CN115340964A - Bacillus strain with swollenin activity and application thereof - Google Patents
Bacillus strain with swollenin activity and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a bacillus strain with swollenin activity and application thereof, wherein fermentation liquor of the bacillus strain has extremely high swollenin protein activity. The strain is a bacillus strain CFFSH013, an original strain is separated from the surface of tobacco leaves and identified as the bacillus strain through 16srDNA, the strain is preserved in China center for type culture collection, and the number of the preservation center is CCTCCNO: m2020635.
Description
Technical Field
The invention relates to a strain, in particular to a bacillus strain with swollenin activity, belonging to the technical field of strains.
Background of the invention:
swollenins (expansins, also known as dilators or expansins) are proteins that cause the relaxation of plant cell walls and play a key role in plant cell expansion and a series of vital activities involving cell wall modification. Swollenins are encoded by a multigene family, and studies have shown that the swollenin superfamily consists of four gene subfamilies.
The cell wall plays a vital role in various cellular activities, such as differentiation, transport and communication, senescence, abscission, plant-pathogen interactions and ultimately plant growth. It provides both the mechanical strength required by the plant and the plasticity required for the development of plant tissues and organs. Although the biochemical mechanism of swollenin operation is not completely understood, it is widely believed that the action of swollenin on the cell wall, which is not an enzyme in itself, causes this demanding plasticity, but that swollenin can relax the cell wall, causing a sustained reduction in the tensile strength of the cell wall, which in turn leads to cell swelling.
Swollenins were first discovered by S McQueen-Mason in cucumber (DOI: 10.1105/tpc.4.11.1425), and later were found ubiquitous in a variety of plants, including Arabidopsis, tobacco, rice, maize, soybean, and the like. However, the swollenins derived from plants have low expression yield and are difficult to produce on a large scale.
In addition to plant origin, the prior art has found that microorganisms which partially colonize the surface of plants also have the ability to secrete and produce microbial swollenin proteins. Including expansin-like proteins from dictyostelium discodermatum (Kawata et al, 2015), bsExlx1 from Bacillus subtilis (Kerff et al, 2008), hcExlx2 from the marine bacterium Hahella chejuensis (Lee et al, 2010), pectobacterium carotovorum from a plant pathogenic bacterium (Olarte-Lozano et al, 2014) and ScExlx1 from the family Basidiomycetes fungi Schizophyllum (Tovar-Herrera et al, 2015). These microbial expansins have demonstrated a variety of capabilities that can be used to attach and colonize plants with microorganisms. In addition, several researchers are evaluating the potential use of these microbial expansins in the conversion of cellulosic biomass for biofuel production as a means of breaking down the cellulose structure (Georgelis et al, 2015).
Although the swollenin does not hydrolyze glycosidic bonds in cellulose molecules, the swollenin has a CBD binding domain similar to cellulase and a sequence similar to phytoswollenin, and can play a role in swelling and disintegrating cellulose and cell walls and improve the action efficiency of cellulase. The swollenin and biomass degrading enzymes such as cellulase and the like have synergistic effect, so that the lignocellulose biomass substrate is effectively converted, and the economic benefit of fermentation production is improved.
Patent application fungus-derived swollenin protein and gene and application thereof (CN 201710269769.7) disclose a swollenin protein derived from Talaromyces leycettanus. However, the production of the protein still needs to be expressed by recombinant expression of Pichia pastoris (Pichia pastoris) cells, saccharomyces cerevisiae (Saccharomyces cerevisiae) cells or Hansenula polymorpha (Hansenula polymorpha) cells.
The cheap and fast large-scale production of the swollenin protein is an important condition for the industrial application of the swollenin protein. The bacillus strain grows rapidly, has high protein expression quantity, is an important probiotic, has high safety, and is very suitable for large-scale production of the swollenin protein. Meanwhile, the bacillus itself is a probiotic of many plants, and the bacillus can produce the bacterial swollenin protein BsExlx1 which is firstly found in the bacillus subtilis in 2008. The search for strains that produce high levels of the bacterial swollenin protein is a key technology lacking in the prior art.
Disclosure of Invention
The invention aims to provide a bacillus strain with swollenin activity, and a fermentation liquid of the bacillus strain has extremely high swollenin protein activity. The strain is a bacillus strain CFFSH013, an original strain is separated from the surface of tobacco leaves by an inventor and identified as the bacillus strain through 16srDNA, and the strain is preserved in China center for type culture Collection with the address: wuhan university in Wuhan City; and (3) classification and naming: bacillus CFFSH013 (Bacillus sp.cffsh 013); the number of the preservation center is CCTCC NO: m2020635; the preservation date is as follows: 26/10/2020. The culture was tested by the culture collection on day 10 at 11 months 2020 and was found to be viable.
The purpose of the invention is realized by the following technical scheme.
A bacillus strain with swollenin activity is characterized in that the strain has a preservation number of CCTCC NO: strain of M2020635.
A product having swollenin activity, which comprises any one of a Bacillus strain CFFSH013, a Bacillus strain CFFSH013 broth supernatant, a Bacillus strain CFFSH013 fermentation extract, and a Bacillus strain CFFSH013 protein product.
A method for accelerating the degradation process of biomass is to combine the Bacillus strain CFFSH013 (CCTCC NO: M2020635) with other biomass degrading enzymes.
Furthermore, the method of using the Bacillus strain CFFSH013 (CCTCC NO: M2020635) in combination with other biomass-degrading enzymes includes using the fermentation product of the Bacillus strain CFFSH013 in combination with other biomass-degrading enzymes.
A microbial inoculum for accelerating the degradation process of biomass contains Bacillus strain CFFSH013 (preservation number is CCTCC NO: M2020635).
Furthermore, the microbial inoculum for accelerating the biomass degradation process also contains other bacterial strains capable of producing biomass degrading enzymes.
The term "biomass" in the present application refers to various organisms produced by photosynthesis using the atmosphere, water, land and the like, that is, all living organic substances capable of growing are generally called biomass, and includes all plants, microorganisms, animals using plants and microorganisms as food, and waste products produced by the same, such as crops, crop waste, wood waste, and biologically derived components such as biologically derived cellulose, starch, lignin and the like.
The concept of the biomass degrading enzyme in the application of the invention refers to degrading enzymes such as cellulase, protease, pectinase, laccase and the like which can decompose various biomasses and components thereof.
Detailed Description
The technical features of the present invention will be further described with reference to specific embodiments.
Example 1: cultivation of Bacillus Strain CFFSH013
Preparing LB liquid culture medium (5 g/L yeast extract and 10g/L peptone), sterilizing at 115 deg.C for 20min, cooling to room temperature, inoculating 1% (v/v) selected Bacillus strain CFFSH013, culturing at 37 deg.C for 24 hr, and storing the fermentation broth at 4 deg.C.
To facilitate protein activity determination, the fermentation broth of the bacillus strain CFFSH013, which was used for the different fermentation products, was treated as follows:
(1) Fermentation broth of CFFSH 013: storing at 4 deg.C after fermentation, and shaking before use;
(2) CFFSH013 inactivation fermentation broth: heating the fermentation liquor to 100 ℃ after fermentation is finished, and then cooling to 4 ℃ for storage for later use;
(3) And (3) fermenting supernatant of CFFSH013, namely centrifuging the fermentation liquor by using a refrigerated centrifuge (4 ℃,8000 rpm) after the fermentation is finished, discarding the lower-layer thallus, and collecting the supernatant and storing the supernatant at 4 ℃ for later use.
(4) And (3) fermenting the supernatant freeze-dried powder by CFFSH013, namely centrifuging the fermentation liquor by using a refrigerated centrifuge (4 ℃,8000 rpm) after the fermentation is finished, discarding the lower-layer thallus, collecting the supernatant, preserving at-80 ℃ for 12 hours to pre-freeze into ice, sublimating and freeze-drying the fermentation liquor into powder by using a vacuum freeze-dryer, and collecting the powder.
(5) CFFSH013 thallus lysate (cell lysate of CFFSH 013) is prepared by centrifuging the fermentation liquid with a refrigerated centrifuge (4 deg.C, 8000 rpm) after fermentation, resuspending the lower layer thallus with distilled water, crushing with an ultrasonic cell crusher, and collecting and storing at 4 deg.C.
Example 2: biomass degradability (Jerusalem artichoke powder)
The method comprises the steps of taking jerusalem artichoke slices as a substrate for biomass degradation, taking beta-glucanase (produced by Yizhi biotechnology limited in Henan) and beta-glucanase (produced by Baijia biotechnology limited in Guangzhou), and measuring the contents of glucose and fruit carbon released in hydrolysate by using a high performance liquid chromatography evaporative light scattering detection analysis method (HPLC-ELSD, chromatographic column: philomen MARS MCa) after hydrolysis. The Blank (BK) of the experiment was prepared by directly adding inulin to water to prepare an aqueous solution having a final concentration of 5% (w/w), and the glucosylfructose content thereof was directly measured. The blank of the experiment adopts inulin added with water to prepare an aqueous solution with a final concentration of 5% (w/w), and then the reaction is carried out in a water bath shaker at 40 ℃ for 30min, and then the contents of glucose and fructose in the measuring period are measured. The measurement conditions and treatment conditions for the other experimental groups are listed in table 1.
The results (table 1) show that the fermentation product of CFFSH013 strain alone has no significant biomass degradation effect, but when it was complexed with other biomass degradation enzymes, it could greatly increase the efficiency of the β -levanase and β -glucanase action, indicating a synergistic effect similar to swollenin.
TABLE 1 Biomass degradation of Jerusalem artichoke meal
Example 3: biomass degradation (straw foam)
The wheat straw foam obtained by physically crushing waste wheat straw into fine foam is used as the treatment substrate of the embodiment, and the treatment process comprises the steps of taking 200g of wheat straw foam, spraying 200g of water, adding CFFSH013 fermentation product and other enzyme or bacteria agent according to the treatment method listed in Table 2, and uniformly mixingHomogenizing, storing in 30 deg.C incubator with humidity of 60% for 7 days, adding 0.1% HCL solution 200g, shaking at 25 deg.C for 12 hr to leach out glucose, collecting the leachate, and detecting with high performance liquid chromatography evaporative light scattering detection (HPLC-ELSD, chromatographic column: firema MARS M) Ca ) Glucose content was measured. The cellulase used in this example was purchased from Biotechnology (Shanghai) Inc., and the laccase was purchased from Shandongxuan Yangyi Biotech Co., ltd. Trichoderma reesei is derived from a purified strain of Trichoderma reesei strain from Kangfeng brand of Shandong Zibo Yuan kang Biotech Co.
As a result, as shown in table 2, the efficiency of the CFFSH013 fermented product for biomass dissociation by the enzyme preparation/microbial inoculum was greatly improved.
TABLE 2 Biomass degradation treatment conditions of straw foam
Claims (6)
1. A strain of bacillus having swollenin activity, characterized by: the strain is a strain with a preservation number of CCTCC NO: strain of M2020635.
2. A product having swollenin activity, wherein: the product is any one of a bacillus strain CFFSH013, a fermentation broth of the bacillus strain CFFSH013, a supernatant of the fermentation broth of the bacillus strain CFFSH013, a fermentation extract of the bacillus strain CFFSH013, and a protein product of the bacillus strain CFFSH 013.
3. A method of accelerating a biomass degradation process, comprising: the Bacillus strain CFFSH013 (CCTCC NO: M2020635) is used in combination with other biomass degrading enzymes.
4. A method of accelerating a biomass degradation process according to claim 3, wherein: the method of using the Bacillus strain CFFSH013 (with the preservation number of CCTCC NO: M2020635) in combination with other biomass-degrading enzymes includes using the fermentation product of the Bacillus strain CFFSH013 together with other biomass-degrading enzymes.
5. A microbial inoculum for accelerating the degradation process of biomass is characterized in that: the microbial inoculum contains Bacillus strain CFFSH013 (CCTCC NO: M2020635).
6. The microbial inoculum for accelerating the biomass degradation process according to claim 5, which is characterized in that: the microbial inoculum also contains other strains which can produce substance degrading enzymes.
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