Bacillus velezensis ZZ04 and application thereof

Abstract

The invention discloses a bacillus velezensis ZZ04, which is classified and named as bacillus velezensis, is preserved in China General Microbiological Culture Collection Center on August 23, 2023, and has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 28229. According to the invention, a culture medium and fermentation conditions for producing cellulase by the bacillus velezensis ZZ04 are optimized, the enzyme activity of carboxymethyl cellulase (CMCase) in the obtained crude enzyme can reach 222.1 U.mL <-1 >, and the enzyme activity of filter paper enzyme (FPA) can reach 171.2 U.mL <-1 >, so that the strain ZZ04 has relatively high cellulose degradation capability under a normal temperature condition, can be used for biodegradation of cellulose, and can be used for preparing cellulose biologically. And a theoretical and practical application foundation is laid for treatment and utilization of wastes such as straws and fruit peels in agricultural production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, and more particularly to a bacillus velezensis ZZ04 and application thereof. BACKGROUND

[0002] Cellulose is an important carbohydrate, which is the main component of plant cell walls, and is the most widely distributed and largest storage polysaccharide in nature, and its smallest constituent unit is glucose. Cellulose is the most abundant renewable resource in the world, but has not been fully utilized.

[0003] Cellulose-degrading bacteria are a kind of microorganisms that can produce cellulase and exist in large quantities in nature. After isolation and screening, these microorganisms have great application potential. In nature, cellulose is mainly decomposed into oligosaccharides and cellobiose by cellulose-degrading bacteria, and finally degraded into glucose. Using cellulose-degrading bacteria to treat cellulose has the advantages of high decomposition efficiency and no pollution, and has broad application prospects in the resourceization of biomass solid waste.

[0004] Therefore, screening high-yield cellulase-producing strains and applying them to degrade biomass solid waste such as straw and fruit peels is of great significance for cellulose resource utilization. SUMMARY

[0005] To solve the above problems, the present application provides a bacillus velezensis ZZ04 and application thereof.

[0006] To achieve the above purpose, the present application adopts the following technical solutions:

[0007] The bacillus velezensis ZZ04 is classified and named as Bacillus velezensis, and was preserved in the China General Microbiological Culture Collection Center on August 23, 2023, with a preservation number of CGMCC No.28229 and a preservation address of No.3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard.

[0008] Another object of the present application is to provide a crude enzyme liquid, which is obtained by inoculating the bacillus velezensis ZZ04 seed liquid into an enzyme-producing culture medium, centrifuging, and taking the supernatant.

[0009] Preferably, the OD value of the bacillus velezensis ZZ04 seed liquid is 1. 600

[0010] Preferably, the inoculation amount of the bacillus velezensis ZZ04 seed liquid is 3%.

[0011] ​Preferably, the composition of the enzyme-producing culture medium is: 2% CMC-Na, 2% proteose peptone, 2% yeast powder, 0.5% NaCl, 0.1% KH2PO4, 0.02% MgSO4·7H2O, water to 1L, pH 7, by mass of 1000mL of the culture medium.

[0012] Preferably, the conditions of the shaking culture are: 35°C, 150rpm, and 48h.

[0013] Preferably, the centrifugation conditions are: 10000r / min, 4°C, and 10min.

[0014] Another object of the present application is to provide the application of the above-mentioned Bacillus velezensis ZZ04 or crude enzyme liquid in degrading straw and peel.

[0015] Through the above technical solution, the present application discloses a strain of Bacillus velezensis ZZ04, optimizes the culture medium and fermentation conditions for producing cellulase, and the enzyme activity of carboxymethyl cellulase (CMCase) in the obtained crude enzyme liquid can reach 222.1U·mL -1 , and the enzyme activity of filter paper enzyme (FPA) can reach 171.2U·mL -1 , which indicates that the strain ZZ04 has a high cellulose degradation capacity under normal temperature conditions, and can be used for biological degradation of cellulose, thereby laying a theoretical and practical application foundation for the treatment and utilization of agricultural production such as straw, peel and other wastes. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : Hydrolysis circle diagram of 4 strains with obvious hydrolysis circle obtained through CMC-Na culture medium screening;

[0017] Figure 2 : Glucose standard curve;

[0018] Figure 3 : Colony morphological characteristics of ZZ04 strain;

[0019] Figure 4 : Construction of multigene phylogenetic tree of 16s rDNA, gyrA and gyrB sequences of ZZ04 strain;

[0020] Figure 5 : Effect of different carbon sources on cellulase activity of ZZ04 strain;

[0021] Figure 6 : Effect of different nitrogen sources on cellulase activity of ZZ04 strain;

[0022] Figure 7 : Effect of different carbon source and nitrogen source dosage ratios on cellulase activity of ZZ04 strain;

[0023] Figure 8 Effect of different inoculation amount (v / v) on cellulase activity of ZZ04 strain;

[0024] Figure 9 Effect of different initial pH value of enzyme production medium on cellulase activity of ZZ04 strain;

[0025] Figure 10 Effect of different culture temperature on cellulase activity of ZZ04 strain;

[0026] Figure 11 Effect of different culture time on cellulase activity of ZZ04 strain;

[0027] wherein, Figures 5-11 Different small letters in the table represent significant difference at P<0.05 level. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0029] The reagents involved in the embodiments of the present application are all purchased from the market channel, and the methods not mentioned are conventional experimental methods, which will not be described one by one here.

[0030] The following reagents are illustrative:

[0031] Sodium carboxymethyl cellulose screening medium (g / L): CMC-Na 10 g, KH2PO4 1 g, (NH4)2SO4 1 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, yeast powder 1 g, agar 15 g, distilled water 1000 mL.

[0032] Enzyme production medium with CMC-Na as the only carbon source: CMC-Na 10 g, peptone 5 g, (NH4)2SO4 2 g, KH2PO4 2 g, yeast powder 1 g, MgSO4 0.5 g, CaCl2 0.1 g, NaCl 0.5 g, FeSO4·7H2O 0.05 g, MnSO4 0.02 g, water to 1000 mL.

[0033] LB liquid medium: containing 1% of trypsin, 0.5% of yeast extract, 1% of sodium chloride, and the rest of water, with a pH of 7.0, based on the mass of 1000 mL of medium.

[0034] Isolation, screening and identification of strains of Example 1

[0035] 1. Isolation, screening of strains

[0036] (1) Isolation of strains

[0037] Soil samples were collected from the humus layer of a corn planting base in Chaoyang Town, Longwen District, Zhangzhou City, Fujian Province, at a depth of 10-15 cm. 1.00 g of the soil sample was weighed, 100 mL of sterile water was added, and it was stirred and then left to stand. After standing, 1 mL of the supernatant was taken, and it was diluted with sterile water to 10 4 , 10 5 , 10 6 times the volume, and the diluted solution was spread on a CMC-Na screening medium plate. Sterile water was spread as a control, and 3 replicates were set for each dilution. All the medium plates were incubated at 28°C for 1-2 days, and fast-growing strains were picked and purified by three-zone streaking to obtain single colonies. A total of 13 single colonies were selected, and they were named ZZ01, ZZ02, ZZ03,..., ZZ13, respectively. They were stored in glycerol at -80°C for later use.

[0038] (2) Screening of strains

[0039] The 13 purified strains described above were inoculated in the center of a CMC-Na plate medium, and after being incubated at 28°C for 2 days, the plates were stained with 20 mL of 1.0 g·L -1 Congo red dye for 30 min. After the dye was discarded, the plates were decolorized with 20 mL of 1 mol·L -1 NaCl solution for 30 min, and then washed with sterile water for 2-3 times. Whether a yellow hydrolysis ring was produced on the medium was observed, the diameter of the hydrolysis ring (D) and the diameter of the colony (d) were measured, and the ratio of the hydrolysis ring to the colony diameter (D / d) was calculated. Four strains with obvious hydrolysis rings were obtained (see Figure 1 ), and the results are shown in Table 1.

[0040] Table 1 Determination of Congo red staining hydrolysis rings of the four strains ZZ01-ZZ04

[0041]

[0042] As can be seen from Table 1, the yellow hydrolysis ring of strain ZZ04 was the largest, and the ratio of the hydrolysis ring to the colony diameter (D / d) was the largest.

[0043] The crude enzyme solution of the four strains in Table 1 was subjected to determination of cellulase activity, and the specific operation was as follows:

[0044] ① Determination of carboxymethyl cellulase (CMCase) activity of the crude enzyme solution of the four strains ZZ01-ZZ04

[0045] A. Preparation of standard curve:

[0046] 0.5 g of analytical reagent anhydrous glucose was weighed and dissolved in distilled water to prepare glucose standard solutions with concentrations of 0.04, 0.06, 0.08, 0.10 and 0.12 mg / L, respectively; 2.5 mL of each standard solution was taken and placed in 5 15 mL test tubes, 2.5 mL of DNS reagent was added to each test tube, after boiling water bath for 10 min, the test tubes were quickly cooled to room temperature with running water, and the volume was made up to 5 mL with distilled water, the absorbance of the solution in the test tube was measured at a wavelength of 540 nm, and distilled water was used as a blank control instead of the glucose standard solution; the standard glucose concentration (mg / mL) was taken as the abscissa, and the corresponding absorbance was taken as the ordinate to draw the glucose standard curve, as shown in FIG. 1. Figure 2 .

[0047] According to the concentration of the glucose standard solution and the corresponding absorbance, the glucose standard curve equation was fitted, and the standard curve y = 0.0111x-0.0786 was obtained, with a linear correlation coefficient R 2 = 0.9875, which was good for linear and could be used for cellulase activity determination.

[0048] B. Preparation of crude enzyme solution:

[0049] After activation, 6 mL of each of the above four strains was inoculated into 200 mL of LB liquid medium, and cultured at 28°C and 180 r / min for 24 h; when OD600 = 1 was measured by using an ultramicro UV spectrophotometer, the bacterial suspension at this time was used as a seed solution for subsequent experiments.

[0050] 5 mL of bacterial suspension was inoculated into 100 mL of enzyme-producing culture medium with CMC-Na as the sole carbon source, and incubated at 37°C and 175 r / min for 4 d; the obtained supernatant was the crude enzyme solution after centrifugation at 10,000 r / min and 4°C for 10 min.

[0051] C. Determination of CMCase enzyme activity:

[0052] In the test tube, 1.5 mL of citric acid buffer solution (pH 4.8) containing 1% CMC-Na was added as a substrate, after preheating, 0.5 mL of crude enzyme solution was added and shaken to mix, and then placed in a 50°C water bath for reaction for 30 min, then 2 mL of DNS solution was immediately added to terminate the reaction, and then placed in a boiling water bath for 10 min, quickly cooled and made up to 10 mL with distilled water, and the absorbance was measured at a wavelength of 540 nm; the control group was treated by adding 2 mL of DNS solution first and then adding the crude enzyme solution, and the glucose content obtained by enzyme hydrolysis was calculated according to the glucose standard curve to obtain the enzyme activity value. Enzyme activity is defined as: 1 μg of glucose produced by 1 mL of crude enzyme solution per minute under the above determination conditions is 1 enzyme activity unit.

[0053] (2) Enzymatic activity of the crude enzyme solution of the four strains ZZ01, ZZ02, ZZ03 and ZZ04

[0054] A. Preparation of the standard curve: same as ①A.

[0055] B. Preparation of the crude enzyme solution: same as ①A.

[0056] C. Enzymatic activity determination of FPA:

[0057] In a test tube, add a piece of filter paper (1 cm x 6 cm, about 50 mg) and 1.5 mL of sodium citrate buffer (pH 4.8) as a substrate, preheat, then add 0.5 mL of crude enzyme solution and shake well, place in a 50°C water bath for 60 min, then immediately add 2 mL of DNS solution to terminate the reaction, place in a boiling water bath for 10 min, then quickly cool and dilute to 10 mL with distilled water, measure the absorbance at 540 nm, and determine the glucose content produced by enzymatic hydrolysis by comparing with the control group which is first added with 2 mL of DNS solution and then added with the crude enzyme solution, and then convert to enzyme activity value. Enzyme activity definition: 1 μg of glucose produced by 1 mL of crude enzyme solution per minute under the above determination conditions is 1 unit of enzyme activity.

[0058] The results of the determination of cellulase activity of the crude enzyme solutions of the above four strains are shown in Table 2.

[0059] Table 2 Determination of cellulase activity of the crude enzyme solutions of the four strains ZZ01- ZZ04

[0060]

[0061] As can be seen from Table 2, the enzyme activities of carboxymethyl cellulase and filter paper enzyme of the crude enzyme solution of ZZ04 are the highest among the four strains, which are 96.82 U·mL -1 and 65.29 U·mL -1 , respectively.

[0062] 2. Identification of the strain

[0063] (1) Identification of colony morphological characteristics

[0064] ZZ04 strain was inoculated into NA medium and cultured at 37°C for 24 h, and grew well, with light yellow colonies, irregular edges, smooth surface and dryness (see Figure 3 ), which is a gram-positive bacterium, with bacilli-shaped cells, length of 2.2 μm-9.7 μm, width of 0.2 μm-0.6 μm, flagellum and intercalary spores. According to the comparison with the "Common Bacteria System Identification Manual", this strain is preliminarily classified as Bacillus.

[0065] (2) Molecular biology identification

[0066] The DNA of the ZZ04 strain was amplified by 16s rDNA, gyrA and gyrB, and then sequenced by Shengong Bioengineering (Shanghai) Co., Ltd. to obtain 1516 bp (as shown in SEQ ID NO. 1), 989 bp (as shown in SEQ ID NO. 2), and 1160 bp (as shown in SEQ ID NO. 3) sequence fragments. After Blast comparison, the strain ZZ04 had 100% similarity with the 16s rDNA gene sequence of Bacillus velezensis in Genbank (accession number: MK641661), 99.90% similarity with the gyrA gene (accession number: MZ357346), and 100% sequence similarity with the gyrB gene (accession number: MT119762). The sequencing results of 16s rDNA, gyrA and gyrB of the strain ZZ04 were compared with the sequences of species with higher homology in GenBank to construct a multigene phylogenetic tree. It was found that the strain ZZ04 and Bacillus velezensis were in one branch, and their genetic distance was the closest (see Figure 4 ), and it was determined to be Bacillus velezensis.

[0067] Example 2 Optimization of enzyme production fermentation conditions of ZZ04 strain

[0068] After the ZZ04 strain was activated, 6 mL was inoculated into 200 mL of LB liquid medium, and cultured at 28°C and 180 r / min for 24 h. When the OD600 measured by ultraviolet spectrophotometer was 1, it was used as seed liquid for the following process optimization.

[0069] 1. Optimization of enzyme production nutritional conditions

[0070] (1) Selection of optimal carbon source

[0071] The basal culture medium with the same nitrogen source but different carbon sources was prepared by using 2% peptone as the nitrogen source and adding 1% CMC-Na, glucose, sucrose, starch, beef extract and corn stalk chips as the carbon source (based on the mass of 1000 mL of culture medium, in addition to the carbon source, it also contained 2% peptone, 0.5% NaCl, 0.1% KHPO4, 0.02% MgSO4·7H2O, and the rest was water, pH 7.0). The seed liquid of ZZ04 strain was inoculated into 40 mL of the above-mentioned basal culture medium at an inoculum volume of 2% (v / v) in a triangular flask (250 mL), sealed with a vent plug, placed in a water bath shaker, and cultured at 37°C and 150 rpm for 36 h. The crude enzyme solution was then taken out and centrifuged at 10,000 r / min and 4°C for 10 min to obtain the crude enzyme solution. The CMC enzyme activity (determination method is the same as in Example 1.1 (2) ①C) and the filter paper enzyme activity (determination method is the same as in Example 1.1 (2) ②C) of the crude enzyme solution cultured with different carbon sources were determined. The results are shown in FIG. Figure 5 shown.

[0072] from Figure 5 It can be seen that strain ZZ04 has a wide range of carbon source utilization. However, in the culture medium with CMC-Na and beef extract as carbon sources, the enzyme activities of CMCase and FPA in the crude culture medium after culture are much higher than those of other tested carbon sources, especially when CMC-Na is used as the carbon source, with the highest enzyme activity values ​​of CMCase being 84.7 U·mL -1 The enzyme activity of FPA was 70.6 U·mL -1 Therefore, the experiment determined that CMC-Na was the optimal carbon source for cellulase from strain ZZ04.

[0073] (2) Selection of the best nitrogen source

[0074] The basal culture medium with the same carbon source but different nitrogen sources was prepared by using 1% CMC-Na as the carbon source and adding 2% peptone, yeast powder, (NH4)2SO4, KNO3, NH4CL and NH4NO3 as nitrogen sources respectively (based on the mass of 1000 mL of culture medium, in addition to the nitrogen source, it also contained 1% CMC-Na, 0.5% NaCl, 0.1% KHPO4, 0.02% MgSO4·7H2O, and the rest was water, pH 7.0). The seed liquid of the ZZ04 strain was inoculated at an inoculum rate of 2% (v / v) into a 250 mL Erlenmeyer flask containing 40 mL of the above-mentioned basal culture medium. The flask was sealed with a vent plug and placed in a water bath shaker. After culturing at 37°C and 150 rpm for 36 h, the flask was taken out and centrifuged at 10,000 r / min and 4°C for 10 min to obtain a crude enzyme solution. The CMC enzyme activity (the determination method was the same as in Example 1.1(2)①C) and the filter paper enzyme activity (the determination method was the same as in Example 1.1(2)②C) of the crude enzyme solution cultured with different nitrogen sources were determined. The results are shown in FIG.Figure 6 shown.

[0075] Depend on Figure 6 It can be seen that the types of nitrogen sources that can be used by strain ZZ04 to produce cellulase are less diverse than the types of carbon sources that can be used. Under conditions of inorganic nitrogen sources such as (NH4)2SO4, the activity of cellulase produced by this strain is relatively low. When peptone and yeast powder are used as nitrogen sources, the cellulase activity of strain ZZ04 is relatively high. However, the relative maximum values ​​of CMCase and FPA enzyme activities are not obtained under the same nitrogen source conditions. When peptone is used as the nitrogen source, the enzyme activity of CMCase is the highest, at 94.3 U·mL -1 (At this time, the enzyme activity value of PFA is 71.8U·mL -1 ), when yeast powder was used as nitrogen source, the enzyme activity of FPA was the highest, which was 77.7 U·mL -1 (At this time, the enzyme activity value of CMCase is 73.9U·mL -1 In order to balance the cellulase components produced by this strain, the optimal nitrogen source was determined to be an equal mixture of peptone and yeast powder.

[0076] (3) Optimization of the ratio of carbon source to nitrogen source

[0077] In the above carbon and nitrogen source selection experiments, the optimal carbon and nitrogen source types were determined. However, they were based on the classic dosage and ratio in the preservation medium (broth medium), which were 1% and 2%, respectively. These were not necessarily applicable to the cellulase production of strain ZZ04. Therefore, the carbon and nitrogen source dosage ratios were optimized. The experiment was as follows:

[0078] The basic medium with same carbon and nitrogen source composition but different amount ratio was prepared by using CMC-Na as carbon source and equal mixture of peptone and yeast powder as nitrogen source, and changing the amount ratio of carbon source and nitrogen source in the medium (1% / 1%, 1% / 2%, 1% / 3%, 1% / 4%, 2% / 1%, 2% / 2%, 2% / 3%, 2% / 4%, 3% / 1%, 3% / 2%, 3% / 3%, 3% / 4%, 4% / 1%, 4% / 2%, 4% / 3%, 4% / 4%) (1000 mL medium contains 0.5% NaCl, 0.1% KH2PO4, 0.02% MgSO4-7H2O, and the rest is water, pH 7.0, except for carbon source and nitrogen source). The seed liquid of strain ZZ04 was inoculated into a triangular flask (250 mL) containing 40 mL of the above basic medium at an inoculation amount of 2% (v / v), and then the flask was sealed with an air vent plug and placed in a water bath shaker for incubation at 37°C and 150 rpm for 36 h. Then the crude enzyme liquid was obtained by centrifugation at 10000 r / min and 4°C for 10 min, and the CMCase activity (determination method same as Example 1.1(2)①C) and filter paper activity (determination method same as Example 1.1(2)②C) of the crude enzyme liquid under each amount ratio condition were determined, and the results are shown in Table 1. Figure 7

[0079] As can be seen from Table 1, the carbon and nitrogen content in the medium has a great influence on the production of cellulase by strain ZZ04. The enzyme activity values of CMCase and FPA in the crude enzyme liquid generally increase with the decrease of C / N ratio, and reach the maximum when CN is 2% / 4%, and then decrease. Therefore, the optimal amount ratio of C source and N source for the production of cellulase by strain ZZ04 should be 2% / 4%. Figure 7

[0080] Based on the above analysis, it is determined that the optimal enzyme production medium composition of strain ZZ04 should be: 1000 mL medium contains 2% CMC-Na, 2% peptone, 2% yeast powder, 0.5% NaCl, 0.1% KH2PO4, 0.02% MgSO4-7H2O, and the rest is water.

[0081] 2. Optimization of enzyme production culture conditions

[0082] (1) Inoculation amount selection

[0083] ​​The seed liquid of ZZ04 strain was inoculated into 40 mL of the above-mentioned optimal enzyme production medium at an inoculum size of 1%, 2%, 3%, 4%, 5%, 6% and 7% (v / v) respectively. The flask was sealed with a vent plug and placed in a water bath shaker. After incubation at 37°C and 150 rpm for 36 h, the flask was taken out and centrifuged at 10000 r / min and 4°C for 10 min to obtain a crude enzyme solution. The CMC enzyme activity (determination method is the same as that of Example 1.1(2)①C) and the filter paper enzyme activity (determination method is the same as that of Example 1.1(2)②C) of the crude enzyme solution under each inoculum size were determined. The results are shown in FIG. Figure 8 shown.

[0084] Depend on Figure 8 It can be seen that with the increase of inoculum size, the activity of cellulase produced by strain ZZ04, CMCase and FPA, both increase first and then decrease after reaching a certain inoculum size. Among them, the activity of CMCase is relatively stable and increases within the inoculum size range of 3% to 4%, reaching its highest value of 158.3 U·mL at 4%. -1 The enzyme activity of FPA also showed a stable downward trend within the inoculum range of 3% to 5%, with a peak of 125.3 U·mL at 3%. -1 In order to maximize the enzyme activity, the optimal inoculation amount should be determined to be 3%.

[0085] (2) Selection of initial pH of culture medium

[0086] The optimal enzyme production medium described in 1(3) was adjusted with 1 mol / L hydrochloric acid (HCl) and sodium bicarbonate (NaHCO3), and pH test paper was used for real-time pH detection. A gradient culture medium of pH 4, pH 5, pH 6, pH 7, pH 8, and pH 9 was prepared. The seed liquid of the ZZ04 strain was inoculated at a 3% inoculation amount (v / v) into a triangular flask (250 mL) containing 40 mL of enzyme production culture medium of different pH values. The flask was sealed with a vent plug and placed in a water bath shaker. After culturing at 37°C and 150 rpm for 36 h, the flask was taken out and centrifuged at 10000 r / min and 4°C for 10 min to obtain a crude enzyme solution. The CMC enzyme activity of the crude enzyme solution under different initial pH values ​​of the enzyme production culture medium (the determination method is the same as that of Example 1.1(2) ①C) and the filter paper enzyme activity (the determination method is the same as that of Example 1.1(2) ②C) were determined, and the highest value of each was taken as 100%. Each treatment was repeated 3 times. The results are shown in FIG. Figure 9 shown.

[0087] Depend on Figure 9It can be seen that in the crude enzyme solution of the enzyme production medium of strain ZZ04 with initial pH value of 4-10, the enzyme activities of CMCase and FPA are detected. When the pH value is 4-7, the enzyme activities show an increasing trend, and when the pH value is 7, the enzyme activities are the strongest, and the enzyme activities of CMCase and FPA reach the maximum of 197.7 U·mL -1 and 160.4 U·mL -1 ; when the pH value is 8-9, the enzyme activities show a decreasing trend. The results show that the cellulase activity of strain ZZ04 is higher in neutral environment, and the optimum pH is 7.

[0088] (2) Temperature selection

[0089] The seed liquid of ZZ04 strain is inoculated into a triangular flask (250 mL) containing 40 mL of pH 7 enzyme production medium (composition: CMC-Na 2%, peptone 2%, yeast powder 2%, NaCl 0.5%, KH2PO4 0.1%, MgSO4·7H2O 0.02%, and the rest is water) with a 3% inoculation amount, and the air plug is sealed. It is placed in a water bath shaker, and after 36 h of culture at different temperature gradients (25°C, 30°C, 35°C, 40°C, 45°C) and 150 rpm, it is taken out, centrifuged at 10000 r / min and 4°C for 10 min to obtain a crude enzyme solution. The enzyme activities of CMCase (determination method same as Example 1.1 (2)①C) and FPA (determination method same as Example 1.1 (2)②C) in the crude enzyme solution at different culture temperatures are determined, and the highest one of each is taken as 100%, and each treatment is repeated 3 times, and the results are shown in Table 1. Figure 10

[0090] From Figure 10 It can be seen that the culture temperature has a greater impact on the production of cellulase by ZZ04. In the crude enzyme solution of the enzyme production medium of strain ZZ04 cultured at a temperature of 25-45°C, the enzyme activity is detected; when the culture temperature is 25-35°C, the cellulase activity shows an increasing trend; at 35°C, the enzyme activities of CMCase and FPA reach the maximum, which are 203.4 U·mL -1 and 164.1 U·mL -1 ; when the temperature is higher than 35°C, the enzyme activity decreases. The results show that the optimum culture temperature for the cellulase activity of strain ZZ04 is 35°C.

[0091] (3) Optimum culture time

[0092] ​The seed liquid of strain ZZ04 was inoculated at an inoculum volume of 3% into a 250 mL Erlenmeyer flask containing 40 mL of pH 7 enzyme-producing culture medium (composition: CMC-Na 2%, peptone 2%, yeast powder 2%, NaCl 0.5%, KHPO4 0.1%, MgSO4·7H2O 0.02%, and the rest water). The flask was sealed with a vent stopper and placed in a water bath shaker. The flask was cultured at 35°C and 150 rpm for 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 96 h, respectively. Samples were taken after each culture, and the crude enzyme solution was centrifuged at 10000 r / min and 4°C for 10 min to obtain the crude enzyme solution. The CMCase activity (determination method is the same as in Example 1.1(2)①C) and the filter paper enzyme activity (determination method is the same as in Example 1.1(2)②C) in the crude enzyme solution at different culture times were determined. The highest value of each was taken as 100%. Each treatment was repeated 3 times. The results are shown in FIG. Figure 11 shown.

[0093] Depend on Figure 11 It can be seen that with the extension of culture time, the cellulase activity of strain ZZ04 also increased, and an inflection point appeared at 48 h. The enzyme activity values ​​of CMCase and FPA both reached the highest values, which were 222.1 U·mL -1 and 171.2 U·mL -1 After 48 hours, the activity of CMCase decreased slightly with time, but the difference was not significant. However, FPA remained stable from 48 to 72 hours. Therefore, the optimal culture time for cellulase production by strain ZZ04 should be 48 hours.

[0094] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0095] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus velezensis ZZ04, characterized in that: The classification name is Bacillus velezensis, and it was deposited in the General Microbiology Center of China Culture Collection Administration on August 23, 2023. The deposit number is CGMCC No. 28229. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A crude enzyme solution, characterized in that The crude enzyme solution is prepared by inoculating the seed liquid of Bacillus velezensis ZZ04 into an enzyme production culture medium, culturing on a shaking table, centrifuging, and taking the supernatant.

3. A crude enzyme solution according to claim 2, characterized in that: The OD of the seed solution of Bacillus velez ZZ04 600 The value is 1.

4. A crude enzyme solution according to claim 2, characterized in that: The inoculation amount of the Bacillus Velez ZZ04 seed liquid is 3%.

5. A crude enzyme solution according to claim 2, characterized in that: The composition of the enzyme production culture medium is as follows: based on the mass of 1000 mL of the culture medium, it contains 2% CMC-Na, 2% peptone, 2% yeast powder, 0.5% NaCl, 0.1% KHPO4, 0.02% MgSO4·7H2O, and the rest is water, with a pH of 7.

0.

6. A crude enzyme solution according to claim 2, characterized in that: The shaking culture conditions are: 35°C, 150 rpm, and culture for 48 h.

7. A crude enzyme solution according to claim 2, characterized in that: The centrifugal conditions are: 10000 r / min, 4° C., 10 min.

8. Use of the Bacillus Velez ZZ04 according to claim 1 or the crude enzyme solution according to claims 2-7 in degrading straw and peel.

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