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KR-102963092-B1 - RECOMBINANT PLASMID COMPRISING THE SECRETION TYPED GLYCOSIDE HYDROLASE GH50A BETA-AGARASE GENE AND USE THEREOF

KR102963092B1KR 102963092 B1KR102963092 B1KR 102963092B1KR-102963092-B1

Abstract

The present invention relates to a recombinant plasmid containing a secretory glycoside hydrolase GH50A β-agarasase gene and its use, and more specifically, to a recombinant plasmid containing a glycoside hydrolase GH50A β-agarasase gene that can be transformed into Bacillus subtilis and expressed as a secretory form, a cell transformed with said recombinant plasmid, a method for producing secretory glycoside hydrolase GH50A β-agarasase using said transformed cell, and a method for producing neoagarobiose (NA2) from a polysaccharide substrate such as agar using said transformed cell. The recombinant plasmid containing the glycoside hydrolyzing enzyme GH50A β-agarasase gene of the present invention and the GH50A β-agarasase enzyme obtained from cells transformed with said plasmid are produced from GRAS strains, are safe, are effectively secreted from transformed cells, and have excellent activity of the produced enzyme, so they can efficiently produce neoagarobiose from polysaccharides such as agar, and thus can be usefully applied in the fields of medicine, food, cosmetics, and livestock.

Inventors

  • 배영석
  • 김영호
  • 전도연
  • 김기윤
  • 박신영

Assignees

  • 주식회사 에이티삼일바이오

Dates

Publication Date
20260512
Application Date
20231201

Claims (7)

  1. A recombinant plasmid in which a nucleotide sequence consisting of SEQ ID NO. 11 or SEQ ID NO. 12 as a signal sequence and a nucleotide sequence consisting of SEQ ID NO. 13 as a structural gene for the glycoside hydrolase GH50A β-agarasase are operably linked.
  2. A recombinant plasmid according to claim 1, characterized in that the recombinant plasmid is represented by the cleavage map described in FIG. 9.
  3. Transformed cell transformed with the recombinant plasmid described in claim 1 or 2.
  4. In claim 3, the transformed cell is characterized in that the transformed cell is Bacillus subtilis .
  5. delete
  6. delete
  7. delete

Description

Recombinant plasmid comprising the secretion-type glycoside hydrolase GH50A beta-agarasese gene and use thereof The present invention relates to a recombinant plasmid containing a secretory glycoside hydrolase GH50A β-agarasase gene and its use, and more specifically, to a recombinant plasmid containing a glycoside hydrolase GH50A β-agarasase gene that can be transformed into Bacillus subtilis and expressed as a secretory form, a cell transformed with said recombinant plasmid, a method for producing secretory glycoside hydrolase GH50A β-agarasase using said transformed cell, and a method for producing neoagarobiose (NA2) from a polysaccharide substrate such as agar using said transformed cell. Agar is a Generally Recognized As Safe (GRAS) natural material listed by the FDA as a functional food ingredient (PB 265502) and is a complex polysaccharide that constitutes the cell walls of marine red algae. Its main components are agarose (60–70%) and agaropectin (30–40%). Agarose is a polymer composed of alternating α-1,3-linked 3,6-anhydro-L-galactose (LAHG) and β-1,4-linked D-galactose, which is hydrolyzed into L-AHG and D-galactose. Agaropectin consists of the same agarobiose repeating units, but some sugar residues are substituted with sulfate esterification, pyruvate, and ethers (Duckworth and Yaphe, Carbohydr. Res.16. 189-197, 1971). Recently, as the diverse biological activities of products (NAOS, AOS, NA2, and L-AHG) generated using agarase from agar-degrading microorganisms have become known, the development of enzymes involved in agar degradation is actively underway targeting microorganisms derived from seawater, freshwater, soil, and the human body. Bacterial agarases are classified into the glycoside hydrolase (GH) family, broadly consisting of GH16, GH50, GH86, and GH118, which act as β-agarases (Michel G., Appl. Microbiol. Biotechnol. 102, 6847-6863, 2006; Cantarel B., Nucleic Acids res. 37, D233-338, 2009; Chi W., Appl. Microbiol. Biotechnol. 94, 917-930, 2012), and GH96 and GH117, which act as α-agarases (Flatment D., Appl. Environ. Microbiol. 73, 4691-4694, 2007; Ha S., Biochem. Biophys. Res. Commun. 412, 238-244, They are classified as such (2011; Jang W., Appl. Microbiol. Biotechnol. 105, 4621-4634, 2021). Among these, the GH50 family of β-agaranases can yield neoagarobiose (NA2), NA4, or NA2/NA4 as final products from agarose when co-treated with β-agaranase GH16 B. Among these products, neoagarobiose has been reported to have skin whitening (Kobayashi R., Biosci. Biotechnol. Biochem. 61, 162-163, 1997) and anti-obesity and anti-diabetic activities (Hong S., Mar. Drugs. 15, 90, 2017), so the mass production of NA2 is expected to be very useful industrially. Major hosts used for the production of industrially useful proteins include Escherichia coli , Bacillus subtilis , and Saccharomyces cerevisiae . Among these, Bacillus subtilis is a GRAS bacterium recognized by the FDA, possessing the advantages of being non-endotoxin-free and secreting large amounts of protein externally. In particular, it is known as a bacterium suitable for the production of food and pharmaceutical proteins because it is free from codon shifting and allows for genetic engineering and mass culture (Schallmey M., Can. J. Microbiol. 50:1-17, 2004). Figure 1 is a figure showing the vector map (A) and the base sequence (B) of the cloning site used in the present invention. Figure 2 is a diagram showing the electrophoresis of DNA cut with restriction enzyme SacI/XbaI from GH50A DNA (A) amplified by PCR in the process of the present invention and a recombinant plasmid (pBES-50A) produced through cloning. Figure 3 is a figure confirming the nucleotide sequence of pBES-50A produced in the present invention. Figure 4 is a diagram showing the location (A) where a signal sequence is inserted into the vector in the present invention and the DNA (B) cut with restriction enzymes Eco52I and MluI after pBES-50A is electrophoresed. Figure 5 is a diagram showing the pattern of a colony (A) transformed into E. coli after inserting various SP sequences into pBES-50A in the present invention and a DNA library (pBES-50A+SP) purified from them, cut with SacI and XbaI. Figure 6 is a figure showing colonies (A) transformed into Bacillus subtilis using pBES-50A+SP produced in the present invention and the agar-degrading ability of these clones stained with Lugol solution (B). Figure 7 is a figure showing the protein expression levels of the finally selected clones investigated by Western blot. Figure 8 is a thin-layer chromatography (TLC) diagram investigating the NA2 production capacity of the finally selected clone. Figure 9 shows the plasmid cleavage map of pBES-50A+SP of the present invention. The present invention will be described in detail below. The inventors of the present invention constructed a recombinant plasmid by linking the aprE promoter derived from Bacillus subtilis and the GH50A β-agarasase gene derived from the heat-stable Cell