CN116064289A - Endophytic olive bacillus and application thereof - Google Patents
Endophytic olive bacillus and application thereof Download PDFInfo
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- CN116064289A CN116064289A CN202211063786.2A CN202211063786A CN116064289A CN 116064289 A CN116064289 A CN 116064289A CN 202211063786 A CN202211063786 A CN 202211063786A CN 116064289 A CN116064289 A CN 116064289A
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- imb007
- endophytic
- olivibacter
- endophytica
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Abstract
The invention discloses endophytic olive bacillus and application thereof. The registration number of the endophytic olive bacillus (Olivibacter endophytica) IMB007 provided by the invention in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.25273. The endophytic olive bacillus (Olivibacter endophytica) IMB007 provided by the invention represents a new species of olive bacillus, and the detection of endoglucanase activity also proves that the strain has cellulose degradation activity. The strain has wide application prospect in the aspects of food processing, garbage disposal, soil restoration, soil improvement and the like.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to endophytic olive bacillus and application thereof.
Background
Olive genus (Olivibacter) belongs to the bacterial domain (Bacteria) -Bacteroides (Bacteria) -Sphingobacteriales (Sphingobacteriales) -Sphingobacteriales (Sphingobacteriaceae). In 2007, ntougias et al isolated strain AW-6 from alkaline olive oil milling waste. 16S rRNA gene sequence information of strain AW-6 showed that its nearest strain was Sphingobacterium thalpophilum DSM 11723 T The strain is belonging to Sphingobacteriaceae and diverges from Sphingobacterium (Sphingobacterium) and Geobacillus (Pedobacterium) strains, and forms separate branches, and combines physiological and biochemical characteristics and cytochemical classification indexes to establish Oliviacterium (Olivibacterium) with a model of Ceylobacter sibiricus (Olivibacter sitiensis) and Olivibacter sitiensis AW-6 T Is a model strain and is described. (Ntool S, fasseas C, zervakis GI.Olivibacter sitens gen.nov., sp.nov., isolated from alkaline olive-oil mill wastes in the region of Sitia, crete.int J System Evol Microbiol.2007Feb;57 (Pt 2): 398-404.Doi:10.1099/ijs.0.64561-0.PMID: 17267986.).
At present, the olive bacillus contains 9 species of O.sitensis AW-6 which are effectively described T Isolated from alkaline olive oil milling waste, o.sol 034 T And O.ginsengisol 060 T Isolated from ginseng field soil, O.domestcus DC186 T 、O.composti CC-KYC063 T 、O.ginsenosidimutans BS18 T And O.terrae Jip13 T Isolated from compost, O.jilunii 14-2A T Isolation from DDT contaminated soil, O.oleidegradans TBF2/20.2 T Drainage separated from hydrocarbon contaminationAnd (3) a device. The Olivibacter strain has been reported to have a variety of active functions or biological potential. Ginsenosidimutans DC186 T Has beta-glucosidase activity, and can incubate Rb at 30deg.C for 72h 1 Three forms of ginsenoside Rc, rd are converted into PPDOL type ginsenoside (Siddiqi MZ, liu Q, lee SY, choi KD, im WT. Olivibacterium ginsenosidimutans sp nov., with ginsenoside converting activity isolated from compost, and reclassification of Pseudosphingobacterium domesticum as Olivibacter domesticus comb. Nov. Int J Syst Evol Microbiol.2018Aug;68 (8): 2509-2514. Doi:10.1099/ijsem.0.002819.Epub 2018Jun 26.PMID:29944109.); for O.sitensis DSM 17696 T Whole genome DNA sequencing and mining have found that the strain may have heavy metal resistance and organic solvent degradation potential. The genome of the strain contains related genes for encoding protocatechuic acid dioxygenase (protocathoate 3, 4-dioxygenase), which indicates that the strain has potential benzoate and 2, 4-dichlorobenzoate degradation capability; genes encoding carboxymethylbutene lactonase contained in the strain suggest that the strain has the potential to degrade hexachlorocyclohexane and 1, 4-dichlorobenzene; at the same time, aryl alcohol dehydrogenase-related oxidoreductase was also predicted in the genome, suggesting O.sitensis DSM 17696 T It is also possible to participate in the degradation of biphenyl and toluene/xylene (Ntool S, lapidus A, han J, mavromatis K, pati A, chen A, klenk HP, woyke T, fasseas C, kyrpsides NC, zervakis GI. High quality draft genome sequence of Olivibacter sitiensis type strain (AW-6 (T)), a diphenol degrader with genes involved in the catechol path.stand Genomic Sci.2014Mar 20;9 (3): 783-93.Doi:10.4056/sigs.5088950.PMID: 25197463.). Furthermore, in the study of the microbial remediation of soil samples contaminated with weathered oil, it was found that the presence of O.ginsengisol strains was detected throughout the remediation, and that the presence of O.ginsengisol, O.jilunii strains was detected in soil with higher microbial abundance as the degree of remediation increased (Ali N, dashti N, salamah S, sorkhoh N, al-Awadhi H, radwans S.Dynamics of bacterial populations during bench-scale bioremediation of oily seawater and desert soil bio)augmented with coastal microbial mats.microb biotechnol.2016 Mar;9 (2) 157-71.Doi:10.1111/1751-7915.12326.Epub 2016Jan 11.PMID: 26751253. In addition, when isolated culture of potato rhizosphere and phyllospheric microorganisms was performed using nitrogen-free medium, the isolated bacteria contained a large amount of Olivibacter strain, suggesting that Olivibacter strain might promote growth and colonisation of other microorganisms by exerting nitrogen fixation (Turnbull A L, liu Y, lazarovits G. Isolation of Bacteria from the Rhizosphere and Rhizoplane of Potato (Solanum tuberosum) Grown in Two Distinct Soils Using Semi Selective Media and Characterization of Their Biological Properties [ J)].American Journal of Potato Research,2012,89(4):294-305.)。
Disclosure of Invention
The object of the present invention is to provide a novel species of the genus olive and its use.
In a first aspect, the invention claims a novel species of the genus olive.
The novel species of the olive bacillus claimed by the invention is specifically plant endophytic olive bacillus (Olivibacter endophytica) IMB007, and the registration number of the novel species of the olive bacillus in the China general microbiological culture Collection center (CGMCC) is CGMCC No.25273.
The endophytic olive bacillus (Olivibacter endophytica) IMB007 is a gram-negative aerobic heterotrophic bacterium, has no sporulation, does not move, is spherical, has the cell diameter of (0.2-0.3) mu m, and has yellow-white colony, moist surface, smoothness and complete edge; after 5 days of culture on PYG solid medium at 28 ℃, the strain has a growth tolerance range of 10-40 ℃, 0-3% NaCl and pH 6-8.0, and the optimal growth conditions are 28 ℃, 0% NaCl and pH 7.0; alkaline phosphatase, esterase C4, lipid esterase C8, leucine arylamidase, valine arylamidase, cystine arylaminase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, alpha-galactosidase, beta-uronic acid glycosidase, alpha-glucosidase, beta-glucosidase, N-acetyl-glucosamine enzyme and alpha-mannosidase production were positive. The 16S rRNA sequence of the strain is shown as SEQ ID No. 1.
In a second aspect, the invention claims a culture.
The culture claimed in the present invention is a culture of the endophytic olive bacillus (Olivibacter endophytica) IMB007 described in the first aspect, in particular a substance obtained by culturing the endophytic olive bacillus (Olivibacter endophytica) IMB007 in a bacterial culture medium.
In the above culture, the substance includes the endophytic olive bacillus (Olivibacter endophytica) IMB007 (cell itself) and a metabolite of the endophytic olive bacillus (Olivibacter endophytica) IMB007.
In the above culture, the bacterial culture medium may be a solid culture medium or a liquid culture medium.
The term "culture" refers to a collective term for a liquid or solid medium in which a population of microorganisms has grown after artificial inoculation and cultivation. I.e. the product obtained by growing and/or amplifying the microorganism, which may be a biologically pure culture of the microorganism, or may contain a certain amount of medium, metabolites or other components produced during the culture. The term "culture" also includes subcultures obtained by passaging microorganisms, which may be a culture of a certain generation or a mixture of several generations.
In a specific embodiment of the invention, the bacterial culture medium is specifically a PYG culture medium.
In a third aspect, the invention claims a metabolite.
The claimed metabolite of the invention is a metabolite of the endophytic olive bacillus (Olivibacter endophytica) IMB007 of plants as described in the first aspect hereinbefore.
The term "metabolite" refers to a primary metabolite and/or a secondary metabolite produced during metabolism of a microorganism. Primary metabolism refers to a process in which microorganisms absorb various nutrients from the outside and produce substances and energy that maintain vital activities through catabolism and anabolism. The primary metabolite is primary metabolite such as monosaccharide or monosaccharide derivative, nucleotide, vitamin, amino acid, fatty acid, and various macromolecular polymers composed of the same, such as protein, nucleic acid, polysaccharide, and lipid. Secondary metabolism refers to the process of synthesizing substances which have no definite function on the life activities of microorganisms by taking primary metabolites as precursors in a certain growth period of microorganisms. The secondary metabolite is the secondary metabolite, and most of the secondary metabolites are compounds with relatively complex molecular structures. Depending on their action, they can be classified into antibiotics, hormones, alkaloids, toxins, etc.
In a fourth aspect, the invention claims a microbial agent.
The claimed microbial inoculum contains the endophytic olive bacillus (Olivibacter endophytica) IMB007 described in the first aspect, the culture described in the second aspect and/or the metabolite described in the third aspect.
The microbial inoculum is a microbial inoculum for hydrolyzing cellulose.
In the microbial inoculum, the microbial inoculum contains a carrier in addition to the active ingredient. The carrier may be a carrier commonly used in the pesticide arts and which is biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier can be mineral material, plant material or high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, soy flour and starch; the polymer compound may be polyvinyl alcohol and/or polyglycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and/or dodecane.
Among the above-mentioned bacterial agents, the formulation of the bacterial agent can be various formulations, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.
Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.
In a fifth aspect, the invention claims the use of an endophytic olivetobacter plantarum (Olivibacter endophytica) IMB007 as described in the first aspect hereinbefore or a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a microbial inoculum as described in the fourth aspect hereinbefore for any one of the following:
(A1) Degrading cellulose;
(A2) Preparing a product for degrading cellulose;
(A3) Preparing cellulase;
(A4) Preparing a product with cellulase activity;
(A5) A product having endoglucanase activity is prepared.
In a sixth aspect, the invention claims the use of an endophytic olivetobacter plantarum (Olivibacter endophytica) IMB007 as described in the first aspect hereinbefore or a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a microbial inoculum as described in the fourth aspect hereinbefore, in any one of the following:
(B1) Processing food;
(B2) Waste treatment;
(B3) Repairing soil;
(B4) Soil quality improvement.
In a seventh aspect, the invention claims a product for degrading cellulose.
The product for degrading cellulose claimed in the present invention, the active ingredient of which is olive bacillus endophyte (Olivibacter endophytica) IMB007 as described in the first aspect above or a culture as described in the second aspect above or a metabolite as described in the third aspect above or a microbial inoculum as described in the fourth aspect above.
In an eighth aspect, the invention claims a product having cellulase activity.
The product with cellulase activity claimed in the present invention has as active ingredient the endophytic olive bacillus (Olivibacter endophytica) IMB007 described in the first aspect above or the culture described in the second aspect above or the metabolite described in the third aspect above or the microbial inoculum described in the fourth aspect above.
In a ninth aspect, the invention claims a method of degrading cellulose.
The method for degrading cellulose claimed in the invention can comprise the following steps: treatment of a sample to be treated with an endophytic olive bacillus (Olivibacter endophytica) IMB007 as described in the first aspect hereinbefore or a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a bacterial agent as described in the fourth aspect hereinbefore.
In a tenth aspect, the invention claims the use of an endophytic olive bacillus (Olivibacter endophytica) IMB007 as described in the first aspect hereinbefore for the preparation of a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a microbial inoculum as described in the fourth aspect hereinbefore.
Experiments prove that the endophytic olive bacillus (Olivibacter endophytica) IMB007 provided by the invention has a plurality of remarkable difference characteristics with the existing olive bacillus strain in the aspects of phenotype, physiological biochemistry, cytochemistry and the like. Meanwhile, the systematic evolution analysis based on the gene level further proves that the strain is different from the existing strain published by the olive bacillus, so that the endophytic olive bacillus (Olivibacter endophytica) IMB007 provided by the invention represents a new species of the olive bacillus, and the detection of endoglucanase activity also proves that the strain, namely the endophytic olive bacillus (Olivibacter endophytica) IMB007, has cellulose degradation activity. The strain has wide application prospect in the aspects of food processing, garbage disposal, soil restoration, soil improvement and the like.
Preservation description
Classification naming: olive bacillus endophytes (Olivibacter endophytica);
biological materials according to: IMB007;
preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection);
the preservation organization is abbreviated as: CGMCC;
address: beijing, chaoyang area, north Chenxi Lu No.1, 3;
preservation date: 2022, 7, 11;
accession numbers of the preservation center: CGMCC No.25273.
Drawings
FIG. 1 is a photograph showing colony morphology of strain IMB007 cultured at 28℃for 4 days in PYG medium.
FIG. 2 is a diagram showing the screening of cellulose degradation ability of the strain IMB007 and the control strain KCTC 12646.
FIG. 3 is an N-J phylogenetic tree constructed based on the 16S rRNA gene sequences of the strain IMB007 and various representative strains of the genus Olive.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 isolation and characterization of endophytic Olive bacillus (Olivibacter endophytica) IMB007
1. Isolation of Strain IMB007
The strain IMB007 of the invention is separated from the roots of the Chinese taxillus herb, and the sample is collected in Chuxiong in Yunnan province. The specific separation operation is as follows:
washing the collected plant sample with flowing clean water to remove impurities attached to the surface of the plant; then, ultrasonic (40 kHz, 3 min/time) cleaning is carried out for 3 times, so as to further remove impurity particles tightly combined with plants; placing the cleaned plant sample in a ventilated shade place to evaporate and dry the surface moisture; the dried plants were treated with reference to surface disinfection methods employed for mangrove endophyte isolation (Wei Yuzhen, zhang Yuqin, zhao Lili, etc. isolation, screening, and preliminary identification of mangrove endophytes in Guangxi mountain and forest [ J ]. Microbiological notification, 2010 (6): 6.). Placing the sterilized plant sample into a sterile culture dish paved with filter paper, drying in a sterile ventilation environment, and crushing plant tissues for standby use by a crusher.
Strain isolation medium: the components of the isolation medium were as follows: yeast powder 0.25g/L, K 2 HPO 4 0.5g/L, 0.38g/L of marine trace salt, trace vitamin complex, 15g/L of agar, pH of 7-8 and deionized water which are complemented by 1L.
The separation method comprises the following steps: uniformly dispersing and fixing the treated plant tissue fragments on a separation culture medium, and culturing for 3 weeks at 28 ℃. Picking single colony with good growth and different morphological characteristics, and culturing in PYG agar solid culture medium (formula: peptone 3 g.L) -1 Yeast extract 5 g.L -1 Glycerol 10 g.L -1 Betaine 1.25 g.L -1 Sodium pyruvate 1.25 g.L -1 Agar 15 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the pH 7) plates were repeatedly subjected to purification culture to obtain pure cultures for subsequent studies. Meanwhile, the obtained pure strain is preserved at ultralow temperature of liquid nitrogen and frozen at-80 ℃ by taking 20% (v/v) glycerol as a protective agent. Obtaining a strain of the control bacterium Olivibacter ginsengisoli KCTC12646 with the nearest edge T The potential new species with a similarity of 97.9% was strain number IMB007.
2. Identification of Strain IMB007
Strain IMB007 was isolated from PYG (formulation: peptone 3 g.L at 28 ℃ C.) -1 Yeast extract 5 g.L -1 Glycerol 10 g.L -1 Betaine 1.25 g.L -1 Sodium pyruvate 1.25 g.L -1 Agar 15 g.L -1 The method comprises the steps of carrying out a first treatment on the surface of the The balance of deionized water is 1L; pH 7) medium for 4d, and carrying out morphological, physiological and biochemical, cytochemical and gene level studies, other special cases will be described.
1. Physiological and biochemical feature detection of IMB007 strain
Strain IMB007 was cultured on PYG solid medium at 28℃for 4-7 days and colony morphology was observed using a full automatic colony counter (flash) and a full-body microscope (OLYMPUS SZX 7). The growth temperature detection ranges are 4, 10, 15, 20, 25, 28, 30, 37, 42 and 45 ℃; 12 (0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10 and 15) concentration gradients ranging from 0-10% and 15% (0-10 g/100mL and 15g/100 mL) of growth salt concentration (NaCl); the growth pH detection range was 7 (4, 5, 6, 7, 8, 9, 10) gradients between 4 and 10 (Xu P, li WJ, tang SK, zhang YQ, chen GZ, et al Naxibacter alkilitolians gen. Nov., sp. Nov., a novel member of the family Oxalobacteraceae isolated from China. Int J Syst Evol Microbiol 2005; 55:1149-1153). The physiological and biochemical functions of the strain were performed using the detection kit API 50CH, API ZYM, manufactured by Meriera, france, and the detection system GEN III, manufactured by BiOLOG, USA. Other strain physiological characteristics, including gram stain properties, motility, oxygen demand, indole production, H2S production and cellulolytic activity, etc., are mainly described in the actinomycetes systems identification handbook (Xu L H. Actinomycete systems: principles, methods and practices [ M ]. Beijing: science Press,2007,93-108.).
The identification result shows that the strain IMB007 is a gram-negative aerobic heterotrophic bacterium, has no sporulation, does not move, is spherical, has the cell diameter of (0.2-0.3) mu m, and has yellow-white color, moist and smooth surface and complete edge, and is shown in figure 1. After the strain IMB007 is cultured on PYG solid medium at 28 ℃ for 5 days, the growth tolerance range of the strain is 10-40 ℃, 0-3% NaCl and pH 6-8.0, and the optimal growth condition is 28 ℃, 0% NaCl and pH7.0. Alkaline phosphatase, esterase C4, lipid esterase C8, leucine arylamidase, valine arylamidase, cystine arylaminase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, alpha-galactosidase, beta-uronic acid glycosidase, alpha-glucosidase, beta-glucosidase, N-acetyl-glucosamine enzyme and alpha-mannosidase production were positive. The physiological and biochemical characteristics of strain IMB007 are shown in Table 1 as compared to the most recently derived strain of olive genus.
TABLE 1 strain IMB007 T Physiological and biochemical characteristic difference table of its kindred strain
Note that: positive/growth; negative/no growth; w, weak positive.
As can be seen from the results shown in Table 1, the strain IMB007 of the present invention was different from the reported near-source strain in terms of physiological and biochemical characteristics.
2. Cytochemical characterization of strain IMB007
The cell chemistry components of the strain IMB007, such as respiratory quinone type, polar lipids, fatty acids, etc., were detected by HPLC liquid chromatography, TLC thin layer chromatography, and GC gas chromatography techniques (Minnikin DE, O' Donnell AG, goodfeldow M, alderson G, athalyE M et al an integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids.J Microbiol Methods 1984;2:233-241.Sasser M.Identification of bacteria by gas ghromatography of cellular fatty acids,MIDI Technical Note 101.Newark,DE:MIDI inc;1990.).
The results show that: in the strain IMB007 of the present invention, MK-9 (H 4 ) Is the major respiratory quinone type in the respiratory chain, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), biphospholipid glycerol (dpp), phosphatidylinositol (PI) and phosphatidylinositol mannosides (PIMs) are the major polar lipid components. Comparing the inventive strain IMB007 with the proximal strain Olivibacter ginsengisoli KCTC12646 T The fatty acid composition of (2) is shown in Table 2. As can be seen, the main fatty acid of the strain IMB007 of the present invention is Sum Feature 3 (C 16:1 ω7c/C 16:1 Omega 6 c) in an amount of 46.5% of the total content, this major component being present in combination with the proximal strain Olivibacter ginsengisoli KCTC12646 T Is consistent in fatty acid content. However, the strain IMB007 of the present invention has a different identity from other closely related species, such as iso-C 16:0 3OH and C 16:0 2OH, etc. are unique components thereof, and as shown in Table 2, the content of many fatty acid components of the strain IMB007 is also different from other closely related species, so the strain IMB007 of the present invention is a novel species of Olive genus.
TABLE 2 strain IMB007 T Lipid of related strainFatty acid component (%)
| Fatty acid component [ ]>1％) | IMB007 T | KCTC 12646 T |
| C 16:0 | 1.9 | 1.7 |
| iso-C 15:0 | 35.5 | 39.0 |
| iso-C 15:0 3OH | 3.2 | 2.4 |
| iso-C 17:0 3OH | 1.7 | 3.2 |
| iso-C 16:0 3OH | 1.7 | Tr |
| C 16:0 2OH | 1.0 | Tr |
| Sum Feature 3 | 46.5 | 39.3 |
| Sum Feature 9 | 3.3 | 3.7 |
Note that: sum features 3, C 16:1 ω7c/C 16:1 ω6c；Sum Feature 9,iso-C 17:1 Omega 9c; tr, trace.
3. Endoglucanase Activity detection of Strain IMB007
Endoglucanase-producing activity of the strain IMB007 of the present invention was measured using CMC-Na screening plate method. Preparing CMC-Na screening culture medium, which comprises the following specific components: (NH 4) 2SO 4g/L, naCl 0.1g/L, mgSO4.7H2O 0.1g/L, caCl2 0.1g/L, yeast extract 0.5g/L, fe (III) EDTA 0.033g/L, sodium carboxymethylcellulose 2g/L, agar 15g/L. pH7.0. Inoculating the strain in logarithmic growth phase on CMC-Na screening culture medium, and culturing at 28deg.C. After the completion of the culture, 6mL of Congo red staining solution (1 mg/mL) was added to the plate, and the plate and colonies were completely covered. After dyeing for 10-15min, the dyeing solution is poured off, treated with 10mL of NaCl solution (1 mol/L) for 30min and poured off. The colony was observed for a clear hydrolytic circle around it against a white background. If there is a hydrolysis circle, the endoglucanase is positive, otherwise the endoglucanase is negative.
After 7 days of incubation at 28℃in CMC-Na medium, a distinct hydrolytic circle was visible around the experimental strain IMB007 and the reference strain KCTC12646 after staining with Congo red and decolorizing with NaCl solution, indicating that both strains had distinct endoglucanase-producing activity (FIG. 2). The genes related to endoglucanase degradation were also further retrieved from the genome annotation results of strain IMB007 and the nearest strain KCTC12646 (table 3). 6 cellulose degrading enzymes were retrieved in total in strain IMB007, 3 of which encoded the beta-glucosidase and 3 of which encoded the endoglucanase; 12 cellulose degrading enzymes were retrieved in total in the strain KCTC12646 genome, 5 of which code for the beta-glucosidase and 7 for endoglucanases, see Table 3. Taken together, both phenotypically and genotypically, the strain of the invention, IMB007, was shown to have endoglucanase-producing activity.
TABLE 3 endoglucanase genes in the genome of strain IMB007 and its related strains
4. Determination of the phylogenetic status of strains
Genomic DNA of the strain IMB007 of the present invention was extracted and sequenced, and the 16S rRNA gene sequence (SEQ ID No. 1) therein was aligned on-line in the International authoritative bacterial taxonomic analysis database (http:// www.ezbiocloud.net /) (KimOS, cho YJ, lee K, et al 2012, introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species.Int J Syst Evol Microbiol,62:716-721). The results show that the 16S rRNA gene sequence of the strain IMB007 of the invention has the highest similarity with the species of the genus Olive, wherein the species with the highest similarity in pairs of sequences represents the strain Olivibacter ginsengisoli KCTC12646 T Similarity (97.9%), which is significantly below the defined threshold of 98.7% for different species of bacteria, was initially deduced to be a new species of olive bacillus. 16S rRNA gene sequences of representative strains of all species of Olive genus were selected to construct phylogenetic tree (FIG. 3).
To further clarify the phylogenetic status of strain IMB007, the average nucleotide similarity (ANI value) of the whole genome sequence of strain IMB007 and the whole genome sequence of the closely related control strain was compared and calculated on Ezbiocloud, and strain IMB007 and closely related control strain Olivibacter ginsengisoli KCTC12646 were calculated T Is less than the ANI value (95%) of the two divided gene species (Yoon SH, ha SM, lim J, kwon S, chun J.A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie van Leeuwenhoek 2017; 110:1281-1286.). And then the physiological and biochemical characteristics and chemical characteristics are combined, and the strain IMB0 of the invention07 is identified as a new species of the genus olivetobacter.
In conclusion, the strain IMB007 of the present invention has a number of significant differences from the existing olive bacillus strains in terms of phenotype, physiological biochemistry, cytochemistry, etc. Meanwhile, the phylogenetic analysis based on the gene level further illustrates the difference between the strain and each species of the existing olive bacillus, and fully proves that the strain IMB007 of the invention represents a new species of the genus olive bacillus and is named as endophytic olive bacillus (Olivibacter endophytica). Meanwhile, through the detection of endoglucanase activity, the strain provided by the invention has cellulose degradation activity, and has wide application prospects in the aspects of food processing, garbage treatment, soil remediation, soil improvement and the like.
The endophytic olive bacillus (Olivibacter endophytica) IMB007 has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.25273 in the 7 th month 11 year 2022.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. The registration number of the endophytic olive bacillus (Olivibacter endophytica) IMB007 in the China general microbiological culture Collection center is CGMCC No.25273.
2. A culture of the endophytic olivetobacter (Olivibacter endophytica) IMB007 of claim 1, which is obtained by culturing the endophytic olivetobacter (Olivibacter endophytica) IMB007 of claim 1 in a bacterial medium.
3. A metabolite of the endophytic oliver (Olivibacter endophytica) IMB007 of claim 1.
4. The microbial inoculum is characterized in that: the microbial inoculum comprises the endophytic olive bacillus (Olivibacter endophytica) IMB007 of claim 1, the culture of claim 2 and/or the metabolite of claim 3.
5. The microbial agent of claim 4, wherein: the microbial inoculum is a microbial inoculum for degrading cellulose.
6. Use of the endophytic olivetobacter (Olivibacter endophytica) IMB007 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial agent of claim 4 or 5 in any of the following:
(A1) Degrading cellulose;
(A2) Preparing a product for degrading cellulose;
(A3) Preparing cellulase;
(A4) Preparing a product with cellulase activity;
(A5) A product having endoglucanase activity is prepared.
7. Use of the endophytic olivetobacter (Olivibacter endophytica) IMB007 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial agent of claim 4 or 5 in any of the following:
(B1) Processing food;
(B2) Waste treatment;
(B3) Repairing soil;
(B4) Soil quality improvement.
8. A product for degrading cellulose, the active ingredient of which is the endophytic olivetobacter (Olivibacter endophytica) IMB007 of the plant of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial agent of claim 4 or 5;
or (b)
A product having cellulase activity or endoglucanase activity, the active ingredient of which is the endophytic olivetobacter (Olivibacter endophytica) IMB007 of the plant of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial agent of claim 4 or 5.
9. A method of degrading cellulose comprising the steps of: treatment of a sample to be treated with an endophytic olive bacillus (Olivibacter endophytica) IMB007 of claim 1 or a culture of claim 2 or a metabolite of claim 3 or a microbial inoculum of claim 4 or 5.
10. Use of endophytic olivetobacter (Olivibacter endophytica) IMB007 according to claim 1 for the preparation of a culture according to claim 2 or a metabolite according to claim 3 or a microbial agent according to claim 4 or 5.
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