CN118185841A - Vibrio cellulosae strain NN19 and application thereof - Google Patents
Vibrio cellulosae strain NN19 and application thereof Download PDFInfo
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- 241000607598 Vibrio Species 0.000 title claims abstract description 25
- 229920002101 Chitin Polymers 0.000 claims abstract description 48
- 230000000593 degrading effect Effects 0.000 claims abstract description 13
- 241001124860 Cellvibrio sp. Species 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 3
- 244000005700 microbiome Species 0.000 abstract description 5
- 239000001963 growth medium Substances 0.000 abstract description 4
- 239000002699 waste material Substances 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 13
- 238000000855 fermentation Methods 0.000 description 11
- 230000004151 fermentation Effects 0.000 description 11
- 230000000694 effects Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 241000863387 Cellvibrio Species 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
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- 229940088598 enzyme Drugs 0.000 description 5
- 241001266036 Cellvibrio fontiphilus Species 0.000 description 4
- 239000008186 active pharmaceutical agent Substances 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
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- 238000000034 method Methods 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
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- 210000004027 cell Anatomy 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
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- 102100035882 Catalase Human genes 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
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- 230000005526 G1 to G0 transition Effects 0.000 description 1
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- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- HLCFGWHYROZGBI-JJKGCWMISA-M Potassium gluconate Chemical compound [K+].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O HLCFGWHYROZGBI-JJKGCWMISA-M 0.000 description 1
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- BJHIKXHVCXFQLS-UYFOZJQFSA-N keto-D-fructose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of microorganisms, in particular to a vibrio fibrinolyticus strain NN19 and application thereof. The invention provides a novel chitin degrading strain, which is a novel strain of vibrio. The strain can ferment and degrade colloidal chitin in a low-cost culture medium. Provides a new high-quality strain resource for industrially degrading chitin waste.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a vibrio fibrinolyticus strain NN19 and application thereof.
Background
Chitin (Chitin), also known as Chitin, is a polymer formed by connecting N-acetyl-D-glucosamine (GlcNAc) units through beta-1, 4-glycosidic bonds. Chitin is the most abundant natural aminopolysaccharide, which is found mainly in crustacean, insect exoskeletons and fungal cell walls. Chitin exists in crystalline form in nature mainly through intramolecular hydrogen bonds. Chitin oligosaccharides, a degradation derivative of chitin, have a variety of excellent functions. However, it is extremely difficult to degrade due to its complex structure, and it is often used in industry to degrade with concentrated acids. Compared with the chemical method of hydrolyzing chitin with strong acid, the microbial method is more environment-friendly.
Chitin is the second largest renewable resource in nature except cellulose, however chitin resources in nature are not stacked in large quantities, suggesting that nature is implying abundant microorganisms that utilize chitin growth, the most important chitin degrading microorganisms being bacteria that can evolve an efficient enzymatic system to degrade intractable crystalline chitin. Chitin degrading bacteria that have been reported so far mainly include Serratia (Serratia), bacillus (Bacillus), streptomyces (Streptomyces), vibrio (Vibrio), and the like. The microorganisms have the problems of poor degradation capability, long growth period and the like. Therefore, the method screens excellent novel chitin degrading strains from the environment with rich chitin resources, and plays an important role in degrading and utilizing industrial chitin resource wastes.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a chitin degrading bacterium, vibrio fibroicus strain NN19.
The invention provides a vibrio (Cellvibrio sp.) strain NN19 which is preserved in China general microbiological culture collection center, the preservation number is CGMCC No. 29911, the 16S rDNA sequence of the strain is compared in a Ezbiocloud database, the strain with the closest similarity is Cellvibrio fontiphilus MVW-40 T, the similarity is 97.87 percent and is smaller than a new species judgment critical value of 98.65 percent, and the strain is determined to be a new species of vibrio (Cellvibrio) through the comparison of morphology, molecular biology and physiological and biochemical properties with the related mode strain.
The invention also provides an application of the vibrio fibrinolyticus (Cellvibrio sp.) strain NN199 in degrading chitin, wherein the chitin is colloidal chitin, the vibrio fibrinolyticus (Cellvibrio sp.) strain NN19 is inoculated into a fermentation culture medium taking the colloidal chitin as a unique carbon source, and the content of the colloidal chitin is 10 g/L.
The invention has the following beneficial effects: the invention provides a novel chitin degrading strain, which is a novel strain of vibrio. The strain can ferment and degrade colloidal chitin in a low-cost culture medium. Provides a new high-quality strain resource for industrially degrading chitin waste.
Drawings
FIG. 1 shows the change in chitinase activity of the fermentation supernatant of the strains screened for chitin-degrading activity;
FIG. 2 is a schematic diagram of a phylogenetic tree of strain NN 19;
FIG. 3 is a cell transmission electron microscope image of Vibrio cellulosae (Cellvibrio sp.) strain NN 19;
FIG. 4 is a growth curve of Vibrio cellulosis (Cellvibrio sp.) strain NN19 in chitin fermentation medium;
FIG. 5 shows the culture of Vibrio cellulosis (Cellvibrio sp.) strain NN19 in chitin fermentation medium.
The vibrio (Cellvibrio sp.) strain NN19 is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (address: north Star Xway No. 1,3 of the university of China, proc. Acad. Sci. Bay. In China, mail code 100101) at 2024, and is classified and named as vibrio Cellvibrio sp, with the preservation number of CGMCC No. 29911.
Detailed Description
In the following examples:
Preparation of colloidal chitin: dissolving 4g chitin powder in 40 mL concentrated hydrochloric acid, standing for 50 min, slowly adding 1L precooled distilled water overnight after all the chitin powder is dissolved, centrifuging 8000 rpm for 20min to collect precipitate, re-suspending with distilled water, centrifuging (8000 rpm,20 min), repeatedly washing until pH is 7.0, and then keeping with distilled water to constant volume of 100 mL at 4 deg.C;
The chitin liquid fermentation medium comprises ：Na2HPO4,12.8 g/L; KH2PO4 ,3 g/L;NaCL,0.5 g/L;NH4CL,1 g/L;10 g/L colloid chitin; mgSO 4 ,0.002 mol/L;CaCl2, 0.0001 mol/L, 1000X trace elements （（NH4）6（MO7）24,3×10-6 M;H3BO3,4×10-4 M;CoCl2,3×10-5 M;CuSO4,10-5M;MnCl,8×10-5 M;ZnSO4,10-5 M）.
EXAMPLE 1 chitin degrading strain isolation and identification
(1) Chitin degrading strain isolation
The seashore mud and sand was collected from fishing villages in the city of thunder, guangdong, the samples were made into bacterial suspensions in sterile water, inoculated in an inoculum size of 5% to a fermentation medium with 1% (w/v) of colloidal chitin as the sole carbon source, and subjected to enrichment culture by shaking at 180rpm for 5 days at 30 ℃. The enriched bacterial liquid is taken, diluted by 0.85 percent physiological saline in a gradient way, and then is coated into a1 percent colloidal chitin solid culture medium (2 percent agar is added into a chitin liquid fermentation medium), and is subjected to stationary culture for 5 days at 30 ℃. The strain with strong growth was picked up as a monoclonal, inoculated into chitin liquid fermentation medium, cultured for 5 days with shaking at 30 ℃, the chitinase activity of the supernatant was measured by DNS method, and one unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under the enzyme assay conditions. As shown in FIG. 1, the strain with NN19, which has the highest enzyme activity of 80U/L at 48h, is selected as the dominant strain for chitin degradation.
(2) Molecular biological identification of Strain NN19
16S rDNA sequence analysis of strain NN19 showed that it had the highest sequence similarity to model strain Cellvibrio fontiphilusMVW-40 T (97.87%), and that strain NN19 formed a single branch with C. fontiphilus MVW-40 T according to the phylogenetic tree results (FIG. 2). The NN19 strain had a genome sequence accession number JAYKKN010000000, a genome size of 4.46Mb and a DNA G+C content of 47.82 mol%. The ANI and dDDH values between strains NN19 and C. fontiphilus MVW-40 T were 75.47% and 20.4%, respectively, and lower than the ANI (95-96%) and dDDH cutoff (70%) of the species identification standard. The above results confirm that the NN19 strain is a new species of Cellvibrio.
(3) Morphological and physiological biochemical identification of strain NN19
The cellular morphology of NN19 is short rod-like cells, which move through unipolar flagellum. After 48 hours of culture on TSA medium at 30℃the colonies were pale yellow, slightly viscous, round and the average cell size was 0.5 μm in diameter and 1.2-1.4 μm in length (FIG. 3). Gram staining was negative, the strain grew at 15-37 ℃ (optimal temperature 30 ℃), pH 6.5-8.0 (optimal pH 7.0) and 0-3% NaCl (optimal concentration 0.5-1%), positive for oxidase and catalase. The strain is subjected to physiological and biochemical characteristic identification by referring to an experimental method in an eighth edition of Bojie's bacteria identification manual and a common bacteria identification manual. The carbon source utilization condition is identified by using a Biolog GEN III microplate, the substrate metabolism is identified by using an API 20NE reagent strip, and the enzyme activity experiment is measured by using an API ZYM kit. Fatty acid composition determination was analyzed by gas chromatography (Agilent Technologies 6850,680) and identified using the TSBA 6.0.0 database of the microbiological identification system. Cellvibrio fontiphilus KCTC 52237 T and Cellvibrio mixtus subsp. mixtus LMG 21544 T as reference strains. Table 1 shows the differences between strain NN19 and the reference strain. In the table, "+" indicates positive, "W" indicates weak positive, and "-" indicates negative.
Strain NN19 grows in chitin fermentation medium, and is in incubation period of 0-24h, growth log phase of 36-84h, and stationary phase of 84-120 h.
TABLE 1 physiological and biochemical property differences between strain NN19 and reference strain
| Cellvibriosp.NN19 | Cellvibrio fontiphilusKCTC 52237T | Cellvibrio mixtussubsp. mixtus LMG 21544T | |
| Temperature range (optimum temperature) (°c) | 15-37 (30) | 15–40 (20–30) | 10–37 (20–30) |
| NaCl tolerance (optimal) (%) | 0-3 (0.5-1) | 0-2 (0) | 0-4 (0-2) |
| Growth conditions of different media: | |||
| LB medium | - | + | + |
| TSA medium | + | - | - |
| NA medium | + | + | + |
| API 20 NE experimental results: | |||
| Nitrate reduction | + | + | - |
| Mannose | + | W | + |
| Potassium gluconate | W | - | W |
| API ZYM experimental results: | |||
| lipase (C14) | W | + | + |
| Cystine arylamine enzyme | W | W | + |
| Trypsin, trypsin and its preparation method | - | W | + |
| Chymotrypsin | W | - | + |
| Acid phosphatase | + | W | W |
| Alpha-galactosidase | W | + | + |
| Beta-galactosidase | - | W | W |
| Beta-uronic acid glycosidase | - | - | W |
| Alpha-glucosidase | - | + | + |
| Beta-glucosidase | W | + | + |
| Beta-fucosidase | - | - | W |
| Biolog GEN III experimental results: | |||
| d-fructose | - | + | - |
| L-rhamnose | + | + | - |
| Pectin | - | + | + |
| L-malic acid | - | + | - |
。
The main fatty acid type of the strain NN19 is Feature 3 (C 16:1ω7c/C16:1ω6c）,C18:1 ω7c and C 16:0), which is similar to other strains of Vibrio, and NN19 is proved to be the Vibrio, from Table 1, it is clear that the strain NN19 has obvious differences from two reference strains, and NN19 is a new strain of the Vibrio through comprehensive molecular biological identification analysis.
Example 2 Strain NN19 for chitin degradation
Vibrio fibrosus NN19 monoclonal was picked from the plate, inoculated into 5mL of TSA medium, cultured at 30℃for 2 days, transferred to 5mL of chitin fermentation medium at 2% inoculum size, pH was adjusted to 7.0, cultured at 30℃with shaking at 220rpm, and OD 600 of the supernatant was measured every 12 hours. As shown in FIG. 4, strain NN19 gradually grew from 36 hours to 96 hours and became stable. As shown in fig. 5, strain NN19 completely degraded colloidal chitin at 120 hours of fermentation compared to the control.
The above embodiments are only for understanding the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (2)
1. The vibrio (Cellvibrio sp.) strain NN19 is characterized in that the preservation number of the vibrio (Cellvibrio sp.) strain NN19 is CGMCC No. 29911.
2. Use of the vibrio fibrinolyticus (Cellvibrio sp.) strain NN19 as claimed in claim 1 for degrading chitin.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118185907A (en) * | 2024-05-17 | 2024-06-14 | 中国农业科学院北京畜牧兽医研究所 | Chitinase Chi18D from vibrio fibrosus, and coding gene and application thereof |
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| US20050112750A1 (en) * | 2003-06-27 | 2005-05-26 | Michael Howard | Chitin degradative systems |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1998008940A1 (en) * | 1996-08-26 | 1998-03-05 | Novo Nordisk A/S | A novel endoglucanase |
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|---|
| NYOK-SEAN LAU等: "Polysaccharide degradation in Cellvibrionaceae: Genomic insights of the novel chitin-degrading marine bacterium, strain KSP-S5-2, and its chitinolytic activity", SCI TOTAL ENVIRON, vol. 20, no. 912, 7 December 2023 (2023-12-07), pages 10 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118185907A (en) * | 2024-05-17 | 2024-06-14 | 中国农业科学院北京畜牧兽医研究所 | Chitinase Chi18D from vibrio fibrosus, and coding gene and application thereof |
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