Agar-digesting enzyme and utilization thereof

A technology for decomposing enzymes and agar, applied in the direction of application, hydrolytic enzymes, enzymes, etc., can solve problems such as difficult application in industry, high price, and inability to be widely used

Inactive Publication Date: 2006-05-03
JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the previously known agar decomposing enzyme (for example, refer to JP-A No. 6-284888) has a low productivity and high price, so there is a problem that it is difficult to apply to industry, and its enzymatic activity and heat resistance are not sufficient, so it cannot be widely used. Applied to the industry

Method used

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  • Agar-digesting enzyme and utilization thereof
  • Agar-digesting enzyme and utilization thereof
  • Agar-digesting enzyme and utilization thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0212] Screening of agar decomposing bacteria:

[0213] The seabed soil samples stored in the Marine Science and Technology Center were appropriately diluted with Marine broth 2216 culture solution (manufactured by Difco), inoculated on Marine agar plate medium, and cultured at various temperatures from 15°C to 55°C for 16 to 48 hours. The bacteria that decomposed the agar around the colony and formed depressions on the applied plate medium were inoculated on another Marine agar plate medium, and cultured under the temperature conditions suitable for the bacteria. Then, streak culture was repeatedly performed on the same medium to isolate agar-decomposing bacteria.

[0214] The 1325-A7 strain, the 1325-A3 strain, and the A94 strain were obtained from the agar-degrading bacteria screened as above, and these strains are microorganisms capable of producing agar-degrading enzymes with higher agar-degrading enzyme activities than conventionally known. Using the CLUSTAL×Multiple Se...

Embodiment 2

[0216] (1) Analysis of agarase gene (1)

[0217] The chromosomal DNA of the Microglobosa sp.1325-A7 strain (hereinafter abbreviated as "1325-A7 strain") was digested with HindIII and EcoRI to obtain a DNA fragment. This DNA fragment was purified using a High Purity PCR Product Purification Kit (manufactured by Roche) to obtain a purified DNA fragment. This purified DNA fragment was ligated with the plasmid vector pUC18 (manufactured by TaKaRa) previously digested with HindIII and EcoRI using DNA Ligation Kit ver.2.0 (manufactured by TaKaRa). E. coli HB101 (F'supE44 hsdS20 recA13 ara-14 proA2lacY1 galK2 rpsL20 xyl-5mtl-1 leuB6 thi-1) was transformed with this ligation mixture to prepare a transformant.

[0218] The transformant prepared as above was inoculated on an agar medium, and the colonies forming depressions on the agar medium were selected as clones having agar-decomposing activity. Mark the selected clones with agar decomposing activity on LB agar medium (1% bactocul...

Embodiment 3

[0259] (1) Analysis of agarase gene (2)

[0260] The chromosomal DNA of the Microglobulina sp.1325-A3 strain (hereinafter abbreviated as "1325-A3 strain") was digested with HindIII to obtain a DNA fragment. This DNA fragment was purified using a High Purity PCR Product Purification Kit (manufactured by Roche) to obtain a purified DNA fragment. This purified DNA fragment was ligated with plasmid vector pUC18 (manufactured by TaKaRa) previously digested with HindIII and treated with shrimp alkaline phosphatase (manufactured by Roche) using DNA Ligation Kit ver.2.0 (manufactured by TaKaRa). E.coliHB101 (F'supE44 hsdS20 recA13 ara-14proA2 lacY1 galK2 rpsL20 xyl-5mtl-1 leuB6 thi-1) was transformed with this ligation mixture to prepare a transformant.

[0261] The transformant prepared as above was inoculated on an agar medium, and the colonies forming depressions on the agar medium were selected as clones having agar-decomposing activity. Place the selected clones with agar decom...

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Abstract

An agar-digesting enzyme having the following properties. (1) Function: Hydrolyzing beta-1,4 bond of agarose to form neoagarooligosaccharides. (2) Substrate specificity: Acting at least on polysaccharides having a skeleton in which D-galactose units and 3,6-anhydro-L-galactose units are bonded to each other alternately via beta-1,4 bond and alpha-1,3 bond (for example, agar and agarose) and oligosaccharides having the above skeleton to form agar-origin oligosaccharides. (3) Effect of temperature: Sustaining its activity even after heating to 54°C or higher for 30 minutes. Because of having a high activity at a high temperature compared with the existing agar-digesting enzymes, this enzyme can elevate the digestion speed of agar and thus is advantageously usable for industrial purposes.

Description

technical field [0001] The present invention relates to agar decomposing enzyme and application thereof, more specifically, the present invention relates to a novel agar decomposing enzyme with higher activity and stronger heat resistance than previous agar decomposing enzyme and application thereof. Background technique [0002] Agar is a polysaccharide obtained from red algae such as Geliflower and Nostocs, and its main component is agarose. In addition, there is also a small amount of polysaccharides collectively called agar gum in agar, which is formed by the esterification of agarose with sulfuric acid, sulfuric acid pyruvic acid, etc. [0003] Agarose can be hydrolyzed with agar decomposing enzyme β-agarase to obtain new agar oligosaccharides (neoagaroooligo sugar), which have strong anti-aging effect on starch, antibacterial effect after heating, and low heat. In the field of food, it is very useful as a raw material of high-performance food (see, for example, Toshia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N1/15C12N1/19C12N1/21C12N5/10C12N9/38C12N15/56C12P19/00C12P19/12C12P19/14C12Q1/68
CPCC12Y302/01089C12N9/2468C12P19/00
Inventor 大田优佳莉秦田勇二能木裕一伊藤进掘越弘毅
Owner JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY
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