A cold-resistant cellulase-producing bacterium and its application

Abstract

The present invention discloses a cold-resistant cellulase-producing bacterium and its application, belonging to the field of microorganisms and fermentation technology. The bacterium is Acinetobacter lwoffii L1, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCCNO.24216. The strain can produce highly active cellulase under the conditions of 5 ℃-25 ℃, and the produced cellulase is active at 3.378IU / mL-3.419IU / mL, which can be used to ferment rice straw, corn straw and cottonseed hulls to prepare cellulase, and the purpose of efficiently producing cellulase using cheap raw materials is achieved, and the production cost of cellulase is effectively reduced. At the same time, it also solves the problem that winter crops in northern regions cannot be efficiently utilized directly in the fields and agricultural wastes such as rice straw, corn straw and cottonseed hulls are difficult to efficiently handle.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and fermentation, in particular to a cold-resistant cellulase-producing bacterium and application thereof. Background Art

[0002] Efficient fiber-degrading bacteria have broad application prospects in both industrial production and agricultural waste treatment. As the most abundant high-molecular carbohydrate in the world, how to process cellulose is a hot topic and difficulty in modern research.

[0003] In nature, there are many microorganisms that can produce cellulase to degrade cellulose, including fungi, bacteria, and actinomycetes, among which fungi are considered to be the main degraders of cellulose. The most studied are molds, especially Trichoderma viride, Trichoderma reesei, Trichoderma reesei, and Trichoderma koningii. However, since the fermentation liquid of molds is very viscous during deep fermentation, it is easy to cause insufficient oxygen supply, resulting in slow cell growth or even death, which limits its use and development. And because fungi are eukaryotic organisms, editing at the genetic level is more complicated. Compared with fungi, bacteria have the advantages of easy cultivation, fast growth, strong tolerance, and easy genetic level operation.

[0004] Acinetobacter lwoffii belongs to the genus Acinetobacter, a coccobacillus. It does not decompose any sugars and is commonly found in the environment. Currently, most studies on Acinetobacter lwoffii focus on bacterial infections caused by it. There are also related studies that use the ability of Acinetobacter lwoffii to degrade α-solanine for biological control. There are currently no reports that Acinetobacter lwoffii can produce cellulase. Summary of the invention

[0005] The purpose of the present invention is to provide a cold-resistant cellulase-producing bacterium and its application to solve the problems existing in the above-mentioned prior art. The Acinetobacter lofivi L1 obtained by the present invention can ferment rice straw, corn straw and cottonseed hulls to prepare cellulase, achieving the purpose of efficiently producing cellulase using cheap raw materials and effectively reducing the production cost of cellulase. At the same time, it also solves the problem that agricultural wastes such as rice straw, corn straw and cottonseed hulls are difficult to efficiently treat.

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

[0007] The invention provides a cold-resistant cellulase-producing strain, which is Acinetobacter lwoffii L1, and has been deposited in the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures, with a deposit number of CGMCCNO.24216.

[0008] The invention also provides a bacterial agent, comprising the strain.

[0009] The invention also provides application of the strain in producing cellulase.

[0010] The invention also provides a method for producing cellulase, comprising the steps of fermenting and culturing the strain to produce cellulase.

[0011] Furthermore, the fermentation temperature is 5°C-25°C.

[0012] Furthermore, the fermentation temperature is 20°C.

[0013] The present invention also provides an application of the strain or the bacterial agent in degrading agricultural waste containing cellulose.

[0014] Furthermore, the agricultural waste includes rice straw, corn straw and cottonseed hulls.

[0015] The present invention also provides a method for degrading agricultural waste containing cellulose, comprising the step of adding the strain or the bacterial agent to the agricultural waste containing cellulose for fermentation.

[0016] Furthermore, the fermentation temperature is 5°C-25°C.

[0017] The present invention discloses the following technical effects:

[0018] The present invention isolates a cold-resistant cellulase-producing strain Acinetobacter lwoffii L1 from wild yak dung at high altitudes in Linzhi City, Tibet. The strain can produce highly active cellulase at 5°C-25°C, and the produced cellulase activity is 3.378IU / mL-3.419IU / mL, which can be used to ferment rice straw, corn straw and cottonseed hulls to prepare cellulase, achieving the purpose of efficiently producing cellulase using cheap raw materials and effectively reducing the production cost of cellulase. At the same time, it also solves the problem that winter crops in northern regions cannot be efficiently utilized directly in the fields and agricultural wastes such as rice straw, corn straw and cottonseed hulls are difficult to efficiently treat. 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 1Phylogenetic tree of Acinetobacter lwoffii L1 constructed;

[0021] Figure 2 The cellulase activity of Acinetobacter lwoffii L1 at different temperatures;

[0022] Figure 3 The cellulase activity of Acinetobacter lwoffii L1 when rice straw, corn straw and cottonseed hull were used as substrates;

[0023] Figure 4 is the degradation rate of Acinetobacter lwoffii L1 on rice straw, corn straw and cottonseed hull. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The culture medium and its formula used in the embodiments of the present invention are as follows:

[0030] CMC sodium medium: Each 100 ml of CMC sodium medium contains: 1 g sodium carboxymethyl cellulose, 1 g peptone, 0.5 g beef extract, 0.4 g ammonium sulfate, 0.2 g dipotassium hydrogen phosphate, 0.05 g magnesium sulfate heptahydrate and 1.6 g agar. The medium is sterilized by high temperature and high pressure.

[0031] Rice straw enzyme production medium: Each 100 mL of rice straw enzyme production medium contains 0.5 g peptone, 0.5 g NaCl, 0.2 g calcium carbonate, 0.3 g yeast powder and 0.5 g rice straw (crushed into short stalks of about 5 cm in advance). Natural pH, high temperature and high pressure sterilization of rice straw enzyme production medium, set aside;

[0032] Corn straw enzyme production medium: Each 100 mL of enzyme production medium contains 0.5 g peptone, 0.5 g NaCl, 0.2 g calcium carbonate, 0.3 g yeast powder and 0.5 g corn straw (the corn straw is crushed into short stalks of about 5 cm in advance). Natural pH, high temperature and high pressure sterilization of corn straw enzyme production medium, set aside;

[0033] Cottonseed hull enzyme production medium: Each 100 mL of the enzyme production medium contains 0.5 g of peptone, 0.5 g of NaCl, 0.2 g of calcium carbonate, 0.3 g of yeast powder and 0.5 g of cottonseed hull. Natural pH, high temperature and high pressure sterilization of the cottonseed hull enzyme production medium for later use.

[0034] The temperatures involved in the embodiments of the present invention are all fixed values. In practical applications, the same effect of the present invention can be achieved within the range of ±1°C.

[0035] Example 1 Isolation and identification of strain L1

[0036] 1. Isolation of strains

[0037] The strain L1 of the present invention is isolated from wild yak dung at high altitude in Linzhi City, Tibet. The specific isolation method is:

[0038] The dung of wild yaks at high altitude in Linzhi City, Tibet was inoculated into rice straw enzyme production medium for enrichment culture at 20°C. The enriched culture solution was spread on CMC sodium medium plates and cultured at 20°C. After 2 days of culture, Congo red staining was performed, and the single colony with the largest transparent circle was selected, and the strain was named L1.

[0039] 2. Morphological identification

[0040] The strain L1 was inoculated onto a CMC sodium medium plate, the plate was inverted and cultured at 20°C for 2 days, a single colony was picked for Gram staining, and observed under a microscope. L1 was a Gram-negative bacillus.

[0041] 3.16S rDNA sequence analysis

[0042] The genomic DNA of this strain was extracted and 16S rDNA sequencing was performed. The 50 μL PCR reaction system included: genomic DNA (20 ng / μl) 1.0 μl, 10× Buffer (containing 2.5 mM Mg 2+ ) 5.0μL, Taq polymerase (5U / μL), dNTP (10mM) 1.0μL, primer 27F (SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3', 10μM), primer 1492R (SEQ ID NO.2: 5'-GGTTACCTTGTTACGACTT-3', 10μM) 1.0μL, ddH2O 41μL. PCR conditions were: initial denaturation at 94℃ for 4min, denaturation at 94℃ for 1min, annealing at 55.5℃ for 2min, extension at 72℃ for 1min, for a total of 30 cycles, and finally extension at 72℃ for 10min. The quality of the amplified PCR product was detected by 1% agarose gel electrophoresis.

[0043] After the amplified band was single, it was sent to a sequencing company (Beijing Aoke Dingsheng Company) for sequencing. The accession number of the obtained 16S rDNA sequence in GenBank is MZ277422.1. The sequence was compared with the GenBank database by BLAST sequence homology to construct a phylogenetic tree (see Figure 1 ). The strain was identified as Acinetobacter lwoffii, named L1, and deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.24216 and the deposit date December 30, 2021.

[0044] Example 2 Determination of cellulase production by strain L1

[0045] 1. Preparation of crude enzyme solution

[0046] The screened L1 strain was inoculated into the culture medium. After cultivation, 5 mL of the fermentation liquid was centrifuged and the supernatant was the crude enzyme liquid.

[0047] The culture medium is CMC sodium culture medium.

[0048] 2. Glucose standard curve drawing

[0049] Weigh 1.0 g of glucose that has been dried in an oven at 80°C to a constant weight, and dilute to a 100 mL volumetric flask to obtain a 10 mg / mL glucose standard solution. Take 18 25 mL stoppered test tubes, 3 in a group, and add 2.50, 2.45, 2.40, 2.35, 2.30, and 2.25 mL of phosphate buffer solution, and also add 0, 0.05, 0.10, 0.15, 0.20, and 0.25 mL of glucose standard solution. Finally, add 2 mL of DNS solution to each test tube, mix well, heat in a boiling water bath for 5 min, take out the test tubes, cool to room temperature with running water, dilute to 25 mL, and measure OD with an enzyme marker. 540 Draw the glucose standard curve.

[0050] 3. CMCase activity assay

[0051] Take 0.5mL crude enzyme solution and 2mL 0.5% sodium CMC solution in a 25mL stoppered test tube, incubate in a 50℃ water bath for 30min, then add 2mL DNS solution, heat in a boiling water bath for 5min, cool to room temperature with running water, make up to volume, and measure OD 540 The crude enzyme solution heated in a boiling water bath for 10 min was used as the blank sample.

[0052] Calculation of CMCase activity: 1 mL of crude enzyme solution hydrolyzes the corresponding substrate per minute to produce 1 μmol of glucose as reducing sugar, which is defined as one enzyme activity unit (IU / mL).

[0053] 4. CMCase activity of strain L1 under different temperature conditions

[0054] Take 3 mL of the pre-cultured L1 fermentation liquid in 1. and inoculate it into 300 mL of rice straw enzyme production medium. Cultivate it in a shaking incubator at 120 rpm at a temperature of 5°C-25°C. After 10 days of cultivation, take 5 mL of the fermentation liquid and centrifuge it. Take the supernatant as the crude enzyme liquid. The precipitate is used to measure the weight loss rate. Determine the CMCase activity. The results are as follows Figure 2 As shown, the activity of CMCase produced by Acinetobacter lwoffii L1 at 5℃-25℃ was between 3.378IU / ml-3.419IU / ml.

[0055] Example 3: Degradation of rice straw by strain L1

[0056] 3 mL of the pre-cultured L1 fermentation liquid in 1. of Example 2 was inoculated into 300 ml of rice straw enzyme production medium. After culturing at 5°C and 120 rpm in a shaking incubator for 25 days, 5 mL of the fermentation liquid was centrifuged and the supernatant was taken as the crude enzyme liquid. The CMCase activity was measured. The results are as follows Figure 3-4The activity of CMCase produced by Acinetobacter lwoffii L1 using rice straw was 3.376 IU / ml. The weight loss rate of rice straw reached 46%.

[0057] Example 4: Degradation of corn stalks by strain L1

[0058] 3 mL of the pre-cultured L1 fermentation liquid in 1. of Example 2 was inoculated into 300 ml of corn straw enzyme production medium. After culturing at 5°C and 120 rpm in a shaking table for 25 days, 5 mL of the fermentation liquid was centrifuged and the supernatant was taken as the crude enzyme liquid; the precipitate was used to measure the weight loss rate. The CMCase activity was measured. The results are as follows Figure 3-4 The activity of CMCase produced by Acinetobacter lwoffii L1 using corn stalks was 3.289 IU / ml. The weight loss rate of corn stalks reached 42%.

[0059] Example 5 Degradation of cottonseed hulls by strain L1

[0060] 3 mL of the pre-cultured L1 fermentation liquid in 1. of Example 2 was inoculated into 300 ml of corn straw enzyme production medium. After culturing at 5°C and 120 rpm in a shaking table for 25 days, 5 ml of the fermentation liquid was centrifuged and the supernatant was taken as the crude enzyme liquid; the precipitate was used to measure the weight loss rate. The CMCase activity was measured. The results are as follows Figure 3-4 Acinetobacter lwoffii L1 produced 2.186 IU / ml of CMCase using cottonseed hulls. The weight loss rate of cottonseed hulls in water reached 33%.

[0061] 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 cold-resistant cellulase-producing strain, characterized in that: The strain is Acinetobacter lofi Acinetobacter lwoffii )L1, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC NO.24216.

2. A bacterial agent, characterized in that The invention comprises the strain described in claim 1.

3. Use of the strain as claimed in claim 1 in producing cellulase.

4. A method for producing cellulase, characterized in that: The method comprises the steps of fermenting and culturing the strain according to claim 1 to produce cellulase.

5. The method according to claim 4, characterized in that The fermented substrates include rice straw, corn straw and cottonseed hulls.

6. The method according to claim 4, characterized in that The fermentation temperature is 5°C-25°C.

7. Use of the strain according to claim 1 or the bacterial agent according to claim 2 in degrading agricultural waste containing cellulose.

8. The use according to claim 7, characterized in that: The agricultural waste includes rice straw, corn straw and cottonseed husk.

9. A method for degrading agricultural waste containing cellulose, characterized in that: The method comprises the steps of adding the strain according to claim 1 or the bacterial agent according to claim 2 to the cellulose-containing agricultural waste for fermentation.

10. The method according to claim 9, characterized in that The fermentation temperature is 5°C-25°C.

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