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Food grade thermophilic arabinose isomerase expressed from gras, and tagatose manufacturing method by using it

A technology of arabinose and isomerase, which is applied in the field of food-grade thermophilic arabinose isomerase expressed by GRAS and its use in the preparation of tagatose, which can solve the problems of inability and low expression level

Active Publication Date: 2010-01-27
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The expression level of the isomerase derived from the microorganism Geobacillus is too low to be used in industry

Method used

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  • Food grade thermophilic arabinose isomerase expressed from gras, and tagatose manufacturing method by using it
  • Food grade thermophilic arabinose isomerase expressed from gras, and tagatose manufacturing method by using it
  • Food grade thermophilic arabinose isomerase expressed from gras, and tagatose manufacturing method by using it

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Cloning of arabinose isomerase

[0036] Cultivation of Geobacillus stearothermophilus DSM 22 under aerobic conditions. Centrifuge at 8000xg for 10 minutes to recover the cultured cells. Genomic DNA was extracted from the obtained cells by using the cell culture DNA Midi Kit (Qiagen, USA). By using oligonucleotides 5′-TCTAGAATGATGCTGTCATTACGTCCTTATGAATTTTG-S′ (SEQ.ID.NO:1) and 5′-TCTAGATTACCGCCCCCGCCAAAACACTTCGTTCC-S′ (SEQ.ID.NO: 2) inserted by using the oligonucleotides having the sequence of Xbal and BamHi restriction enzyme sites As a primer, genomic DNA was used for polymerase chain reaction (PCR). PCR product 1 was obtained by amplifying a 1494 bp DNA containing the arabinose isomerase gene from Geobacillus stearothermophilus. Again by using the oligonucleotides 5′-CCCGAT ATCATGCTGT-CATT ACGTCCTTATG-S′ (SEQ.ID.NO: 3) and 5′-TGCACTGCAGTTACCGCCCCCG CCAAAACAC-3′ (SEQ. ID.NO: 3) inserted into the oligonucleotides with EcoRV and PstI restriction enzyme site s...

Embodiment 2

[0040] Example 2: Expression of arabinose isomerase in Bacillus

[0041] The recombinant strain Bacillus subtilis GSAlB-1 (preservation number: KCCM10788P) prepared in the above Example 1 was inoculated on LB medium (Bacto-tryptone 10g / L, Bacto-yeast extract) containing 20 μg / mL chloramphenicol 5g / L, NaCl 10g / L), and then shake flask culture at 230rpm at 37°C for 12 hours. Produce pre-culture solution. Then the pre-culture broth was inoculated into the main medium with the same composition at a concentration of 0.1%, followed by shaking flask culture at 37°C at 230rpm until the OD 600 Reach 1 to induce the expression of recombinant arabinose isomerase. In order to determine the enzymatic activity of the expressed arabinose isomerase, the culture solution was centrifuged at 12000×g for 10 minutes, and the cells were recovered. The cells were suspended in 50 mM Tris-HCl (pH 8.2) buffer, followed by sonication (170 watts, cooling on ice at intervals of 1 second / 2 minutes) to lyse...

Embodiment 3

[0044] Example 3: Expression of recombinant arabinose isomerase in coryneform bacteria

[0045] The recombinant strain Corynebacterium glutamicum GSAIC-1 (preservation number: KCCM10789P) prepared in Example 1 was inoculated into MB medium (Bacto-tryptone 10g / L, Bacto) containing 10 μg / mL kanamycin -Yeast extract 5g / L, NaCl 10g / L, soy peptone 5g / L), and then shake flask culture at 30°C at 200 rpm for 24 hours to prepare a pre-culture solution. The obtained pre-culture solution was inoculated into the main medium with a concentration of 1%, and then cultured in a shake flask at 30°C at 200 rpm until the OD 600 Achieve 0.1 to induce the expression of recombinant arabinose isomerase. In order to determine the enzymatic activity of the expressed arabinose isomerase, the culture solution was centrifuged at 8000xg for 10 minutes, and the cells were recovered. The cells were suspended in 50 mM Tris-HCl (pH 7.0) buffer, followed by ultrasonic lysis. Centrifuge again at 8000xg for 12 m...

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Abstract

The present invention relates to a thermophilic arabinose isomerase and a method of manufacturing tagatose using the same, and more precisely, a gene encoding arabinose isomerase originating from the thermophile Geobacillus stearothermophilus DSM22, a recombinant expression vector containing the gene, a method of preparing a food grade thermophilic arabinose isomerase from the recombinant GRAS (Generally Recognized As Safe) strain transformed with the said expression vector, and a method of preparing tagatose from galactose using the said enzyme.

Description

Technical field [0001] The present invention relates to a thermophilic arabinose isomerase and a method for preparing tagatose using the same. More precisely, it relates to a gene encoding arabinose isomerase from Geobacillus stearothermophilus DSM22, containing A recombinant expression vector of the gene, a method for preparing a food-grade thermophilic arabinose isomerase from a recombinant GRAS (Generally Recognized As Safe) strain transformed from the expression vector, and by using the enzyme Method for preparing tagatose from galactose. Background technique [0002] With the increasing interest in a comfortable or healthy life, tagatose has been suggested as a substitute for sugar because it has fewer side effects and sugar is one of the main factors that cause various adult diseases. Tagatose is an isomer of galactose and is known to have physiochemical properties similar to fructose. Tagatose is a natural low-calorie sugar, and has recently been certified as GRAS (Gener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90
CPCC12N9/90C12P19/24C12N9/00
Inventor 金成俌李英美朴承源金政勋宋相勋李康杓
Owner CJ CHEILJEDANG CORP
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