KR19990085427A - Novel recombinant 2,5-diketo-di-gluconate reductase and its genes - Google Patents

Abstract

The present invention provides a 2,5-diketo-D-gluconate reductase gene isolated from Escherichia coli and a recombinant 2,5-diketo- recombinant by the gene. It relates to a di-gluconate reductase.

The present invention isolates the yqhE gene, which is a gene of 2,5-diketo-di-gluconate reductase from E. coli, and encodes only a protein of 2,5-diketo-di-gluconate reductase, among the genes. Was inserted into the plasmid to prepare a recombinant plasmid pTY-YQH.E. Coli ER2566 (pTY-TQH) was obtained by transfecting E. coli DH5α with the recombinant plasmid, followed by culturing the transformed E. coli ER2566 (pTY-TQH). There is an excellent effect of mass production of 5-diketo-di-gluconate reductase.

Description

Novel recombinant 2,5-diketo-di-gluconate reductase and its genes

The present invention relates to a novel recombinant 2,5-diketo-D-gluconate reductase and its genes. More specifically, the gene of 2,5-diketo-di-gluconate reductase is isolated from E. coli, and the gene is expressed in E. coli in a large amount to produce recombinant 2,5-diketo-di-gluconate reductase. It is about a mass production method.

2,5-diketo-di-gluconate reductase is a 2-keto-el-glonate in 2-, 5-diketo-di-gluconate or salt form thereof in the presence of coenzyme NADPH. keto-L-gulonate) or an intracellular enzyme that catalyzes the conversion to its salt form (Sonoyama, T. et al., J. Ferment. Technol. 65: 311-317, 1987). 2-Keto-L-Glonate is a vitamin C synthetic precursor, which is more stable and easier to convert to vitamin C. Therefore, there has been much research on bioconversion by microorganisms (Crawford, TC et al., Adv. Carbohydr. Chem. Biochem. 37: 79-155, 1980). Sonoyama et al. Used an Erwinia herbicola mutant strain from glucose to di-gluconate and 2-keto-D-gluconate to give 2,5- Conversion to diketo-di-gluconate followed by conversion of 2,5-diketo-di-gluconate to 2-keto-el-glonate with Corynebacterium sp. Variants (Sonoyama , T. et al., Appl. Environ.Microbiol. 43: 1064-1069, 1982). Anderson and Grindley et al. Isolated 2,5-diketo-di-gluconate reductase genes from Corynebacterium microorganisms and isolated them from the genus Erwinia, a 2,5-diketo-di-gluconate-producing bacterium. And succeeded in establishing a one-step production process (Anderson, S. et al., Science, 230: 144-148, 1985; Grindley, JF et al., Appl. Environ. Microbiol .. 54: 1770-1775, 1988). However, until now, these enzymes are unstable at temperatures between 39 and 38 ° C. Therefore, studies on the increase in thermostability through gene mutation have been expected. Lazarus et al. (US patent 5583025, 1996) describe a protein tertiary structure of another aldose reductase that shows similarity to the amino acid base sequence of 2,5-diketo-di-gluconate reductase derived from Corynebacterium microorganisms. On the basis of this, an attempt was made to develop an enzyme which improved the thermal stability and catalytic properties of the enzyme by mutating several amino acids. Both existing 2,5-diketo-di-gluconate reductases are believed to have low temperature stability because they are separated from Corynebacterium in relatively low growth temperature.

The present inventors have reported on the production of 2-keto-di-gluconate using recombinant E. coli transforming E. coli with a cell membrane-bound gluconate dehydrogenase gene (Korean Patent 97-48802; Yum et al., J. Bacteriol. 179: 6566-6572, 1997). However, during conversion experiments, we observed a small decrease in 2-keto-di-gluconate produced over time after conversion from recombinant E. coli to gluconate to 2-keto-di-gluconate. Reductase activity of 2-keto-di-gluconate as a substrate was measured from E. coli cell extracts, and it was confirmed that 2-keto aldonate reductase, which was not reported in the past, was present in E. coli. Therefore, it can be seen that the 2-keto-di-gluconate produced by recombinant E. coli is transformed into gluconate and metabolized intracellularly by the action of this enzyme (Yum et al., J. Bacteriol. ). According to these results, E. coli has been reported only in other Corynebacterium genus, Erwinia genus and acetic acid bacteria (Sonoyama et al., Agric. Biol. Chem. 51: 3039-3047, 1987; Truesdell et al., J. Bacteriol. 173: 6651-6656, 1991), it was predicted that the metabolic process of ketogluconate was also present in E. coli. This was confirmed by in vitro conversion experiment using E. coli cell extract as coenzyme solution.

Accordingly, an object of the present invention was conceived in view of the above fact that the recombinant 2,5-diketo-di-gluconate reductase (2,5-diketo-D-gluconate) having an optimum growth temperature at a relatively high temperature reductase) and its genes. Another object of the present invention is to isolate a gene encoding 2,5-diketo-di-gluconate reductase from E. coli, transform the E. coli with the plasmid inserting the gene, and then culture the recombinant 2,5-da. The present invention provides a method for mass production of iketo-di-gluconate reductase.

The object of the present invention is to transform the E. coli with a gene of E. coli protein similar to the amino acid sequence of the 2,5-diketo-di-gluconate reductase obtained from the genus Corynebacterium microorganisms to obtain a gene product The 2,5-diketo-di-gluconate reductase reaction activity of this gene product was measured to confirm that it was a 2,5-diketo-di-gluconate reductase. Subsequently, E. coli was transformed and cultured with only the protein coding region of the culture gene identified by 2,5-diketo-di-gluconate reductase, followed by isolation and purification of pure protein by Protein Purification System. It was. The purified protein was enzymatically reacted with 2,5-diketo-di-gluconate as a substrate and 2,5-diketo-di-gluconate reductase activity was measured to determine the purified protein. This was achieved by identifying a recombinant 2,5-diketo-di-gluconate reductase.

Figure 1 shows the nucleotide sequence of the yqhE gene which is a 2,5-diketo-D-gluconate reductase gene derived from E. coli.

Figure 2 is a schematic diagram showing the process of inserting the protein coding region of the yqhE gene into the pTYB1 plasmid vector.

The present invention comprises the steps of searching for E. coli protein showing amino acid sequence similarity with 2,5-diketo-di-gluconate reductase derived from Corynebacterium microorganisms; The yqhE gene, which encodes an E. coli protein showing similarity to 2,5-diketo-di-gluconate reductase derived from the Corynebacterium microorganism, was amplified by PCR, recombined into a plasmid, and introduced into E. coli. Converting and culturing; The culture product obtained by culturing the transformed Escherichia coli was subjected to enzymatic reaction using 2,5-diketo-di-gloconate as a substrate and NADPH as a coenzyme, followed by 2,5-diketo-di-glucose. Investigating that the culture is a recombinant 2,5-diketo-di-gluconate reductase by measuring nate reductase activity; PCR amplifies only the protein coding genes of the culture product identified as recombinant 2,5-diketo-di-gluconate reductase, recombines into plasmids, introduces them into E. coli, and cultures them. Purely purifying only; The purified protein was subjected to enzymatic reaction using 2,5-diketo-di-gluconate as a substrate and NADPH as a coenzyme, and then 2,5-diketo-di-gluconate reductase activity was measured. Confirming that the purified protein is a recombinant 2,5-diketo-di-gluconate reductase.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples.

Example 1: Search for E. coli protein similar to 2,5-diketo-di-gluconate reductase

Finding other ketogluconate metabolism-related reductase genes in Escherichia coli, the amino acid sequence of the same function is similar among microbial species, and 2,5-diketo derived from Corynebacterium microorganisms Proteins showing amino acid sequence similarities with di-gluconate reductase were searched in the protein database. As shown in Table 1, 49.8% of the similar E. coli proteins showed similarity and the proteins (hypothetical oxidoreductase) encoded by the yqhE gene between metC and sufI genes and similar to other redox enzymes. ; We found a protein (hypothetical oxidoreductase) that encodes the yafB gene that is 42% similar and is present between the aspU and mltD genes and similar to other oxidoreductases.

Escherichia coli protein with amino acid sequence similarity to 2,5-diketo-di-gluconate reductase from Corynebacterium microorganism Similar protein FASTAscore Pencentagematch Size (aa) SP: Q46857: YQHE_ECOLI, Escherichia colihypothetical oxidoreductase in mecC-sufI 1091 49.8 274 PIR: A32950: P100_LEIMALeishmania major probable redutase 958 44.6 284 SP: P15339: 2DKG_CORSP, Corynebacterium sp. 2,5-diketo-D-gluconic acid reductase-B 817 40.5 277 SP: P30863: YAFB_ECOLI, Escherichia colihypothetical oxidoreductase in aspU-mltD 772 42.0 267 SP: Q02198: MORA_PSEPU, Pseudomonas putidamorphine 6-dehydrogenase 616 43.4 295

Example 2: preparation of E. coli transformed with the yqhE gene

In Example 1, the yqhE gene having the highest amino acid sequence similarity with 2,5-diketo-D-gluconate reductase derived from Corynebacterium genus (2,5-diketo-D-gluconate reductase) (FIG. 1). YqhE gene was PCR cloned to confirm that) encodes a 2,5-diketo-di-gluconate reductase. GenBank accession No. A primer of the yqhE gene was designed based on the DNA nucleotide sequence of AE000383. The primers used were as follows and restriction enzyme XbaI recognition sites were made without recognition sites inside the gene for convenience of cloning.

Primers for yqhE Gene Cloning

Primer yqhE-5 ': 5-TGAACGCGTCTAGAACATCACTG-3

Primer yqhE-3 ': 5-CTTCCGGCTCTAGATGATGATGT-3 (complementary arrangement)

(In bold type indicates the base substituted for artificially making the restriction enzyme recognition site XbaI (underlined))

To PCR clone the yqhE gene, Takara's ExTaq DNA polymerase and Perkin Elmer's GeneAmp PCR system 2400 were used, and the genomic DNA of Escherichia coli as template DNA was denatured at 95 ° C for 30 seconds, annealed at 65 ° C for 30 seconds, The polymerization reaction was repeated 30 times at 72 ° C. for 1 minute. The PCR product was digested with restriction enzyme XbaI and then plasmid pUC19 and T4 DNA ligase digested with the same restriction enzyme were linked and transformed with E. coli DH5α. The transformed Escherichia coli was cultured in a medium containing antibiotic ampicillin, plasmids were isolated from a transformant resistant to ampicillin, and plasmid DNA was digested with restriction enzyme XbaI to confirm DNA insertion. Finally, the recombinant plasmid into which the yqhE gene was inserted was named pYQHE.

Example 3: Validation of 2,5-diketo-di-gluconate reductase recombinant with yqhE gene

In Example 2, the transformed Escherichia coli DH5α (pYQHE) containing the recombinant plasmid pYQHE was used to determine whether the yqhE gene product obtained by transforming E. coli DH5α was 2,5-diketo-di-gluconate reductase. After culturing in LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl) liquid medium for 18 hours, centrifugation was performed, and the cells were disrupted by an ultrasonic crusher, and the enzyme activity of the cell extracts was measured. Enzyme activity was carried out according to the method of Miller et al. (J. Biol. Chem. 262: 9016-9020, 1987) and the reductase activity was determined using 2,5-diketo-di-gluconate as a substrate and NADPH as a coenzyme. Measured. As a result, the enzymatic activity of the recombinant E. coli DH5α (pYQHE) cell extract was increased by 10 times more than that of the host microbial E. coli DH5α. Therefore, it was confirmed that the yqhE gene product was 2,5-diketo-di-gluconate reductase and the reaction product was confirmed by HPLC to confirm that the reaction product was 2-keto-L-glonate. The column used HPX-87C from Bio-Rad at 30 ° C. and the solvent (0.008NH ₂ SO ₄ ) rate was 0.5 mL / min. The enzymatic product was finally identified as 2-keto-L-glonate, so that the yqhE gene converted 2,5-diketo-di-gluconate to 2-keto-L-glonate under NADPH coenzyme. It was found that it was a diketo-di-gluconate reductase.

Example 4: Isolation and purification of recombinant 2,5-diketo-di-gluconate reductase produced by transformed Escherichia coli DH5α

In order to more accurately identify that the yqhE gene product identified in Example 3 as 2,5-diketo-di-gluconate reductase is 2,5-diketo-di-gluconate reductase, The enzyme was purified purely.

In order to purify and purified 2,5-diketo-di-gluconate reductase, New England BioLabs Impact ^TM T7 One-Step Protein Purification System was used. This system uses intein protein, a protein splicing element encoded by the Saccharomyces cerevisiae VMA1 gene (Chong et al., Gene, 192: 277-281, 1997; Chong et al., J. Biol. Chem., 271: 22159-22168). Intein is a protein of amino acid at low temperature in the presence of thiols such as 1,4-dithiothreitol (DTT), β-mercaptoethanol or cysteine. It causes a self-cleavage reaction at the end. Therefore, the gene encoding the protein to be purely separated is inserted in-frame before the gene encoding the intein. Since the pTYB vector used in this system has a chitin binding domain at the intein C-terminus, only the fusion protein of the intein-chitin binding domain is affinity using a chitin column. Affinity The bound protein of interest can be cleaved with DTT and isolated purely from the bound fusion protein. In addition, since gene expression is regulated under the T7 promoter and the lac operator, the protein is maximized after a certain time of incubation by isopropyl-beta-di-thiogalactoside (IPTG). Can be expressed as.

Experimental Example 1: Preparation of E. coli transformed with only the protein coding region of the yqhE gene

Oligonucleotide primers were synthesized to prepare plasmids for purifying 2,5-diketo-di-gluconate reductase.

Primer yqhE-Nde: 5-aggaacatATGGCTAATCCAACCGTTATTAAG-3

Primer yqhE-Sap: 5-gagaagctcttccgcaGCCGCCGAACTGGTCAGGATC-3 (complementary base sequence)

(The underlined portions indicate restriction sites NdeI and SapI recognition sites, respectively, and the capital letter indicates the protein-encoding base.)

For PCR cloning only the protein coding region of the yqhE gene, the primers, Takara's ExTaq DNA polymerase and Perkin Elmer's GeneAmp PCR system 2400 were used, and the genomic DNA of Escherichia coli as a template DNA was denatured at 95 ° C for 30 seconds, at 65 ° C. The polymerization reaction was repeated 30 times for 30 minutes by annealing for 30 seconds and 1 minute at 72 degreeC. The PCR product was digested with restriction enzymes NdeI and SapI, and then ligated using plasmids pTYB1 and T4 DNA ligase digested with the same two restriction enzymes and transformed with E. coli DH5α. This is shown in FIG. The transformed E. coli was cultured in a medium containing ampicillin to select a transformant resistant to the antibiotic ampicillin, and to isolate the plasmid of the selected transformant, and then to cut the plasmid DNA with restriction enzyme XbaI to insert DNA. Confirmed. Finally, the recombinant plasmid containing the yqhE gene was named pTY-YQH and E. coli transformed with the plasmid was named E. coli ER2566 (pTY-TQH). Deposited with accession number KCTC 2895 as of date.

Experimental Example 2: Validation of 2,5-diketo-di-gluconate reductase recombined with yqhE gene protein coding gene

Inoculated with E. coli ER2566 (pTY-YQH) transformed in Experimental Example 1 in a 400ml LB liquid medium 1% (v / v) and incubated until A ₆₀₀ becomes 0.5 and added IPTG to 0.3mM protein Expression was induced. Cells cultured for 5 hours after induction were suspended in column buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100), sonicated and centrifuged at 10,000 X g to remove cell solids. It was. The cell extract was passed through a column containing 2 mL chitin beads previously equilibrated with column buffer and washed with 100 mL column buffer. In order to cleavage and isolate only 2,5-diketo-di-gluconate reductase protein in the fusion protein bound to the column chitin bead, the buffer of the column was replaced with a cleavage buffer added with 30 mM DTT and left overnight at 4 ° C. It was. The cleaved protein was recovered by fractionation with column buffer. As a result, pure purified protein was obtained, and 2,5-diketo-di-gluconate reductase was subjected to an enzyme reaction using 2,5-diketo-di-gluconate as a substrate in the same manner as in Example 3. Activity was measured and the reaction product was HPLC and recombinant 2,5-diketo-di-gluconate converts 2,5-diketo-di-gluconate to 2-keto-L-gulonate in the presence of NADPH coenzyme. It was finally confirmed that it is a nate reductase.

The present invention isolates the yqhE gene which is a gene of 2,5-diketo-D-gluconate reductase from E. coli as described in the above Examples and Experimental Examples Recombinant plasmid pTY-YQH was prepared by inserting the gene encoding only the protein of 2,5-diketo-di-gluconate reductase into the plasmid and transforming E. coli DH5α with the recombinant plasmid to transform E. coli ER2566 (pTY). -TQH), followed by culturing this transformed E. coli ER2566 (pTY-TQH) to convert 2,5-diketo-di-gluconate to 2-keto-el-glonate, a vitamin C precursor. It is a very useful invention in the biopharmaceutical industry because it has an excellent effect of mass-producing, 5-diketo-di-gluconate reductase.

Claims (7)

YqhE gene encoding 2,5-diketo-di-gluconate reductase derived from E. coli. The 2,5-diketo-di-gluconate reductase gene of claim 1, wherein the yqhE gene has the following nucleotide sequence. The 2,5-diketo-di-gluconate reductase gene according to claim 1 or 2, wherein the yqhE gene has the following amino acid sequence. Recombinant plasmid pTY-YQH inserted with yqhE gene, which is a 2,5-diketo-di-gluconate reductase gene derived from E. coli. E. coli ER2566 (pTY-YQH) strain (accession number KCTC 2895) transformed with recombinant plasmid pTY-YQH inserted with yqhE gene, a 2,5-diketo-di-gluconate reductase gene derived from E. coli. E. coli ER2566 (pTY-YQH) transformed with the recombinant plasmid pTY-YQH inserted with the yqhE gene, a 2,5-diketo-di-gluconate reductase gene derived from E. coli, was recombinant 2,5-da. A method for mass production of iketo-di-gluconate reductase. Recombinant 2,5-diketo-di-gluconate reductase obtained by culturing E. coli ER2566 (pTY-YQH) transformed with a 2,5-diketo-di-gluconate reductase gene derived from E. coli A method for producing 2-keto-L-glonate by enzymatic reaction using 2,5-diketo-di-gluconate as a substrate and NADPH as a coenzyme.