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NOVEL [beta]-1,3-1,6-ENDOGLUCANASE PRODUCING, FROM [beta]-GLUCAN, OLIGOSACCHARIDES OR GLUCOSE

An endoglucanase, oligosaccharide technology, applied to oligosaccharides or glucose. field, able to solve problems such as undisclosed enzyme reactivity

Active Publication Date: 2018-12-21
KOREA UNIV RES & BUSINESS FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As far as β-1,3-endoglucanase is concerned, Korean Patent No. 10-1483182 discloses that laminarin is degraded into glucose and laminarin by β-1,3-endoglucanase, but does not The reactivity of undisclosed enzymes relative to clamskins

Method used

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  • NOVEL [beta]-1,3-1,6-ENDOGLUCANASE PRODUCING, FROM [beta]-GLUCAN, OLIGOSACCHARIDES OR GLUCOSE
  • NOVEL [beta]-1,3-1,6-ENDOGLUCANASE PRODUCING, FROM [beta]-GLUCAN, OLIGOSACCHARIDES OR GLUCOSE
  • NOVEL [beta]-1,3-1,6-ENDOGLUCANASE PRODUCING, FROM [beta]-GLUCAN, OLIGOSACCHARIDES OR GLUCOSE

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0047] Obtaining gly5m gene by cloning technology

[0048] Containing 23g / L (gram / liter) instant ocean sea salt (instant ocean sea salt), 50mM (mmol / liter) Tris-HCl, 2g / L glucose, 2g / L yeast extract and 0.5g / L chloride Proteus strain (Saccharophagus degradans) 2-40 T (ATCC 43961) for 12 hours.

[0049] Saccharophagus degradans 2-40 was obtained using a commercially available DNA isolation kit (Qiagen, Valencia, CA, USA). T (ATCC 43961) Genomic DNA. The target gene gly5m (GenBank ID.ABD82251.1) was amplified using Solg 2×Taq PCR smart mix 2 (SolGent, Daejeon, Korea). The primers used herein were 5'-GCGGGATCCATGAGAGAAAAACTACTGCGCG-3' (forward) and 5'-GCGCTCGAGGTGGTGGTGGTGGTGGTGGTCAACTGCTTCAACACTCCA-3' (reverse), each of which had a BamHI restriction site and an XhoI restriction site at the 5' end . In addition, in order to increase the affinity of the HisTrap column, the base sequence of the gene encoding histidine was added. Both the PCR product and the pET28a+ vector we...

example 2

[0050] Overexpression and purification of Gly5M

[0051] For overexpression of the gene obtained in Example 1, the gene was transformed into a protein expressing host Escherichia coli BL21(DE3). In the Luria-Bertani broth (Luria-Bertani (LB) broth) containing 50 mg / L (mg / liter) kanamycin (Kanamycin) (BD, Sparks, Maryland, USA (BD, Sparks, Bacterial cells were grown at 37°C in MD, USA) until the absorbance at 600 nm (nanometer) reached 0.6. Protein expression was induced using 0.1 mM (mmol / liter) IPTG at 16°C, and thus the recombinant protein was expressed in a water-soluble form. For the isolation of expressed Gly5M protein, cells were disrupted by sonication and centrifugation, followed by purification of the supernatant by using a HisTrap column (GE Healthcare, Piscataway, USA). Purified proteins were concentrated using Amicon Ultra Centrifugal filter (Millipore, Billerica, MA, USA) and bicinchoninic acid (BCA) ) protein assay kit (protein assay kit) (Pierce, Rockford, I...

example 3

[0053] Confirm the substrate specificity and cationic effect of Gly5M

[0054] In order to confirm the substrate specificity of the protein, the protein contained in 20mM (mmol / liter) Tris-HCl (pH 6.0) containing shiurin, laminarin, curdlan (Osaka and Light Co., Ltd. (Wako, Osaka, Japan) , Japan)), carboxymethylcellulose (Sigma-Aldrich, St. Louis, MO, USA) and xylan (Sigma-Aldrich, St. Louis, MO, USA) 1% of different dextrans from the company (Sigma-Aldrich, St Louis, MO, USA) were reacted with 10.5 μM (micromole / liter) Gly5M protein at 40° C. for 30 minutes. The resulting reducing sugars were quantified by the DNS method.

[0055]As shown in Table 1, the Gly5M protein exhibited the highest activity for laminarin, and when shiurin was used as a substrate, the Gly5M protein exhibited approximately 55.6% activity compared to when only laminarin was used as a substrate. relative activity. It has been confirmed that the Gly5M protein does not hydrolyze curdlan, unlike other β-...

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Abstract

The present invention relates to a novel [beta]-1,3-1,6-endoglucanase producing, from [beta]-glucan, oligosaccharides or glucose. More specifically, the present invention provides an effect of producing oligosaccharides or glucose of various degrees of polymerization in high yields by using a [beta]-1,3-1,6-endoglucanase exhibiting [beta]-1,3-endoglucanase and [beta]-1,6-endoglucanase activity on[beta]-glucan and exhibiting transglycosylation activity on laminarioligosaccharide.

Description

technical field [0001] The present invention relates to a novel β-1,3-1,6-endoglucanase produced from β-glucan and produces oligosaccharides or glucose. Background technique [0002] Currently, due to the increased oil price due to the depletion of global oil resources and the strengthening of environmental regulations in accordance with the strengthening of the United Nations Framework Convention on Climate Change (UNFCCC), there are concerns about development that can replace, for example, oil Research on eco-friendly and environment-friendly energy sources such as conventional fossil fuels such as coal and coal are actively progressing. Among them, brown algae composed of various polysaccharides grow faster than lignocellulosic and herbaceous biomass and have the advantage of high productivity per cultivated area. In addition, since brown algae have a low lignin content, they are easily converted into raw materials for producing bioenergy and chemicals, and since carbon ...

Claims

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Application Information

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IPC IPC(8): C12P19/04C12P19/20C12N9/24C12N15/52
CPCC12P19/04C12N9/24C12N15/52C12N9/2405C12P19/02C12P19/14C12Y302/01075C12Y302/01039C12P19/20
Inventor 金京宪王大毛金度亨
Owner KOREA UNIV RES & BUSINESS FOUND
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