Aeromicrobium and its applications

Abstract

The present invention discloses an Aeromicrobium and its application. The present invention provides Aeromicrobium sp. E6 with a preservation number of CGMCC No. 29811. The application of the above Aeromicrobium or its bacterial suspension or its culture solution in at least one of the following 1)-3) is also within the scope of protection of the present invention: 1) preparing amylase; 2) hydrolyzing starch; 3) preparing lipase; 4) hydrolyzing fat; 5) preparing β-glucosidase; 6) hydrolyzing aesculin. The experiments of the present invention have proved that the strain E6 discovered by the present invention is a new species of Aeromicrobium sp., which has the functions of hydrolyzing starch, hydrolyzing fat and hydrolyzing aesculin.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an Aeromicrobium and its application. Background Art

[0002] There are currently 23 valid species in the genus Aeromicrobium. Amylase is a general term for enzymes that hydrolyze starch and glycogen and is an important carbohydrate-hydrolyzing enzyme. Lipase can catalyze reactions such as lipolysis, transesterification, and ester synthesis. Cellulase is a class of enzymes that can hydrolyze cellulose into cellobiose and glucose. However, during the hydrolysis process of cellulase, a large amount of cellobiose and oligosaccharides produced will strongly inhibit the activity of cellulase, resulting in a sharp decline in the efficiency of cellulose hydrolysis. β-Glucosidase can efficiently catalyze the conversion of cellobiose and oligosaccharides into glucose, relieve product inhibition, and thus ensure the continuity and stability of the entire hydrolysis process. Summary of the Invention

[0003] One object of the present invention is to provide Aeromicrobium sp. E6.

[0004] The Aeromicrobium sp. E6 provided by the present invention has a deposit number of CGMCC No. 29811.

[0005] The application of the above-mentioned Aeromicrobium or its bacterial suspension or its culture solution in at least one of the following is also within the scope of protection of the present invention:

[0006] 1) Preparation of amylase;

[0007] 2) Hydrolysis of starch;

[0008] 3) Preparation of lipase;

[0009] 4) Hydrolysis of fat;

[0010] 5) Preparation of β-glucosidase;

[0011] 6) Hydrolysis of esculin.

[0012] Another object of the present invention is to provide a product.

[0013] The product provided by the present invention comprises the above-mentioned Aeromicrobium or its bacterial suspension or its culture solution.

[0014] The above product has at least one of the following functions 1)-3):

[0015] 1) Hydrolysis of starch;

[0016] 2) Hydrolysis of fat;

[0017] 3) Hydrolysis of esculin.

[0018] Another object of the present invention is to provide the following method.

[0019] The present invention provides a method for hydrolyzing starch, comprising the following steps: hydrolyzing a test sample containing starch under the action of the above-mentioned termite fungus or its bacterial suspension or its culture solution or its fermentation product. In the embodiments of the present invention, soluble starch is used as the test sample containing starch.

[0020] Or, a method for hydrolyzing fat, comprising the following steps: hydrolyzing a test sample containing fat under the action of the above-mentioned termite fungus or its bacterial suspension or its culture solution or its fermentation product. In the embodiments of the present invention, a fat source (a mixture of 2% polyvinyl alcohol aqueous solution and glyceryl tributyrate in a ratio of 1:9) is used as the test sample containing fat.

[0021] Or, a method for hydrolyzing aesculin, comprising the following steps: hydrolyzing a test sample containing aesculin under the action of the above-mentioned termite fungus or its bacterial suspension or its culture solution or its fermentation product. In the embodiments of the present invention, aesculin is used as the test sample containing aesculin.

[0022] The experiments of the present invention prove that the present invention discovers that strain E6 is a new species of the genus Aeromicrobium sp., which has the functions of hydrolyzing starch, hydrolyzing fat and hydrolyzing aesculin.

[0023] Depository Instructions

[0024] Species name: Aeromicrobium

[0025] Latin name: Aeromicrobium sp.

[0026] Strain number: E6

[0027] Depository institution: China General Microbiological Culture Collection Center

[0028] Abbreviation of depository institution: CGMCC

[0029] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences

[0030] Date of deposit: January 30, 2024

[0031] Registration number in the depository center: CGMCC No. 29811 Description of the drawings

[0032] Figure 1 It is a phylogenetic tree of E6 and valid species of the same genus Aeromicrobium.

[0033] Figure 2 Phylogenetic trees of E6, Aeromicrobium, NCBI and patent strains Detailed implementation manners

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods

[0035] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels

[0036] Example 1: Isolation and identification of Aeromicrobium sp. E6

[0037] I. Isolation of Aeromicrobium sp. E6

[0038] The pure strain E6 was isolated from the produced fluid sample of an oil well in Songyuan City, Jilin Province in August 2022

[0039] II. Identification

[0040] 1. Morphological, physiological and biochemical identification

[0041] The above-isolated strain E6 formed round, regular and moist cyan colonies on the LB solid medium (formula: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, pH 7 - 8, agar powder 1.5%). According to the "Manual for Systematic Identification of Common Bacteria", it was determined that E6 could grow weakly using sucrose or glucose under aerobic conditions and could grow weakly under anaerobic conditions

[0042] 2. 16S rDNA gene identification

[0043] The activated E6 was inoculated into the LB liquid medium (formula: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, pH 7 - 8), and cultured with shaking at 30 °C and 150 rpm for 48 h. Take 1 ml of fresh culture broth, centrifuge at 4 °C and 8000 rpm for 5 min, collect the cells in a 2 ml centrifuge tube, and extract DNA using a DNA extraction kit. After electrophoresis detection, PCR amplification was carried out using the universal primers 8F / 1492R. After electrophoresis detection of the PCR product, the 16S rDNA gene sequence was determined, and the specific sequence is shown as Sequence 1 in the sequence listing

[0044] After aligning the 16S rDNA gene sequence of the above-mentioned E6 strain using the Ezbiocloud database, it was found that this strain belongs to the genus Aeromicrobium, but has a low similarity with valid species strains, with similarities of only 98.98%, 98.91%, 98.76%, and 98.03% to Aeromicrobium tamlense, Aeromicrobium choanae, Aeromicrobium phoceense, and Aeromicrobium flavum, respectively. The relevant sequences of the genus Aeromicrobium in the Ezbiocloud database were used to construct a phylogenetic tree with the software MEGA as shown in Figure 1 , and E6 is in an independent branch on the phylogenetic tree, being significantly different from the above 4 valid species.

[0045] The relevant sequences of the genus Aeromicrobium in the Ezbiocloud database, the sequence KJ191077.1 with the highest similarity (only 99.27%) in NCBI, and the sequences in patents CN111778188A and CN104805042A were used to construct a phylogenetic tree with the software MEGA as shown in Figure 2 , and E6 is still in an independent branch on the phylogenetic tree.

[0046] Therefore, the E6 strain is a new species of a newly discovered Aeromicrobium sp.

[0047] The Aeromicrobium sp. E6 was deposited on January 30, 2024, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and the deposit registration number is CGMCC No. 29811.

[0048] Example 2. Functional study of Aeromicrobium sp. E6

[0049] 1. Detection of amylase activity of E6 CGMCC No. 29811

[0050] Starch solid medium: Add 0.2% soluble starch and 1.5% agar to the LB liquid medium, and pour solid plates after sterilization.

[0051] The activated E6 bacterial liquid was inoculated into the LB liquid medium and cultured with shaking at 30 °C and 150 rpm for 48 h to obtain a bacterial liquid (about 2×10 8 cfu / ml).

[0052] Take 5 μl of fresh bacterial liquid and spot it on the starch solid medium, then incubate it statically at 30 °C for 48 h, and add Lugol's iodine solution for color development.

[0053] Observation showed that an obvious transparent circle appeared around the E6 colony, and the ratio of the diameter of the transparent circle to that of the colony was 12 / 7 = 1.71, indicating that strain E6 had good amylase activity.

[0054] 2. Detection of lipase activity of E6 CGMCC No.29811

[0055] Solid medium: Add 2% fat source (a mixture of 2% polyvinyl alcohol aqueous solution (volume percentage) and tributyrin in a ratio of 1:9) and 1.5% agar to the LB liquid medium, and pour a solid plate after sterilization.

[0056] Inoculate the activated E6 bacterial liquid into the LB liquid medium, and incubate it with shaking at 30 °C and 150 rpm for 48 h to obtain a bacterial liquid (about 2×10 8 cfu / ml).

[0057] Take 5 μl of fresh bacterial liquid and spot it on the fat solid medium, then incubate it statically at 30 °C for 48 h.

[0058] Observation showed that an obvious transparent circle appeared around the E6 colony, and the ratio of the diameter of the transparent circle to that of the colony was 15 / 6 = 2.5, indicating that strain E6 had good lipase activity.

[0059] 3. Detection of β-glucosidase activity of E6 CGMCC No.29811

[0060] Solid medium: Add 0.05% aesculin, 0.25% ferric ammonium citrate and 1.5% agar to the LB medium, and pour a solid plate after sterilization.

[0061] Inoculate the activated E6 bacterial liquid into the LB liquid medium, and incubate it with shaking at 30 °C and 150 rpm for 48 h to obtain a bacterial liquid (about 2×10 8 cfu / ml).

[0062] Take 5 μl of fresh bacterial liquid and spot it on the above solid medium, then incubate it statically at 30 °C for 48 h.

[0063] Observation showed that an obvious black precipitate circle appeared around the E6 colony, and the ratio of the diameter of the black precipitate circle to that of the colony was 13 / 7 = 1.86, indicating that strain E6 had good β-glucosidase activity.

Claims

1. Aeromicrobium Aeromicrobium sp. )E6, whose accession number is CGMCC No.29811.

2. Use of the Aeromicrobium or its bacterial suspension or its culture solution according to claim 1 in at least one of the following: 1) preparing amylase; 2) Hydrolyzed starch; 3) preparing lipase; 4) Hydrolyze fat; 5) preparing β-glucosidase; 6) Hydrolyze esculin.

3. A product comprising the Aeromicrobium or its bacterial suspension or culture solution as described in claim 1.

4. The product according to claim 3, characterized in that: The product has at least one of the following functions 1)-3): 1) Hydrolyzed starch; 2) Hydrolyze fat; 3) Hydrolyze esculin.

5. A method for hydrolyzing starch, comprising the following steps: subjecting a sample to be tested containing starch to hydrolyze under the action of the Aeromicrobium or its bacterial suspension or its culture solution as claimed in claim 1.

6. A method for hydrolyzing fat, comprising the following steps: subjecting a sample containing fat to be tested to hydrolyze under the action of the Aeromicrobium or its bacterial suspension or culture solution as claimed in claim 1.

7. A method for hydrolyzing aesculin, comprising the following steps: subjecting a sample to be tested containing aesculin to hydrolysis under the action of the Aeromicrobium or its bacterial suspension or culture solution as claimed in claim 1.

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