Application of a Pseudomonas aeruginosa and its enzyme preparation in cellulose degradation

Abstract

This invention provides the application of *Pseudomonas* and its enzyme preparations in cellulose degradation, relating to the fields of microbial resource utilization and ecological agriculture. The *Pseudomonas* strain AHhStB30 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025628, and the deposit date is March 31, 2025. The *Pseudomonas* strain of this invention is derived from autumn grassland soil and can significantly improve cellulose degradation efficiency under low-temperature conditions. It can efficiently decompose agricultural waste and industrial cellulose waste, accelerate carbon cycling in synergy with the microbial system, maintain high enzyme activity at low temperatures, and reduce pretreatment energy consumption. The *Pseudomonas* strain of this invention has a long-lasting activity retention time and can significantly improve the cellulose degradation rate. Cellulose treated with the *Pseudomonas* strain of this invention or its enzyme preparations degrades more rapidly at low temperatures and exhibits higher activity at low temperatures.

Description

Technical Field

[0001] This invention belongs to the field of microbial resource utilization and ecological agriculture technology, and more specifically, it relates to the application of a Pseudomonas aeruginosa and its enzyme preparation in cellulose degradation. Background Technology

[0002] Traditional agricultural production leaves behind large amounts of straw and plant residues, which are difficult to degrade naturally, leading to soil compaction, obstructed nutrient cycling, pH imbalance, and increased pests and diseases. These straw and plant residues are mainly composed of cellulose, hemicellulose, and lignin, and their treatment methods include burning, composting, and direct return to the field. While burning can quickly clean up farmland, it releases large amounts of pollutants and damages soil structure. Composting and direct return to the field can increase soil organic matter, but because cellulose, hemicellulose, and lignin are difficult to degrade quickly, they can easily lead to a short-term decrease in soil permeability, microbial imbalance, and even affect crop growth. Therefore, microbial degradation, as an environmentally friendly and efficient method, has become an important research direction for sustainable agricultural development.

[0003] Currently, various cellulose-degrading microorganisms have been studied and applied, such as Trichoderma, Actinomycetes, and other bacteria. However, most known cellulose-degrading bacteria have high environmental requirements, especially at low temperatures (below 20°C), where their degradation efficiency slows down, limiting the rate of cellulose degradation under low-temperature conditions. Therefore, finding and screening microorganisms capable of efficiently degrading cellulose at low temperatures is of great significance for promoting the resource utilization of agricultural waste and improving soil quality.

[0004] Pseudomonas, a widely distributed environmental microorganism, has attracted attention due to its strong environmental adaptability and the ability to secrete multiple enzyme systems. Some Pseudomonas strains have been shown to have promising applications in environmental remediation and soil improvement. However, research on Pseudomonas strains capable of efficiently degrading cellulose under low-temperature conditions is still limited, and the related mechanisms and application value require further exploration.

[0005] Therefore, in response to the need for cellulose degradation in farmland under low-temperature conditions, this invention provides a novel Pseudomonas strain and studies its degradation ability under low-temperature conditions, aiming to provide a new solution for ecological agriculture and the resource utilization of farmland straw. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an application of Pseudomonas aeruginosa and its enzyme preparations in cellulose degradation, thereby solving the problem of slow cellulose degradation rate in farmland under low-temperature conditions.

[0007] In a first aspect, the present invention provides a Pseudomonas bacterium, namely B30, classified and named Pseudomonas sp. AHhStB30, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2025628 and deposit date of March 31, 2025.

[0008] In a second aspect, the present invention provides an enzyme preparation, characterized in that: the enzyme preparation comprises the Pseudomonas bacteria described in the first aspect.

[0009] Preferably, the enzyme preparation is in solid or liquid form. The effective enzyme activity of Pseudomonas in the preparation is ≥104 U / g or 104 U / mL.

[0010] Furthermore, the effective enzyme activity is ≥106 U / g or ≥106 U / mL.

[0011] Thirdly, the present invention provides a composition comprising the Pseudomonas aeruginosa or its fermentation product as described in the first aspect or the enzyme preparation as described in the second aspect.

[0012] Fourthly, the present invention provides an enzyme preparation comprising agent A, agent B, and agent C: Agent A includes the Pseudomonas bacteria and a protectant, Agent B includes EDTA, and Agent C is water.

[0013] Preferably, the mass ratio of agent A, agent B and agent C is 0.2~0.4:1~5:50~100, for example: 0.2:1:50, 0.3:1.5:75, 0.4:5:100.

[0014] Preferably, the effective enzyme activity of the cellulase in agent A is 10⁴-10⁶ U / g.

[0015] Preferably, the protective agent in agent A is glycerol. The protective agent is mainly used to ensure the activity of the enzyme in the enzyme preparation.

[0016] Furthermore, the mass concentration of the glycerol is 25-40%, for example 25%, 27%, 30%, 32%, 35%, or 40%.

[0017] Agent B in this invention is EDTA, which reacts with heavy metal ions such as Pb. 2+ Hg 2+ It binds to prevent it from inhibiting enzyme activity; however, it should be noted that excessive amounts may chelate essential metal ions.

[0018] Fifthly, the present invention provides a method for preparing the enzyme preparation described in the fourth aspect, comprising the following steps: (1) The *Pseudomonas* strain described in the first aspect was inoculated into liquid enzyme-producing medium at an inoculum size of 4%, and cultured with shaking at 18°C ​​for 3 days at 180 rpm. The culture medium was then centrifuged at 4°C, and the supernatant was used as the crude enzyme solution to obtain Agent A.

[0019] (2) Prepare the enzyme preparation by mixing agent A, agent B and agent C or by packaging agent A, agent B and agent C separately.

[0020] In a sixth aspect, the present invention provides the application of any one of the Pseudomonas aeruginosa described in the first aspect, the enzyme preparation described in the second aspect, the composition described in the third aspect, or the enzyme preparation described in the fourth aspect in the degradation of cellulose in farmland under low-temperature conditions.

[0021] Preferably, the application includes at least one of the following: (1) Application in soil improvement; (2) Application of cellulose degradation in farmland; (3) Applications in improving feed utilization; Fifthly, the application of a Pseudomonas aeruginosa and its enzyme preparation in cellulose degradation is derived from autumn grassland soil. It can significantly improve the efficiency of cellulose degradation under low temperature conditions, efficiently decompose agricultural waste and industrial cellulose waste, accelerate carbon cycling in synergy with the microbial system, maintain high enzyme activity under low temperature conditions, and reduce pretreatment energy consumption.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. The Pseudomonas strain of the present invention has a long period of active retention and can significantly improve the cellulose degradation rate.

[0023] 2. Cellulose treated with the Pseudomonas aeruginosa or enzyme preparations containing it according to the present invention degrades more rapidly at low temperatures and has higher activity at low temperatures. Attached Figure Description

[0024] Figure 1 Bacterial colony composition (genus level); Figure 2 Phylogenetic analysis of cellulose-degrading bacteria Figure 3 Filter paper degradation ability Figure 4 CMCase enzyme activity; Culture preservation for patent procedures: The Pseudomonas aeruginosa (B30) of the present invention; Deposit date: March 31, 2025; Preservation institution: China Center for Type Culture Collection (CCTCC); Address of the depository: Wuhan University, Wuhan, China, 430072, China; Accession number: CCTCCNO:M2025628; Classification and naming: Pseudomonas sp. AHhStB30. Detailed Implementation

[0025] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0026] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0027] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0028] The culture medium used in the examples is as follows: (1) Sodium carboxymethyl cellulose solid culture medium: CMC-Na 15.0g, yeast powder 1.0g, MgSO4·7H2O 0.5g, KH2PO4 1.0g, agar 20.0g, distilled water 1000mL, sterilized at 121°C for 20min (2) Sodium carboxymethyl cellulose liquid culture medium: CMC-Na 15.0g, yeast powder 1.0g, MgSO4·7H2O 0.5g, KH2PO4 1.0g, distilled water 1000mL, sterilized at 121°C for 20min (3) Hutchison liquid culture medium: NaNO3 2.5g, KH2PO4 1.0g, NaCl 0.1g, MgSO4·7H2O 0.3g, FeCl3 0.01g, CaCl2 0.1g, H2O 1000ml Example 1: Identification of the strain Sodium carboxymethyl cellulose solid culture medium was used to perform preliminary isolation and screening of cellulose-degrading strains from collected soil samples.

[0029] (1) Take two 10g soil samples and add them into two 100ml conical flasks containing 90mL of sterile physiological saline. Place them in a shaker at 18℃ and shake for 30min to mix thoroughly, so as to prepare a suspension with a concentration of 10-1. (2) Dilute the above bacterial solution with sterilized physiological saline to form suspensions of 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶. (3) In the clean bench, take 90 μL of bacterial suspension diluted to 10⁻⁴, 10⁻⁵ and 10⁻⁶ concentrations respectively, and spread them evenly on sodium carboxymethyl cellulose solid medium with a sterile spreader. Set up three replicates for each concentration gradient, and mark the room temperature and low temperature. After standing for 10 min, incubate upside down in an 18℃ constant temperature incubator for 48 h and observe the colony growth.

[0030] (4) After colonies grow on the plate, observe the growth of the colonies, select single colonies with different morphologies, streak them on sodium carboxymethyl cellulose medium, and incubate them upside down in a constant temperature incubator at 18℃ for 48h. Repeat this process three times to obtain purified single strains. Strand and isolate different morphologies of the strains and record the results.

[0031] (5) PCR identification of the strain (6) Perform pure culture of the strain (7) Inoculate the pure culture into the preservation solution and store at -80°C.

[0032] Strain identification results:

[0033] Example 2: Degradation ability of Congo red staining method The principle of the Congo red staining method for cellulose is based on the specific binding between Congo red dye and cellulose. Congo red dye can form a red complex with cellulose, but it cannot bind to cellulose hydrolysis products (such as cellobiose and glucose). When cellulose-degrading bacteria break down cellulose, the cellulose is broken down into cellobiose and glucose by cellulase, disrupting the binding of Congo red to cellulose and preventing the formation of the red complex. Therefore, in cellulose-containing culture media, a clear zone appears around the cellulose-degrading bacteria.

[0034] The degradation efficiency of cellulose-degrading bacteria can be preliminarily assessed by observing the size of the clear zone. A larger clear zone indicates a stronger cellulose-degrading ability of the strain. This method is simple, intuitive, and widely used for the screening and identification of cellulose-degrading bacteria.

[0035] Results of degradation ability determination using Congo red staining method:

[0036] Example 3: Filter paper strip disintegration experiment The isolated bacterial strain was cultured in Hutchison medium for filter paper strip degradation experiments: 3 mL of bacterial suspension was inoculated into a centrifuge tube containing 45 mL of filter paper strip disintegration medium (Hutchison medium), and a 1 cm × 6 cm Xinhua No. 1 filter paper strip was placed in the tube. The tube was incubated at 18°C ​​with shaking for 15 days, during which the tube was gently shaken, and the disintegration of the filter paper strip was observed periodically. The control group consisted of disintegration medium without bacterial inoculation but containing filter paper strips, and three replicates were designed. The degradation and disintegration of the filter paper strips in each tube were observed to further screen for strains with better cellulose degradation effects. The criteria for judgment were the degree of filter paper strip disintegration: (+) partial degradation of the filter paper surface; (++) swelling of the filter paper edge; (+++) amorphous filter paper edge; (++++) pasty filter paper edge.

[0037] Results of filter paper strip disintegration experiment:

[0038] Example 4: Enzyme Activity Assay The low-temperature cellulose-degrading strains obtained from the initial screening were inoculated into liquid enzyme-producing medium at an inoculation rate of 4%, and cultured at 18°C ​​with shaking for 3 days at 180 rpm. 1 mL of liquid was taken from the enzyme-producing medium and centrifuged at 4°C. The supernatant after centrifugation was used as the crude enzyme solution.

[0039] (2) Preparation of the standard curve: 0.1% Standard Glucose Solution: Accurately weigh 1000 mg of anhydrous glucose, dissolve it in distilled water, and bring the volume to 1000 mL. Plotting the Standard Curve: Take a clean centrifuge tube or test tube, follow the steps in the table below, zero the tube with 0, and measure the absorbance at 540 nm. Plot the absorbance value on the ordinate and the concentration of each standard (mg / mL) on the abscissa to obtain the standard curve.

[0040]

[0041] Formula for calculating enzyme activity:

[0042] In the formula, 5.56 represents the number of μmol per mg of glucose (1000 / 180=5.56). Results of enzyme activity assay for the strain: The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A type of Pseudomonas, characterized in that: The Pseudomonas species mentioned is AHhStB30, classified as Pseudomonas sp. AHhStB30, and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M2025628, on March 31, 2025.

2. An enzyme preparation, characterized in that: The enzyme preparation comprises the Pseudomonas aeruginosa or its fermentation product as described in claim 1.

3. A composition, characterized in that: The composition comprises the Pseudomonas aeruginosa of claim 1 or its fermentation product, or the enzyme preparation of claim 2.

4. The enzyme preparation according to claim 2, characterized in that, In the enzyme preparation, the enzyme activity of Pseudomonas aeruginosa is greater than or equal to 105 U / g.

5. The enzyme preparation according to claim 4, characterized in that: The protective agent is glycerol.

6. The enzyme preparation according to claim 4 or 5, characterized in that: The effective enzyme activity of the cellulase in Agent A is 105-109 U / g.

7. The method for preparing the enzyme preparation according to any one of claims 4-6, characterized in that: The preparation method includes the following steps: (1) The Pseudomonas aeruginosa described in claim 1 is activated, cultured in liquid culture medium, and the cells are collected by centrifugation. Then the enzyme is extracted, or the collected enzyme is added with a protectant to prepare a liquid enzyme preparation to obtain Agent A. (2) Prepare the enzyme preparation by mixing agent A, agent B and agent C or by packaging agent A, agent B and agent C separately.

8. The application of the Pseudomonas aeruginosa of claim 1, the enzyme preparation of claim 2, the composition of claim 3, or the enzyme preparation of any one of claims 4-6 in the degradation of cellulose in farmland under low temperature conditions.

9. The application according to claim 8, characterized in that: The application includes at least one of the following: (1) Application in soil improvement; (2) Application of cellulose degradation in farmland; (3) Application in improving feed utilization.

10. A method for cellulose degradation, characterized in that: Farmland residues and litter can be treated with the Pseudomonas bacteria of claim 1, the enzyme preparation of claim 2, the composition of claim 3, or any of the enzyme preparations of claims 4-6.

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