Acinetobacter juni. X8 and application thereof in preparing algin lyase

A technology of Acinetobacter angnei and alginate lyase, which is applied in the direction of lyase, bacteria, and microorganism-based methods, and can solve the problems of difficult separation, poor storage stability, and low production of alginate lyase

Inactive Publication Date: 2009-12-02
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alginate lyase has a wide range of sources, including seaweed extracts, marine molluscs, digestive gl...

Method used

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  • Acinetobacter juni. X8 and application thereof in preparing algin lyase
  • Acinetobacter juni. X8 and application thereof in preparing algin lyase
  • Acinetobacter juni. X8 and application thereof in preparing algin lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Enrichment, domestication and isolation and screening of strains

[0049] Inoculate the spoiled Hijiki leaves and bacterial suspension into a 500mL Erlenmeyer flask filled with 100mL No. 1 medium, and enrich and culture them at 30°C and 180rpm for 72 hours. If the growth is good, pipette 5mL of the culture medium to inoculate Cultivate in No. 2 medium at 30°C and 180 rpm for the same time, otherwise continue to inoculate into No. 1 medium for cultivation. Repeat this for many times, and then move to the culture medium with sodium alginate as the only carbon source for separation and screening.

[0050] The formula of No. 1 and No. 2 liquids and separation and screening medium is as follows:

[0051] ① Medium 1: 0.5% sodium alginate, 0.4% peptone, 2.5% NaCl, 1.0% MgSO 4 ·7H 2 O, 0.2%K 2 HPO 4 ·3H 2 O, 0.001% FeSO 4 ·7H 2 O, the solvent is water, the pH value is 7.2 to 7.4;

[0052] ② Medium 2: 0.5% sodium alginate, 0.5% (NH 4 ) 2 SO 4 , 2.5% NaCl, ...

Embodiment 2

[0055] Embodiment 2: Preparation of alginate lyase enzyme liquid

[0056] The preparation method of alginate lyase is as follows:

[0057] (1) Inoculate the strain Acinetobacter juni X8 stored in a slant test tube into an isolation medium plate, culture for 48-72 hours, and transfer several times in this way to activate the strain; the isolation medium is prepared according to the following composition: sodium alginate 5g, (NH 4 ) 2 SO 4 5g, NaCl 25g, K 2 HPO 4 ·3H 2 O2g, MgSO 4 ·7H 2 O 1g, 20g agar, make up to 1000mL with water, pH 7.2-7.4, heat to dissolve the agar, cool to get a plate;

[0058] (2) The activated strain is inoculated into a 250mL Erlenmeyer flask containing 50mL liquid seed medium, cultivated at 30°C and 150rpm for 8h to obtain a seed liquid; the liquid seed medium formula is: 0.5% sodium alginate, 0.5 %(NH 4 ) 2 SO 4 , 2.5% NaCl, 0.2% K 2 HPO 4 ·3H 2 O, the solvent is water, pH7.5;

[0059] (3) Inoculate 1.0 mL of seed liquid into a 250 mL E...

Embodiment 3

[0068] Embodiment 3: Preparation of fucoidan oligosaccharides

[0069]The preparation process of alginate oligosaccharides is as follows: add 1mL 0.75% (w / w) alginate substrate solution, 1mL 0.1moL / L pH7.5 sodium phosphate buffer solution, and 1mL enzyme solution prepared in Example 2 to the test tube in sequence , and react at 40.0°C for 5 minutes to obtain unpurified alginate oligosaccharides. As determined by the 3,5-dinitrosalicylic acid method, about 1395 μg of alginate can be obtained per mg of purified alginate lyase Fucoidan oligosaccharides.

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Abstract

The invention provides a novel algin lyase producing strain, namely Acinetobacter juni. X8 (which is preserved in the China Center for Type Culture Collection, wherein the preservation number is CCTCCC No: M 209110 and the preservation time is May 20, 2009) and application thereof in preparing algin lyase. The Acinetobacter juni. X8 has the following advantages: (1) the nutritional requirement is simple, the cultivation is easy, the generation time is short, and the fermentation cultivation time is short; (2) intracellular crude enzyme solution is simple to prepare, and a centrifugal collected thallus can be shattered directly after being added into buffer solution; (3) the algin lyase prepared from the Acinetobacter juni. X8 has higher enzyme activity, and unseparated and unpurified crude enzyme solution can reach 53.9 U/mg (157.3 U/mL); (4) the reaction conditions required by the enzyme enzymolysis algin is mild, and the acting time is short; and (5) the algin lyase has good stability, can keep higher enzyme activity (more than 97 percent) after being preserved for 5 days at a temperature of less than or equal to 4 DEG C, and is stable for 8 hours under a condition of which the pH is between 7.7 and 9.0.

Description

(1) Technical field [0001] The invention relates to a new alginate lyase-producing bacterium—Acinetobacter juni X8, and its application in preparing the alginate lyase. (2) Background technology [0002] Alginate is a linear polysaccharide polymer present in the cell walls of algae such as Laminaria japonica, Gulfweed, and Sargassum fusiforme. It is composed of β-D-1,4-mannuronic acid ( β-D-mannuronic acid, referred to as M) and α-L-1,4-guluronic acid (α-L-guluronic acid, referred to as G) two monomers composed of acidic polysaccharides, with the glycobiology and With the in-depth study of sugar chemistry, it has been found that fucoidan oligosaccharides with different degrees of polymerization have many good biological activities, and have a wide range of application values ​​in the development of new drugs and health food. There are many methods for preparing algal oligosaccharides, such as acid degradation, alkali degradation, oxidative degradation, enzymatic degradation...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/88C12R1/01
Inventor 丁玉庭刘书来张建友侯保兵
Owner ZHEJIANG UNIV OF TECH
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