CN117568224A - Salt-tolerant alkali-resistant enzyme-producing bacterium and application thereof - Google Patents

Abstract

The invention aims to provide a salt-tolerant alkali-tolerant enzyme-producing bacterium and application thereof. The provided strain is named as W13, and the preservation number of the China general microbiological culture Collection center is CGMCC No.28520. The strain can produce lipase and beta-glucosidase under high salt and high alkali, can be heterotrophically nitrified, is a new functional species of the Niston Lian Keshi strain, and has wide application prospects in the aspects of environmental management and the like.

Description

Salt-tolerant alkali-resistant enzyme-producing bacterium and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a salt-tolerant alkali-tolerant enzyme-producing bacterium and application thereof.

Background

The lipase can catalyze reactions such as lipolytic, transesterification and ester synthesis, is an important industrial enzyme preparation variety, and is widely applied to industries such as environmental protection, feed, daily chemicals and the like. Cellulases are a class of enzymes that hydrolyze cellulose to glucose oligomers and glucose, and many microorganisms in nature contain cellulases. As the cellulase can be widely applied to industries such as textile, papermaking, food, biofuel and the like. However, during the hydrolysis of cellulases, the large amounts of cellobiose and oligosaccharides produced strongly inhibit the activity of cellulases, resulting in a drastic decrease in the efficiency of cellulose hydrolysis. Beta-glucosidase can efficiently catalyze cellobiose and oligosaccharides to convert into glucose, and can remove product inhibition, so that the whole hydrolysis process is ensured to be continuous and stable. Ammonia nitrogen can consume a large amount of dissolved oxygen in the water body, so that the water body is anoxic, and water eutrophication can be caused. The traditional nitrifying bacteria are autotrophic bacteria, the required growth environment is strict, the growth speed is low, and the nitrifying bacteria are difficult to cultivate in a large quantity, so that the application is limited. Heterotrophic nitrifying bacteria can directly utilize organic matter nutrition in water body to grow and simultaneously degrade ammonia nitrogen, and has strong adaptability and high growth speed, and COD and ammonia nitrogen can be removed simultaneously.

Disclosure of Invention

It is an object of the present invention to provide a strain of nigella (nestenkonia sp.) W13.

The invention provides a Fresnel Lian Keshi bacterium (Nesterenkonia sp.) W13, and the preservation number is CGMCC No.28520.

The use of the above-described fresnel Lian Keshi bacterium or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof in at least one of the following is also within the scope of the present invention:

1) The application in producing lipase or the application in preparing lipase products;

2) The application in the production of beta-glucosidase or the application in the preparation of products for producing beta-glucosidase;

3) The application in degrading ammonia nitrogen or in preparing a product for degrading ammonia nitrogen.

It is another object of the invention to provide a product.

The product provided by the invention comprises the above-mentioned Fresnel Lian Keshi bacterium or a bacterium suspension thereof or a culture solution thereof or a fermentation product thereof.

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

1) The use of said product in the production of lipases;

2) The use of said product in the production of beta-glucosidase;

3) The product is applied to degrading ammonia nitrogen.

The culture of the above-mentioned Nidset Lian Keshi strain W13 also falls within the scope of the present invention. The culture of the strain of the genus Nicotiana Lian Keshi, W13, is obtained by culturing the strain of the genus Nicotiana Lian Keshi, W13 in a microorganism culture medium (e.g., a fermentation broth containing the strain of the genus Nicotiana Lian Keshi, W13 secreted into a liquid culture medium, or a solid culture medium containing the strain of the genus Nicotiana Lian Keshi, W13).

The culture of the above-mentioned Nidst Lian Keshi strain W13 has at least one of the following functions 1) to 3):

1) Producing lipase;

2) Producing beta-glucosidase;

3) Degrading ammonia nitrogen.

Experiments prove that the strain provided by the invention is named W13, and the preservation number of the China general microbiological culture Collection center (CGMCC) is CGMCC No.28520. The strain can produce lipase and beta-glucosidase under high salt and high alkali, can be heterotrophically nitrified, and is a new functional species of the Niestre Lian Keshi strain.

Preservation description

Strain name: nidset Lian Keshi bacterium

Latin name: nesterenkonia sp.

Strain number: w13

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, the region of Chaoyang, north Chen Xili, no. 1, 3, china academy of sciences microbiological institute

Preservation date: 2023 9.22 days

Accession numbers of the preservation center: CGMCC No.28520

Drawings

FIG. 1 is a W13 phylogenetic tree.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1, isolation and characterization of Nesterenkonia sp.W13

1. Isolation of Nesterenkonia sp.W13

And 9 months in 2020, separating the pure strain W13 from the Zhangjia saline-alkali soil sample.

2. Authentication

1. Morphological and physiological biochemical identification

The isolated W13 strain 2216E was subjected to solid medium to form fine bluish white colonies. The activated W13 is inoculated into a liquid culture medium with 5g/L peptone, 1g/L yeast powder and 25g/L, pH sodium chloride of which the values are adjusted to 9-10, and the culture medium is subjected to shaking culture at 30 ℃ for 24 hours at 150rpm to obtain a subsequent fresh seed bacterial liquid. pH values of liquid culture mediums of 5g/L peptone, 1g/L yeast powder and 25g/L sodium chloride are respectively adjusted to 7, 8, 9, 10, 11 and 12, 1% (v/v) of fresh bacterial liquid of W13 is respectively inoculated, shake culture is carried out at 30 ℃ and 150rpm, W13 grows at pH values of 8-10, and the optimal pH value is 9. pH values of liquid culture mediums of 5g/L peptone and 1g/L yeast powder are respectively adjusted to 9, sodium chloride with final concentrations of 0, 2.5, 5, 7.5, 10, 15 and 20 percent are respectively added, 1% (v/v) of fresh bacterial liquid of W13 is respectively inoculated, shaking culture is carried out at 30 ℃ and 150rpm, and W13 grows in 0-15 percent sodium chloride.

2. 16S rDNA Gene identification

The activated W13 is inoculated into a liquid culture medium with 5g/L peptone, 1g/L yeast powder and 25g/L, pH sodium chloride of which the values are adjusted to 9-10, and the culture is carried out at 30 ℃ for 24 hours under shaking at 150 rpm. 1ml of fresh culture bacterial liquid is taken and centrifuged for 5min at the temperature of 4 ℃ and at the speed of 8000rpm, bacterial cells are collected in a 2ml centrifuge tube, and DNA is extracted by adopting a DNA extraction kit. After electrophoresis detection, PCR amplification was performed using the universal primer 8F/1492R. After the PCR product is electrophoretically detected, the gene sequence of the 16S rDNA is determined, and the specific sequence is shown as a sequence 1 in a sequence table.

The 16S rDNA gene of the above W13 strain was found to belong to the genus Nesterenkonia but to have a low similarity with the existing model strain by using the Ezbiocloud database and the NCBI database. The highest similarity to the Ezbiocloud database is only 97.04% and the highest similarity to the NCBI database is only 97.12%.

The phylogenetic tree was constructed using software MEGA as shown in fig. 1, W13 in a separate branch on the phylogenetic tree using the nestenkonia related sequences in the Ezbiocloud database and the sequences disclosed in patent CN108239612A, CN109439562a as suspected new species.

Thus, the W13 strain was a new species of the New Nesterenkonia genus of the newly discovered Nesterte Lian Keshi strain.

The strain of Nesterenkonia sp.Africa Lian Keshi (Nesterenkonia sp.) W13 was deposited at the China General Microbiological Culture Center (CGMCC) with accession number CGMCC No.28520, which was deposited at the North Chen West Lu No. 1, 3, the university of China center of science, which was the Korean area of Beijing, at day 9 and 22.

Example 2 Lipase Activity of W13 CGMCC No.28520 Strain

Solid medium: adding 2% fat source (2% polyvinyl alcohol solution and tributyrin mixed according to 1:9) and 1.5% agar into liquid culture medium with peptone 5g/L, yeast powder 1g/L and sodium chloride 25g/L, pH adjusted to 9-10, sterilizing, and pouring into solid plate.

Inoculating the strain W13 CGMCC No.28520 obtained in example 1 into liquid culture medium with peptone 5g/L, yeast powder 1g/L, and sodium chloride 25g/L, pH adjusted to 9-10, shake culturing at 30deg.C and 150rpm for 24 hr to obtain W13 bacterial liquid (CFU content of 10) ⁹ And/ml).

Mu.l of the fresh W13 bacteria solution was inoculated onto the solid medium and subjected to stationary culture at 30℃for 48 hours.

The results show that: apparent transparent circles appear around the colony of W13, the circle diameter ratio of the transparent circles to the colony is 12/4=3, which indicates that the strain W13 has better lipase activity.

EXAMPLE 3 beta-glucosidase Activity of W13 CGMCC No.28520 Strain

Solid medium: adding esculin 0.05%, ferric ammonium citrate 0.25% and agar 1.5% into liquid culture medium with peptone 5g/L, yeast powder 1g/L and sodium chloride 25g/L, pH adjusted to 9-10, sterilizing, and pouring into solid plate.

Inoculating the strain W13 CGMCC No.28520 obtained in example 1 into liquid culture medium with peptone 5g/L, yeast powder 1g/L, and sodium chloride 25g/L, pH adjusted to 9-10, shake culturing at 30deg.C and 150rpm for 24 hr to obtain W13 bacterial liquid (CFU content of 10) ⁹ And/ml).

Mu.l of the fresh W13 bacteria solution was inoculated onto the solid medium and subjected to stationary culture at 30℃for 48 hours.

The results show that: obvious black precipitation circles appear around the colony of the strain W13, and the circle diameter ratio of the black precipitation circles to the colony is 13/7=1.86, which indicates that the strain W13 has better beta-glucosidase activity.

Example 4 heterotrophic nitrification function detection of W13 CGMCC No.28520 Strain

The formula of the heterotrophic nitrification medium of the high-salt high-alkali liquid comprises the following components: (NH) ₄ ) ₂ SO ₄ 0.5g/L, sodium acetate 2g/L, K ₂ HPO ₄ ·3H ₂ O3.27g/L、KH ₂ PO ₄ 0.3g/L, naCI g/L, pH 9.

Inoculating the strain W13 CGMCC No.28520 obtained in example 1 into liquid culture medium with peptone 5g/L, yeast powder 1g/L, and sodium chloride 25g/L, pH adjusted to 9-10, shake culturing at 30deg.C and 150rpm for 24 hr to obtain W13 bacterial liquid (CFU content of 10) ⁹ And/ml).

Taking 1000 μl of the fresh W13 bacteria liquid, centrifuging at 4deg.C and 8000rpm for 5min, collecting thallus, and suspending with equal volume of sterile water to obtain W13 bacteria suspension (CFU content 10) ⁹ And/ml).

Then, the W13 bacterial suspension was inoculated in a liquid heterotrophic nitrification medium at 1% (volume percentage) and cultured with shaking at 30℃and 150rpm for 7d, and the W13 culture medium was collected.

The culture medium was used as a control without inoculating the W13 bacterial suspension.

Determination of Ammonia nitrogen in Water quality by Standard "HJ 536-2009" detection by salicylic acid spectrophotometry "the ammonia nitrogen content in the W13 culture solution and the control culture solution was 57.57mg/L and 100.6mg/L, respectively.

Ammonia nitrogen removal = (ammonia nitrogen concentration in non-inoculated control culture solution-ammonia nitrogen concentration in inoculated W13 culture solution)/ammonia nitrogen concentration in non-inoculated control culture solution × 100%

Results: the ammonia nitrogen removal rate in the W13 culture solution is 42.77%, which shows that the strain W13 has heterotrophic nitrification function.

Claims (5)

1. The preservation number of the Fresnel Lian Keshi bacteria W13 is CGMCC No.28520.

2. Use of the bacterium of the genus fresnel Lian Keshi according to claim 1, in any of the following:

1) Use of a bacterium of the genus nies Lian Keshi W13 for producing a lipase or for preparing a lipase-producing product;

2) Application of Nidset Lian Keshi strain W13 in the production of beta-glucosidase or in the preparation of beta-glucosidase production products;

3) Application of Ninst Lian Keshi bacteria W13 in degrading ammonia nitrogen or in preparing ammonia nitrogen degrading products.

3. A product, characterized in that: a method of producing a bacterium comprising the step of producing a bacterium W13 containing the Fresnel Lian Keshi bacterium of claim 1.

4. Use of the product of claim 3 for any of the following applications:

1) The use of said product in the production of lipases;

2) The use of said product in the production of beta-glucosidase;

3) The product is applied to degrading ammonia nitrogen.

5. A culture of the bacterium fresnel Lian Keshi according to claim 1, characterised in that: the culture has the functions of any one of the following:

1) Producing lipase;

2) Producing beta-glucosidase;

3) Degrading ammonia nitrogen.