KR20010010091A - Novel trehalose synthase fusion gene and fusion protein and process for preparation of trehalose therefrom - Google Patents

Abstract

PURPOSE: A fusion gene of trehalose synthase, fusion enzyme protein and a method for producing trehalose using the same are provided. The trehalose is effectively produced in higher yield using a fusion gene of BvMTSase and BvMTHase gene that code trehalose biosynthase. CONSTITUTION: The fusion gene is produced by fusing the BvMTSase gene and BvMTHase gene isolated from Brevibacterium helvolum, in which the BvMTSase gene is 2,328bp of Brevibacterium maltooligosyltrehalose synthase gene, and the BvMTHase gene is 1,767bp of Brevibacterium maltooligosyl trehalose trehalohydrolase gene. The method for producing trehalose comprises the steps of: fusing the BvMTSase gene and BvMTHase gene to produce the BvMTSHase gene; inserting the BvMTSHase gene into E. coli expression vector pRSET; transforming E. coli with the transformed expression vector and incubating the transformant for 12 hours; inducing the expression of fusion enzyme by adding IPTG and incubating for 4 hours; and isolating and purifying the fusion enzyme by using Ni2+-NTA-agarose gel and SDS-PAGE; and producing trehalose from soluble starch by using the fusion enzyme BvMTSHase.

Description

Novel trehalose synthase fusion gene and fusion protein and process for preparation of trehalose therefrom

The present invention relates to a method for fusion of trehalose biosynthetic enzyme gene isolated from Brevibacterium helvolum to produce a new gene, and to produce trehalose using a new trehalose biosynthetic fusion enzyme expressed therefrom. More specifically, two genes encoding trehalose biosynthesis expressed in Brevibacterium helvolum ATCC 11822 were fused at the gene level to prepare a single gene, and the transhalo biosynthetic fusion enzyme translated therefrom and using the same To effectively produce trehalose in various substrates, especially starch.

Trehalose (α-D-glucopyranosyl- [1-1] -α-D-glucopyranose) is a non-reducing disaccharide found in insects, bacteria, fungi, yeasts and some plants (see Elbein, Adv. Carbohydr. Chem. Biochem). , 30: 227-256, (1974)). Trehalose is also a storage form of reducible carbohydrates, but it is believed to play a role in protecting the cells from adverse effects resulting from various types of undesirable physical and chemical external impacts, primarily depletion of nutrients, high temperatures, drying, oxygen deficiency, and osmotic pressure (see : Eleutherio et al., Cryobiology, 30: 591-596, (1993)). Trehalose maintains the cell structure by incorporating hydrogen and phospholipids of proteins and cell membranes during drying, while maintaining the intrinsic flavor, color and texture of foods (Crowe et al., Science, 223). : 701-703, (1984)). Therefore, in the case of dried food, not only can be used as a food additive for preserving color and flavor, but also it can improve the shelf life of the food by reducing the available moisture. Trehalose biosynthetic reaction pathways are best known in yeast and Escherichia coli, whose genes have already been isolated and their structure 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 synthetase activity in the back muscle was determined (Mueller et al., Physiol. Plant, 90: 86-92, (1994)). 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. Therefore, in this study, in order to achieve the purpose of isolating new genes that biosynthesize trehalose, and to increase the efficiency of trehalose synthesis using enzymes obtained by mass expression of this gene, Brevibacterium helvolum ATCC 11822 Trehalose biosynthesis gene was isolated.

The present inventors found that Brevibacterium helvolum ATCC 11822 produced trehalose during the study of trehalose biosynthesis enzymes, and the trehalose biosynthesis present in Brevibacterium was associated with α (1 → 4) binding. It has the property of synthesizing trehalose from the reduced end of several linked maltooligosaccharides, and has already isolated its genes. The first gene is the Brevibacterium maltooligosyltrehalose synthase (BvMTSase) gene, and the translated Brevbacterium maltooligosyltrehalose synthase (BvMTSase) is a reduced end of maltooligosaccharide. The α (1 → 4) glycosidic bonds present are converted to α (1 → 1) glycosidic bonds to produce maltooligosyl trehalose. The second gene is the Brevibacterium maltooligosyl trehalose trehalohydrolase (BvMTHase) gene, from which the Brevibacterium maltooligosyl trehalose hydrolase (BvMTHase) is produced by BvMTSase. Hydrolysis of the α (1 → 4) glycosidic linkage between the maltooligosil site and the trehalose site of oligosil trehalose yields maltooligosaccharide and trehalose with two fewer glucose units. However, they are composed of two genes, and for the production of trehalose, two enzymes must be produced and reacted, respectively.

When one or more enzymes are used as above, each enzyme is produced and reacted to produce the final product. Since each enzyme must be produced at a constant concentration necessary for the reaction, there may be a limitation in the amount of the final product. It is difficult to maintain a positive consistency. The recent development of genetic recombination technology allows the fusion of two genes into a single gene, whereby two enzymes are fused into one enzyme protein. In many cases, however, fusion proteins are made from fusion genes, but fusion proteins have two enzyme activities at the same time, and the reaction rate is superior to each enzyme reaction. In the present invention, two genes were prepared as one new gene using a genetic manipulation method, and 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 one step compared to the case of synthesizing trehalose using each enzyme, and the trehalose synthesis efficiency was higher than that of each enzyme. Increased.

The main object of the present invention was to fuse two genes of trehalose biosynthesis isolated from Brevibacterium helvolum ATCC 11822 into a new gene, and to provide new trehalose biosynthetic fusion enzyme proteins obtained from this fusion gene. will be. Still another object of the present invention is to provide a method for effectively synthesizing trehalose using a fusion enzyme protein obtained from a synthetic fusion gene through the present invention.

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

1 is a diagram illustrating a method for producing a BvMTSHase fusion gene by fusing the BvMTSase gene and the BvMTHase gene.

Figure 2 shows the nucleotide sequence of the BvMTSHase fusion gene and the amino acid sequence of the fusion enzyme protein translated therefrom.

3 is a result of mass expression of BvMTSHase fusion gene in Escherichia coli, and then the fusion enzyme protein was isolated and confirmed by SDS-PAGE method.

FIG. 4 shows the results of trehalose synthesis using the fusion enzyme protein isolated from FIG. 3 and the synthesis of trehalose by thin layer chromatography.

FIG. 5 shows the results of trehalose synthesis using starch as a substrate using the fusion enzyme protein purely separated from FIG. 3 and confirming trehalose synthesis by thin layer chromatography.

FIG. 6 shows the results of trehalose synthesis using starch as a substrate using the fusion enzyme protein in the reaction of FIG. 5, and the reaction was confirmed and quantified by the HPIC method for 24 hours.

FIG. 7 is a result of trehalose synthesis reaction using starch as a substrate using a fusion enzyme protein and alpha-amylase enzyme in the reaction of FIG. 5, and the reaction was confirmed and quantified by the HPIC method for 24 hours.

The present invention comprises the steps of gene fusion; Expression and pure separation of the fusion protein from the fusion gene; Trehalose synthesis using a fusion protein; And synthesizing trehalose from starch.

We have already isolated two trehalose biosynthetic enzyme genes from Brevibacterium helvolum ATCC 11822. 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. . However, they are composed of two genes, and for the production of trehalose, the two enzymes must be produced separately and produced in different reactions. Therefore, trehalose production efficiency is thought to be lower than that of using one enzyme. Therefore, in the present invention, two genes were prepared as one gene by using polymerase chain reaction (PCR) and gene recombination method, and trehalose biosynthesis synthesized therefrom was prepared as one fusion enzyme (BvMTSHase).

In order to confirm whether trehalose can be synthesized using BvMTSHase obtained by translation from the fusion gene, the fusion gene was expressed in Escherichia coli, and the purified fusion enzyme protein was purified purely and assayed for trehalose synthesis. The structural gene portion of the fusion gene was isolated and inserted into the pRSET plasmid, an expression vector in E. coli, to induce the expression of the fusion enzyme protein. The expressed fusion enzyme protein was purified by Ni ²⁺ -NTA-agarose adsorption gel chromatography, and the fusion enzyme protein was confirmed by SDS-PAGE electrophoresis. In order to assay the trehalose synthesis ability of the isolated fusion enzyme protein, 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. In order to examine how the purely isolated fusion enzyme protein reacts with soluble starch, trehalose synthesis was performed using starch as a substrate. As a result, it was confirmed that trehalose was synthesized from starch, and in this case, it was confirmed that the yield of trehalose was higher than that of the reaction using each enzyme protein. Therefore, this method was developed to mass produce trehalose from starch.

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 Genes

We have already isolated two trehalose biosynthetic enzyme genes from Brevibacterium helvolum ATCC 11822. 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 maltooligosyltrehalose. The second gene is the Brevibacterium maltooligosyltrehalose trehalohydrolase (BvMTHase) gene, whose structural gene portion is 1,767 bp, from which the Brevibacterium maltooligosiltrehalase hydrolase (BvMTHase) 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 maltooligolic and trehalose sites of maltooligolic trehalose produced by BvMTSase, resulting in maltooligosaccharides and trehalose with two fewer glucose units. . However, they are composed of two genes, and for the production of trehalose, two enzymes must be produced and produced in different reactions. Therefore, in the present invention, two genes were prepared as one gene by using polymerase chain reaction (PCR) and gene recombination method, and trehalose biosynthesis synthesized therefrom was prepared as one fusion enzyme (BvMTSHase).

Figure 1 shows a process for producing a single BvMTSHase fusion gene for the BvMTSase and BvMTHase gene using polymerase chain reaction (PCR) and genetic recombination techniques. The BvMTSase and BvMTHase genes have a gene structure overlapping one base. When the gene is expressed, the BvMTSase enzyme protein can be expressed as a protein, but the BvMTHase enzyme protein produces a completely different protein, and also intermediates the protein synthesis. This incomplete enzyme protein is synthesized. Therefore, there is a difficulty in preparing each enzyme protein by separating each gene. In order to prepare a single fusion gene that does not overlap two genes, oligonucleotide primers as shown in FIG. 1 were synthesized, and a fusion gene in which one more A base was inserted was prepared by using a PCR method. The BvMTSHase fusion gene has a structure of a fusion gene in which the translation end region of the BvMTSase gene is lost and the translation start region of the BvMTHase gene is linked after the last amino acid of the BvMTSase gene. Figure 2 shows the nucleotide sequence of the prepared BvMTSHase fusion gene and the amino acid sequence translated therefrom. The structural gene portion of the trehalose biosynthetic enzyme fusion gene prepared in the present invention is 4,095 bp, and the two enzyme proteins are encoded in a fused form. The first gene region of the fusion gene is the BvMTSase gene portion, and the second gene region is the BvMTHase gene. The fusion enzyme protein translated therefrom is maltooligosiltrehalose synthetase (BvMTSHase), which is a fusion enzyme protein having a molecular weight of about 150 KDa composed of 1,365 amino acids.

Example 2 Expression and Pure Separation of BvMTSHase Fusion Gene in Escherichia Coli

In order to confirm whether trehalose can be synthesized using the BvMTSHase fusion enzyme protein translated from the synthetic fusion gene in Example 1, the fusion gene is expressed in E. coli, the expressed fusion enzyme protein is purely isolated, and the trehalose synthesis ability is assayed. It was. The structural gene portion of the fusion gene was isolated and inserted into the pRSET plasmid, which is an expression vector in E. coli. E. coli BL21 was transformed with the recombinant plasmid thus prepared (pRBvMTSH), incubated for 12 hours, and IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 1 mM and incubated for 4 hours to express the fusion enzyme protein. Induced. The expressed fusion enzyme protein was purified by Ni ²⁺ -NTA-agarose adsorption gel chromatography. 3 is a result confirmed by SDS-PAGE electrophoresis of the expressed fusion enzyme protein. FIG. 3 is a protein molecular weight marker, and 1,2 of FIG. 3 induces and expresses pRBvMTH and pRBvMTS plasmids including BvMTHase and BvMTSase genes in E. coli, and expresses each enzyme protein expressed by Ni ²⁺ -NTA-agarose. This is the result of pure separation using gel chromatography. 3 is the result of pure expression of the pRBvMTSH plasmid containing the BvMTSHase gene in Escherichia coli and the expressed fusion enzyme protein by Ni ²⁺ -NTA-agarose adsorption gel chromatography. As shown in 3 of FIG. 3, when the pRBvMTSH plasmid containing the BvMTSHase fusion gene was induced in Escherichia coli, the BvMTSHase fusion enzyme protein was specifically expressed in a large amount, and the expressed fusion enzyme protein was Ni ²⁺ -NTA-agarose adsorption gel chromatography. Pure water separation was possible using chromatography.

Example 3 Trehalose Synthesis and Substrate Specificity Using BvMTSHase Fusionase Protein

In order to investigate trehalose synthesis ability and substrate specificity of purely isolated BvMTSHase fusion enzyme protein, trehalose synthesis experiments were conducted using various maltooligosaccharides as substrates. Under the 50mM phosphate buffer solution (pH 7.0), various maltooligosaccharides were added at a concentration of 1 mM and 500ng of pure enzyme was added to make the total reaction volume 100ul. It was reacted at 37 ° C. for 1 hour and treated at 95 ° C. for 10 minutes to terminate the reaction. 4 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. S of FIG. 4 represents glucose (G1), trehalose (T), maltose (G2), maltotriose (G3), maltotetraose (G4), and maltopentose as reference materials. (maltopentaose, G5). From G3 to M in FIG. 4, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooligosaccharide mixtures were used as substrates, respectively, to conduct trehalose synthesis experiments. As shown in FIG. 4, maltooligosaccharides (figure 4, G5 and G7) consisting of five or more odd glucose units are the final reactants trehalose and maltotriose (G3), and maltotriose is no longer hydrolyzed. Did. Malto-oligosaccharides (figure 4 G6) 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 It can be seen that the switch to.

Example 4 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 24 hours. Under the conditions of 50mM phosphate buffer solution (pH 7.0), 1% water-soluble starch was added as a substrate, and 1 µg of purely isolated fusion enzyme (BvMTSHase) was added to make the total reaction volume 100ul. The reaction was carried out at 37 ° C. for 24 hours and the reaction was terminated by treatment at 95 ° C. for 10 minutes. 5 shows the results of trehalose synthesis using starch as a substrate and the synthesis of trehalose by thin layer chromatography. S of FIG. 5 represents glucose (G1), trehalose (T), maltose (G2), maltotriose (G3), maltopentaose (G4) and maltopentaose as reference materials. (maltopentaose, G5). As shown in FIG. 5 [-Amylase], when the reaction was carried out for the trehalose synthesis reaction up to 24 hours, it was confirmed that the amount of trehalose synthesized from starch increases with the reaction time. FIG. 6 is a result of identifying the trehalose synthesized by HPIC method and quantifying the amount of trehalose synthesized using the 24-hour reactant of FIG. 5 [-Amylase]. As shown in FIG. 6, after about 24 hours, it was confirmed that about 30% of the starch was converted to trehalose.

Example 5 Enhancement of Trehalose Synthesis Efficiency from Starch Using Alpha-amylase

In order to increase the production efficiency of trehalose from starch, soluble starch was treated with alpha-amylase and the reaction time was extended to 24 hours. Under the conditions of 50mM phosphate buffer solution (pH 7.0), 1% water-soluble starch was added as a substrate, and 0.05 μl of alpha-amylase and 1 μg of purified pure fusion enzyme (BvMTSHase) were added to make the total reaction volume 100ul. . It was reacted at 37 ° C. for 24 hours and treated at 95 ° C. for 10 minutes to terminate the reaction. As shown in FIG. 5 [+ Amylase], the addition of alpha-amylase and trehalose synthesis reaction up to 24 hours result in the amount of trehalose synthesized from starch according to the reaction time without addition of alpha-amylase (Fig. 5 , -Amylase) was found to increase significantly. FIG. 7 shows the results of identifying the trehalose synthesized by the HPIC method using the 24-hour reaction product of FIG. 5 [+ Amylase] and quantifying the amount of trehalose synthesized. As shown in FIG. 7, it was confirmed that about 70% of the starch was converted to trehalose after the reaction for 24 hours, and the trehalose conversion efficiency of about 230% or more was increased compared to the case where no alpha-amylase was added (FIG. 6).

Therefore, by using the BvMTSHase fusion enzyme protein used in the present invention, trehalose can be effectively produced in one reaction as compared with the case of using the respective enzymes of BvMTSase and BvMTHase. In addition, water-soluble starch may be first treated with alpha-amylase enzyme to be converted to maltooligosaccharide and then used as a substrate, thereby increasing the production efficiency of trehalose by more than two times, thereby mass-producing trehalose.

As described and demonstrated in detail above, the present invention relates to a novel fusion gene in which two genes encoding trehalose biosynthesis in Brevibacterium helvolum ATCC 11822, and a new trehalose biosynthetic fusion enzyme protein synthesized therefrom. . In addition, to provide a trehalose synthesis method using a fusion enzyme protein synthesized from the fusion gene produced by the present invention. The structural gene portion of the trehalose biosynthetic enzyme fusion gene prepared in the present invention is 4,095 bp, and the two enzyme proteins are encoded in a fused form. The first gene region of the fusion gene is the maltooligosiltrehalose synthase gene portion, and the second gene region is the maltooligosiltrehalose hydrolase gene. The fusion enzyme protein translated therefrom is a fusion protein having a molecular weight of about 150 kDa consisting of 1,365 amino acids. The fusion enzyme protein translated therefrom is maltooligosiltrehalose synthetase, a fusion enzyme protein having a molecular weight of about 150 kDa composed of 1,365 amino acids. This fusion enzyme protein is a fusion enzyme protein capable of simultaneously synthesizing maltogosiltrehalose and hydrolysis reaction. After recombination of the fusion gene into the expression vector, it was overexpressed in E. coli and purified from the recombinant fusion enzyme protein to confirm that they acted as enzymes for producing trehalose from maltooligosaccharides and starch under laboratory conditions. The fusion gene prepared in the present invention and the fusion enzyme protein produced by translation therefrom are new ones that do not exist in nature and, unlike the enzymes that biosynthesize trehalose using two previously reported enzymes, can simultaneously perform two reactions. One new fusion enzyme protein.

Sequence Listing

〈110〉 CHOI, Yang-Do, KIM, Chung-Ho

〈120〉 Novel trehalose synthase fusion gene and fusion protein

and process for preparation of trehalose therefrom

〈210〉 1

<211> 4098

<212> DNA

213 Brevibacterium helvolum

<400> 1

One

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tcg ctg acg gcg ctg cgc cgc gag cat ccg gac ctg gca gat ctc ggc ttt ggc cag acg 3900

gag gtt tcg ttc gac gac gac gcc ggc tgg ctg cgc ttc agg ccg gtc tcc gtg gag gtg 3960

ctc gtg aac ctg tca gac gcc aag gta cgg ctg gat gat gcg gca ggt gac ctc ctt ctg 4020

gcc acg gac gaa ggg aac cct ctg gac ggc ggg tcc ctc gcc ctg gtg ccg tgg agt gcc 4080

gcg gtc ctc aag tcc tga 4098

〈210〉 2

〈211〉 1365

<212> PRT

213 Brevibacterium helvolum

<400> 2

Met Lys Thr Pro Val Ser Thr Tyr Arg Phe Gln Ile Arg Thr Ser Phe

1 5 10 15

Thr Leu Phe Asp Ala Ala Glu Gln Val Pro Tyr Leu Lys Asp Leu Arg

20 25 30

Val His Trp Val Phe Leu Ser Pro Ile Leu Thr Ala Glu Lys Gly Ser

35 40 45

Glu His Gly Tyr Asn Ser Thr Asp Pro Ser Pro Val Asp Pro Asp Arg

50 55 60

Gly Gly Pro Lys Ala Leu Gln Ala Leu Ser Lys Val Ala Arg Lys His

65 70 75 80

Gly Met Gly Val Leu Leu Asp Ile Val Thr Asn His Val Gly Val Ala

85 90 95

Thr Pro Val Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg

100 105 110

Lys Ser Pro Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu Gly Gly

115 120 125

Gly Lys Val Arg Leu Pro Met Leu Gly Ser Asp Asn Asn Leu Asp Asn

130 135 140

Leu Glu Val Lys Asp Gly Lys Leu Arg Tyr Tyr Asn His Arg Ser Phe

145 150 155 160

Arg Leu Gly Lys Glu Asn Arg Glu Gly Asp Ser Leu Gln Glu Val His

165 170 175

Thr Arg Gln His Tyr Gln Leu Met Asp Trp Arg Arg Ala Asp Ala Glu

180 185 190

Leu Asn Tyr Arg Arg Phe Leu Ala Val Thr Thr Leu Ala Gly Ile Arg

195 200 205

Val Glu Glu Pro Ser Val Phe Glu Lys Val His Ala Glu Val Gly Arg

210 215 220

Trp Phe Thr Glu Gly Leu Val Asp Gly Phe Arg Val Asp His Pro Asp

225 230 235 240

Gly Phe Ala Asp Pro Asp Arg Tyr Phe Arg Trp Phe Lys Asp Val Ser

245 250 255

Gly Gly Ala Tyr Val Leu Val Glu Lys Ile Leu Glu Pro Gly Glu Val

260 265 270

Leu Pro Gln Asp Phe Ala Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu

275 280 285

Ala Asp Val Asp Arg Val Phe Val Asp Pro Ala Gly Gln Gln Ala Leu

290 295 300

Asp Ala Leu Asp Ala Ser Leu Arg Gly Thr Ser Glu Pro Ala Asp Tyr

305 310 315 320

Ala Glu Met Ile Arg Gly Thr Lys Arg Met Ile Ala Asp Gly Ile Leu

325 330 335

Arg Ser Glu Val Leu Arg Leu Ala Arg Leu Val Pro Glu Ser His Gly

340 345 350

Phe Ser Val Asp Gln Ala Ala Asp Ala Ile Ala Glu Ile Ile Ala Ser

355 360 365

Phe Pro Val Tyr Arg Ser Tyr Leu Pro Val Gly Ala Asp Val Leu Lys

370 375 380

Glu Ala Cys Glu Ser Ala Ala Ala His Arg Pro Asp Leu Glu Val Ala

385 390 395 400

Val Gly Thr Leu Gln Pro Leu Leu Leu Asp Pro Ala Lys Pro Ile Ala

405 410 415

Ile Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu

420 425 430

Asp Thr Ala Phe Tyr Arg Tyr Thr Arg Leu Asp Thr Leu Thr Glu Val

435 440 445

Gly Ala Glu Pro Thr Glu Phe Ala Val Ser Pro Gln Glu Phe His Gln

450 455 460

Arg Met Glu Arg Arg Gln Gln Glu Leu Pro Leu Ser Met Thr Thr Leu

465 470 475 480

Ser Thr His Asp Thr Lys Arg Ser Glu Asp Ala Arg Ala Arg Ile Ser

485 490 495

Val Ile Ala Glu Leu Pro Glu Glu Trp Ala Glu Thr Leu Ala Glu Leu

500 505 510

Arg Lys Leu Ala Pro Ile Pro Asp Gly Pro Phe Glu Asn Leu Leu Trp

515 520 525

Gln Ala Ile Val Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Gly

530 535 540

Tyr Ala Glu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Lys Trp Thr

545 550 555 560

Asp Pro Asn Glu Asp Phe Glu Ser Lys Val Gln Ala Ala Val Asp Ala

565 570 575

Val Phe Asp Asp Ala Lys Val Ala Lys Val Leu Thr Asp Phe Val Ala

580 585 590

Arg Ile Ala Ala Phe Ser Ala Ala Asn Ser Val Ser Ala Lys Leu Val

595 600 605

Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Ser Glu Leu

610 615 620

Trp Glu Arg Ser Leu Thr Glu Pro Asp Asn Arg Arg Pro Leu Asp Phe

625 630 635 640

Gly Ala Arg Gln Glu Ala Leu Ala Lys Leu Gln Pro Arg Cys Leu Ala

645 650 655

Arg Thr Arg Ala Gln Lys Arg Thr Lys Leu Leu Val Thr Ser Arg Ala

660 665 670

Leu Arg Leu Arg Arg Asp Arg Pro Glu Leu Phe Gln Gly Tyr Ser Pro

675 680 685

Val Asn Ala Ser Gly Ala Ala Ala Asp His Leu Leu Ala Phe Ser Arg

690 695 700

Gly Thr Asp Ala Asp Ser Gly Ala Leu Thr Leu Ala Thr Arg Leu Pro

705 710 715 720

Ala Gly Leu Gln Ala Gly Gly Gly Trp Arg Asp Thr Ala Val Asp Leu

725 730 735

Pro Thr Ala Met Arg Asp Glu Leu Thr Gly Ala Ser Tyr Gly Pro Gly

740 745 750

Gln Val Ser Val Ala Glu Val Leu Gly Thr Tyr Pro Val Ala Leu Leu

755 760 765

Ala Pro Val Asp Gly Glu Lys Ala Met Thr Leu Val Asn Val Gly Pro

770 775 780

Glu Arg Phe Asp Val Trp Ala Pro Asp Val Ser Ser Val Val Leu Val

785 790 795 800

Ala Asp Gly Arg Gln Tyr Pro Met Gln Lys Lys Glu Thr Ala Pro Gly

805 810 815

Ser Glu Gly Trp Trp Thr Ala Ser Asp Ala Pro Pro Asn Gly Asp Val

820 825 830

Asp Tyr Gly Tyr Leu Leu Asp Gly Asn Thr Thr Pro Val Pro Glu Pro

835 840 845

Arg Ser Arg Arg Leu Pro Ala Gly Val His Asn His Ser Arg Thr Tyr

850 855 860

Asn Pro Pro Pro Tyr Arg Trp Gln Asp Ser Arg Trp Arg Gly Lys Glu

865 870 875 880

Leu Gln Gly Thr Leu Ile Tyr Gln Leu His Val Gly Thr Ser Thr Pro

885 890 895

Asp Gly Thr Leu Asp Ala Ala Gly Glu Lys Leu Ser Tyr Leu Val Asp

900 905 910

Leu Gly Ile Asp Phe Ile Glu Leu Leu Pro Val Asn Gly Phe Asn Gly

915 920 925

Thr His Asn Trp Gly Tyr Asp Gly Val Gln Trp Tyr Thr Val His Glu

930 935 940

Gly Tyr Gly Gly Pro Ala Ala Tyr Gln Arg Phe Val Asp Ala Ala His

945 950 955 960

Ala Ala Gly Leu Gly Val Ile Gln Asp Val Val Tyr Asn His Leu Gly

965 970 975

Leu Arg Gly Asn Tyr Phe Pro Lys Leu Gly Pro Asn Leu Lys Gln Gly

980 985 990

Asp Ala Asn Thr Leu Gly Asp Ser Val Asn Leu Asp Gly Ala Gly Ser

995 1000 1005

Asp Val Phe Arg Glu Tyr Ile Leu Asp Asn Ala Ala Leu Trp Val Gly

1010 1015 1020

Asp Tyr His Val Asp Gly Val Gly Phe Asp Ala Val His Ala Val Arg

1025 1030 1035 1040

Asp Glu Arg Ala Val His Ile Leu Glu Asp Leu Gly Ala Leu Gly Asp

1045 1050 1055

Ala Ile Ser Gly Glu Thr Gly Leu Pro Lys Thr Leu Ile Ala Glu Ser

1060 1065 1070

Asp Phe Asn Asn Pro Arg Leu Ile Tyr Pro Arg Asp Val Asn Gly Tyr

1075 1080 1085

Gly Leu Ala Gly Gln Trp Ser Asp Asp Phe His Thr Ala Val His Val

1090 1085 1100

Ser Val Ser Gly Glu Thr Thr Gly Tyr Tyr Ser Asp Phe Glu Ser Leu

1105 1110 1115 1120

Ala Val Leu Ala Lys Val Leu Lys Asp Gly Phe Leu His Asp Gly Ser

1125 1130 1135

Tyr Ser Ser Phe Arg Gly Arg His His Gly Arg Pro Ile Asn Pro Ser

1140 1145 1150

Leu Ala Asn Pro Ala Ala Leu Val Val Cys Asn Gln Asn His Asp Gln

1155 1160 1165

Ile Gly Asn Arg Ala Thr Gly Asp Arg Leu Ser Gln Ser Leu Ser Tyr

1170 1175 1180

Gly Gln Leu Ala Val Ala Ala Val Leu Thr Leu Thr Ser Pro Phe Thr

1185 1190 1195 1200

Pro Met Leu Phe Met Gly Glu Glu Tyr Gly Ala Ser Thr Pro Trp Gln

1205 1210 1215

Phe Phe Thr Ser His Pro Glu Pro Glu Leu Gly Lys Ala Thr Ala Glu

1220 1225 1230

Gly Arg Ile Lys Glu Phe Glu Arg Met Gly Trp Asp Pro Ala Val Val

1235 1240 1245

Pro Asp Pro Gln Asp Pro Glu Thr Phe Asn Arg Ser Lys Leu Asp Trp

1250 1255 1260

Ser Glu Ala Ser Thr Gly Asp His Ala Arg Leu Leu Glu Leu Tyr Lys

1265 1270 1275 1280

Ser Leu Thr Ala Leu Arg Arg Glu His Pro Asp Leu Ala Asp Leu Gly

1285 1290 1295

Phe Gly Gln Thr Glu Val Ser Phe Asp Asp Asp Ala Gly Trp Leu Arg

1300 1305 1310

Phe Arg Pro Val Ser Val Glu Val Leu Val Asn Leu Ser Asp Ala Lys

1315 1320 1325

Val Arg Leu Asp Asp Ala Ala Gly Asp Leu Leu Leu Ala Thr Asp Glu

1330 1335 1340

Gly Asn Pro Leu Asp Gly Gly Ser Leu Ala Leu Val Pro Trp Ser Ala

1345 1350 1355 1360

Ala Val Leu Lys Ser

1365

000

Claims (5)

Trehalose biosynthetic enzyme fusion structural genes having the following nucleotide sequences prepared by fusing the BvMTSase gene and BvMTHase gene isolated from Brevibacterium helvolum: Maltotoolyl trehalose synthetic hydrolase (BvMTSHase) protein having the following amino acid sequence translated from the structural gene of the fusion gene of claim 1: Method for mass production of fusion enzyme protein of claim 2 using the fusion gene of claim 1 A method for producing trehalose from starch using the fusion enzyme protein produced in claim 3 The method of claim 4, characterized in that the treatment of alpha-amylase in the starch to increase trehalose production efficiency