Pseudoeuonymus psychrophilus and application of pseudoeuonymus psychrophilus in degrading straw in low-temperature environment

Abstract

The invention discloses Pseudoeuonymus psychrophilus and application thereof to degradation of straw in a low-temperature environment, and belongs to the technical field of microorganisms, the preservation number of Pseudoeuonymus psychrophilus JM11 is CGMCC No.41455, the preservation date is September 14, 2024, the preservation unit is China General Microbiological Culture Collection Center, the preservation number is CGMCC No.41455, the preservation number is CGMCC No.41455, the preservation number is CGMCC No.41455, the preservation number is CGMCC No.41455, the preservation number is CGMCC No.41455, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The optimum growth temperature of the pseudoeuglena psychrophila JM11 provided by the invention is 15 DEG C, the pseudoeuglena psychrophila JM11 can quickly generate cellulase, the activity of the cellulase is as high as 583.3 U / mL, straws can be degraded in a low-temperature environment, the degradation rate is 31.2%, and a new method is provided for degrading the straws in the low-temperature environment.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a psychrophilic pseudogymna and application of the psychrophilic pseudogymna in degrading straw in a low-temperature environment. Background Art

[0002] Straw is mainly composed of cellulose, pectin, lignin, etc., among which cellulose has the highest content, accounting for 40% to 50% of the total amount of straw. Due to the tight binding between cellulose molecules and stable structure, it is difficult to be naturally degraded. However, through specific biotechnology and the action of degrading enzymes, the cellulose in straw can be effectively degraded. Using cellulose degrading enzymes, cellulose can be degraded into glucose, providing scientific basis and technical support for the high-value utilization of straw.

[0003] Low-temperature cellulase is a class of enzymes that can efficiently degrade cellulose under low temperature conditions. Compared with conventional cellulases, low-temperature cellulases have higher catalytic efficiency and wider substrate specificity. Low-temperature cellulases are usually composed of multiple enzyme components, including endoglucanase (CMCase), exoglucanase (cellobiohydrolase) and β-glucosidase. These enzyme components work synergistically to degrade cellulose into small molecules such as glucose.

[0004] Therefore, finding new strains that can degrade cellulose to improve the degradation of straw under low temperature conditions is the development direction of biodegradation research. Summary of the invention

[0005] The purpose of the present invention is to provide a psychrophilic Pseudomonas sp. and its application in degrading straw in a low-temperature environment to solve the problems existing in the above-mentioned prior art. The psychrophilic Pseudomonas sp. can quickly produce cellulase with a cellulase activity of up to 583.3 U / mL, and can degrade straw in a low-temperature environment.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a strain of Pseudogymnoascus pannorum JM11, wherein the preservation number of the Pseudogymnoascus pannorum JM11 is CGMCC No.41455, the preservation date is September 14, 2024, the preservation unit is the General Microbiology Center of China National Microbiological Culture Collection Committee, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides the use of the psychrophilic Pseudomonas sp. JM11 in preparing cellulase.

[0009] The present invention also provides a method for preparing cellulase, comprising the steps of fermenting the psychrophilic Pseudomonas sp. JM11 and collecting the fermentation liquid.

[0010] Further, the psychrophilic Pseudomonas sp. JM11 is inoculated into an enzyme production medium, cultured for 2-9 days, and the fermentation liquid is centrifuged to obtain the cellulase;

[0011] The components of the enzyme production culture medium include: 10g bran, 10g corn cob powder, 20g microcrystalline cellulose, 10g peptone and 1.5g potassium dihydrogen phosphate, and water is added to 1L.

[0012] Furthermore, the culture is carried out at 15° C. and 180 rpm for 2-4 days.

[0013] The present invention also provides the cellulase prepared according to the method.

[0014] The present invention also provides the use of the psychrophilic Pseudomonas sp. JM11 or its fermentation liquid or the cellulase in degrading straw.

[0015] The present invention also provides a straw-degrading preparation, comprising the psychrophilic Pseudomonas sp. JM11 or its fermentation liquid or the cellulase.

[0016] The present invention discloses the following technical effects:

[0017] The present invention screened and obtained a strain that can grow in a low-temperature environment, which was identified as a psychrophilic Pseudogymnoascus strain, and its classification name was Pseudogymnoascus pannorum. It was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on September 14, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No.41455.

[0018] The optimum growth temperature of the psychrophilic pseudogymna JM11 provided by the present invention is 15° C. It can quickly produce cellulase with a cellulase activity of up to 583.3 U / mL, and can degrade straw in a low-temperature environment with a degradation rate of 31.2%, thereby providing a new method for degrading straw in a low-temperature environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 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 work.

[0020] Figure 1is the growth graph of strain JM11 at different temperatures;

[0021] Figure 2 The structure of the spore of strain JM11 under scanning electron microscope;

[0022] Figure 3 The bar graph shows the cellulase concentration produced by strain JM11 on different days;

[0023] Figure 4 This is the activity electrophoresis (Native PAGE) experiment of strain JM11. The leftmost lane is the marker, and lanes 1-14 are crude enzyme solutions from days 1 to 14, respectively;

[0024] Figure 5 This is the result of testing the degradation efficiency of straw by strain JM11. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1 Isolation and Identification of Strain

[0031] 1. Screening and separation

[0032] 1.1 Preliminary screening

[0033] About 25 g of soil sample was mixed in 75 mL of sterile water at 170 rpm for 30 minutes. Then, it was placed in a refrigerator at 4 ° C for 4 hours. After 4 hours, 300 μL of supernatant was spread on potato dextran agar (PDA) plates supplemented with 100 μg / mL streptomycin sulfate to prevent bacterial growth and isolate only fungi. The culture plates were incubated at 10 ° C or 4 ° C for several days until single colonies of different morphological types appeared. The strains were isolated and inoculated into new PDA plates and incubated at 10 ° C for 10 days for subsequent experiments.

[0034] 1.2 Rescreening

[0035] The strains obtained in the initial screening were inoculated into PDA medium and cultured in constant temperature incubators at 5°C, 10°C, 15°C, and 20°C for 8 days. The colony sizes of the strains were measured and the growth rates were calculated. The growth rate represents the high-temperature growth ability of the strains. The greater the growth rate, the stronger the growth ability of the strains at this temperature.

[0036] PDA culture medium: 200g potato juice, add 20g glucose, dilute to 1L with pure water, and then add 1.5% agar powder.

[0037] According to the results of the rescreening, the strain JM11 with the fastest growth rate at 15°C was selected as the target bacteria. Its optimal growth temperature is 15°C and its growth rate is Figure 1 shown.

[0038] 2. Morphological characteristics of strain JM11

[0039] The diameter of the colony on the PDA culture medium is 20-25mm. The colony is velvety, with white edges and light pink in the middle. After growth, the colony becomes gray-green in the middle with amber water droplets, the back is yellow-brown, and there are many white spores.

[0040] 3. Spore structure of strain JM11

[0041] The results were taken using a scanning electron microscope. Figure 2 As shown, the spores of strain JM11 are oval in shape with a circular bottom.

[0042] 4. Molecular biological identification of strain JM11

[0043] The SEQ ID NO.1 sequence obtained by 16S rDNA sequencing was compared with the NCBI GenBank database and found to have 99% homology with Pseudogymnoascus pannorum. Molecular biology and morphological identification confirmed that strain JM11 was a psychrophilic Pseudogymnoascus pannorum.

[0044] SEQ ID NO.1:

[0045] .

[0046] The above-mentioned strain JM11 was identified as a psychrophilic Pseudogymnoascus strain, and its classification name was Pseudogymnoascuspannorum. On September 14, 2024, it was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41455.

[0047] Example 2 Determination of cellulase activity produced by strain JM11

[0048] Activation of strain JM11: The preserved Pseudogycetes were inoculated on PDA medium and cultured in a 15°C incubator for 7-9 days to obtain culture fluid.

[0049] Prepare enzyme production medium: bran 10g, corncob powder 10g, microcrystalline cellulose 20g, peptone 10g, potassium dihydrogen phosphate 1.5g, add water to 1L. Take the culture solution at 10% inoculation amount and inoculate it into the enzyme production medium, culture at 15°C and 180rpm for 1-14 days, and take the fermentation solution every day to determine the enzyme activity.

[0050] Enzyme activity determination method: Take 2.0mL of fermentation liquid in a sterile bench, centrifuge at 10000rpm for 5min in a low-temperature refrigerated centrifuge. After centrifugation, take 50μL of crude enzyme solution and put it into a new centrifuge tube. Add 200μL of 2% CMC-Na solution (different cellulases choose different substrates) to the centrifuge tube in advance, pH = 4.8. Place the centrifuge tube in a 50℃ metal bath for reaction for 30min. After the reaction is completed, add an equal volume of DNS solution to the centrifuge tube, heat the metal bath at 95℃ for 10min, and quickly take it out and cool it in an ice water bath after heating. Measure the absorbance value at 540nm. Use the inactivated enzyme solution as a control.

[0051] Definition of cellulase activity: The amount of enzyme required for 1 mL of original enzyme solution to catalyze the substrate to produce 1 μg of glucose per minute is defined as one enzyme activity unit (U / mL).

[0052] The results are as follows Figure 3 As shown, at 15°C, the maximum cellulase activity of JM11 can reach 583.3 U / mL, and the crude enzyme solution obtained after 3 days of culture has the highest cellulase activity.

[0053] Enzyme activity detection method: conduct activity electrophoresis experiment and prepare activity gel; run the gel at 80 volts for 100 minutes; soak in CMC for 2 hours; stain with Congo red for 1 hour; decolorize with sodium chloride for 30 minutes.

[0054] The results are as follows Figure 4 As shown, strain JM11 showed clear bands from the 3rd to the 7th day, indicating that the crude enzyme solution obtained from 3-7 days of culture had high cellulase activity.

[0055] Example 3 Ability of strain JM11 to degrade cellulose in straw under low temperature conditions

[0056] Prepare straw liquid culture medium: straw: 5g, corn cob powder 2g, nutrient solution 30mL (urea 3g, ammonium sulfate 6g, peptone 3g, calcium chloride 0.1g, magnesium sulfate 0.5g, potassium dihydrogen phosphate 1g, sodium chloride 0.1g, ferrous sulfate 0.05g, manganese sulfate 0.016g, zinc sulfate 0.014, cobalt chloride 0.037g), add water to 1L.

[0057] Determination of straw degradation rate: Pick 5 JM11 bacteria blocks with a diameter of 5 mm and inoculate them into a 100mL triangular flask containing 20mL PDA liquid culture medium, and culture them in a shaking incubator (180rpm) at 16℃ for 48h. Then, inoculate the culture solution into a 100mL triangular flask containing 20mL straw liquid culture medium at a 10% inoculation amount, place it in a 16℃ incubator for culture, and shake it twice a day (shaking condition: 100rpm). From the 1st to the 15th day, filter and collect the straw in the bottle every day and rinse it with distilled water 3 times, dry it at 85℃ to constant weight, weigh the remaining corn straw, and calculate the degradation rate, with 3 replicates per day.

[0058] The results are as follows Figure 5 As shown, the straw degradation rate reached the highest level of 31.2% at 14 days of cultivation.

[0059] 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 modifications and improvements 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 Pseudogymnoascus pannorum JM11, characterized in that: The preservation number of the psychrophilic Pseudomonas aeruginosa JM11 is CGMCC No.41455, the preservation date is September 14, 2024, the preservation unit is the General Microbiology Center of the China Culture Collection Administration, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the psychrophilic Pseudomonas sp. JM11 according to claim 1 in the preparation of cellulase.

3. A method for preparing cellulase, characterized in that: The method comprises the steps of fermenting the cold pseudogymna JM11 described in claim 1 and collecting the fermentation liquid.

4. The method according to claim 3, characterized in that The psychrophilic Pseudomonas sp. JM11 is inoculated into an enzyme production medium, cultured for 2-9 days, and the fermentation liquid is centrifuged to obtain the cellulase; The components of the enzyme production culture medium include: 10g bran, 10g corn cob powder, 20g microcrystalline cellulose, 10g peptone and 1.5g potassium dihydrogen phosphate, and water is added to 1L.

5. The method according to claim 4, characterized in that The culture is carried out at 15° C. and 180 rpm for 2-4 days.

6. Cellulase prepared by the method according to any one of claims 3 to 5.

7. Use of the psychrophilic Pseudomonas sp. JM11 or its fermentation liquid as claimed in claim 1 or the cellulase as claimed in claim 6 in degrading straw.

8. A straw-degrading preparation, characterized in that: It comprises the psychrophilic Pseudomonas sp. JM11 or its fermentation broth as described in claim 1 or the cellulase as described in claim 6.

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