KR20000000845A - Novel gene encoding trehalose biosynthetic enzyme and production method of trehalose thereby - Google Patents

Abstract

PURPOSE: Genes encoding Brevibacterium maltooligosyltrehalose synthease (BvMTase) and Brevibacterium maltooligosyl trehalose trehalohydrolase (BvMTHase) are isolated to obtain trehalose in large quantities which is used as food additives or food preservatives. CONSTITUTION: Trehalose is produced by following steps of: isolating gene encoding BvMTase and BvMTHase from Brevibacterium helvolum (ATCC 11822); cloning the gene into an expression vector which consists of 4,515 bp and encodes two enzyme proteins; massively expressing the recombinant proteins (enzymes) in E. coli; purifying the enzymes; and producing trehalose from maltooligosaccharide or starch by the enzymes. The structural gene of BvMTase gene consists of 2,328 bp; BvMTase consists of 776 amino acids. and has a molecular weight of about 85.8 kDa; the structural gene of BvMVHase consists of 1,767 bp; BvMTHase consists of 589 amino acids. and has a molecular weight of about 64.2 kDa.

Description

Novel trehalose biosynthetic enzyme gene and production method of trehalose using same

The present invention relates to a novel trehalose biosynthesis gene isolated from Brevibacterium helvolum and a method for producing trehalose using the same. More specifically, the present invention provides a gene encoding trehalose biosynthesis expressed in Brevibacterium helvolum ATCC 11822 and a trehalose biosynthetic enzyme translated therefrom, and using the same to effectively prepare trehalose in various substrates, especially starch. It is about how to.

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 reducible carbohydrates, but it is known to protect cells from the adverse effects of 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 keeps the cell structure intact by phospholipids and hydrogen bonds of proteins and cell membranes during drying process. It also allows food to maintain its 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 pathways are best known in yeast and Escherichia coli, whose genes have already been isolated and their structures are known (De Virgilio et al., Eur. J. Biochem., 212: 315-323, (1993). Kaasen et al., Gene, 145: 9-15, (1994)). In addition, Rhizobium sp. Trehalose synthetic activity was measured in the back mycorrhizal fungus (Mueller et al., Physiol. Plant, 90: 86-92, (1994)). At present, trehalose is extracted from the yeast culture and used as a food additive, but the price is expensive and there is a problem that has not been put to practical use.

Accordingly, the present invention has been made to solve the above problems, focusing on the production of trehalose by Brevibacterium helvolum ATCC 11822, trehalose biosynthesis gene from Brevibacterium helvolum ATCC 11822 The trehalose synthesis efficiency was increased by using the enzyme obtained by the isolation and mass expression of this gene.

It is an object of the present invention to provide novel trehalose biosynthesis genes isolated from Brevibacterium helvolum ATCC 11822 and trehalose biosynthetic enzyme proteins obtained from these genes. Another object of the present invention is to provide an efficient production method of trehalose using an enzyme protein obtained from the gene of the trehalose biosynthetic enzyme of the present invention.

The above object of the present invention is Brevibacterium maltooligosiltrehalose synthetase (hereinafter referred to as BvMTSase) and Brevibacterium maltooligo encoding trehalose biosynthesis expressed in Brevibacterium helvolum ATCC 11822 The siltrehalose hydrolase (hereinafter referred to as BvMTVHase) gene is isolated, these BvMTSase and BvMTVHase genes are recombined into expression vectors, expressed in Escherichia coli, and the expressed recombinant protein is purely separated, and then trehalose is synthesized from various starches. Achieved by doing so.

Hereinafter, the configuration and operation of the present invention will be described in detail.

1 is a photograph showing the results of genomic DNA of Brevibacterium digested with restriction enzymes of BamHI, EcoRI, HindIII, KpnI, and PstI, respectively, and Southern blot analysis.

Figure 2 shows the relative position and restriction map of the DNA fragment corresponding to the BvMTSase and BvMTHase gene.

Figure 3 shows the nucleotide sequence of the BvMTSase and BvMTHase gene and amino acid sequence translated therefrom.

Figure 4 is a schematic diagram of the production of the expression vector pBvMTSase and pBvMTHase plasmid.

5 is a result of mass-expressing BvMTSase and BvMTHase genes in Escherichia coli, and then separating the respective enzyme proteins and confirming them by SDS-PAGE method.

FIG. 6 shows the results of trehalose synthesis using the enzyme proteins isolated from FIG. 5 and the synthesis of trehalose by thin layer chromatography.

FIG. 7 shows trehalose synthesis reaction using maltopentaose (G5) as a substrate, and confirms and quantifies trehalose synthesis by HPLC.

FIG. 8 shows the results of trehalose synthesis using starch as a substrate using each enzyme protein purified in FIG. 5, and the synthesis of trehalose by thin layer chromatography.

FIG. 9 is a result of quantifying the synthesis of trehalose for 72 hours in the reactants of FIG. 8 by HPLC method.

FIG. 10 shows the results of trehalose synthesis using starch as a substrate using a mixture of enzyme proteins and alpha-amylase enzymes purely separated in FIG. 5, and confirming trehalose synthesis by thin layer chromatography.

FIG. 11 is a result of confirming and quantifying the synthesis of trehalose by HPLC method for 72 hours from the reactants of FIG. 10.

The present invention comprises the steps of separating genomic DNA from Brevibacterium; Isolation of trehalose biosynthetic genes from these genomic DNAs; Expression and pure separation of BvMTSase and BvMTVHase genes in E. coli; Synthesizing trehalose using the BvMTSase and BvMTVHase enzyme proteins; And synthesizing trehalose from various starches.

In order to separate genomic DNA from Brevibacterium helvolum ATCC 11822, the method of Samburk et al. Was modified to isolate genomic DNA from Brevibacterium (Sambrook, J. et al., Molecular Cloning: A Laboratory Mannual). , 2nd Ed., Cold Spring Harbor Laboratory, 1989). Brevibacterium cultures were centrifuged to collect Brevibacterium, and reacted after adding lysozyme, 10% SDS, 5M NaCl and CTAB solution (10% CTAB, 0.7M NaCl). After the reaction was completed, a mixed solution of phenol, chloroform and isoamyl alcohol was mixed at 25: 24: 1 parts by weight, and the supernatant was taken by centrifugation, and then the same volume of isopropyl alcohol as the supernatant was added. And precipitated DNA was isolated. RNase A and a proteinase K-containing solution were added to the isolated DNA, and then treated at 37 ° C. for 30 minutes. After the reaction was completed, a mixed solution containing phenol, chloroform and isoamyl alcohol in a weight part of 25: 24: 1 was added thereto, followed by centrifugation to obtain a supernatant. After taking the supernatant, NaCl was added to form a concentration of 200 mM, and then doubled volume of ethyl alcohol was added to precipitate DNA.

The probe used for isolation of trehalose synthetic gene from genomic DNA of Brevibacterium used the trehalose synthase gene of Mycobacterium tuberculosis H37Rv, which the present inventors have previously isolated. To isolate trehalose synthetic genes from Brevibacterium genomic DNA, genomic DNA of Brevibacterium was digested with restriction enzymes of BamHI, EcoRI, HindIII, KpnI and PstI, and then agarose gel electrophoresis, ³² Southern blot analysis was performed using P-labeled Mycobacterium trehalose synthase gene DNA fragment as a probe (Sambrook, J. et al., Molecular Cloning: A Laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory). , 1989). As a result, a DNA fragment of about 4.3kbp hybridized with the probe DNA when digested with EcoRI. 4.3 kbp sized EcoRI DNA fragments were isolated and cloned. As a result, the size of the DNA fragment that determined the base sequence is 4,715bp, encoding two trehalose biosynthesis-related enzymes. The first gene is the Brevibacterium maltooligosyltrehalose synthase (BvMTSase) gene with a structural gene portion of 2,328 bp, and the Brevibacterium maltooligosiltrehalose synthase (BvMTSase) translated therefrom is amino acid 776 It is a protein with a molecular weight of about 85.8kDa consisting of dogs. 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 Brevibacterium maltooligosyl trehalose trehalohydrolase (BvMTHase) gene, whose structural gene is 1,767 bp, and is translated from the Brevibacterium maltooligosyltrehalose hydrolase (BvMTHase). Is a protein having a molecular weight of about 64.2 kDa consisting of 589 amino acids. This enzyme hydrolyzes α (1 → 4) glycosidic bonds between the maltooligolic and trehalose sites of maltooligolic trehalose produced by BvMTSase, resulting in maltooligosaccharides and trehalose with two fewer glucose units. .

To confirm whether trehalose can be synthesized using two enzyme proteins BvMTSase and BvMTHase obtained from the isolated gene, each gene was expressed in Escherichia coli, and the two enzyme proteins expressed were purely separated and assayed for trehalose synthesis. . Each structural gene was isolated and inserted into pRSET plasmid, which is an expression vector in E. coli, to induce the expression of each enzyme protein. Each enzyme protein expressed was purified by Ni ²⁺ -NTA-agarose adsorption gel chromatography, and each enzyme protein was confirmed by SDS-PAGE electrophoresis. In order to assay the trehalose synthesis ability of the two enzyme proteins, trehalose synthesis reaction was carried out using various maltooligosaccharides as substrates. As a result, maltooligosaccharides in odd glucose units were the final reactants trehalose and maltotriose (G3), and maltotriose was no longer hydrolyzed. Maltooligosaccharides, which are even glucose units, were found to be trehalose and maltotetraose (G4), and only a small amount of maltotetraose was converted to trehalose and maltose when maltotetraose was reacted for a long time. When BvMTSase was reacted with maltopentaose, it was confirmed that about 80% of maltopentaose was converted to maltotriosyl trehalose. However, when BvMTHase was reacted with maltopentaose, the enzyme did not react directly with maltopentaose. When maltopentaose was reacted with both BvMTSase and BvMTHase, maltopentaose was converted to trehalose and maltotriose by 100%. Trehalose synthesis was performed using starch as a substrate to see how the two enzyme proteins reacted with starch. As a result, it was confirmed that trehalose was synthesized from starch, and a method for mass production of trehalose from starch was developed by this method.

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. Genomic DNA Isolation from Brevibacterium

To isolate genomic DNA from Brevibacterium helvolum ATCC 11822, Sambrook et al. (Sambrook, J. et al., Molecular Cloning: A Laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory, 1989) Modifications isolated genomic DNA from Brevibacterium. Brevibacterium was incubated with 50 mL of LB medium for 18 hours at 37 ° C. The Brevibacterium was collected by centrifugation of the culture medium, and lysozyme was prepared at a concentration of 2 mg / mL using TE buffer (10 mM Tris-HCl / pH8.0, 1 mM EDTA / pH8.0), and then 5.5mL. Was added. After 1 hour of reaction at 37 ° C., 760 μl of 10% SDS was added and reacted at 65 ° C. for 10 minutes. After adding 1 mL of 5M NaCl and 800 μl of CTAB solution (10% CTAB, 0.7M NaCl) at 65 ° C. The reaction was carried out for 10 minutes. After the reaction, a mixed solution of 8 mL of phenol, chloroform and isoamyl alcohol in a volume ratio of 25: 24: 1 was added thereto, and the supernatant was taken by centrifugation. After adding the same volume of isopropyl alcohol (isopropyl alcohol) to the supernatant slowly mixed well to separate the precipitated DNA. After dissolving the isolated DNA in 500 μl of TE buffer, RNase A was added at a concentration of 10 μg / mL and treated at 37 ° C. for 30 minutes, and a solution containing proteinase K (5 mg / mL) was added. ) 10 μl was added and then treated at 37 ° C. for 30 minutes. After the reaction was completed, a mixed solution containing 500 µl of phenol, chloroform, and isoamyl alcohol in a weight part of 25: 24: 1 was added thereto, and the supernatant was collected by centrifugation. After taking the supernatant, NaCl was added to form a concentration of 200 mM, and then 1 mL of ethyl alcohol was added to precipitate DNA.

Example 2 Isolation of Trehalose Biosynthesis Genes

The probe used for isolation of trehalose synthetic gene from genomic DNA of Brevibacterium used the trehalose synthase gene of Mycobacterium tuberculosis H37Rv, which the present inventors have previously isolated. To isolate trehalose synthetic genes from Brevibacterium genomic DNA, genomic DNA of Brevibacterium was digested with restriction enzymes of BamHI, EcoRI, HindIII, KpnI and PstI, and then agarose gel electrophoresis, ³² Southern blot analysis was performed using P-labeled Mycobacterium trehalose synthase gene DNA fragment as a probe (Sambrook, J. et al., Molecular Cloning: A Laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory). , 1989). Experimental results, as shown in Figure 1, lane M1 is a DNA size marker as a result of cleavage of λ DNA with HindIII, M2 is a DNA size marker as a result of cleavage of pUC18 plasmid DNA with EcoRI and HindIII and lanes 1 to 5 are Brevibac Genomic DNA of terium is digested with restriction enzymes of BamHI, EcoRI, HindIII, KpnI and PstI, respectively. At this time, the DNA fragment of about 4.3kbp was hybridized with the probe DNA when digested with EcoRI.

The 4.3 kbp sized EcoRI DNA fragment was then isolated and inserted into plasmid pUC18 digested with EcoRI. E. coli MC1061 was transformed with the recombinant plasmid thus prepared, and colony hybridization was performed to select positive E. coli. The plasmid was separated from these E. coli, and a restriction enzyme map was prepared to determine the base sequence. The isolated EcoRI DNA fragment was named pBvR4.3, and the base sequence was determined. As a result, pBvR4.3 contained only a portion of the 5 'region of trehalose synthase gene. Southern blot analysis was performed using DNAs treated with other restriction enzymes to isolate whole genes. As a result of cleavage of Brevibacterium genomic DNA with SalI, a DNA fragment of about 6.0 kbp was hybridized with the probe DNA. SalI DNA fragments of 6.0 kbp size were isolated and inserted into plasmid pUC18 digested with SalI. The recombinant plasmid thus prepared was subjected to colony hybridization in the same manner as described above to select positive E. coli. The plasmid was isolated from these E. coli, and a restriction enzyme map was prepared to determine the base sequence. The isolated SalI DNA fragment was named pBvS6.0 and the nucleotide sequence was determined. As a result, pBvS6.0 overlaps about 1 kbp of the 3 'region of pBvR4.0 and includes all 3' regions of trehalose synthase gene. there was.

The relative positions and restriction maps of the DNA fragments corresponding to pBvR4.3 and pBvS6.0 obtained above are shown in FIG. 2. In Figure 2, E represents EcoRI, S represents SalI, B represents BamHI, H represents HindIII restriction sites, and arrows indicate the direction of DNA fragment and sequencing determined.

The nucleotide sequence of the entire trehalose biosynthesis gene and the amino acid sequence translated therefrom are shown in FIG. 3. In FIG. 3, the underlined portion shows the ribosome binding site, the shadowed portion shows the amino acid sequence commonly found in enzymes of the alpha-amylase line, and the (mark) indicates the translation termination site, respectively. As a result, the DNA fragment having the base sequence was 4,715 bp in size, encoding two trehalose biosynthesis related enzymes. The first gene is the Brevibacterium maltooligosyltrehalose synthase (BvMTSase) gene with a structural gene portion of 2,328bp, and the Brevibacterium maltooligosiltrehalose synthase (BvMTSase) translated therefrom is amino acid 776 It is a protein with a molecular weight of about 85.8kDa consisting of dogs. This enzyme converts α (1 → 4) glycosidic bonds at the reduced end of maltooligosaccharide to α (1 → 1) glycosidic bonds to produce maltooligosyltrehalose. The second gene is the Brevibacterium maltooligosyltrehalose trehalohydrolase (BvMTHase) gene whose structural gene portion is 1,767 bp, and the translated Brevbacterium maltooligosyltrehalose hydrolase (BvMTHase) is It is a protein having a molecular weight of about 64.2 kDa consisting of 589 amino acids. This enzyme hydrolyzes α (1 → 4) glycosidic bonds between the maltooligosil and trehalose sites of maltooligosiltrehalose produced by BvMTSase to produce maltooligosaccharides and trehalose with two fewer glucose units. .

Example 3 Expression and Pure Separation of BvMTSase and BvMTHase Genes in Escherichia Coli

In order to confirm whether trehalose can be synthesized using two enzyme proteins BvMTSase and BvMTHase which are translated from the gene isolated in Example 2, each gene is expressed in Escherichia coli, and the two expressed enzyme proteins are purely separated, and then the trehalose sum is combined. Performance was assayed. A cleavage site cut by treatment of the BvMTSase structural gene portion with the restriction enzymes EcoRI and EcoRv was inserted into the pRSET plasmid, which is an expression vector of E. coli, to prepare a pBvMTSase plasmid. The cleaved cleavage site was inserted into the pRSET plasmid, which is an expression vector in Escherichia coli, to prepare a pBvMTHase plasmid (FIG. 4). E. coli BL21 was transformed with each of the recombinant plasmids thus prepared, and cultured for 12 hours, followed by addition of IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 1 mM and incubation for another 4 hours to express each enzyme protein. Induced. The expressed enzyme protein was purified by Ni ²⁺ -NTA-agarose adsorption gel chromatography, and the expressed enzyme protein was subjected to SDS-PAGE electrophoresis. Experimental results, as shown in Figure 5, lane M is the protein molecular weight marker, C is a control experiment to extract the entire protein from E. coli containing only pRSET plasmid and SDS-PAGE electrophoresis, lanes 1, 3 are BvMTSase and PRMTS and pRMTH plasmid containing the BvMTHase gene were induced in E. coli, and the entire protein was extracted and SDS-PAGE electrophoresis was performed. Lanes 2 and 4 are the result of pure separation of each expressed protein using Ni ²⁺ -NTA-agarose adsorption gel chromatography. In this case, when pRMTS and pRMTH plasmids including BvMTSase and BvMTHase genes were induced in E. coli, BvMTSase and BvMTHase enzymes were specifically expressed in large amounts. The present inventors named the transformed Escherichia coli as Escherichia coliBL21 (pVvMTHase) and Escherichia coliBL21 (pVvMTSase), respectively, and deposited with KCTC 0474BP and KCTC 0475BP on May 12, 1998 to the Korea Institute of Science and Technology. Each was deposited.

Example 4 Trehalose Synthesis and Substrate Specificity Using BvMTSase and BvMTHase Enzyme Proteins

In order to investigate trehalose synthesis ability and substrate specificity of the purely isolated BvMTSase and BvMTHase enzyme proteins in Example 3, trehalose synthesis experiments were performed using various maltooligosaccharides as substrates. Under the conditions of 50 mM phosphate buffer solution (pH 7.0), various maltooligosaccharides were added at a concentration of 1 mM, and 500 ng of pure enzyme was added to make the total reaction volume 50 µl. The reaction was carried out at 37 ° C. for 1 hour, treated at 95 ° C. for 10 minutes to terminate the reaction, and then confirmed trehalose synthesis by thin layer chromatography. Experimental results, as shown in Figure 6 lane 1 is a reference material trehalose (trehalose (T), maltotriose (G3), maltotetraose (maltotetraose (G4), maltopentaose (G5), maltohexa) Maltohexaose (G6), maltoheptaose (maltoheptaose, G7) is a mixture. Lanes 2 to 8 are the results of trehalose synthesis experiments using maltotriose, maltotetraose, maltopentose, maltohexaose, maltoheptaose, maltooligosaccharide, and water-soluble starch as substrates, respectively. Maltooligosaccharides (lanes 2, 4, 6), consisting of five or more odd glucose units, were the final reactants trehalose and maltotriose (G3), and maltotriose was no longer hydrolyzed. Malto-oligosaccharides (lanes 3 and 5) consisting of 6 or more even glucose units are the final reactants trehalose and maltotetraose (G4), and only a small amount of trehalose and maltose when maltotetraose is reacted for a long time Switched to However, trehalose synthesis using starch as a substrate resulted in very low levels of trehalose production (lane 8).

In addition, maltopentaose was treated with BvMTSase and BvMTHase enzyme proteins among various maltooligosaccharides used in the above experiment, trehalose synthesis was performed, and then synthesized trehalose was subjected to HPLC method. As a result, as shown in FIG. 7A, when BvMTSase was reacted with maltopentaose, about 80% of maltopentaose (G5) was converted into maltotriosyltrehalose (G3-T) after 1 hour. At this time, Figure 7a is the result of HPLC analysis after the reaction of BvMTSase to maltopentaose, Figure 7b is the result after the reaction of BvMTHase to maltopentaose and Figure 7c is the result after the reaction of both BvMTSase and BvMTHase to maltopentaose. . However, when BvMTHase was reacted with maltopentaose, the enzyme did not directly react with maltopentaose (FIG. 7B). In addition, when both BvMTSase and BvMTHase were reacted with maltopentaose, maltopentaose was 100% converted into trehalose and maltotriose (G3) (FIG. 7C).

Example 5. Trehalose Synthesis from Starch

In order to develop trehalose synthesis method using starch, soluble starch was used as a substrate and the reaction time was extended to 72 hours. Under the conditions of 50mM phosphate buffer solution (pH 7.0), 1% water-soluble starch was added as a substrate, and 8 µg each of purely separated enzymes (BvMTSase and BvMTHase) was added to make the total reaction volume 100µl. The reaction was completed at 37 ° C. for 24, 48, and 72 hours, and the reaction was terminated by treatment at 95 ° C. for 10 minutes, followed by trehalose synthesis using starch as a substrate, and synthesis of trehalose by thin layer chromatography. Confirmed. As a result, as shown in Figure 8 lane M represents a mixture of glucose (glucose, G1), trehalose (T), maltotriose (G3), maltopentaose (G5) as a reference material, In addition, when the trehalose synthesis reaction proceeded up to 72 hours, the amount of trehalose synthesized from starch increased with the reaction time. At this time, the water-soluble starch was treated with purely separated BvMTSase and BvMTHase. In addition, in the above experiments, the 72-hour reaction identified the trehalose synthesized by HPLC method and quantified the amount of trehalose synthesized. As a result, as shown in Figure 9 18.0% of the starch was converted to trehalose after 72 hours.

Example 6 Enhancement of Trehalose Synthesis Efficiency from Starch Using Alpha-amylase

To increase the production efficiency of trehalose from starch, soluble starch was treated with alpha-amylase and the reaction time was extended to 72 hours. Under the conditions of 50mM phosphate buffer solution (pH 7.0), 1% water-soluble starch was added as a substrate, and 0.1 μl of alpha-amylase and 8 μg of purified enzymes (BvMTSase and BvMTHase) were respectively added. Μl. The reaction was carried out at 37 ° C. for 24, 48 and 72 hours and the reaction was terminated by treatment at 95 ° C. for 10 minutes. As a result, as shown in FIG. 10, when the trehalose synthesis reaction was performed after adding alpha-amylase to 72 hours, the amount of trehalose synthesized from starch according to the reaction time was not compared with that without adding alpha-amylase (FIG. 8). Significantly increased. In addition, the reaction product treated with alpha-amylase and reacted for 72 hours to the aqueous starch was identified trehalose synthesized by HPLC method and the amount of synthesized trehalose was quantified. As a result, as shown in FIG. 11, after 72 hours of reaction, 61.9% of starch was converted to trehalose, and the trehalose conversion efficiency was increased by about 344% or more compared to the case where no alpha-amylase was added (FIG. 8).

Therefore, in order to effectively produce trehalose using BvMTSase and BvMTHase used in the present invention, water-soluble starch is first treated with alpha-amylase enzyme to be converted to maltooligosaccharide and then used as a substrate to efficiently produce trehalose in large quantities.

As can be seen from the above embodiment, the present invention has the effect of providing a novel trehalose biosynthesis gene isolated from Brevibacterium helvolum ATCC 11822 and an enzyme protein translated therefrom. In addition, the present invention has the effect of recombining a novel trehalose biosynthetic gene into an expression vector and expressing it in E. coli and purely separating the recombinant protein therefrom, thereby producing a large amount of trehalose from malto-oligosaccharides and starch. Since it is effective to provide a food preservative is a very useful invention in the food industry.

Claims (11)

Trehalose biosynthetic gene having the following nucleotide sequence isolated from Brevibacterium helvolum: 1 GAATTCACCG CCTTCGTGAA CAAGCTGCGC CACGACCACC CCACGTTCCG CCGCAGCCGC 61 TTCTTCGACG GCCGCCCGGT CCGCCGTGGT GAAGGCGAAA AGCTGCCGGA CATCGTCTGG 121 CTGAAGACCG ACGGCACCGA GATGCTGCCC GAGGGACTGG GGGAGTGGCT TCGGCCGGAC 181 CATCGGCGTG TTCTACAACG GTGACGGCAT CCAGGAACAG GATTCCCGCG GCCGCCGGAT 241 CACCGATGAC AGCTTCATCA TGGCCTTCAA CGCCCATGAC GATGCCGTGG ACTTCTGCCT 301 TCCCGCCGAG GAGTACTCCC AGTACTGGGA GGTCCAATTC GATACCGCCG CCCACGCGGA 361 CGACTACCAA CCGCTCAAGG CCGGTGCCAC GCTGAAGCTG GACGCGAAAT CCATGGTGGT 421 CCTGCGCGCC TACTCCGGCC CCGAAGAAGA AGTCGACTAC TCCGCCAGCG GCCTCCATTG 481 CGTCCATGGC CGAACCAGGA AGAAACCCCA CGAAGAAATG GTGGGAAACC CCAAACCAAG 541 GCTGCCGAAA CCCACCAAAG CAAGGGCAAC GGGCGCCGAC GAAAGACGCC CAGGCATGAA 601 GACTCCGGTC TCCACTTACC GCTTTCAAAT CCGCACCAGC TTCACCCTGT TCGACGCCGC 661 TGAACAGGTC CCGTATTTGA AGGACCTCCG CGTCCACTGG GTGTTCCTCT CGCCCATCCT 721 CACCGCGGAA AAAGGTTCGG AACACGGTTA CAACTCAACC GATCCCTCCC CCGTGGACCC 781 CGACCGTGGC GGGCCGAAGG CCCTGCAGGC ATTGTCCAAG GTGGCCCGCA AACACGGAAT 841 GGGCGTCCTG CTGGACATCG TGACCAACCA CGTCGGTGTG GCCACTCCCG TGCAGAATCC 901 CTGGTGGTGG TCCCTGCTCA AGGAGGGCCG CAAATCGCCC TACGCCGAAG CGTTCGACGT 961 CGACTGGGAC CTGGGCGGCG GAAAGGTCCG GCTGCCCATG CTGGGCTCGG ACAACAACCT 1021 GGACAACCTG GAGGTCAAGG ACGGCAAACT CCGCTACTAC AACCACCGGT CGTTCCGGTT 1081 GGGGAAGGAG AACAGGGAAG GCGATTCCCT GCAGGAGGTG CACACCCGCC AGCACTACCA 1141 GCTGATGGAC TGGCGCCGCG CGGACGCCGA GCTGAATTAC CGTCGTTTTT TGGCGGTGAC 1201 CACGCTGGCC GGCATCCGGG TGGAGGAACC GTCTGTCTTC GAGAAGGTTC ATGCCGAGGT 1261 GGGCCGGTGG TTCACCGAGG GCCTGGTGGA CGGGTTCCGC GTGGACCACC CGGACGGATT 1321 CGCCGATCCC GACCGGTACTTCCGGTGGTT CAAGGACGTC AGCGGGGGCG CATACGTCCT 1381 GGTGGAGAAA ATCCTGGAGC CGGGCGAAGT GCTGCCGCAG GACTTCGCCT GCGAAGGCAC 1441 CACCGGATAC GACGCACTGG CTGACGTGGA CCGGGTCTTC GTTGACCCGG CGGGGCAGCA 1501 GGCGCTGGAC GCACTGGATG CTTCCCTGCG GGGCACCTCC GAACCCGCCG ACTACGCCGA 1561 AATGATCCGC GGCACCAAGC GCATGATCGC CGACGGCATC CTGCGCTCCG AGGTGCTGCG 1621 GCTGGCCCGG CTGGTACCTG AATCCCACGG TTTCAGCGTT GACCAGGCAG CGGATGCGAT 1681 CGCGGAAATC ATCGCATCGT TCCCGGTGTA CCGGTCCTAC CTGCCGGTGG GCGCCGACGT 1741 CCTCAAGGAG GCGTGCGAGT CCGCCGCCGC GCACCGGCCG GACCTGGAGG TGGCGGTGGG 1801 AACCCTCCAG CCGCTGCTGC TGGATCCCGC CAAACCCATC GCCATCCGGT TCCAGCAGAC 1861 CTCCGGCATG GTCATGGCCA AGGGCGTGGA GGACACCGCG TTCTACCGCT ACACCCGGCT 1921 GGACACGCTG ACCGAAGTGG GCGCTGAGCC TACCGAGTTC GCCGTGTCTC CGCAGGAGTT 1981 CCACCAGCGG ATGGAGCGCC GTCAGCAGGA GCTGCCGCTG TCCATGACCA CGTTGTCCAC 2041 CCACGACACC AAGCGCAGCG AGGATGCCAG GGCCCGGATC TCGGTCATCG CTGAACTGCC 2101 GGAGGAGTGG GCGGAAACGC TGGCGGAACT GCGTAAACTG GCGCCGATCC CGGACGGCCC 2161 GTTCGAGAAC CTGCTGTGGC AGGCAATCGT CGGCGCCTGG CCGGCAAGCC GGGAACGGCT 2221 TCAGGGTTAC GCCGAAAAGG CAGCCCGGGA GGCCGGCAAC TCCACCAAGT GGACCGACCC 2281 CAACGAGGAC TTCGAGTCCA AGGTGCAGGC CGCCGTCGAT GCAGTCTTCG ACGACGCCAA 2341 GGTCGCCAAG GTTCTCACGG ACTTCGTGGC CCGGATCGCT GCCTTTTCCG CGGCCAACTC 2401 GGTTTCCGCC AAGCTGGTCC AGCTGACCAT GCCCGGCGTG CCTGATGTGT ACCAGGGCAG 2461 CGAACTCTGG GAACGCTCGC TCACGGAACC GGACAACCGC CGGCCCCTGG ACTTCGGTGC 2521 CCGGCAGGAA GCACTGGCAA AGCTCCAACC CCGGTGCCTT GCCCGAACGC GGGCACAGAA 2581 GCGCACCAAG CTTCTGGTCA CCTCGCGGGC ACTGCGCCTG CGCCGGGACC GGCCGGAGCT 2641 GTTCCAGGGG TACTCGCCGG TGAACGCCAG CGGTGCCGCG GCGGACCACC TGCTCGCGTT 2701 CAGCCGCGGA ACAGACGCTG ACTCCGGTGC CCTTACGCTG GCCACCCGGC TCCCCGCCGG 2761 ACTGCAGGCC GGCGGCGGCT GGCGGGACAC CGCCGTCGAC CTTCCCACTG CCATGCGCGA 2821 CGAACTCACC GGGGCCAGCT ACGGACCCGG CCAGGTTTCG GTCGCGGAGG TGCTGGGTAC 2881 CTACCCGGTG GCCCTGCTGG CACCTGTGGA TGGAGAAAAG GCATGACCTT GGTCAACGTT 2941 GGACCCGAAC GCTTTGATGT GTGGGCGCCG GATGTTTCGT CCGTGGTGTT GGTGGCTGAC 3001 GGCCGGCAGT ACCCCATGCA AAAAAAGGAA ACGGCGCCCG GCTCTGAAGG ATGGTGGACG 3061 GCGTCCGACG CCCCCCCGAA CGGTGATGTG GACTACGGCT ACCTGCTGGA CGGCAACACC 3121 ACCCCTGTCC CGGAACCCCG CTCCCGCCGG CTCCCCGCCG GCGTCCACAA TCATTCCCGG 3181 ACCTACAATC CCCCCCCCTA CCGTTGGCAG GATTCCCGGT GGCGCGGCAA GGAACTGCAG 3241 GGAACCCTCA TCTACCAACT CCATGTGGGC ACCTCCACGC CCGATGGGAC CTTGGACGCC 3301 GCAGGGGAGA AGCTCAGCTA CCTGGTGGAC CTGGGCATCG ACTTCATCGA ACTGCTGCCG 3361 GTCAACGGCT TCAACGGAAC CCACAACTGG GGCTACGACG GCGTCCAGTG GTACACCGTC 3421 CACGAAGGCT ATGGCGGCCC TGCTGCGTAC CAGCGGTTCG TCGACGCCGC CCACGCCGCA 3481 GGACTGGGCG TCATCCAGGA CGTGGTGTAC AACCACCTGG GACTTAGGGG CAACTACTTC 3541 CCAAAGTTGG GCCCGAACCT GAAACAGGGC GACGCCAACA CCTTGGGTGA TTCGGTGAAC 3601 TTGGACGGGG CCGGTTCGGA TGTGTTCCGG GAATACATCC TGGACAACGC CGCCCTGTGG 3661 GTGGGGGACT ACCACGTGGA CGGGGTGGGA TTCGATGCCG TGCACGCGGT GCGGGACGAG 3721 AGGGCCGTGC ACATCTTGGA GGACCTGGGA GCCTTGGGCG ACGCTATTTC GGGTGAGACC 3781 GGGCTGCCCA AGACCCTCAT CGCGGAATCG GACTTCAACA ACCCGCGCCT GATCTACCCC 3841 CGCGACGTGA ACGGGTACGG TCTGGCCGGG CAGTGGAGTG ACGACTTCCA CACCGCGGTG 3901 CACGTCAGCG TCAGCGGCGA AACCACCGGT TACTACTCGG ACTTCGAATC CCTTGCCGTG 3961 CTGGCCAAGG TGCTCAAGGA CGGGTTCCTG CACGACGGCA GCTACTCCAG CTTCCGCGGA 4021 CGGCACCACG GCCGGCCCAT CAACCCATCG TTGGCCAACC CGGCGGCGCT GGTGGTCTGC 4081 AACCAGAACC ATGACCAGAT CGGCAACCGG GCCACGGGGG ACAGGCTGTC GCAGTCGCTG 4141 TCCTACGGGC AGCTGGCTGT GGCGGCGGTG CTTACGCTGA CCTCGCCGTT CACGCCCATG 4201 CTGTTCATGG GTGAGGAATA CGGCGCTTCC ACGCCCTGGC AGTTTTTCAC CTCGCACCCC 4261 GAACCGGAGC TTGGTAAGGC CACCGCGGAA GGCCGCATCA AAGAATTCGA GCGCATGGGG 4321 TGGGATCCCG CCGTCGTGCC TGACCCGCAG GACCCGGAAA CCTTCAACCG CTCCAAGCTG 4381 GACTGGTCCG AGGCCTCCAC GGGTGACCAT GCGCGGCTGC TGGAGCTGTA CAAGTCGCTG 4441 ACGGCGCTGC GCCGCGAGCA TCCGGACCTG GCAGATCTCG GCTTTGGCCA GACGGAGGTT 4501 TCGTTCGACG ACGACGCCGG CTGGCTGCGC TTCAGGCCGG TCTCCGTGGA GGTGCTCGTG 4561 AACCTGTCAG ACGCCAAGGT ACGGCTGGAT GATGCGGCAG GTGACCTCCT TCTGGCCACG 4621 GACGAAGGGA ACCCTCTGGA CGGCGGGTCC CTCGCCCTGG TGCCGTGGAG TGCCGCGGTC 4681 CTCAAGTCCT GAATCAGCGG TTACCCGGGG GCATG The trehalose biosynthesis gene according to claim 1, wherein the trehalose biosynthesis gene is isolated by cleaving the Brevibacterium genomic DNA with restriction enzymes EcoRI and SalI. Structural genes of maltooligosiltrehalose synthetase (BvMTSase) having the following nucleotide sequence isolated from Brevibacterium helvolum: 1 ATG AAG ACT CCG GTC TCC ACT TAC CGC TTT CAA ATC CGC ACC AGC TTC 49 ACC CTG TTC GAC GCC GCT GAA CAG GTC CCG TAT TTG AAG GAC CTC CGC 97 GTC CAC TGG GTG TTC CTC TCG CCC ATC CTC ACC GCG GAA AAA GGT TCG 145 GAA CAC GGT TAC AAC TCA ACC GAT CCC TCC CCC GTG GAC CCC GAC CGT 193 GGC GGG CCG AAG GCC CTG CAG GCA TTG TCC AAG GTG GCC CGC AAA CAC 241 GGA ATG GGC GTC CTG CTG GAC ATC GTG ACC AAC CAC GTC GGT GTG GCC 289 ACT CCC GTG CAG AAT CCC TGG TGG TGG TCC CTG CTC AAG GAG GGC CGC 337 AAA TCG CCC TAC GCC GAA GCG TTC GAC GTC GAC TGG GAC CTG GGC GGC 385 GGA AAG GTC CGG CTG CCC ATG CTG GGC TCG GAC AAC AAC CTG GAC AAC 433 CTG GAG GTC AAG GAC GGC AAA CTC CGC TAC TAC AAC CAC CGG TCG TTC 481 CGG TTG GGG AAG GAG AAC AGG GAA GGC GAT TCC CTG CAG GAG GTG CAC 529 ACC CGC CAG CAC TAC CAG CTG ATG GAC TGG CGC CGC GCG GAC GCC GAG 577 CTG AAT TAC CGT CGT TTT TTG GCG GTG ACC ACG CTG GCC GGC ATC CGG 625 GTG GAG GAA CCG TCT GTC TTC GAG AAG GTT CAT GCC GAG GTG GGC CGG 673 TGG TTC ACC GAG GGC CTG GTG GAC GGG TTC CGC GTG GAC CAC CCG GAC 721 GGA TTC GCC GAT CCC GAC CGG TAC TTC CGG TGG TTC AAG GAC GTC AGC 769 GGG GGC GCA TAC GTC CTG GTG GAG AAA ATC CTG GAG CCG GGC GAA GTG 817 CTG CCG CAG GAC TTC GCC TGC GAA GGC ACC ACC GGA TAC GAC GCA CTG 865 GCT GAC GTG GAC CGG GTC TTC GTT GAC CCG GCG GGG CAG CAG GCG CTG 913 GAC GCA CTG GAT GCT TCC CTG CGG GGC ACC TCC GAA CCC GCC GAC TAC 961 GCC GAA ATG ATC CGC GGC ACC AAG CGC ATG ATC GCC GAC GGC ATC CTG 1009 CGC TCC GAG GTG CTG CGG CTG GCC CGG CTG GTA CCT GAA TCC CAC GGT 1057 TTC AGC GTT GAC CAG GCA GCG GAT GCG ATC GCG GAA ATC ATC GCA TCG 1105 TTC CCG GTG TAC CGG TCC TAC CTG CCG GTG GGC GCC GAC GTC CTC AAG 1153 GAG GCG TGC GAG TCC GCC GCC GCG CAC CGG CCG GAC CTG GAG GTG GCG 1201 GTG GGA ACC CTC CAG CCG CTG CTG CTG GAT CCC GCC AAA CCC ATC GCC 1249 ATC CGG TTC CAG CAG ACC TCC GGC ATG GTC ATG GCC AAG GGC GTG GAG 1297 GAC ACC GCG TTC TAC CGC TAC ACC CGG CTG GAC ACG CTG ACC GAA GTG 1345 GGC GCT GAG CCT ACC GAG TTC GCC GTG TCT CCG CAG GAG TTC CAC CAG 1393 CGG ATG GAG CGC CGT CAG CAG GAG CTG CCG CTG TCC ATG ACC ACG TTG 1441 TCC ACC CAC GAC ACC AAG CGC AGC GAG GAT GCC AGG GCC CGG ATC TCG 1489 GTC ATC GCT GAA CTG CCG GAG GAG TGG GCG GAA ACG CTG GCG GAA CTG 1537 CGT AAA CTG GCG CCG ATC CCG GAC GGC CCG TTC GAG AAC CTG CTG TGG 1585 CAG GCA ATC GTC GGC GCC TGG CCG GCA AGC CGG GAA CGG CTT CAG GGT 1633 TAC GCC GAA AAG GCA GCC CGG GAG GCC GGC AAC TCC ACC AAG TGG ACC 1681 GAC CCC AAC GAG GAC TTC GAG TCC AAG GTG CAG GCC GCC GTC GAT GCA 1729 GTC TTC GAC GAC GCC AAG GTC GCC AAG GTT CTC ACG GAC TTC GTG GCC 1777 CGG ATC GCT GCC TTT TCC GCG GCC AAC TCG GTT TCC GCC AAG CTG GTC 1825 CAG CTG ACC ATG CCC GGC GTG CCT GAT GTG TAC CAG GGC AGC GAA CTC 1873 TGG GAA CGC TCG CTC ACG GAA CCG GAC AAC CGC CGG CCC CTG GAC TTC 1921 GGT GCC CGG CAG GAA GCA CTG GCA AAG CTC CAA CCC CGG TGC CTT GCC 1969 CGA ACG CGG GCA CAG AAG CGC ACC AAG CTT CTG GTC ACC TCG CGG GCA 2017 CTG CGC CTG CGC CGG GAC CGG CCG GAG CTG TTC CAG GGG TAC TCG CCG 2065 GTG AAC GCC AGC GGT GCC GCG GCG GAC CAC CTG CTC GCG TTC AGC CGC 2113 GGA ACA GAC GCT GAC TCC GGT GCC CTT ACG CTG GCC ACC CGG CTC CCC 2161 GCC GGA CTG CAG GCC GGC GGC GGC TGG CGG GAC ACC GCC GTC GAC CTT 2209 CCC ACT GCC ATG CGC GAC GAA CTC ACC GGG GCC AGC TAC GGA CCC GGC 2257 CAG GTT TCG GTC GCG GAG GTG CTG GGT ACC TAC CCG GTG GCC CTG CTG 2305 GCA CCT GTG GAT GGA GAA AAG GCA TGA The pBvMTSase plasmid of claim 3 prepared by treating the maltooligosiltrehalose biosynthetic enzyme (BvMTSase) gene with restriction enzymes EcoRI and EcoRV and inserting into EcoRI, PvuII sites of the pRSET plasmid. According to claim 3, Maltooligosiltrehalose biosynthesis (BvMTSase) protein having the following amino acid sequence translated from the structural gene: 1 Met Lys Thr Pro Val Ser Thr Tyr Arg Phe Gln Ile Arg Thr Ser Phe 17 Thr Leu Phe Asp Ala Ala Glu Gln Val Pro Tyr Leu Lys Asp Leu Arg 33 Val His Trp Val Phe Leu Ser Pro Ile Leu Thr Ala Glu Lys Gly Ser 49 Glu His Gly Tyr Asn Ser Thr Asp Pro Ser Pro Val Asp Pro Asp Arg 65 Gly Gly Pro Lys Ala Leu Gln Ala Leu Ser Lys Val Ala Arg Lys His 81 Gly Met Gly Val Leu Leu Asp Ile Val Thr Asn His Val Gly Val Ala 97 Thr Pro Val Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg 113 Lys Ser Pro Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu Gly Gly 129 Gly Lys Val Arg Leu Pro Met Leu Gly Ser Asp Asn Asn Leu Asp Asn 145 Leu Glu Val Lys Asp Gly Lys Leu Arg Tyr Tyr Asn His Arg Ser Phe 161 Arg Leu Gly Lys Glu Asn Arg Glu Gly Asp Ser Leu Gln Glu Val His 177 Thr Arg Gln His Tyr Gln Leu Met Asp Trp Arg Arg Ala Asp Ala Glu 193 Leu Asn Tyr Arg Arg Phe Leu Ala Val Thr Thr Leu Ala Gly Ile Arg 209 Val Glu Glu Pro Ser Val Phe Glu Lys Val His Ala Glu Val Gly Arg 225 Trp Phe Thr Glu Gly Leu Val Asp Gly Phe Arg Val Asp His Pro Asp 241 Gly Phe Ala Asp Pro Asp Arg Tyr Phe Arg Trp Phe Lys Asp Val Ser 257 Gly Gly Ala Tyr Val Leu Val Glu Lys Ile Leu Glu Pro Gly Glu Val 273 Leu Pro Gln Asp Phe Ala Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu 289 Ala Asp Val Asp Arg Val Phe Val Asp Pro Ala Gly Gln Gln Ala Leu 305 Asp Ala Leu Asp Ala Ser Leu Arg Gly Thr Ser Glu Pro Ala Asp Tyr 321 Ala Glu Met Ile Arg Gly Thr Lys Arg Met Ile Ala Asp Gly Ile Leu 337 Arg Ser Glu Val Leu Arg Leu Ala Arg Leu Val Pro Glu Ser His Gly 353 Phe Ser Val Asp Gln Ala Ala Asp Ala Ile Ala Glu Ile Ile Ala Ser 369 Phe Pro Val Tyr Arg Ser Tyr Leu Pro Val Gly Ala Asp Val Leu Lys 385 Glu Ala Cys Glu Ser Ala Ala Ala His Arg Pro Asp Leu Glu Val Ala 401 Val Gly Thr Leu Gln Pro Leu Leu Leu Asp Pro Ala Lys Pro Ile Ala 417 Ile Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu 433 Asp Thr Ala Phe Tyr Arg Tyr Thr Arg Leu Asp Thr Leu Thr Glu Val 449 Gly Ala Glu Pro Thr Glu Phe Ala Val Ser Pro Gln Glu Phe His Gln 465 Arg Met Glu Arg Arg Gln Gln Glu Leu Pro Leu Ser Met Thr Thr Leu 481 Ser Thr His Asp Thr Lys Arg Ser Glu Asp Ala Arg Ala Arg Ile Ser 497 Val Ile Ala Glu Leu Pro Glu Glu Trp Ala Glu Thr Leu Ala Glu Leu 513 Arg Lys Leu Ala Pro Ile Pro Asp Gly Pro Phe Glu Asn Leu Leu Trp 529 Gln Ala Ile Val Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Gly 545 Tyr Ala Glu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Lys Trp Thr 561 Asp Pro Asn Glu Asp Phe Glu Ser Lys Val Gln Ala Ala Val Asp Ala 577 Val Phe Asp Asp Ala Lys Val Ala Lys Val Leu Thr Asp Phe Val Ala 593 Arg Ile Ala Ala Phe Ser Ala Ala Asn Ser Val Ser Ala Lys Leu Val 609 Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Ser Glu Leu 625 Trp Glu Arg Ser Leu Thr Glu Pro Asp Asn Arg Arg Pro Leu Asp Phe 641 Gly Ala Arg Gln Glu Ala Leu Ala Lys Leu Gln Pro Arg Cys Leu Ala 657 Arg Thr Arg Ala Gln Lys Arg Thr Lys Leu Leu Val Thr Ser Arg Ala 673 Leu Arg Leu Arg Arg Asp Arg Pro Glu Leu Phe Gln Gly Tyr Ser Pro 689 Val Asn Ala Ser Gly Ala Ala Ala Asp His Leu Leu Ala Phe Ser Arg 705 Gly Thr Asp Ala Asp Ser Gly Ala Leu Thr Leu Ala Thr Arg Leu Pro 721 Ala Gly Leu Gln Ala Gly Gly Gly Trp Arg Asp Thr Ala Val Asp Leu 737 Pro Thr Ala Met Arg Asp Glu Leu Thr Gly Ala Ser Tyr Gly Pro Gly 753 Gln Val Ser Val Ala Glu Val Leu Gly Thr Tyr Pro Val Ala Leu Leu 769 Ala Pro Val Asp Gly Glu Lys Ala *** Structural genes of maltooligosiltrehalose hydrolase (BvMTHase) having the following base sequence isolated from Brevibacterium helvolum: 1 ATG ACC TTG GTC AAC GTT GGA CCC GAA CGC TTT GAT GTG TGG GCG CCG 49 GAT GTT TCG TCC GTG GTG TTG GTG GCT GAC GGC CGG CAG TAC CCC ATG 97 CAA AAA AAG GAA ACG GCG CCC GGC TCT GAA GGA TGG TGG ACG GCG TCC 145 GAC GCC CCC CCG AAC GGT GAT GTG GAC TAC GGC TAC CTG CTG GAC GGC 193 AAC ACC ACC CCT GTC CCG GAA CCC CGC TCC CGC CGG CTC CCC GCC GGC 241 GTC CAC AAT CAT TCC CGG ACC TAC AAT CCC CCC CCC TAC CGT TGG CAG 289 GAT TCC CGG TGG CGC GGC AAG GAA CTG CAG GGA ACC CTC ATC TAC CAA 337 CTC CAT GTG GGC ACC TCC ACG CCC GAT GGG ACC TTG GAC GCC GCA GGG 385 GAG AAG CTC AGC TAC CTG GTG GAC CTG GGC ATC GAC TTC ATC GAA CTG 433 CTG CCG GTC AAC GGC TTC AAC GGA ACC CAC AAC TGG GGC TAC GAC GGC 481 GTC CAG TGG TAC ACC GTC CAC GAA GGC TAT GGC GGC CCT GCT GCG TAC 529 CAG CGG TTC GTC GAC GCC GCC CAC GCC GCA GGA CTG GGC GTC ATC CAG 577 GAC GTG GTG TAC AAC CAC CTG GGA CTT AGG GGC AAC TAC TTC CCA AAG 625 TTG GGC CCG AAC CTG AAA CAG GGC GAC GCC AAC ACC TTG GGT GAT TCG 673 GTG AAC TTG GAC GGG GCC GGT TCG GAT GTG TTC CGG GAA TAC ATC CTG 721 GAC AAC GCC GCC CTG TGG GTG GGG GAC TAC CAC GTG GAC GGG GTG GGA 769 TTC GAT GCC GTG CAC GCG GTG CGG GAC GAG AGG GCC GTG CAC ATC TTG 817 GAG GAC CTG GGA GCC TTG GGC GAC GCT ATT TCG GGT GAG ACC GGG CTG 865 CCC AAG ACC CTC ATC GCG GAA TCG GAC TTC AAC AAC CCG CGC CTG ATC 913 TAC CCC CGC GAC GTG AAC GGG TAC GGT CTG GCC GGG CAG TGG AGT GAC 961 GAC TTC CAC ACC GCG GTG CAC GTC AGC GTC AGC GGC GAA ACC ACC GGT 1009 TAC TAC TCG GAC TTC GAA TCC CTT GCC GTG CTG GCC AAG GTG CTC AAG 1057 GAC GGG TTC CTG CAC GAC GGC AGC TAC TCC AGC TTC CGC GGA CGG CAC 1105 CAC GGC CGG CCC ATC AAC CCA TCG TTG GCC AAC CCG GCG GCG CTG GTG 1153 GTC TGC AAC CAG AAC CAT GAC CAG ATC GGC AAC CGG GCC ACG GGG GAC 1201 AGG CTG TCG CAG TCG CTG TCC TAC GGG CAG CTG GCT GTG GCG GCG GTG 1249 CTT ACG CTG ACC TCG CCG TTC ACG CCC ATG CTG TTC ATG GGT GAG GAA 1297 TAC GGC GCT TCC ACG CCC TGG CAG TTT TTC ACC TCG CAC CCC GAA CCG 1345 GAG CTT GGT AAG GCC ACC GCG GAA GGC CGC ATC AAA GAA TTC GAG CGC 1393 ATG GGG TGG GAT CCC GCC GTC GTG CCT GAC CCG CAG GAC CCG GAA ACC 1441 TTC AAC CGC TCC AAG CTG GAC TGG TCC GAG GCC TCC ACG GGT GAC CAT 1489 GCG CGG CTG CTG GAG CTG TAC AAG TCG CTG ACG GCG CTG CGC CGC GAG 1537 CAT CCG GAC CTG GCA GAT CTC GGC TTT GGC CAG ACG GAG GTT TCG TTC 1585 GAC GAC GAC GCC GGC TGG CTG CGC TTC AGG CCG GTC TCC GTG GAG GTG 1633 CTC GTG AAC CTG TCA GAC GCC AAG GTA CGG CTG GAT GAT GCG GCA GGT 1681 GAC CTC CTT CTG GCC ACG GAC GAA GGG AAC CCT CTG GAC GGC GGG TCC 1729 CTC GCC CTG GTG CCG TGG AGT GCC GCG GTC CTC AAG TCC TGA 7. The pBvMTHase plasmid of claim 6 prepared by treating the maltooligosiltrehalose hydrolase (BvMTHase) gene with restriction enzymes Hind III and KpnI and inserting into Hind III, KpnI sites of the pRSET plasmid. According to claim 6, Maltooligosiltrehalose hydrolase (BvMTHase) protein having the following amino acid sequence translated from the structural gene: 1 Met Thr Leu Val Asn Val Gly Pro Glu Arg Phe Asp Val Trp Ala Pro 17 Asp Val Ser Ser Val Val Leu Val Ala Asp Gly Arg Gln Tyr Pro Met 33 Gln Lys Lys Glu Thr Ala Pro Gly Ser Glu Gly Trp Trp Thr Ala Ser 49 Asp Ala Pro Pro Asn Gly Asp Val Asp Tyr Gly Tyr Leu Leu Asp Gly 65 Asn Thr Thr Pro Val Pro Glu Pro Arg Ser Arg Arg Leu Pro Ala Gly 81 Val His Asn His Ser Arg Thr Tyr Asn Pro Pro Pro Tyr Arg Trp Gln 97 Asp Ser Arg Trp Arg Gly Lys Glu Leu Gln Gly Thr Leu Ile Tyr Gln 113 Leu His Val Gly Thr Ser Thr Pro Asp Gly Thr Leu Asp Ala Ala Gly 129 Glu Lys Leu Ser Tyr Leu Val Asp Leu Gly Ile Asp Phe Ile Glu Leu 145 Leu Pro Val Asn Gly Phe Asn Gly Thr His Asn Trp Gly Tyr Asp Gly 161 Val Gln Trp Tyr Thr Val His Glu Gly Tyr Gly Gly Pro Ala Ala Tyr 177 Gln Arg Phe Val Asp Ala Ala His Ala Ala Gly Leu Gly Val Ile Gln 193 Asp Val Val Tyr Asn His Leu Gly Leu Arg Gly Asn Tyr Phe Pro Lys 209 Leu Gly Pro Asn Leu Lys Gln Gly Asp Ala Asn Thr Leu Gly Asp Ser 225 Val Asn Leu Asp Gly Ala Gly Ser Asp Val Phe Arg Glu Tyr Ile Leu 241 Asp Asn Ala Ala Leu Trp Val Gly Asp Tyr His Val Asp Gly Val Gly 257 Phe Asp Ala Val His Ala Val Arg Asp Glu Arg Ala Val His Ile Leu 273 Glu Asp Leu Gly Ala Leu Gly Asp Ala Ile Ser Gly Glu Thr Gly Leu 289 Pro Lys Thr Leu Ile Ala Glu Ser Asp Phe Asn Asn Pro Arg Leu Ile 305 Tyr Pro Arg Asp Val Asn Gly Tyr Gly Leu Ala Gly Gln Trp Ser Asp 321 Asp Phe His Thr Ala Val His Val Ser Val Ser Gly Glu Thr Thr Gly 337 Tyr Tyr Ser Asp Phe Glu Ser Leu Ala Val Leu Ala Lys Val Leu Lys 353 Asp Gly Phe Leu His Asp Gly Ser Tyr Ser Ser Phe Arg Gly Arg His 369 His Gly Arg Pro Ile Asn Pro Ser Leu Ala Asn Pro Ala Ala Leu Val 385 Val Cys Asn Gln Asn His Asp Gln Ile Gly Asn Arg Ala Thr Gly Asp 401 Arg Leu Ser Gln Ser Leu Ser Tyr Gly Gln Leu Ala Val Ala Ala Val 417 Leu Thr Leu Thr Ser Pro Phe Thr Pro Met Leu Phe Met Gly Glu Glu 433 Tyr Gly Ala Ser Thr Pro Trp Gln Phe Phe Thr Ser His Pro Glu Pro 449 Glu Leu Gly Lys Ala Thr Ala Glu Gly Arg Ile Lys Glu Phe Glu Arg 465 Met Gly Trp Asp Pro Ala Val Val Pro Asp Pro Gln Asp Pro Glu Thr 481 Phe Asn Arg Ser Lys Leu Asp Trp Ser Glu Ala Ser Thr Gly Asp His 497 Ala Arg Leu Leu Glu Leu Tyr Lys Ser Leu Thr Ala Leu Arg Arg Glu 513 His Pro Asp Leu Ala Asp Leu Gly Phe Gly Gln Thr Glu Val Ser Phe 529 Asp Asp Asp Ala Gly Trp Leu Arg Phe Arg Pro Val Ser Val Glu Val 545 Leu Val Asn Leu Ser Asp Ala Lys Val Arg Leu Asp Asp Ala Ala Gly 561 Asp Leu Leu Leu Ala Thr Asp Glu Gly Asn Pro Leu Asp Gly Gly Ser 577 Leu Ala Leu Val Pro Trp Ser Ala Ala Val Leu Lys Ser *** Enzyme protein production method characterized in that for producing the enzyme protein corresponding to each using the gene of maltooligosiltrehalose synthase (BvMTSase) or maltooligosiltrehalose hydrolase (BvMTHase). 10. The method according to claim 9, wherein the two enzyme proteins are used to produce trehalose from starch. The method for producing trehalose according to claim 9 or 10, wherein the starch is treated with alpha-amylase in producing trehalose from the starch using the two enzyme proteins.