One alternate monolithic HT1 and its culture method and application

A technology of Enteromorpha polysaccharides and lyases, which is applied in the field of microorganisms, can solve the problems of lack of industrial enzyme strains of Enteromorpha polysaccharides, and achieve the effects of easy heterologous expression and purification, high-efficiency degradation, and mild enzymolysis reaction conditions

Active Publication Date: 2022-08-05
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Enteromorpha polysaccharide lyase is mainly derived from marine bacteria, and currently there are few strains available. The only reported strains capable of producing Enteromorpha polysaccharide lyase are Alteromonas macleodii B7 and Alteromonas sp.A321. industrial enzyme strains

Method used

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  • One alternate monolithic HT1 and its culture method and application
  • One alternate monolithic HT1 and its culture method and application
  • One alternate monolithic HT1 and its culture method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Screening of Example 1 Strain

[0041] In the ultra-clean workbench, add 2 g of prolifera samples to the prepared seawater culture medium, put it into a 25°C shaker and shake for one week. Dilute the prolifera suspension to 10 -1 ~10 -5 Concentration gradient, respectively coated in TYS solid medium, cultured in a constant temperature incubator at 25°C for 48 hours, and then selected a single colony for continuous streaking until purified to obtain a single colony.

[0042] A single colony purified on TYS solid medium was picked, respectively inoculated into TYS liquid medium, and cultured in a shaker at 25°C overnight to obtain seed liquid. Take the seed liquid and inoculate it in the liquid medium with the polysaccharide as the sole carbon source at 1% of the inoculum, and culture it on a shaker for 3 days at 25° C. and 180 rpm to obtain the supernatant of the extracellular fermentation liquid of the strain. Take 1 mL of extracellular fermentation broth supernatant...

Embodiment 2

[0044] Example 2 Identification of bacterial strains

[0045] (1) Strain characteristics

[0046] Strain HT1 is a Gram-negative bacteria such as figure 1 As shown, the colonies are round, white, moist and smooth. The cells of strain HT1 are rod-shaped and have flagella (2.0-2.5 μm in length and 1.0-1.5 μm in width).

[0047] (2) Identification of strains

[0048] Use the BioTeke bacterial genome extraction kit to extract the DNA in the strains screened in Example 1. For the specific extraction method, refer to the instructions of the kit. Use 27F and 1492R primers to carry out PCR amplification of the 16S rRNA gene of the HT1 strain, and connect the amplified products. onto the pMD19-T vector (TaKaRa, China) and sequenced on an Applied Biosystems 3730xl DNA sequencer. .

[0049] PCR primers use universal primers:

[0050] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3',

[0051] 1492R: 5'-GGTTACCTTGTTACGACTTC-3',

[0052] PCR reaction conditions were: pre-denaturation at 95°C for 5 mi...

Embodiment 3

[0056] Cultivation of Example 3 Strain

[0057] The culture method of above-mentioned Altomonas sp. HT1 bacterial liquid, the steps are as follows:

[0058] (1) The Alteromonas sp. HT1 stored in the glycerol tube was streaked and inoculated in TYS solid medium, and cultured upside down for 1 day at 25°C;

[0059] (2) picking a single colony of Alteromonas HT1 from the TYS solid medium, inoculating it into the TYS liquid medium, and under the conditions of 25° C. and 180 rpm, shaking the culture overnight to obtain the seed liquid;

[0060] (3) take the seed liquid obtained in step (2), inoculate it into a liquid fermentation medium with Prolifera polysaccharide as the sole carbon source at a volume percentage of 1%, and cultivate it on a shaking table at 25° C. and a rotating speed of 200 rpm for 3 days to obtain alternating single Bacterial fluid of Bacillus HT1.

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Abstract

The present invention relates to a strain of Altomonas HT1 and its cultivation method and application. Altomonas sp. HT1 in the present invention is preserved in the China Center for Type Culture Collection on March 9, 2021, and the preservation address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO. M2021217. Alteromonas sp. HT1 can grow in the medium with Prolifera as the sole carbon source. in the form of sugar. The strain and the prolifera polysaccharide lyase secreted by the strain can be applied to the degradation of marine green algae and the preparation of functional oligo-oligosaccharides, and have good application potential in medicine, cosmetics and food industries.

Description

technical field [0001] The invention relates to a strain of Altomonas HT1, a culture method and application thereof, and belongs to the technical field of microorganisms. Background technique [0002] Enteromorpha is widely distributed in coastal areas all over the world. It belongs to the Chlorophyta phylum Chlorophyta, Ulvae, Ulvae, and is also an important algae that affects my country's summer coastal environment in recent years. Enteromorpha polysaccharide accounts for about 43.4-60.2% of the dry weight of Enteromorpha. It is a sulfated polysaccharide composed of 3-sulfated rhamnose, glucuronic acid and xylose. It has a variety of biological functions such as virus, blood lipid regulation, and blood lipid lowering functions, and has good application potential in the fields of food and medicine. However, the characteristics of high viscosity and high molecular weight of Prolifera polysaccharides seriously affect people's absorption and utilization of Prolifera polysacch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/88C12N15/60C12P19/00C12P19/12C12R1/01
CPCC12N1/20C12N9/88C12P19/00C12P19/12C12Y402/02
Inventor 张玉忠陈秀兰耿玉慧宋晓妍张熙颖于春梅赵芳
Owner SHANDONG UNIV
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