture process conditions: tank pressure 0.05 MPa, culture temperature 37°C, stirring speed 400 rpm, inoculation amount 2%, pH control 7.2;

[0076] (4) Seed tank transplanting conditions: The bacteria are darkly stained, strong, and free of foreign bacteria.

[0077] 3. Liquid fermentation to produce cellulase

[0078] (1) Fermentation tank culture medium: CMC-Na 1%, glucose 1%, bran 2%, corn steep liquor 3%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.2%, ammonium sulfate 2.0%, and the rest is water, pH 7.2 (“%” indicates mass volume percentage).

[0079] (2) Fermentation tank sterilization process conditions: 121℃, 0.12 MPa, sterilization for 30 min.

[0080] (3) Fermentation tank process conditions: tank pressure 0.05 MPa, culture temperature 37°C, rotation speed 200-800 rpm (by adjusting the rotation speed, the dissolved oxygen is controlled at 30-35%), inoculation amount 2%, feed when pH rises to 7.6, control pH 7.1-7.2 (initial feed amount is 10 g / L / h), reduce feed amount when pH is lower than 7.2, increase feed amount when pH is higher than 7.4, adjust feed amount appropriately according to pH conditions, and release the tank when fermentation reaches serious autolysis of the bacteria and no significant increase in enzyme activity.

[0081] 4. Feed

[0082] (1) Feed medium: CMC-Na 5%, glucose 50%, corn steep liquor 3.5%, ammonium sulfate 3%, dipotassium hydrogen phosphate 4%, and the rest water, pH 7.2 (% indicates mass volume percentage).

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

[0084] 5. Place the can

[0085] After 70-80 hours of cultivation, the enzyme activity increases slowly and the bacteria begin to autolyze, then they can be put into the tank.

[0086] 6. Extraction and purification of alkaline cellulase

[0087] After the fermentation is completed, 40% water and 0.3% calcium chloride are added according to the volume of the fermentation liquid, and 3% perlite filter aid is added to perform plate and frame filtration; the clarified filtered enzyme liquid is ultrafiltered and concentrated using an ultrafiltration membrane; 20% glycerol as a stabilizer, 10% sodium chloride, and 0.15% potassium sorbate as a preservative are added to the concentrate, and then diatomaceous earth is used for filtration and sterilization to obtain the finished alkaline cellulase enzyme preparation.

[0088] Fermentation was carried out using the mutant strain of Bacillus amyloliquefaciens CGMCC No. 26079 (MN101202) and culture medium. Table 2 shows the fermentation cycle and 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 2.50L small tank fermentation test results

[0090]

[0091] As can be seen from Table 2, the fermentation level of the induced strain MN101202 was relatively stable, and the alkaline cellulose fermentation enzyme activity reached more than 1100 U / mL.

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

[0093] Example 4 Analysis of Enzymatic Properties

[0094] (1) Optimum reaction temperature

[0095] The cellulase product prepared in Example 3 was taken and the method described in Example 2 was used to measure the cellulase activity at 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60°C at normal pH 9.0. The relative enzyme activity was calculated with the highest enzyme activity as 100%. The results are shown in FIG. Figure 1 As shown, the optimal reaction temperature of the cellulase produced by the mutant strain MN101202 is 30°C, and it still has more than 85% enzyme activity at 20°C, while the wild type has only about 50% enzyme activity at 20°C, indicating that the cellulase produced by the mutant strain is more suitable for washing under natural low temperature conditions and is more in line with the requirements of normal temperature washing.

[0096] (2) Thermal stability

[0097] The cellulase product prepared in Example 3 was taken and the enzyme solution was placed in 50, 55, 60, 65, 70, 75, and 80°C for 2 hours. The enzyme activity was determined by the method described in Example 2, and the relative enzyme activity was calculated with the original enzyme activity without treatment as 100%. The experimental results are shown in FIG. Figure 2As shown, the cellulase produced by the mutagenic strain MN101202 has basically no loss of enzyme activity when kept at 50°C for 2 hours, and can still maintain more than 85% of the enzyme activity when kept at 60°C for 2 hours, 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 in the solid detergent molding process.

[0098] (3) Optimal reaction pH

[0099] The cellulase product prepared in Example 3 was taken, and the cellulase activity was measured at 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 at a temperature of 40°C according to the enzyme activity determination method described in Example 2. The relative enzyme activity was calculated with the highest enzyme activity as 100%. The determination results are as follows: Figure 3 As shown in the figure, the cellulase activity produced by the mutant strain MN101202 is highest when the pH is around 10.0, which is basically the same as the wild type. Under pH 12.0 conditions, the relative enzyme activity can still be maintained at more than 80%, while the wild type is only 63%. Since the pH value in the washing liquid is basically in the alkaline region of 10-11, the enzyme used as a detergent ingredient needs to adapt to the alkaline environment, and the cellulase produced by this strain just meets this requirement.

[0100] (4) Acid and alkali resistance

[0101] The cellulase product prepared in Example 3 was placed 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, and the enzyme activity was measured after standing at room temperature for 2 hours. The relative enzyme activity was calculated with the original untreated enzyme activity as 100%. The measurement results are shown in FIG. Figure 4 As shown, the relative enzyme activity of the cellulase produced by the mutagenic strain MN101202 remained above 85% after being treated at pH 12 for 2 h, and it had better alkali resistance than the wild type.

[0102] Example 5 Determination of enzyme substrate specificity

[0103] The cellulase product prepared in Example 3 was taken and the enzyme activity was measured using 1% CMC-Na, 1% microcrystalline cellulose (Avicel), 1% cellulose powder and filter paper strips (0.5×0.5 cm) as substrates according to the enzyme activity measurement method described in Example 2 (replacing the "1% CMC-Na solution" in step ② of the enzyme activity measurement method in Example 2). The relative enzyme activity was calculated with the highest enzyme activity as 100%. The measurement results are as follows: Figure 5As shown, the cellulase produced by the mutant strain has stronger substrate specificity than the wild type. The cellulase CMCase produced by the mutant strain MN101202 has stronger activity and has basically no hydrolysis ability for cellulose with higher crystallinity, such as Avicel, cellulose powder, and filter paper strips. This shows that the cellulase produced by the mutant strain is mainly endo-cellulase, which has higher hydrolysis activity only for cellulose in the non-crystalline region, but very low hydrolysis activity for cellulose in the crystalline region. Therefore, it will not cause a significant decrease in fabric strength or damage to the fabric, so the cellulase is an ideal additive for detergents.

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

[0105] Alkaline protease is generally added to detergents at present, which may hydrolyze other types of enzymes added to the detergent to a certain extent and inactivate them, so it is necessary to measure the resistance of the cellulase to alkaline protease. Take the cellulase product prepared in Example 3, add 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 U / mL of alkaline protease, oscillate and react at 40°C for 1 hour, and measure the residual enzyme activity of cellulose according to the enzyme activity determination method described in Example 2, and calculate the relative enzyme activity with the untreated original enzyme activity as 100%. The measurement results are as follows: Figure 6 As shown, for the cellulase produced by the mutagenic strain MN101202, adding a low concentration of alkaline protease has little effect on its enzyme activity. When the concentration increases to 6 U / mL, the enzyme activity begins to decrease. When the concentration increases to 10 U / mL, the relative enzyme activity of the mutant can still be maintained above 70%, while the relative enzyme activity of the wild type decreases to 52%. Compared with the wild type, the alkaline cellulase of the present invention has better resistance to degradation by alkaline protease and is more suitable for use in detergents.

[0106] Example 7 Effects of Commonly Used Surfactants and Additives in Detergents on Cellulase Activity

[0107] At present, the surfactants added to detergents are mainly LAS and AEO-9, and the auxiliary agents used are mainly 4A zeolite, sodium polyacrylate, sodium silicate, sodium carbonate, EDTA, sodium sulfate, and flavors are also added. Take the finished cellulase produced by the mutagenic strain MN101202 prepared in Example 3, add 0.2, 0.4, 0.6, 0.8, 1g / L of the above surfactants and auxiliary agents respectively, react at 40°C for 1h, measure the residual enzyme activity, and calculate the residual enzyme activity with the untreated original enzyme activity as 100%. The measurement results are as follows Figure 7 As shown, the surfactants and adjuvants used above have little effect on the cellulase activity produced by MN101202 within a wide range of concentrations, and can be widely used in detergent products.

[0108] Example 8 Determination of the Decontamination Efficacy of Cellulase

[0109] Take the finished cellulase produced by the mutagenic strain MN101202 prepared in Example 3, and add it to the corresponding test solution at 30U / L. The detergency determination method is carried out in accordance with the method specified in GB / T13174-2021, using three kinds of dirty cloth test pieces JB-01, JB-02, and JB-03, and using a whiteness meter to read the whiteness F1 of each test piece before washing at 457nm. Determine the decontamination efficiency of cellulase on these three kinds of dirty cloth. The washing test was carried out in a vertical decontamination machine. During the test, 250mg / kg hard water was used to prepare the test sample and the standard detergent into a test solution with a concentration of 0.2%, and 1L was poured into the corresponding decontamination bathtub, the bathtub was placed in the corresponding position and the stirring impeller was installed, the instrument was adjusted to keep the washing test temperature at 30°C, and the test pieces for measuring whiteness were put into each bathtub one by one, stirring was started, and the speed was maintained at 120rpm, and the washing process lasted for 20min and then stopped. Take out the test pieces from each cleaning bathtub, put them into the rinser for rinsing and dehydration, repeat the 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 each type of dirty cloth. The detergency calculation formula is as follows:

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

[0111] Where: R—determination power;

[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 standard solution, %;

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

[0116] The results of the test are as follows Figure 8 As shown, under the same detergent and the same washing conditions, adding a small amount of cellulase produced by MN101202 to the detergent can significantly improve the decontamination ability. In addition, the decontamination ability of the enzyme-added detergent varies with the type of dirty cloth, but its decontamination ability is significantly improved compared with the detergent without enzyme, indicating that the mutant alkaline cellulase has good decontamination ability and is suitable for the detergent industry.

[0117] 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 Bacillus amyloliquefaciens strain, characterized in that: Specifically, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) MN101202, the deposit number is CGMCC No. 26079.

2. The Bacillus amyloliquefaciens according to claim 1 ( Bacillus amyloliquefaciens ) The application of MN101202, characterized in that, It is used in the field of detergents.

3. The Bacillus amyloliquefaciens according to claim 1 ( Bacillus amyloliquefaciens ) The application of MN101202, characterized in that, It is used in the production of cellulase.

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