Pyrolysis protein anaerobic bacillus and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pyrolytic protein anaerobic bacillus and application thereof. The specific technical scheme is as follows: the bacterial strain XW-4 is preserved in the China General Microbiological Culture Collection Center on August 30, 2024, and the preservation number of the bacterial strain XW-4 is CGMCC (China General Microbiological Culture Collection Center) No.31784. According to the invention, the bacterial strain XW-4 can be used for preparing the bacillus amyloliquefaciens XW-4, and the bacillus amyloliquefaciens XW-4 can be used for preparing the bacillus amyloliquefaciens XW-4. The invention provides a high-temperature-resistant cellulose degrading bacterium which can tolerate the high temperature of 70 DEG C, can secrete cellulase and efficiently degrade cellulose, provides a new efficient degradation means for treating waste with high cellulose content, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a pyrolytic protein anaerobic bacillus and application thereof. Background Art

[0002] The cellulose content in straw is about 40%, which has great application potential. The traditional way to deal with straw is burning, but it will cause serious air pollution and waste a lot of resources.

[0003] In order to realize the resource utilization of straw, especially the utilization of cellulose in straw, common treatment methods include physical method, chemical method and biological method. The physical method requires the cellulose to be cut into powder first, which consumes a lot of energy and manpower. The chemical method requires acid and alkali soaking, which is easy to cause environmental pollution. The biological method mainly uses the cellulase secreted by microorganisms to degrade cellulose. It has low energy consumption and is environmentally friendly. It is the main research direction now.

[0004] For biological utilization of cellulose, currently most of them use eukaryotic microorganisms represented by white rot fungi to biodegrade straw at room temperature. However, studies have shown that high temperature promotes the degradation of cellulose, mainly because high temperature can accelerate the destruction of the crystallinity of straw cellulose, making its structure loose and accelerating the degradation rate of cellulose.

[0005] Therefore, if new high-temperature cellulose-degrading bacteria can be provided, they will have great application potential. Summary of the invention

[0006] The invention aims to provide a pyrolytic protein anaerobic bacillus and application thereof.

[0007] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a strain of Anoxybacillus caldiproteolyticus XW-4, strain XW-4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 30, 2024, and the deposit number is CGMCC No.31784.

[0008] Correspondingly, a strain of Anoxybacillus caldiproteolyticus XW-4, the 16S rDNA sequence of the strain XW-4 is shown in SEQ NO ID:1.

[0009] Correspondingly, a method for preparing cellulase using the pyrolytic proteolytic anaerobic Bacillus is provided.

[0010] Preferably, the pyrolyzing proteolytic Bacillus is cultured to prepare cellulase at a temperature of 50-70°C.

[0011] Preferably, the anaerobic pyrolytic proteolytic Bacillus is cultured to prepare cellulase at a pH of 5-7.

[0012] Accordingly, a method for degrading cellulose using the pyrolytic proteolytic anaerobic Bacillus is provided.

[0013] Preferably, the pyrolytic protein anaerobic Bacillus is cultured to prepare cellulase to degrade cellulose, and the culture temperature is 50-70°C and the pH is 5-7. There are various methods for degrading cellulose, for example, the prepared cellulase can be extracted and then used to degrade cellulose; the pyrolytic protein anaerobic Bacillus can also be co-cultured with the material to be degraded, and the material to be degraded is used as a culture substrate or one of the substrates; the pyrolytic protein anaerobic Bacillus can also be prepared as a bacterial preparation alone or together with other microorganisms / auxiliary materials, and applied to the material to be degraded.

[0014] The present invention has the following beneficial effects: The present invention provides a high temperature resistant cellulose degrading bacterium: Anoxybacillus caldiproteolyticus. The strain can withstand a high temperature of 70°C, can secrete cellulase, and can efficiently degrade cellulose, providing a new and efficient degradation method for treating waste with high cellulose content, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a photo of the transparent zone of strain XW-4 cultured at 60°C;

[0016] Figure 2 is the OD of strain XW-4 growing at different pH 600nm Schematic diagram of changes;

[0017] Figure 3 is the colony morphology of strain XW-4;

[0018] Figure 4 This is a microscopic observation of strain XW-4 after Gram staining;

[0019] Figure 5 Schematic diagram of molecular evolutionary tree analysis of strain XW-4;

[0020] Figure 6 is the glucose standard curve;

[0021] Figure 7 This is a graph showing the degradation of different celluloses by strain XW-4. DETAILED DESCRIPTION

[0022] The present invention provides a new Anoxybacillus caldiproteolyticus XW-4. Strain XW-4 has strong high temperature resistance, can grow normally in the range of 50-70°C and 5.0-7.0, and can secrete cellulose degrading enzymes, and has good cellulose degrading ability. Strain XW-4 was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on August 30, 2024, with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31784. The 16S rDNA sequence of strain XW-4 is shown in SEQ ID NO: 1.

[0023] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0024] The culture medium and reagents used in the present invention are as follows:

[0025] CMC-Na-LB liquid culture medium (1 L): Add 10.0 g of CMC-Na (sodium carboxymethyl cellulose) to the LB liquid culture medium formula, and adjust to the desired pH (4, 5, 6, 7, 8, 9, 10) using 2 mol / L HCl solution or NaOH solution; sterilize at 121°C for 20 min.

[0026] Congo red-cellulose selection medium (1L): yeast extract 0.1g, CMC-Na 2.0g, (NH4)2SO4 2.0g, MgSO4·7H2O 0.5g, K2HPO4 1.0g, FeSO4·7H2O 0.01g, Congo red 0.2g, agar 15g, fully dissolved in distilled water, pH value natural; sterilize at 121℃ for 20min.

[0027] Enzyme fermentation medium (1L): CMC-Na 5.0g, peptone 3.0g, yeast extract 0.1g, (NH4)2SO42.0g, MgSO4·7H2O 0.5g, K2HPO4 1.0g, FeSO4·7H2O 0.01g, fully dissolved in distilled water, pH value natural; sterilize at 121℃ for 20min.

[0028] Cellulose degradation medium: Add 100 mL of enzyme fermentation medium and 0.5 g of straw to a conical flask, and sterilize at 121°C for 20 min. The cellulose is rice straw (length 2-3 cm, long rod), wheat straw (length 2-3 cm, thin rod), corn straw (length about 1 cm, crushed) or filter paper (3 cm × 1 cm, long strip). For the straw, soak it in distilled water, wash away impurities, filter it with 2 layers of gauze, and dry it at 103°C to constant weight. The filter paper is Xinhua double-circle quantitative filter paper (diameter 9 cm), purchased from Yusi Tuofan Biotechnology (Hangzhou) Co., Ltd., its main component is cellulose, and the ash content is 0.01%. The filter paper is dried at 103°C to constant weight.

[0029] Glucose standard stock solution: Weigh 1.0 g of anhydrous glucose dried to constant weight at 103°C and dilute to 100 mL with distilled water.

[0030] Glucose standard working solution: Pipette 0.00, 1.00, 1.50, 2.00, 2.50, 3.00, and 3.50 mL of glucose standard stock solution into a 10 mL volumetric flask, and dilute to 10 mL with water.

[0031] Phosphate buffer: Weigh 12.1 g of NaH2PO4·H2O and 2.2 g of Na2HPO4·2H2O, dissolve in 1 L of distilled water, and adjust the pH to 6.0.

[0032] CMC-Na solution: Weigh 2 g of CMC-Na and slowly add it into 200 mL of phosphate buffer. Heat (80-90°C) and stir magnetically until the CMC-Na is completely dissolved. After cooling, dilute with phosphate buffer, adjust the pH to 6.0 with 2 mol / L HCl solution or NaOH solution, and make up to 300 mL.

[0033] Seed solution: Pick a single colony and inoculate it into LB liquid medium, culture it at 60℃ and 150rpm for 2 days, and dilute the bacterial solution with sterile saline to a bacterial content of about 1×10 6 CFU / mL, and obtain the inoculum solution.

[0034] Example 1: Screening and identification of microorganisms

[0035] 1. Screening

[0036] Weigh 5g of the sample collected from the compost of garden waste in a vineyard in Chengdu, add it to 100mL LB liquid culture medium, shake and mix, and enrich and culture at 60℃, 150rpm shaker for 1d. Then dilute it with sterile saline in sequence, evenly spread the dilution on Congo red-cellulose selection medium, and obtain single colonies after static culture at 60℃ for 2d. Pick a single colony with a decolorization circle around it, and purify it by continuous streaking on a new Congo red-cellulose selection medium. After the colony morphology is stable, the strain obtained by preliminary separation is stored on a slant culture medium. A strain with an obvious transparent circle of cellulose degradation was screened and obtained, and the strain number is XW-4.

[0037] 2. Cellulose degradation ability test

[0038] The seed liquid was spotted on Congo red-cellulose selective medium and cultured at 40, 50, 60 and 70°C for 2 days. The transparent zone and colony diameter were observed and measured, and the HC value (the ratio of the transparent zone diameter to the colony diameter, the larger the value, the stronger the ability of the strain to degrade cellulose) was calculated.

[0039] Strain XW-4 can grow normally on Congo red-cellulose selective medium at 50, 60 and 70°C, and transparent circles of different sizes appear, indicating that the strain can produce cellulase and degrade cellulose at 50-70°C. When strain XW-4 was inoculated into an environment of 80°C or even higher, the strain did not show obvious discomfort and could still survive normally, but the solid culture medium quickly dried and cracked, so no subsequent tests were performed. The results of HC value determination are shown in Table 1. The HC value of XW-4 at 60°C is the largest, and the growth condition is as follows Figure 1 shown.

[0040] Table 1 HC value determination results of strains on selective medium

[0041] Temperature / ℃ Transparent ring diameter / mm Colony diameter / mm HC value 40 - - - 50 33 6 5.5 60 32 5 6.4 70 18 8 2.3

[0042] 3. Identification and physiological and biochemical characteristics

[0043] XW-4 seed solution was inoculated into CMC-Na-LB liquid medium with different pH values ​​(pH = 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and original pH = 5.64), cultured at 60 ° C and 150 rpm for 2 days, and the OD of the culture medium was measured every 1 hour. 600nm , observe the growth. Figure 2 As shown, XW-4 can grow rapidly and normally in the pH range of 5.0 to 7.0.

[0044] The strain XW-4 was streaked on LB slant medium and cultured at 60℃ for 2 days. The colony characteristics were as follows: approximately circular in shape, translucent, with irregular edges, smooth and moist surface, easy to pick up, and growth conditions were as follows: Figure 3 Strain XW-4 is a Gram-positive bacterium with a long rod-shaped body, growing singly or in pairs, with spores, a diameter of about 0.5 μm, and a length of about 2 to 4 μm. Figure 4 shown.

[0045] The genomic DNA of XW-4 was extracted using a bacterial genomic DNA rapid extraction kit (Shanghai Sangon Biotechnology, product number: B518225). The isolated genomic DNA was commissioned to Shanghai Sangon Biotechnology for PCR amplification and sequencing. Its 16S rDNA sequence is shown in SEQ NO ID: 1. The sequences were compared using the BLAST program on NCBI, and the phylogenetic tree was constructed using MEGA 7.0. The results are shown in Figure 5 As shown. The sequence similarity between strain XW-4 and strain Anoxybacillus caldiproteolyticus strain R35652 was 99.65%. Therefore, strain XW-4 was identified as Anoxybacillus caldiproteolyticus and named Anoxybacillus caldiproteolyticus.XW-4.

[0046] Example 2: Demonstration of cellulase activity of strain XW-4

[0047] Take 1mL of seed liquid and inoculate it into 100mL of enzyme fermentation medium, shake the flask at 60℃ and 150rpm for 2d, centrifuge the fermentation liquid at 4000rpm for 10min, take the supernatant and get the crude enzyme liquid. Use DNS colorimetric method to determine the enzyme activity. Enzyme activity is defined as: each mL of crude enzyme liquid hydrolyzes CMC-Na at 60℃ and pH=6.0 for 1h to produce reducing sugar equivalent to 1μg of glucose, which is one enzyme activity unit, expressed as U / mL.

[0048] According to Table 2, glucose standard solution, phosphate buffer and DNS reagent are respectively taken into 25mL stoppered test tubes and mixed. Place each group of test tubes in a boiling water bath at the same time, take them out after reacting for 10 minutes, quickly cool them to room temperature with running cold water, make up to 25mL, cover the stopper, mix well and measure the absorbance at 540nm. Draw a standard curve with the amount of glucose as the horizontal axis and the absorbance as the vertical axis to obtain the linear regression equation. The plotting results are shown in Figure 6 , the measured equation is y = 0.1178x + 0.0674, R 2 It is 0.9982.

[0049] Table 2 Glucose standard curve

[0050]

[0051] Take four 25mL stoppered test tubes (one blank tube and three sample tubes); accurately add 2.00mL of CMC-Na solution to each of the four tubes; accurately add 0.5mL of crude enzyme solution to each of the three sample tubes, but not to the blank tube, mix well with a vortex mixer, and cover the stopper; place the four test tubes in a 60℃ water bath at the same time, react for 30 minutes, then take them out; quickly and accurately add 3.0mL of DNS reagent to each test tube, accurately add 0.5mL of crude enzyme solution to the blank tube, shake well, and place the four test tubes in a boiling water bath at the same time, accurately time, heat for 10 minutes, take them out, quickly cool to room temperature with running cold water, make the volume up to 25mL, cover the stopper and mix well; measure the absorbance of the reaction solution at 540nm, calculate the amount of glucose in each tube through the standard curve, subtract the amount of reducing sugar produced by the reaction from the sample tube to the blank tube, and take the average value X to calculate the cellulase activity (CMCA). Calculation formula: CMCA = X × 1 / 0.5 × 2. X represents the amount of reducing sugar produced by the reaction, 1 / 0.5 is converted into 1 mL of enzyme solution, and 2 is the time conversion factor. After calculation, the cellulase activity of strain XW-4 is 324.8 U / mL.

[0052] Example 3: Demonstration of the ability of strain XW-4 to degrade cellulose

[0053] Take 1mL of seed liquid and inoculate it into 100mL of 4 different cellulose degradation culture media (filter paper, rice straw, wheat straw and corn straw), and shake the flask at 60℃ and 150rpm for 7 days. After filtering the degraded filter paper and straw with 2 layers of gauze, dry them at 103℃, and weigh them to calculate the degradation rate. The calculation formula is: Degradation rate = (bc) / a×100%. Among them, a is the original weight of the straw or filter paper, b is the weight after 7 days of blank, and c is the weight after 7 days of degradation. Blank refers to keeping the corresponding cellulose degradation culture medium under the same conditions for 7 days and the subsequent treatment is exactly the same, except that the seed liquid is not inoculated.

[0054] The results of cellulose degradation ability of XW-4 are shown in Table 3. Figure 7 shown. Figure 7 In the figure, ①-④ are filter paper, rice straw, wheat straw and corn straw, respectively; A is the corresponding untreated cellulose before degradation, and B is the cellulose after degradation; in Table 1, a is the original weight of the straw or filter paper; b is the blank weight after 7 days; c is the weight after 7 days of degradation (all in g).

[0055] Table 3 Cellulose degradation ability test results

[0056] Cellulose a b c Degradation rate / % filter paper 0.500 0.492 0.326 33.2 Rice straw 0.500 0.482 0.360 24.4 Wheat straw 0.500 0.475 0.380 19.0 Corn stalks 0.500 0.471 0.364 21.4

[0057] The results showed that after 7 days of degradation, the filter paper changed from long strips to fragments, with a degradation rate of 33.2%; after 7 days of degradation, the coarse rice straw changed into fine fragments, with a degradation rate of 24.4%; after 7 days of degradation, the wheat straw and corn straw both became smaller in volume and looser in structure, with degradation rates of 19.0% and 21.4%, respectively.

[0058] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Anoxybacillus caldiproteolyticus XW-4, characterized in that: Strain XW-4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 30, 2024, with the deposit number CGMCC No.31784.

2. Anoxybacillus caldiproteolyticus XW-4, characterized in that: The 16S rDNA sequence of strain XW-4 is shown in SEQ NO ID:

1.

3. A method for preparing cellulase using the pyrolytic protein anaerobic Bacillus according to claim 1 or 2.

4. The method according to claim 3, characterized in that: The pyrolytic protein-free Bacillus is cultured to prepare cellulase at a temperature of 50-70°C.

5. The method according to claim 3, characterized in that: The pyrolytic protein-free Bacillus is cultured to prepare cellulase, and the culture pH is 5-7.

6. A method for degrading cellulose using the pyroproteolytic anaerobic Bacillus according to claim 1 or 2.

7. The method according to claim 6, characterized in that: The pyrolytic protein-free Bacillus is cultured to prepare cellulase to degrade cellulose, and the culture temperature is 50-70°C.

8. The method according to claim 6, characterized in that: The pyrolytic protein-free Bacillus is cultured to prepare cellulase to degrade cellulose, and the culture pH is 5-7.

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