KR100781062B1 - A novel trehalose synthesizing fusion gene and a process for the trehalose production utilizing a thermostable fusion protein expressed from the gene - Google Patents

Abstract

A trehalose synthesizing fusion protein is provided to synthesize and hydrolyze maltooligosyltrehalose and increase the production efficiency of trehalose due to excellent heat-resistance. A maltooligosyltrehalose synthesis hydrolase(MhMTSHase) protein derived from Metallosphaera hakonesis(JCM8857) includes an amino acid sequence of SEQ ID : NO. 2. An MhMTSH gene encodes the MhMTSHase protein and includes a sequence of SEQ ID : NO. 1. A method for producing the protein comprises a step of culturing E. coli transformed by a recombinant vector including the MhMTSH gene. A method for producing trehalose comprises a step of adding substrate such as a water-soluble starch or a liquid corn starch to the MhMTSHase protein and then reacting it at a temperature of 65-75 deg.C under the pH of 5.0-6.0.

Description

A novel trehalose synthesizing fusion gene and a process for the trehalose production utilizing a thermostable fusion protein expressed from the gene}

1 is a schematic diagram of a method for producing an MhMTSH fusion gene represented by SEQ ID NO: 1 by fusing an MhMTS gene and an MhMTH gene.

Figure 2 shows the results of confirming the biosynthesis of the heat-resistant fusion enzyme protein (MhMTSHase) after the mass expression of the MhMTSH fusion gene in E. coli by the SDS-PAGE method.

3 is a reaction of the fusion enzyme protein (MhMTSHase) obtained by mass expression in Figure 2 and the substrate maltopentaose (G5), and confirmed the synthesis of trehalose by thin layer chromatography (Fig. A) and HPIC (Fig. B). The result is.

Figure 4 is a measure of the optimum temperature and pH (pH) of the trehalose synthesis reaction of the fusion enzyme protein (MhMTSHase). Trehalose synthesis was carried out using maltopentaose (G5) as a substrate at each temperature (Fig. A) and pH (Fig. B), and trehalose was synthesized by thin layer chromatography.

5 is a result of measuring the heat resistance of the fusion enzyme protein (MhMTSHase). Figure A shows the effect of 30 minutes prestorage temperature on enzyme activity. Figure B shows changes in enzyme activity after 48 hours storage at 70 ° C.

6 is a result of confirming the synthesis of trehalose by the reaction with the fusion enzyme protein (MhMTSHase) using a variety of maltooligosaccharide as a substrate.

Figure 7 shows the results of trehalose synthesis using the heat-resistant fusion enzyme protein (MhMTSHase) 1% water-soluble starch (soluble starch) as a substrate, and by the reaction time thin film chromatography method.

The present invention provides a new gene by fusion of two trehalose biosynthetic enzyme genes isolated from a hyperthermophilic archaebacterium Metallosphaera hakonesis (JCM 8857), and a new heat-resistant trehalose biosynthetic fusion enzyme expressed therefrom. To a method for producing trehalose. More specifically, two genes encoding trehalose biosynthesis expressed in Metallospara Hakonesis were fused at the gene level to produce a single gene, and efficiently produced trehalose using the heat-resistant trehalose biosynthetic fusion enzyme translated therefrom. It is.

Trehalose (α-D-glucopyranosyl- [1-1] -α-D-glucopyranose) is a non-reducing disaccharide found in insects, bacteria, fungi, yeast and some plants (Elbein, Adv . Carbohydr . Chem . Biochem . 30 : 227-256, 1974). Trehalose is also a storage form of reducing carbohydrates, but it is known to protect cells from adverse effects resulting from various types of undesirable physical and chemical external impacts, such as depletion of nutrients, high temperatures, drying, oxygen deficiency, and osmotic pressure. Eleutherio et al., Cryobiology 30: 591-596, 1993). Trehalose maintains the structure of cells by hydrogen bonds with phospholipids of proteins and cell membranes during drying, and allows foods to retain their unique flavor, color, and texture (Crowe et al., Science 223: 701-703, 1984). Therefore, in the case of dried food can be used as a food additive to preserve the color and flavor as well as to improve the shelf life of the food by reducing the available water.

Trehalose biosynthetic reaction pathways are best known in yeast and Escherichia coli, whose genes have already been isolated and their structures determined (De Virgilio et al., Eur . J. Biochem . 212: 315-323, 1993; Kaasen et al., Gene 145: 9-15, 1994). In addition, Rhizobium sp. Trehalose synthesis activity was measured in dorsal mycorrhiza (Mueller et al., Physiol . Plant . 90: 86-92, 1994). Trehalose biosynthesis of these microorganisms consists of two stages of enzyme reaction catalyzed by two proteins. The first enzyme is maltooligosyltrehalose synthase (MTSase), which binds α (1 → 4) glycosidic bonds at the reduced end of maltooligosaccharides to α (1 → 1) glycosidic bonds. Conversion to produce maltooligosyltrehalose. The second enzyme is maltooligosyltrehalose trehalohydrolase (MTHase), which hydrolyzes α (1 → 4) glycosidic bonds between the maltooligosil and trehalose sites of maltogosil trehalose produced by MTSase. This results in maltooligosaccharides and trehalose, which have two fewer units of glucose.

When two enzymes from these two genes are put into one reaction vessel at the same time and reacted with the initial substrate, the intermediate product produced from the first enzyme, maltooligosyltrehalose, is once diffused into solution. As a result, it becomes difficult to react with the second enzyme until a certain concentration is reached. Each enzyme should be maintained at the required concentration for a long time, and there may be a limitation in the amount of the final product, thus making it difficult to maintain consistent yields.

If two genes are fused to one gene using genetic recombination technology, it is possible to make a form in which two enzymes are fused to one enzyme protein. In this case, the fusion protein has two enzymatic activities at the same time and can be converted into the final product trehalose by the second enzymatic activity without diffusion of the intermediate produced at the first enzymatic activity site.

Indeed, some researchers of the present invention participated in Brevibacterium helvolum ) has been linked to the genes of two enzymes, BvMTS and BvMTH, to form the fusion protein BvMTSH to confirm trehalose production (Kim et al., Appl . Environ Microbiol . 66: 4620-4624). However, in many cases, fusion proteins are made from fusion genes, but there are few cases where fusion proteins have two enzymatic activities at the same time and the reaction rate is superior to the respective reactions. The reason is that there should be no mutual influence in forming the correct tertiary structure of each domain that constitutes the two enzymatic activities of the fusion protein, and also there should be no influence on the transfer and binding of the substrate and the reaction product at the two sites. Because.

Generally, when trehalose synthetic genes are separated, they must be transformed into E. coli or yeast and expressed as proteins, followed by lysis of microorganisms and purification of these proteins. This is because other proteins derived from the microorganisms themselves react with the substrate to produce unnecessary byproducts. The pure separation of enzymes for industrial use requires expensive facilities and technologies.

When the reaction is carried out at high temperature (above 60 ° C) using heat-resistant trehalose synthase produced by separation from thermophilic microorganism, all enzymes derived from microorganisms can be thermally denatured and inactivated. Therefore, microbial lysates can be used directly in the reaction without a separate pure separation process. At higher temperatures it is also possible to significantly increase the rate of trehalose production reactions. In particular, it increases the rate of dissociation of starch, which is used as the lowest substrate, and at the same time, many enzyme reactions can occur.

Currently, trehalose is extracted from yeast culture and used as a food additive, but its price is expensive and it has not been put to practical use. The present inventors have proposed a new fusion gene for biosynthesis of trehalose, and conducted a study aiming to increase the efficiency of trehalose synthesis using a heat-resistant enzyme obtained by mass expression of this gene.

The main object of the present invention is thermophilic metallospara Hakonesis (Metallosphaera hakonesis, JCM 8857), fused two genes of trehalose biosynthesis isolated fromMhMTSHTo provide new heat-resistant trehalose biosynthetic fusion enzyme proteins from this fusion gene.

Another object of the present invention is the MhMTSH It is to provide a recombinant expression vector containing the gene and E. coli transformed with the vector.

Still another object of the present invention is to provide a method for producing the protein by culturing E. coli and a protein produced by the method.

Another object of the present invention relates to a method for producing trehalose using the protein and to trehalose produced by the method.

In order to achieve the object of the present invention, the present invention provides a metal oligo siltrehalose synthase (MhMTSHase) protein derived from Metallosphaera hakonesis (JCM 8857) having an amino acid sequence of SEQ ID NO: 2.

The invention also MhMTSH encoding the protein MhMTSHase Provide the gene. MhMTSH Los parametric Hakone gene metal sheath (Metallosphaera hakonesis , JCM 8857) is a trehalose biosynthetic enzyme fusion structure gene ( MhMTSH ) having a nucleotide sequence of SEQ ID NO: 1 prepared by fusing the MhMTS gene and MhMTH gene. In addition, variants of such sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, MhMTSH The gene may comprise a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1.

The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to). The% identifies the number of positions where identical nucleic acid bases are present in both sequences, yielding the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison region, and multiplying the result by 100 to display the sequence Calculated by calculating the percent of same sex. Optimal alignment of sequences for comparison is by computerized implementation of known algorithms (e.g. GAP, BESTFIT, FASTA and TFAST in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science). Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information), or by examination.

The structural gene portion of the trehalose biosynthetic enzyme fusion gene prepared in the present invention encodes two enzyme proteins in a fused form. The first gene region of the fusion gene is the maltooligosiltrehalose synthase (MhMTSase) gene portion, and the second gene region is the maltooligosiltrehalose hydrolase (MhMTHase) gene. The heat-resistant fusion enzyme protein translated therefrom is heat-resistant maltooligosiltrehalose synthase (MhMTSHase). The fusion enzyme protein is a fusion enzyme protein capable of simultaneously synthesizing and synthesizing maltogosiltrehalose, and has excellent heat resistance and has an advantage of increasing trehalose production efficiency.

In the present invention, two genes were linked to one new gene using a gene recombination method, and a heat-resistant trehalose biosynthesis synthesized therefrom was prepared as one new fusion enzyme. The synthesized fusion enzyme had all the enzyme activities, and it was possible to reduce the enzymatic reaction in a single step as compared with the case of synthesizing trehalose using each enzyme, and it was analyzed that the heat resistance was high.

The present invention consists of fusion of genes, overexpression of fusion proteins from fusion genes, and trehalose biosynthesis using heat-resistant fusion proteins.

The inventors of the present invention have reported that the hyperthermophilic archaebacterium Metallospara Hakonesis ( Metallosphaera) Two trehalose biosynthesis related enzyme genes were isolated from hakonesis , JCM 8857 (Seo et al., J. Microbiol. Biotechnol . 29 (1), in print, 2007). The first gene is the metallospara Hakonesis maltooligosiltrehalose synthase gene ( MhMTS , GenBank accession number AY444508), whose structural gene portion is 2,142 bp. Metallospara Hakonesis maltooligosiltrehalose synthetase (MhMTSase) translated therefrom is a protein having a molecular weight of about 83.8 kDa consisting of 713 amino acids. This enzyme converts α (1 → 4) glycosidic bonds at the reduced end of maltooligosaccharide to α (1 → 1) glycosidic bonds to produce maltooligosyl trehalose.

The second gene is the maltooligosiltrehalose hydrolase gene ( MhMTH , GenBank accession number AY444509) whose structural gene portion is 1,679 bp. Metallospara Hakonesis maltooligosiltrehalose hydrolase (MhMTHase) translated therefrom is a protein having a molecular weight of about 63.7 kDa consisting of 558 amino acids. The enzyme hydrolyzes α (1 → 4) glycosidic bonds between the maltooligolic and trehalose sites of maltooligolic trehalose produced by MhMTSase to produce maltooligosaccharides and trehalose with two fewer glucose units. .

In order to achieve another object of the present invention, the present invention is MhMTSH Provided is a recombinant expression vector comprising a gene.

For industrial production of trehalose, each gene must be expressed in Escherichia coli and produced separately, and each of two different chain enzyme reactions must be performed. In the present invention, two genes were prepared as one gene using a polymerase chain reaction (PCR) and a gene recombination method (FIG. 1). The DNA construct was constructed by inserting it into the pRSET-B plasmid (Invitrogen Inc.), which is an E. coli expression vector, to biosynthesize one fusion enzyme (MhMTSHase) having two parts of trehalose biosynthetic enzyme activity. MhMTSH of the invention The gene may be expressed in prokaryotic or eukaryotic cells.

Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9 (Qiagen); pBS vector, PHAGESCRIPT vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia; And pRSET-B from Invitrogen Inc. Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Stratagene); And pSVK3, pBPV, pMSG and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art. In the present invention, pRSET-B (Invitrogen Inc.) vector, which is one of E. coli expression vectors, was used.

The expression vector will preferably comprise one or more selectable markers. The markers include dihydrofolate reductase or neomycin resistance to eukaryotic cultures and include tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria.

In order to achieve another object of the present invention, the present invention provides E. coli transformed with the recombinant expression vector of the present invention.

Recombinant constructs can be introduced into cultured host cells using well known techniques such as infection, transduction, transfection, transfection, electroporation, and transformation. Representative examples of hosts include bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium cells; Fungal cells such as yeast cells; Insect cells, such as Drosophila S2 and Spodhodera Sf9 cells; Animal cells such as CHO, COS and Bowes melanoma cells; And plant cells. Suitable culture media and conditions for host cells are known in the art. In the present invention, Escherichia coli BL21 was used as a host cell.

In order to achieve another object of the present invention, the present invention provides a method for producing a protein of the present invention comprising the step of culturing E. coli transformed with the recombinant expression vector of the present invention. The medium, incubation temperature, incubation time, etc. required for culturing E. coli utilize methods well known in the art.

In order to achieve another object of the present invention, the present invention provides a maltooligosiltrehalose synthetase (MhMTSHase) protein produced by the above method. The produced fusion gene was expressed in E. coli, and after lysis (E. coli), it was confirmed that the fusion enzyme protein (MhMTSHase) biosynthesis using SDS-PAGE electrophoresis (Fig. 2). In addition, maltopentaose (G5) was used as a substrate and reacted with the E. coli lysis (total fraction), and then analyzed by thin layer chromatography (TLC) and HPIC (high pH ion chromatography) to produce trehalose. It was confirmed (FIG. 3).

This fusion enzyme protein was analyzed to be very heat resistant. The optimum temperature of trehalose synthesis reaction of this fusion enzyme protein was found to be 65-75 ° C, the optimum pH (pH) was found to be pH 5.0-6.0 (Fig. 4). After the pre-storage of the fusion enzyme protein at various temperatures for 30 minutes, the enzyme reaction was carried out at 70 ° C and pH 5.5. It was shown (Fig. 5). The enzyme activity is maintained at 78% when stored for 48 hours at 70 ° C.

In order to achieve another object of the present invention, the present invention provides a method for producing trehalose comprising the step of reacting by adding a substrate to maltooligosiltrehalose synthase (MhMTSHase) protein produced by the above method do. In the production method, the substrate may be, but is not limited to, water soluble starch or liquid corn starch. In addition, the reaction can be carried out at a temperature of 65-75 ℃ and pH of 5.0-6.0. In order to assay the trehalose synthesis ability of the expressed heat-resistant fusion enzyme protein, trehalose synthesis reaction was carried out using various maltooligosaccharides as substrates. As a result, it was confirmed that the heat-resistant fusion enzyme protein produced trehalose from various maltooligosaccharides (FIG. 6).

Maltodextrin, the most abundant in nature as an industrially available substrate for trehalose production, is starch. Water-soluble starch of the fusion enzyme protein as a substrate was confirmed to produce trehalose (Fig. 7).

The present invention also provides trehalose produced by the method of producing trehalose.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

Example 1 Fusion of Trehalose Biosynthesis Gene

Metallosphaera hakonesis, two heat resistant trehalose biosynthesis genes isolated from JCM 8857) (Seo et al. , J. Microbiol. Biotechnol. 29 (1), in print, 2007) of MhMTS (GenBank accession number AY444508) and MhMTH (GenBank accession number AY444509) was linked to one fusion gene, MhMTSH , using gene recombination technology. Oligonucleotide primers such as those shown in FIG. 1 were synthesized to produce one fusion gene in which two genes do not overlap, and a polymerase chain reaction (PCR) was performed. The base sequences of the four oligonucleotide primers shown in FIG. 1 are as follows.

Primer 1: 5'-CGGAAA GAATTC ATG CTAGTCGCAACCTAT-3 '(SEQ ID NO: 3)

Primer 2: 5'-GAATTTGCGCCAAA CAT ACC TTTCATCAAAACC-3 '(SEQ ID NO: 4)

Primer 3: 5'-GGTTTTGATGAAA GGT ATG TTTGGCGCAAATTC-3 '(SEQ ID NO: 5)

Primer: 5'-CGGAAA GAATTC TTAGGTTTCCTCAACCTC-3 '(SEQ ID NO: 6)

Primer 1 contained a 5 'sequence including ATG (in bold), a translation start codon of the MhMTS gene ORF (open reading frame), and an Eco RI restriction enzyme recognition sequence (underlined) was added to the front. Primer 3 was added to the p sequence, except for three translation stop codon TAA in the ORF of gene MhMTS, the front part I put the portions corresponding to "the 5 MhMTH gene ORF. Primer 2 was prepared with the complementary nucleotide sequence of primer 3. Primer 4 contained the complementary sequence of the 3 'side of the MhMTH gene ORF, and immediately after the translational stop codon TAA of the MhMTH gene, an Eco RI restriction enzyme recognition sequence (underlined) was added.

PCR was performed with primers 1 and 2 using the MhMTS gene as a template to obtain a DNA fragment having an Eco RI recognition sequence on the 5 'side and a 5' portion of the MhMTH on the 3 'side. PCR was carried out using primers 3 and 4 using the MhMTH gene as a template to obtain DNA fragments in which a portion of MhMTS 3 'on the 5' side and Eco RI were connected to the 3 'side. The two PCR products have 33 sequences complementary to each other on the 3 'and 5' sides.

After the PCR products were mixed, PCR was further performed using primers 1 and 4 to prepare MhMTSH fusion genes. By denaturing the two strands of the PCR product by heating, cooling, and reannealing again, a common nucleotide sequence of the 3 'side of MhMTS and the 5' side of MhMTH can be obtained. This was made into two complete DNA strands by Taq DNA polymerase, and PCR amplification using primers 1 and 4 to prepare a MhMTSH fusion gene.

MhMTSH fusion gene will have the nucleotide sequence shown in SEQ ID NO: 1, has the structure of a fusion gene with a translation termination site of the gene MhMTS is gone and the translation start site of the gene MhMTH connected to the last amino acid of the following MhMTS. The structural gene portion of the prepared trehalose biosynthetic enzyme fusion gene is 3,816 bp, encoding the two enzyme proteins in the fused form. The first gene region of the fusion gene is the maltooligosiltrehalose synthase gene ( MhMTS ) portion, and the second gene region is the maltooligosiltrehalose hydrolase gene ( MhMTH ).

Example 2 Biosynthesis of MhMTSHase Fusionase Protein

The DNA construct (pRMhMTSH) was prepared by inserting the MhMTSH fusion gene into E. coli expression vector pRSET-B (Invitrogen Inc.). The recombinant plasmid pRMhMTSH containing the prepared fusion gene was transformed into Escherichia coli BL21, incubated for 12 hours, and then IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.7 mM and further cultured for 4 hours. Expression of the fusion enzyme protein was induced.

Figure 2 is the result confirmed by SDS-PAGE electrophoresis of the expressed fusion enzyme protein. 2, M is a protein molecular weight marker, and C in FIG. 2 is induced expression of only pRSET-B plasmid containing no foreign gene in E. coli. T and S of FIG. 2 are pRMhMTSH plasmids containing the MhMTSH gene in E. coli, induced expression, and separated into total fraction (T) and soluble fraction (S) and analyzed by SDS-PAGE. As shown in the total fraction, when the pRMhMTSH plasmid containing the MhMTSH fusion gene was induced in E. coli, the MhMTSHase fusion enzyme protein was expressed in large quantities.

The fusion enzyme protein produced from the prepared trehalose biosynthesis fusion gene is maltooligosiltrehalose synthase (MhMTSHase) having a molecular weight of about 147.5 kDa composed of 1,271 amino acids. The amino acid sequence of this fusion enzyme protein is shown by SEQ ID NO.

Example 3 Verification of Enzyme Activity of MhMTSHase Fusion Enzyme Protein

To investigate the trehalose synthesis ability of the MhMTSHase fusion enzyme protein expressed in E. coli, trehalose synthesis experiments were carried out using maltopentaose (G5) linked with five glucose chains. The reaction conditions were adjusted to a concentration of 5 mM maltopentaose under 20 mM sodium acetate buffer (pH 5.5) and 10 μl of the total fraction of Escherichia coli co-enzyme (lysate) was added to the total reaction volume. 50 μl. It was reacted at 70 ° C. for 1 hour and treated at 100 ° C. for 10 minutes to terminate the reaction.

3 is a result of confirming trehalose synthesis by thin layer chromatography method and HPIC after conducting trehalose synthesis experiment using maltopentaose as a substrate. 3, M is a standard (glucose, G1), trehalose (trehalose, T), maltose (G2), maltotriose (G3), maltotetraose (maltotetraose (G4), It is a mixture of maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7). Figure 3 T and S are the results of the trehalose synthesis experiment using maltopentaose as a substrate using the total fraction (T) and the soluble fraction (S) obtained by centrifuging the total fraction of Figure 2, respectively. In this experiment, it was confirmed that MhMTSHase fusion enzyme protein produces trehalose using maltopentaose as a substrate.

In this experiment, not only trehalose but also sugars of various lengths remain shortened as the length of the substrate becomes shorter, and the disaccharide maltose (G2) or trisaccharide maltotriose (G3) is no longer used as a substrate for enzymatic reaction. This is because the efficiency to be lowered (see Example 5, Fig. 6). Therefore, the use of long chain glucose polymers such as starch as a substrate can minimize the proportion of these residual polysaccharides (see Example 6, Figure 7).

<Example 4> Optimum conditions for heat resistance and activity of MhMTSHase fusion enzyme protein

Basic reaction conditions of MhMTSHase fusion enzyme protein were investigated. First, the acidity (pH) was set to 5.5 using 20 mM sodium acetate buffer and 1 mM maltopentaose was used as a substrate. The total reaction volume was made 50 μl by adding 10 μl of the ultrasonically disrupted E. coli coenzyme (total fraction) solution. It was reacted for 1 hour and treated at 100 ° C. for 10 minutes to terminate the reaction.

In experiments with varying temperatures, the optimum temperature for trehalose synthesis of MhMTSHase fusion enzyme protein was found to be around 70 ° C, and the optimum acidity (pH) was found to be pH 5.0-6.0 (Figure 4). In this experiment, 0.1 M McIlvaine buffer (pH 4-5), 0.1 M sodium phosphate buffer (pH 5-8), and 0.1 M sodium bicarbonate buffer (pH 8-9) were used as buffers for pH control. .

After pre-storing the fusion enzyme protein at various temperatures for 30 minutes, the enzyme reaction was carried out at 70 ° C and pH 5.5.Therefore, there was no significant change in activity by pre-storage up to 70 ° C. From the case it was shown to decrease significantly (Fig. 5). And the enzyme activity is maintained about 78% when stored for 48 hours at 70 ° C. Therefore, the MhMTSHase fusion enzyme protein produced by fusion of two genes isolated from high temperature bacteria has the heat resistance of each enzyme protein produced from each gene before fusion (Seo et al., J. Microbiol . Biotechnol . 29 (1), in print, 2007).

<Example 5> Substrate specificity of trehalose synthesis using MhMTSHase fusion enzyme protein

To investigate the substrate specificity of trehalose synthesis of MhMTSHase fusion enzyme protein, trehalose synthesis experiments were conducted using various maltooligosaccharides as substrates. Under the conditions of 20 mM sodium acetate buffer (pH 5.5), maltooligosaccharides of various lengths were added at a concentration of 1 mM, and 10 μl of the total fraction of Escherichia coli co-enzyme was added. 50 μl. It was reacted at 70 ° C. for 1 hour and treated at 100 ° C. for 10 minutes to terminate the reaction.

6 is a result of confirming trehalose synthesis by a thin layer chromatography method after the trehalose synthesis experiment using a variety of maltooligosaccharide as a substrate. 6, M represents glucose (glucose, G1), trehalose (T), maltose (G2), maltotriose (G3), maltotetraose (maltotetraose, G4), It is a mixture of maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7). From T3 to T7 of FIG. 6, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose are the results of trehalose synthesis experiments, respectively. As shown in FIG. 6, the MhMTSHase fusion enzyme protein was confirmed to produce trehalose using maltopentaose as well as various maltooligosaccharides as a substrate.

Example 6 Trehalose Synthesis from Water Soluble Starch Using MhMTSHase Fusion Enzyme Protein

Starch is the most abundant maltodextrin in the natural world as an industrially available substrate for trehalose production. Using water soluble starch as a substrate, it was confirmed whether trehalose can be produced using the fusion enzyme protein developed in the present invention (FIG. 7). 1% water-soluble starch was added to 20 mM sodium acetate buffer (pH 5.5), and 10 μl of the solubilized E. coli coenzyme (total fraction) solution was added to make the total reaction volume 50 μl. This was reacted at 70 ° C., and treated at 100 ° C. for 10 minutes for 1 to 24 hours to terminate the reaction.

As shown in Figure 7, the longer the reaction time, the production of trehalose increased. Starch is a long chain maltodextrin, which constantly provides new substrates for each step of producing one molecule of trehalose. In addition, many starch macromolecules (maltooligosaccharides) are branched to the starch macromolecule, so that many enzymatic reactions can occur simultaneously.

For experimental purposes, by-products such as maltose (G2), which is a disaccharide, and maltotriose (G3), which are trisaccharides, were rarely found when using starch as a substrate. (FIG. 7). And even when using a total fraction obtained by lysis of E. coli, the reaction can proceed at high temperature, which is unnecessary by inactivating heat-denatured sugar-modifying enzymes produced by E. coli. It was demonstrated that the production of by-products can be minimized (FIG. 7).

According to the present invention, the present invention is a metallospera Hakonesis (Metallosphaera hakonesis, JCM 8857), a new fusion gene fusion of two genes encoding trehalose biosynthesis and a new heat-resistant trehalose biosynthetic fusion enzyme protein synthesized therefrom. The fusion gene prepared in the present invention and the fusion enzyme protein produced therefrom are new ones that do not exist in nature, and two reactions can be simultaneously performed by improving the method of biosynthesizing trehalose using two previously reported enzymes. To provide new enzyme formulations.

Since the fusion enzyme protein prepared in the present invention has an advantage of excellent heat resistance, the fusion enzyme protein is reacted under high temperature conditions of about 70 ° C. to increase the decomposition efficiency of soluble starch or liquefied corn starch. By proceeding trehalose production efficiency can be significantly increased. In addition, the heat resistance of the fusion enzyme protein can greatly improve the preservation of the enzyme preparation can reduce the cost of trehalose production. As demonstrated in Example 6 (FIG. 7), even when using a total fraction obtained by dissolving E. coli, the reaction can proceed at a high temperature so that E. coli produced a self-producing sugar protein (sugar) Inactivating -modifying enzymes through thermal denaturation can minimize the production of unwanted byproducts.

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Claims (13)

Metallospara Hakonesis having the amino acid sequence of SEQ ID NO: 2 (Metallosphaera hakonesis, JCM 8857) derived maltooligosiltrehalose synthetic hydrolase (MhMTSHase) protein. MhMTSH encoding the MhMTSHase protein of claim 1 gene. The MhMTSH of claim 2 having a nucleotide sequence of SEQ ID NO: 1 gene. delete MhMTSH of claim 2 or 3 Recombinant expression vector comprising a gene. Escherichia coli transformed with the recombinant expression vector of claim 5. The method of producing the protein of claim 1 comprising culturing the E. coli of claim 6. Maltooligosiltrehalose synthase (MhMTSHase) protein produced by the method of claim 7. The protein of claim 8, wherein the optimal temperature of the protein is 65-75 ° C. and the optimal pH is 5.0-6.0. A method of producing trehalose comprising the step of adding a substrate to the protein of claim 8 for reaction. The method of claim 10 wherein the substrate is water soluble starch or liquid corn starch. The method of claim 10, wherein the reaction is carried out at a temperature of 65-75 ° C. and a pH of 5.0-6.0. A trehalose produced by the method of any one of claims 10-12.

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