Arabinose Isomerase Expressed From Corynebacterium Genus and Tagatose Manufacturing Method By Using It

a technology of arabinose isomerase and corynebacterium genus, which is applied in the field of arabinose isomerase gene, can solve the problem that the production of tagatose using recombinant i>e. coli/i> is not suitable for the production of tagatose as a food ingredien

Inactive Publication Date: 2008-05-29
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]It is an object of the present invention to provide a method of preparing GRAS strains expressing arabinose isomerase stably in their cells under the conditions of high temperature and high concentration of galactose by taking advantage of mass-production of the recombinant enzyme in the Corynebacterium sp. strain.

Problems solved by technology

However, the biological production of tagatose using recombinant E. coli is not appropriate for the production of tagatose as a food ingredient.

Method used

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  • Arabinose Isomerase Expressed From Corynebacterium Genus and Tagatose Manufacturing Method By Using It
  • Arabinose Isomerase Expressed From Corynebacterium Genus and Tagatose Manufacturing Method By Using It
  • Arabinose Isomerase Expressed From Corynebacterium Genus and Tagatose Manufacturing Method By Using It

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of the Arabinose Isomerase

[0035]Thermotoga neapolitana DSM 5068 was cultured under anaerobic conditions. Centrifugation was performed at 8,000×g for 10 minutes to recover the cultured cells. Genomic DNA was extracted from the obtained cells by using a Cell culture DNA Midi Kit (Qiagen, U.S.A.). Polymerase Chain Reaction (PCR) was performed with the genomic DNA buy using oligonucleotides 5′-CCCGA TATCATGATCGATCTCAAACAGTATGAG-3′ (SEQ. ID. NO: 1 ) and 5′- TGCACTGCAGTCATCT TTTTAAAAGTCCCC-3′ (SEQ. ID. NO: 2) with the insertion of EcoRV and Pst1 restriction enzyme site sequences as primers. PCR product was obtained by amplifying the 1509 bp DNA containing the Thermotogas neapolitana arabinose isomerase gene. To mass-produce the arabinose isomerase encoded by the amplified gene, two of the Corynebacterium sp. derived vectors exhibiting excellent protein overexpressing capacity were selected. The vectors were introduced into E. coli DH5alpha, which were deposited at the Korean Cultu...

example 2

[0037]Expression of the Arabinose Isomerase in Corynebacterium the recombinant strains Corynebacterium glutamicum CJ-1-TNAI and Corynebacterium glutamicum CJ-7-TNAI (Accession Nos: KCCM10786P and KCCM10787P) prepared in Example 1 were inoculated in MB medium (Bacto-trypton 10 g / L, bacto-yeast extract 5 g / L, NaCl 10 g / L, Soytone 5 g / L) containing 10 μg / Me of kanamycin at the concentration of OD600=0.1, followed by culture at 30° C. for 24 hours to induce expression of the recombinant arabinose isomerase. To measure the enzyme activity of the expressed arabinose isomerase, the culture solution was centrifuged at 8,000×g for 10 minutes and cells were recovered. The cells were resuspended in 50 mM Tris-HCl (pH 7.0) buffer, followed by ultrasonification to lyse the cells. The supernatant was obtained as a crude enzyme solution, with which galactose isomerization was performed. Particularly, for the isomerization, 100 μl of an enzyme solution containing 40 mM of galactose as a substrate w...

example 3

Optimization of the Culture Condition for Mass-Production of the Recombinant Strain

[0038]The recombinant strains prepared in Example 2 were inoculated in MB medium (Bacto-trypton 10 g / L, bacto-yeast extract 5 g / L, NaCl 10 g / L, Soytone 5 g / L) containing 10 μg / Ml of kanamycin at the primary concentration of OD600=0.6. The growth of the strains in two basic media for the culture of Corynebacterium, MB medium (Bacto-trypton 10 g / L, bacto-yeast extract 5 g / L, NaCl 2.5 g / L, Beef extract 5 g / L), was investigated. Temperature dependent (25° C., 30° C., 37° C.), pH dependent, glucose (carbon source) and sucrose concentration dependent growths in the two media were compared. In addition, the growths under the various conditions and the expression levels of the enzyme thereby were measured every hour to judge the optimum expression condition for mass-production of the recombinant arabinose isomerase (Tables 2 and 3).

TABLE 2ModifiedmediumMB mediumAerobicAerobicStationary25° C.30° C.37° C.OD6001...

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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 thermophilic Thermotoga neapolitana DSM 5068, 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 an arabinose isomerase gene expressed in a Corynebacterium sp. strain and a method of preparing tagatose using the same, and more particularly, the present invention provides a gene encoding arabinose isomerase originating from Thermotoga neapolitana DSM 5068, a recombinant expression vector containing the gene, a method of preparing a thermophilic arabinose isomerase by expressing the gene in a food grade GRAS Corynebacterium sp. strain transfected with the recombinant expression vector, and a method of preparing tagatose from galactose using the same.BACKGROUND ART[0002]With the increasing interest in well-being or a healthy life, tagatose has been proposed as an alternative to sugar as it has less side effects and sugar is one of the major factors causing various adult diseases. Tagatose is the isomer of galactose and is known to have fructose-like physiochemical properties. Tagatose is a natural low-calorie sugar, and has re...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P19/24C12N9/92
CPCC12N9/90C12P19/24C12P19/02C12N9/92
Inventor KIM, SEONG-BOLEE, YOUNG-MIPARK, SEUNG-WONKIM, JUNG-HOONSONG, SANG-HOONLEE, KANG-PYOKIM, HYE-WONCHOI, HYE-JIN
Owner CJ CHEILJEDANG CORP
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