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Flavobacterium and application thereof

A technology of Flavobacterium and bacterial liquid, which is applied in the directions of bacteria, microorganisms, biochemical equipment and methods, etc., can solve the problems that the enzyme production capacity has not yet reached the requirements of commercialization, and there is no enzyme, and achieves good market promotion prospects and enzyme production activity. high effect

Inactive Publication Date: 2013-04-17
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there have been some reports on the research on fucoidan sulfatase at home and abroad, but there is no commercial enzyme, mainly because the enzyme production ability of these microorganisms has not yet met the requirements of commercialization. It is an important way to solve the application problem of fucoidan sulfate

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0018] 2. Preparation of Flavobacterium Bacterial Liquid

[0019] Flavobacterium RC2-3 with the preservation number CGMCC No.6932 was inoculated in liquid medium (Fucoidan 0.2%, peptone 1%, ammonium nitrate 5mg / mL, prepared with membrane seawater, pH natural), 25°C, 180rpm Cultivate under the conditions for 12 hours to obtain the seed culture liquid, inoculate the seed culture liquid into a fresh liquid medium according to the inoculum amount of 5%, and cultivate at 25°C and 150 rpm for 72 hours to obtain the Flavobacterium bacterial liquid.

[0020] 3. Production of Flavobacterium intracellular enzymes

[0021] 1. Preparation of intracellular enzymes from Flavobacterium zymogenes

[0022] Centrifuge the bacterium liquid of Flavobacterium RC2-3 at 4°C and 10000g for 5-10 minutes, discard the supernatant, take the precipitate, dissolve the precipitate with a small amount of 20mM Tris-HCl, pH8.0, and carry out the process under the condition of 500w under the protection of ice ...

Embodiment 1

[0025] The intracellular enzyme was mixed with equal volumes of 0.2% Fucoidan (pH 8.0, dissolved in 20mM Tris-HCl), shaken at 25°C and 120rpm for 2 hours, and the reducing sugar content was determined by the Somogyi-Nelson method. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 nanomol of reducing sugar per hour at 25°C and pH 8.0. It was determined that the enzyme production activity of Flavobacterium reached 180U / mL after being cultured for 72 hours.

Embodiment 2

[0027] The enzyme-producing bacteria were inoculated in liquid medium (Fucoidan 0.2%, ammonium nitrate 0.2%, prepared with membrane-passed seawater, with natural pH), cultured at 25°C and 150 rpm for 72 hours, and 1 mL was sampled under sterile conditions every 24 hours. Inactivate the enzyme at 80°C for 15 minutes, and centrifuge at 10,000 rpm for 10 minutes to obtain the supernatant. Use high performance gel size exclusion chromatography (chromatographic conditions are as follows: Agilent 1100 high performance liquid chromatography; TSK-gel G3000 PW×1 (30cm×7.5mm) chromatographic column; 40°C column temperature; 0.2M NaCl as mobile phase; 0.5mL / min flow rate; differential detector) to detect changes in the molecular weight of polysaccharides. The results showed that after culturing for 0h, 24h, 48h, and 72h, the content of Fucoidan with a retention time of 13.6min was 100%, 75%, 33%, and 28%, respectively. It is proved that the bacterial strain screened by the present inve...

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Abstract

The invention relates to flavobacterium and application thereof. The preservation number of the flavobacterium is CGMCC No.6932. The flavobacterium can be used for degrading fucoidan. The flavobacterium with fucoidan enzyme production capacity has high activity in strain enzyme production capacity up to 180 U / ml, and has excellent market application foreground.

Description

technical field [0001] The invention belongs to the technical field of marine microorganisms, and in particular relates to a Flavobacterium and its application, that is, a bacterium with fucoidan sulfatase production capacity and its application. Background technique [0002] Fucoidan sulfate (Fucoidan) is a high-molecular-weight sulfated heteropolysaccharide widely present in brown algae. It is mainly composed of L-fucose and sulfate ester groups, and also contains galactose, xylose, glucose, Mannose, rhamnose, uronic acid, etc. Studies have found that it has a variety of physiological activities, including anti-coagulation, anti-tumor, anti-inflammatory, anti-thrombosis, improving immunity, anti-oxidation, anti-infection, etc. Therefore, fucoidan sulfate has become the main focus of today's natural marine active substances . However, due to the huge molecular weight of fucoidan sulfate, ranging from tens of thousands to hundreds of thousands, it seriously affects its abs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/14C12R1/20
Inventor 王莹李八方朴美子程凡升
Owner QINGDAO AGRI UNIV
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