Bacillus strain as well as application and method of bacillus strain in production of cellulase
By screening and optimizing the culture conditions of Bacillus tequilensis KT-18, the problems of high production cost and poor stability of cellulase are solved, and cellulase with high efficiency secretion and good stability are achieved, suitable for livestock and poultry Application in feed.
Patent Information
- Application Number
- CN202410686146.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-30
- Publication Date
- 2024-12-27
AI Technical Summary
The high production cost and poor stability of cellulases limit their widespread use in livestock and poultry production.
A Bacillus tequilensis KT-18 strain that can efficiently secrete cellulase was selected, and optimized by specific culture and fermentation conditions, including culture at 30-40℃, 120-240rpm, using bran, The reaction system of sodium chloride, peptone and yeast extract, the fermentation time is no less than 72 hours.
This strain can efficiently secrete cellulase, with an enzyme activity of up to 32.23U/ml, and has good passage stability, acid resistance and heat resistance. It can maintain enzyme activity during high-temperature granulation and gastric passage of the feed, effectively degrading the feed in the feed cellulose.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a bacillus strain and an application and method of the strain in producing cellulase. Background Art
[0002] Crop straw is a rich resource and a renewable cellulose resource. However, cellulose is a polysaccharide composed of β-1,4-glycosidic bonds that is widely present in plant cell walls. It is easy to form crystals and fibrils, which increases the viscosity of digestive tract chyme and reduces the digestion and absorption of nutrients by animal intestines. It is a common anti-nutritional factor in plant-based feed for livestock and poultry. This greatly reduces the utilization rate of cellulose resources such as crop straw in feed. The development and utilization of cellulose resources can alleviate the problem of feed resource shortage.
[0003] As a new type of enzyme preparation, cellulase has been widely used in livestock and poultry farming. It is usually used in combination with other carbohydrate enzymes and plays a certain synergistic role. Cellulase can degrade the main structure of cellulose in plant raw materials into glucose, eliminate anti-nutritional factors in feed, and facilitate further digestion and absorption by animals. At present, domestic and foreign scholars have conducted relevant research on the structure and enzymatic characteristics of cellulase, but the disadvantages of high production cost and poor stability of cellulase limit its widespread use in livestock and poultry production. Summary of the invention
[0004] In order to solve the shortcomings of the current cellulase production, such as high cost and poor stability, the present invention screened a strain of Bacillus tekila that can efficiently secrete cellulase from Guizhou Xiang pig feces. The cellulase secreted by it has certain heat resistance and acid resistance, and can retain a certain enzyme activity in the intestine to exert its hydrolysis effect on feed cellulose during the high-temperature pelleting and stomach passage of feed.
[0005] Biomaterial Deposit
[0006] The Bacillus tequilensis KT-18 provided by the present invention has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on March 22, 2024, CGMCC No.30104, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0007] The invention comprises the following contents: a bacillus strain, which is bacillus tequila KT-18 and has a preservation number of CGMCC No.30104.
[0008] The invention discloses an application of Bacillus tequila KT-18 in producing cellulase.
[0009] A method for producing cellulase, comprising the following steps:
[0010] Add the Bacillus tequila strain KT-18 cultured to the logarithmic growth phase to the reaction system, and adjust the OD600 to 0.03-0.06;
[0011] The reaction system contains bran, sodium chloride, peptone and yeast extract;
[0012] The reaction system is cultured at 30-40°C and 120-240 rpm, and the reaction time is not less than 72 hours.
[0013] Preferably, in the method for producing cellulase, the bran is 20 g / L, peptone is 10 g / L, sodium chloride is 5 g / L, and yeast extract is 5 g / L.
[0014] The beneficial effects of the present invention are as follows: the Bacillus tekila KT-18 of the present invention can efficiently secrete cellulase, and the activity of cellulose produced by fermentation at the shake flask level is 32.23U / ml, and has good passage stability. The secreted cellulase has excellent acid resistance and heat resistance, and can retain a certain enzyme activity in the intestine to exert its hydrolysis effect on feed cellulose during the high-temperature pelleting and stomach passage of feed. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
[0016] Figure 1 Colony morphology of Bacillus tequila, left: colony morphology; middle: Gram staining; right: spore staining.
[0017] Figure 2 Phylogenetic tree of Bacillus tequila KT-18.
[0018] Figure 3 Optimal reaction temperature of cellulase secreted by Bacillus tequila KT-18.
[0019] Figure 4 Thermostability of cellulase secreted by Bacillus tequila KT-18.
[0020] Figure 5 The optimal reaction pH of cellulase secreted by Bacillus tequila KT-18.
[0021] Figure 6 pH stability of cellulase secreted by Bacillus tequila KT-18. DETAILED DESCRIPTION
[0022] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.
[0023] (1) Culture medium
[0024] Screening medium: 1.0 g sodium carboxymethyl cellulose, 0.2 g (NH4)2SO4, 0.2 g KH2PO4, 0.02 g MgSO4·7H2O, adjust pH to neutral, 2.0 g agar, add distilled water to 100 mL, sterilize. Solid activation medium: 2.5 g tryptone, 1.25 g yeast extract, 2.5 g sodium chloride, 3.5 g agarose, add distilled water to 250 mL, sterilize.
[0025] Seed culture medium: 1 g of tryptone, 0.5 g of yeast powder, 1 g of sodium chloride, add distilled water to 100 mL and sterilize.
[0026] Fermentation medium: 1 g tryptone, 0.5 g yeast powder, 1 g sodium chloride, 2 g bran, add distilled water to 100 mL and sterilize.
[0027] (2) Determination of cellulase:
[0028] According to the industry standard NY / T 912-2004, carboxymethyl cellulose (CMC) enzyme activity is used to determine the activity of carboxymethyl cellulose. The enzyme activity unit is defined as: the amount of enzyme required to degrade and release 1 μmol of reducing sugar from a solution of 4 mg / ml sodium carboxymethyl cellulose per minute at 37°C and pH 5.5 is one enzyme activity unit (U).
[0029] Extraction of crude enzyme solution: centrifuge the fermentation solution at 4°C, 4000 rpm for 10 min and take the supernatant.
[0030] Add 450μl 4mg / ml sodium carboxymethyl cellulose to each test tube and blank tube, and place in a water bath at a certain temperature for 5 minutes; then add 200μl crude enzyme solution to the test tube and shake well; after accurate reaction for 10 minutes, add 1ml DNS reagent to both test tubes. Add 200μl crude enzyme solution to the blank tube. Place both test tubes in a boiling water bath for 5 minutes, take out the test tubes, and cool to room temperature; then use the blank as a control and measure the absorbance OD of the sample at 540nm 540 The enzyme activity was calculated based on the standard curve.
[0031] Preparation of standard curve: Prepare 10mg / ml glucose mother solution (1g anhydrous glucose dissolved in pH 5.5 acetic acid-sodium acetate buffer, dilute to 100ml). Dilute the mother solution with the corresponding buffer to make a 0.1-1mg / ml glucose standard dilution. Repeat three times for each standard solution. Add 450ul substrate to each test tube, and then add 200μl standard dilution. The subsequent operation steps are the same as the sample determination. Add 200μl of the corresponding buffer to the blank instead of 200μl of the standard solution. Take the glucose concentration as the y-axis, OD 540 Plot a standard curve for the x-axis.
[0032] Enzyme activity calculation formula: X D =[(A E -A B )×K+C0]×1000 / (M×t)
[0033] X D : Cellulase activity of sample dilution (U / ml);
[0034] A E : OD of enzyme reaction solution 540 ;
[0035] A B : OD of enzyme blank solution 540；
[0036] K: slope of standard curve;
[0037] C 0： Standard curve intercept;
[0038] M: molar mass of glucose (180.2 g / mol)
[0039] t: reaction time (min)
[0040] Example 1: Screening and identification of strains
[0041] (1) Screening of cellulase-producing strains:
[0042] Initial screening: Weigh 5g of animal feces, inoculate into a conical flask equipped with 100mL of saline and prepared glass beads, mix thoroughly to disperse the sample, and then transfer to a constant temperature water bath at 80℃ for 5min. Take 10mL of high-temperature treated feces suspension and add it to 90mL of liquid seed culture medium, place it in a shaker at 37℃ and 180r / min for 24h. Dilute the initial sample 20 times, 60 times, 200 times, and 400 times respectively; then take 200μL of the dilution and apply it to the screening plate, place it in a constant temperature box for culture, culture it at 37℃ for 24-72h, and observe the growth of the colony.
[0043] Rescreening: Pick up complete colonies on the screening plate, streak inoculate on the solid plate of seed culture medium and place it in a 37℃ biochemical incubator for 24 hours. After streaking and purification 2-3 times, a pure strain can be obtained. Pick a single colony on the plate and inoculate it into the fermentation medium, place it in a shaker at 30℃ and culture it for 24-72 hours. Observe the growth of the colony, and then determine the activity of cellulase in the culture medium. Select the strain with the highest enzyme activity and the fastest growth rate, inoculate it into the seed slant culture medium, and store it at 4℃.
[0044] After screening, 22 strains with cellulose degradation ability were obtained from animal feces. After inoculation into 100mL fermentation culture medium and culturing on a shaking table for 72 hours, the culture medium was taken to measure the cellulase activity and it was found that the 18th strain had the highest enzyme activity of 32.23U / mL. This strain was named KT-18.
[0045] (2) Strain identification
[0046] Morphological identification
[0047] The strains obtained by screening were streaked on a solid plate of seed culture medium, cultured in a biochemical incubator at 37°C for 24 hours, and colonies were picked for Gram staining. After 48 hours of culture, colonies were picked for spore staining and observed under an optical microscope.
[0048] Sequence Amplification and Analysis of 16S rDNA
[0049] Take 1mL of culture solution sample, centrifuge and discard the supernatant, take the bacteria, extract total DNA with bacterial genomic DNA extraction kit, and perform 16SrDNA sequence amplification. The DNA amplification primers used are 16S universal primers: 27F and 1492R, which were commissioned to be synthesized by Bioengineering (Shanghai) Co., Ltd. The PCR reaction system is a 50μL system: Premix TaqTM (TaKaRa TaqTMVersion 2.0) 25μL, primer 27F 1μL, primer 1492R 1μL, template DNA 2μL, ultrapure water 21μL. PCR amplification conditions: 95℃3min, 95℃30s, 55℃30s, 72℃1.5min, a total of 35 cycles, 72℃, 10min, 4℃ storage. DNA sequence determination was commissioned to Qingke Biotechnology.
[0050] The 16S rDNA sequence of KT-18 strain was compared and analyzed by BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST), and the phylogenetic tree was drawn by MEGA. The obtained sequence was compared and analyzed with the nucleic acid data in GenBank by Blast to construct the phylogenetic tree.
[0051] After culturing at 37°C for 24 hours, large milky white opaque colonies with wrinkled surfaces ( Figure 1 Left); Gram staining microscopy results showed that the bacteria were purple rod-shaped and Gram-positive ( Figure 1 Middle); after 36 hours of incubation at 37°C, observation under a microscope revealed both vegetative cells in the early stages of spore formation and free spores ( Figure 1 right).
[0052] The genome of KT-18 strain was extracted and PCR amplified to obtain the 16S rRNA of the strain. After the sequence was obtained by sequencing, the sequence was compared with BLAST in the NCBI database, and the phylogenetic tree was constructed using MEGA11 software ( Figure 2 ). KT-18 has the smallest genetic distance with Bacillus tequilensis and the closest relationship. Combined with the colony morphology of the strain, it is judged to be Bacillus tequilensis. It was deposited in the China Microbiological Culture Collection Center CGMCC No. 30104 on March 22, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
[0053] Example 2: Shake flask fermentation of Bacillus tequilensis KT-18
[0054] 1) Preparation of culture medium
[0055] Liquid seed culture medium: 1 g of tryptone, 0.5 g of yeast powder, 1 g of sodium chloride, add distilled water to 100 mL and sterilize.
[0056] Solid activation medium: 2.5 g tryptone, 1.25 g yeast extract, 2.5 g sodium chloride, 3.5 g agarose, add distilled water to make up to 250 mL, and sterilize.
[0057] Fermentation medium: 1 g tryptone, 0.5 g yeast powder, 1 g sodium chloride, 2 g bran, add distilled water to 100 mL and sterilize.
[0058] 2) Seed preparation
[0059] Dip the bacterial suspension of the strain, streak it on the solid culture medium, and culture it in a constant temperature incubator at 37°C for about 24 hours to obtain a single colony. Pick a single colony and inoculate it into a 100ml conical flask containing 25ml of liquid seed culture medium, and culture it overnight in a shaker at 37°C, 200rpm for 16 hours (until the logarithmic growth phase) to make a seed solution.
[0060] 3) Shake flask fermentation
[0061] The prepared seed liquid was inoculated into a 500ml conical flask containing 100ml liquid fermentation medium at a 1% inoculation rate, and the fermentation starting OD600 needed to be adjusted to 0.04. Cultured in a shaker at 37°C, 200rpm for 4 days, and samples were taken every 24h. Centrifuged the fermentation broth, and the crude enzyme solution was collected to determine the cellulase content in the fermentation broth. The results showed that the enzyme activities were 12.21U / m L, 22.32U / m L, 32.23U / m L, and 28.57U / m L at 24h, 48h, 72h, and 96h of fermentation, respectively. Therefore, the optimal fermentation time was determined to be 72h.
[0062] Example 3: Study on the enzymatic properties of cellulase secreted by KT-18
[0063] 1) Optimum temperature: Cellulase and substrate were mixed and reacted in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃ for 5 minutes. The effect of temperature on enzyme activity was measured and the optimum temperature was determined with the highest activity as 100%. Figure 3 It shows that from 30℃ to 50℃, the enzyme activity gradually increases with the increase of temperature, and from 50℃ to 90℃, the enzyme activity gradually decreases with the increase of temperature. Therefore, the optimum temperature is around 50℃.
[0064] 2) Thermal stability: The enzyme solution was preheated at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 2 minutes, then cooled in ice for 5 minutes, and then the enzyme activity was measured under the optimal conditions. The highest activity was taken as 100% to measure its high temperature resistance. Figure 4 As shown, from 30℃ to 60℃, the enzyme activity fluctuates between 80%-100%. From 60℃ to 80℃, as the treatment temperature increases, the enzyme activity gradually decreases, but at 80℃ the residual enzyme activity reaches more than 40%. This shows that the cellulase secreted by KT-18 can withstand the high-temperature pelleting process of feed to a certain extent.
[0065] 3) Optimum pH value: In the activity test system at 50°C, the pH value of the substrate was adjusted with the corresponding buffer (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0), and the ability of cellulase to catalyze the hydrolysis of sodium carboxymethyl cellulose was determined to determine its optimal pH value. Figure 5 As shown, there is almost no enzyme activity at pH = 3.0, but the enzyme activity reaches the maximum at pH = 6.0, and then the enzyme activity slowly decreases with the increase of pH. Therefore, its optimal pH is 6.0.
[0066] 4) Determination of pH stability: A series of enzyme solutions with different pH values (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) were prepared with different pH buffers and placed at 25°C for 1 hour. The enzyme activity under the optimal conditions was then determined. The enzyme activity under the optimal conditions was used as the control, and the pH stability was determined with the highest activity as 100%. The results are shown in Table 1. Figure 6 As shown in the figure, when the pH of the enzyme solution is 3.0, the enzyme activity still remains at 19.22%. As the pH of the enzyme solution increases, the enzyme activity gradually increases, and when the pH is 6.0, the enzyme activity is almost not lost. From pH 6.0 to 8.0, as the pH of the enzyme solution increases, the enzyme activity gradually weakens, and when pH = 9.0, the relative enzyme activity is 20%. This shows that the cellulase secreted by KT-18 can tolerate the acidic environment of the stomach to a certain extent. And it can maintain its stable enzyme activity in the intestinal environment, and exert its best hydrolysis performance on feed cellulose.
[0067] In addition, it should be noted that the names of the raw material components of the specific embodiments described in this specification may be different. All equivalent or simple changes made according to the principles described in the patent concept of the present invention are included in the protection scope of the patent of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the specific embodiments described or replace them in a similar manner, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.
Claims
1. A Bacillus strain, characterized in that: The strain is Bacillus tequila KT-18, and its deposit number is CGMCC No.30104.
2. Application of Bacillus tequila KT-18 in the production of cellulase.
3. A method for producing cellulase, characterized in that: The method for producing cellulase comprises the following steps: Add the Bacillus tequila strain KT-18 cultured to the logarithmic growth phase to the reaction system, and adjust the OD600 to 0.03-0.06; The reaction system contains bran, sodium chloride, peptone and yeast extract; The reaction system is cultured at 30-40°C and 120-240 rpm, and the reaction time is not less than 72 hours.
4. The method for producing cellulase according to claim 3, characterized in that The bran is 20 g / L, peptone is 10 g / L, sodium chloride is 5 g / L, and yeast extract is 5 g / L.