A bacterium of the genus porphyromonas and its use

Abstract

The application aims to provide a S. luteinigens and application thereof. A strain provided is named 3ca2, which is obtained by using an autotrophic culture medium with sodium nitrite as a nitrogen source and sodium carbonate as a carbon source, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.29813. The strain can produce a surfactant and beta-glucosidase, and is a functional new S. luteinigens species.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a species of *Gynostemma pentaphyllum* and its applications. Background Technology

[0002] Biosurfactants are primarily surface-active secondary metabolites produced by microorganisms. Common biosurfactants include lipopeptides and glycolipids. With the increasing emphasis on green, environmentally friendly, and sustainable development across various sectors, biosurfactants have received growing attention. Cellulase is a class of enzymes that hydrolyze cellulose into oligosaccharides and glucose; many naturally occurring microorganisms contain cellulase. Cellulase has wide applications in industries such as textiles, papermaking, food, and biofuels. However, during cellulase hydrolysis, the large amounts of cellobiose and oligosaccharides produced strongly inhibit cellulase activity, leading to a sharp decline in cellulose hydrolysis efficiency. β-glucosidase can efficiently catalyze the conversion of cellobiose and oligosaccharides into glucose, relieving product inhibition and ensuring a continuous and stable hydrolysis process. Summary of the Invention

[0003] One object of the present invention is to provide Luteimonas sp. 3ca2.

[0004] The Luteimonas sp. 3ca2 provided by this invention has the accession number CGMCC No. 29813.

[0005] The use of *Gastrodinium glabra* or its bacterial suspension or culture broth or fermentation product in at least one of the following is also within the scope of protection of this invention:

[0006] 1) Applications in the production of biosurfactants or bioemulsifiers or in the preparation of products that produce biosurfactants or bioemulsifiers;

[0007] 2) Applications in the production of β-glucosidase or in the preparation of products that produce β-glucosidase.

[0008] Another object of the present invention is to provide a product.

[0009] The product provided by the present invention includes the above-mentioned *Gynostemma pentaphyllum* or its bacterial suspension or its culture medium or its fermentation product.

[0010] The above-mentioned products have at least one of the following functions: 1)-2)

[0011] 1) The application of the product in the production of biosurfactants or bioemulsifiers;

[0012] 2) Application of the product in the production of β-glucosidase.

[0013] The culture of *Xanthomonas glaucus* 3ca2 described above also falls within the scope of protection of this invention. The culture of *Xanthomonas glaucus* 3ca2 is a substance obtained by culturing *Xanthomonas glaucus* 3ca2 in a microbial culture medium (such as fermentation broth containing *Xanthomonas glaucus* 3ca2 and substances secreted into a liquid culture medium, or substances containing *Xanthomonas glaucus* 3ca2 and substances secreted into a solid culture medium).

[0014] The above-mentioned culture of *Gastrodinium 3ca2* has at least one of the following functions: 1)-2):

[0015] 1) Production of biosurfactants or bioemulsifiers;

[0016] 2) Production of β-glucosidase.

[0017] Experiments of this invention demonstrate that the strain provided by this invention, named 3ca2, has the accession number CGMCC No. 29813 from the China General Microbiological Culture Collection Center. This bacterium is capable of producing surfactants and β-glucosidase, and is a novel species of functional *Glaucoma glabra*.

[0018] Preservation Instructions

[0019] Strain name: *Xanthomonas glaucus*

[0020] Latin name: Luteimonas sp.

[0021] Strain number: 3ca2

[0022] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0023] Collection institution abbreviation: CGMCC

[0024] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences

[0025] Deposit date: January 30, 2024

[0026] CGMCC Registration Number: CGMCC No. 29813 Attached Figure Description

[0027] Figure 1 Phylogenetic tree of effective species in the same genus as 3ca2 and Luteimonas.

[0028] Figure 2 Phylogenetic tree of 3ca2 and Luteimonas, and patented strains. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1: Isolation and identification of Luteimonas sp. 3ca2

[0032] I. Isolation of Luteimonas sp. 3ca2

[0033] In October 2023, pure strain 3ca2 was isolated from aquarium water samples using an autotrophic culture medium (formula: sodium chloride 0.3 g / L, ferrous sulfate 0.03 g / L, magnesium sulfate 0.03 g / L, potassium dihydrogen phosphate 2.18 g / L, sodium nitrite 0.5 g / L, sodium carbonate 1 g / L, pH 7.5-8.0) with sodium nitrite as the nitrogen source and sodium carbonate as the carbon source.

[0034] II. Identification

[0035] 1. Morphological and physiological biochemical identification

[0036] The isolated 3ca2 strain was able to grow on LB solid medium (formulation: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7-8, 1.5% agar powder), forming regular, round, bluish-yellow colonies. Activated 3ca2 was inoculated into LB liquid medium (formulation: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7-8) and cultured at 30°C with shaking at 150 rpm for 24 h to obtain a fresh seed culture. 10 g / L peptone, 5 g / L yeast extract, and a pH of the liquid culture medium adjusted to 7-8 were added. Sodium chloride solutions of 0%, 1%, 2%, 3%, 4%, 5%, and 6% were added respectively, and each solution was inoculated with 1% (v / v) of fresh 3ca2 bacterial culture. The cultures were incubated at 30°C with shaking at 150 rpm. 3ca2 grew in 1-2% sodium chloride solution, showed weak growth in 3% sodium chloride solution, and did not grow in 0%, 4%, 5%, and 6% sodium chloride solutions. According to the literature "Handbook of Systematic Identification of Common Bacteria," 3ca2 could not utilize glucose, arabinose, or maltose as carbon sources for growth under the conditions of 30°C and 150 rpm shaking culture.

[0037] 2. Identification of 16S rDNA gene

[0038] Activated 3ca2 cells were inoculated into LB liquid medium (formulation: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7-8) and cultured at 30°C with shaking at 150 rpm for 24 h. 1 ml of fresh culture was centrifuged at 4°C and 8000 rpm for 5 min, and the cells were collected in a 2 ml centrifuge tube. DNA was extracted using a DNA extraction kit. After electrophoresis, PCR amplification was performed using universal primers 8F / 1492R. After electrophoresis of the PCR product, the 16S rDNA gene sequence was determined; the specific sequence is shown in Sequence Listing 1.

[0039] After comparing the 16S rDNA genes of the above-mentioned 3ca2 strain with the Ezbiocloud and NCBI databases, it was found that the strain belongs to the genus Luteimonas, but has low similarity to existing strains. The strain with the highest similarity to the Ezbiocloud database, Luteimonas marina, has only 99.36% similarity, while the highest similarity sequence to the NCBI database is the Luteimonas marina type strain FR1330. T The sequence similarity was only 99.36%. Differences in sodium chloride growth concentration and carbon source utilization were observed between 3ca2 and its most similar genus, *Luteimonas marina*, as shown in Table 1, where + indicates growth or utilization, and - indicates no growth or utilization. A phylogenetic tree was constructed using MEGA software from related *Luteimonas* sequences in the Ezbiocloud database. Figure 1 3ca2 is on a separate branch in the phylogenetic tree.

[0040] Table 1. Differences between strain 3ca2 and Luteimonas marina

[0041] project 3ca2 Luteimonas marina 0% Sodium Chloride - + 2% sodium chloride + - glucose - + Arabic sugar - + maltose - +

[0042] Using the Ezbiocloud database, sequences related to the genus *Luteimonas*, as well as strain sequences from patents CN117143754A, CN115820462A, CN114574388A, CN111763631A, CN110079479A, CN108893432A, CN105255774A, CN104371941A, and CN103865820A, were used to construct a phylogenetic tree using the MEGA software. Figure 2 3ca2 remains in a separate branch on the phylogenetic tree, and is quite different from the patented strains. Its highest similarity to strain CN115820462A is only 98.07%.

[0043] Therefore, strain 3ca2 is a newly discovered species of the genus Luteimonas.

[0044] Luteimonas sp. 3ca2 was deposited on January 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 29813.

[0045] Example 2: Detection of surfactant production function in strain 3ca2 CGMCC No. 29813

[0046] The 3ca2 CGMCC No. 29813 strain obtained in Example 1 was inoculated into LB liquid medium and cultured at 30°C and 150 rpm for 72 h with shaking to obtain 3ca2 bacterial suspension. The bacterial suspension was centrifuged at 4°C and 8000 rpm for 5 min, and the supernatant was collected to obtain the supernatant culture medium.

[0047] A control culture solution was obtained by using a blank culture medium without 3ca2 bacterial culture as a control.

[0048] The emulsification index (E24) was determined according to the method described in the literature "Study on the properties of rhamnolipids in biosurfactants".

[0049] Emulsification index (E24) = (Emulsion height / Total liquid height) × 100%

[0050] The results showed that the emulsification index (E24) of the 3ca2 supernatant culture medium was 34.6%, while the emulsification index (E24) of the control culture medium was 0, indicating that strain 3ca2 has the function of producing surfactants.

[0051] Example 3: β-glucosidase activity of strain 3ca2 CGMCC No. 29813

[0052] Solid culture medium: Add 0.05% aescin, 0.25% ferric ammonium citrate and 1.5% agar to a liquid culture medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and pH adjusted to 7-8. Sterilize and pour into solid plates.

[0053] The 3ca2 CGMCC No.29813 strain obtained in Example 1 was inoculated into LB liquid medium and cultured at 30°C and 150 rpm for 24 h with shaking to obtain 3ca2 bacterial suspension (CFU content of 10). 9 (pcs / ml).

[0054] Take 5 μl of the above fresh 3ca2 bacterial culture and inoculate it onto the above solid culture medium. Incubate at 30°C for 48 h.

[0055] The results showed that a distinct black precipitate ring appeared around the 3ca2 colony, and the ratio of the black precipitate ring to the colony diameter was 12 / 8 = 1.5, indicating that strain 3ca2 has good β-glucosidase activity.

Claims

1. Luteimonas sp. 3ca2, with accession number CGMCC No. 29813.

2. Any of the following applications of *Xanthomonas glaucus* as described in claim 1: 1) Application of Luteimonas sp. 3ca2 in the production of biosurfactants or bioemulsifiers or in the preparation of products that produce biosurfactants or bioemulsifiers; 2) Application of Luteimonas sp. 3ca2 in the production of β-glucosidase or in the preparation of products that produce β-glucosidase.

3. A product, characterized in that: It contains Luteimonas sp. 3ca2 as described in claim 1.

4. Any of the following applications of the product of claim 3: 1) The application of the product in the production of biosurfactants or bioemulsifiers; 2) Application of the product in the production of β-glucosidase.

5. The culture of *Gastrodinium glabra* according to claim 1, characterized in that: The culture has the following function 1) or 2): 1) Production of biosurfactants or bioemulsifiers; 2) Production of β-glucosidase.

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