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Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof

一种皂苷糖苷酶、地杆菌的技术，应用在糖基化酶、酶、应用等方向，能够解决收率低、没有效率、制备成本高等问题

Inactive Publication Date: 2012-11-14
KOREA RES INST OF BIOSCIENCE & BIOTECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as described in Example 3, the above-mentioned saponin α-glucosidase is produced by adding Aspergillus bacteria to the culture medium containing wheat bran and ginseng powder and then removing the bacteria. Therefore, the yield is low, and it is not available as a mass production method. Efficiency, high preparation cost, loss of target components during preparation, etc.

Method used

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  • Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof
  • Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof
  • Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1. The taxonomic characteristic analysis of new bacterial strain Gsoil3082

[0070] Gsoil3082 was cultured at 30°C in R2A. After the cells were cultured for 3 days, the morphology of the cells was observed with a Nikon light microscope, and Gram staining (gram stain), anaerobic Analysis of conventional physiological or biochemical characteristics such as anaerobic groove and catalase activity. The use of carbon energy and enzyme activity were analyzed using API 20NE, API ID32 GN and API ZYM test kits (bioMerieux). The strains were cultured under different temperature conditions (4, 15, 20, 25, 30, 37, 42 and 45°C) and various pH values ​​(pH changes by 0.5 in the range of pH 4.5-10.0), and then every 5 days. The unit is measured.

[0071] Growth in nutrient agar (nutrient agar) and TSA (trypticase soy agar, trypticase soy agar) was measured at 30°C. Chemotaxonomic analysis was completed by Montero-Barrientos and other methods.

[0072] To confirm the p...

Embodiment 2

[0098] Example 2.fosmid library screening and sequencing

[0099] The CopyControl Fosmid Library Production kit (CopyControl Fosmid Library Production kit) (Epicentre, USA) was completed according to the method of the manufacturing company. Genomic DNA (genomic DNA) of Geobacter Gsoil3082 was arbitrarily cut into fragments of about 40 Kb. Initially, a small amount of electrophoresis (running) based on pulse field gel electrophoresis (Pulse Field Gel Electrophoresis) was used to measure the size of the cut DNA. End-repair. The size of the repaired DNA above 40Kb was selected by LMP (low melting point) agarose gel electrophoresis. The blunt-ended DNA was purified by LMP (low melting point) agarose gel and ligated into pCC1FOS vector. In vitro packaging was accomplished with the MaxPlax lambda packaging extract kit (Epicentre, USA). Finally in 10mM MgSO 4 When the O.D value in the LB broth (broth) is 600, the product is transformed into the cultured Escherichia coli (E.col...

Embodiment 3

[0101] Example 3. Molecular cloning, expression and purification of recombinant BGL-GYP17

[0102] (3-1) Molecular cloning and expression of recombinant BGL-GYP17

[0103] The assembled DNA sequence of pbg1-gin18 was analyzed with ORF FINDER (National Center for Biotechnology Information's ORF FINDER) using genetic code 1 (genetic code 1). BLASTP was used for the expected ORF to find the ORF inferred to be ginsenoside glycosidase, and the pbg1-gin18 DNA template was amplified by PCR using the next oligonucleotide primer.

[0104] Forward primer:

[0105] 5′-CG GAA TTC ATG GAT CCC TAC GAG GAC CCC-3' (SEQ ID NO: 7)

[0106] Reverse primer:

[0107] 5′-CCC AAGC TT ACC CCG GGA CGA CGA GGC3' (SEQ ID NO: 8)

[0108] (The underlined parts in the above primer sequences represent the restriction enzyme cutting sites of EcoRI and HindIII, respectively)

[0109] The amplified fragments were cloned sequentially into the pHis-Paralle1 expression vector (for use by introducing ...

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PUM

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Abstract

The present invention relates to a novel ginsenoside glycosidase protein derived from the genus Terrabacter, , the protein having an activity whereby it converts PPD (protopanaxadiol) type saponins into highly active substances, which can be absorbed inside the body, by selectively hydrolysing specific position bonding bonds of ginsenoside.; More specifically, the invention relates to the amino acid sequence of the protein, the nucleic acid sequence coding for the protein, a recombinant vector comprising the nucleic acid sequence, and a transformant transformed by means of the vector, and concerns a method for producing ginsenoside glycosidase derived from the genus Terrabacter by culturing the transformant, a method for converting PPD type major saponins into the minor saponin form by using the protein, and a composition for converting PPD type saponins into soluble saponins which comprises the protein as an active component.

Description

technical field [0001] The present invention relates to a novel ginsenoside glycosidase protein derived from Terrabacter sp., specifically, an amino acid sequence constituting the above protein, a nucleotide sequence encoding the above protein, and a recombinant vector comprising the above nucleotide sequence , a transformant transformed with the above-mentioned vector, a method for producing Geobacter ginsenoside glycosidase by culturing the above-mentioned transformant, and a composition for converting PPD-like saponins into in vivo absorbable soluble saponins using the above-mentioned protein. Background technique [0002] Saponin is a polycyclic compound composed of non-sugar parts of glycosides that are widely present in the plant kingdom. The saponin component that contains the main physiologically active components of ginseng or red ginseng has a different chemical composition from saponins found in other plants. structure, so it is named ginsenoside, meaning the glyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N1/21C12P21/00
CPCC12P33/00C12Y302/01021C12N9/2445C12N9/24C12N15/52C12N1/20C12P21/00
Inventor 安东善金成健李成宅林完泽李炯宽金善昌
Owner KOREA RES INST OF BIOSCIENCE & BIOTECHNOLOGY
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