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Paenibacillus for producing agarase and application thereof

A bacillus and agarase technology, applied in the directions of enzymes, bacteria, hydrolase, etc., can solve the problems of low enzyme activity and long fermentation cycle, and achieve the effects of short fermentation cycle, high mutation rate and high enzyme production activity.

Active Publication Date: 2013-06-05
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a high-yield strain of agarase—Paenibacillus sp. WL-15, and its application in the preparation of agar oligosaccharides, which can overcome the existing microbial fermentation and prepare agarase. The disadvantages of long fermentation period and low enzyme activity in

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  • Paenibacillus for producing agarase and application thereof

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Embodiment 1

[0024] Example 1: Mutagenesis and Screening of Paenibacillus WL

[0025] Before mutagenesis of Paenibacillus WL, it was first purified. Paenibacillus WL (CCTCC No: M 2010049) stored in a refrigerator at 4°C was inoculated with a slant medium, incubated at a constant temperature of 30°C for 48h, and then picked a little of the above slant cells, filled with dozens of glass beads and 50mL of sterile artificial seawater. The triangular flask was shaken at room temperature for 10 min to obtain a bacterial suspension with a cell number of 1 × 10 8 pcs / mL. Yeast cell suspension was diluted 10 6 After doubling, draw 0.2 mL of coated plate medium, and cultivate the plate at a constant temperature of 30 °C for 48 hours. After the colonies grow, pick a number of colonies in the deeper depressions of the medium and transfer them to the slant medium respectively, and the slant is at 25-30. Cultivated at constant temperature for 36h. The different slant strains were transferred to the ...

Embodiment 2

[0033] Embodiment 2: the method for preparing agarase by Paenibacillus WL-15 fermentation

[0034] With Paenibacillus WL-15 as strain, the preparation method of preferred agarase is as follows:

[0035] (1) Inoculate Paenibacillus WL-15 strains that were freeze-dried or preserved on slant surfaces of test tubes in the slant medium, and the slant surfaces were cultured in a biochemical incubator at 28°C for 36 hours. The slant medium consists of: agar 20g / L, glucose 1g / L, yeast extract powder 5g / L, NaCl 20g / L, MgSO 4 ·7H 2 O 5g / L; KCl 1g / L, FeSO 4 ·7H 2 O 0.02g / L, CaCl 2 0.2g / L, NaH 2 PO 4 0.78g / L, the solvent is water, pH 7.5;

[0036] (3) Use an inoculation loop to pick the Paenibacillus WL-15 thallus after step (1) activation and culture into the liquid seed medium, and the liquid seed culture is based on 28°C and 200r / min shaking conditions for 24 hours to obtain the seed liquid ; The liquid seed medium consists of: agar 2g / L, yeast extract powder 5g / L, peptone 10...

Embodiment 3

[0039] Embodiment 3: the preparation of agar oligosaccharide

[0040] With pH = 7.0, the concentration is 0.2mol / L Na 2 HPO 4 -NaH 2 PO 4 Buffer preparation 3g / L agar substrate solution (heating and dissolving), adorn 40mL agar substrate solution in 250mL conical flask, add 5mL embodiment 2 agarase crude enzyme solution, conical flask seals with preservative film at 45 ℃, 200r / min shaking conditions, enzymatic hydrolysis reaction for 120min, unseparated and purified agarose oligosaccharides can be obtained. The main components of oligosaccharides are new agarosetose and new agarosexose, and the yield of oligosaccharides can reach 80%. above.

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Abstract

The invention provides paenibacillus (Paenibacillus sp.) WL-15 and application thereof in agarase preparation by microbial fermentation, wherein an agarase high-yield bacterial strain is obtained through ion implantation mutation. The bacterial strain is preserved in the China center for type culture collection, the preservation number of the bacterial strain is China center for type culture collection number: M2011357 (CCTCC No: M2011357), the preservation date of the bacterial strain is October 19th, 2011. The paenibacillus and the application of the paenibacillus have the advantages that 1a low energy N+ ion implantation method is adopted to perform mutation to produce agarase microorganisms, and has the advantages of being high in mutation rate, simple to operate and the like; 2 the agarase generated by the bacterial strain through mutation breeding is good in genetic stability, and a high-yield characteristic is not prone to degenerate; and 3 the bacterial strain has the advantages of being high in enzyme production activity, low in nutrition requirements, short in fermentation period and capable of being applied to agarase preparation by industrial fermentation. The agarase has various purposes in the field of agaro-oligo saccharide preparation and the like.

Description

(1) Technical field [0001] The invention relates to a high-yielding strain of agarase - Paenibacillus sp. WL-15 obtained by ion injection and mutagenesis, and its application in the preparation of agarase by microbial fermentation. (2) Background technology [0002] Agar is a kind of water-soluble polysaccharide extracted from red algae such as Jiangli, Lily and other red algae. It is mainly composed of agarose and sulfur agar. A chain-like neutral sugar in which galactopyranose and 1,4-linked 3,6-endether-α-L-galactopyranose residues are repeatedly alternately linked. Studies in recent years have shown that the degradation products of agarose, oligosaccharides with a degree of polymerization of 2 to 10, have various physiological activities, such as antioxidant activity, hypolipidemic activity, immunomodulatory activity, anti-allergic activity, and glycosidase inhibitory activity. . Agarase is an enzyme that can degrade agar into oligosaccharides. It has the advantages of...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12N15/01C12N9/24C12R1/01
Inventor 应国清唐中秀梅建凤王鸿易喻陈建澍
Owner ZHEJIANG UNIV OF TECH
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