KR20000037638A - New recombinant trehalose synthase, expression vector including its gene, method for production of trehalose using recombinant trehalose synthase - Google Patents

Abstract

PURPOSE: New recombinant trehalose synthase and the expression vector including its gene are provided. Nucleotide sequences of the trehalose synthase are isolated from thermophilic strain, Thermus caldophilus GK24, and a method for production of trehalose using recombinant enzyme is also provided. CONSTITUTION: A trehalose synthase is the enzyme which converts maltose to alpha, alpha-trehalose and alpha, beta-trehalose. The recombinant trehalose synthase is over-expressed in Escherichia coli using the expression vector including its gene which is isolated from Thermus caldophilus GK24. The trehalose is obtained by the reaction of the purified recombinant trehalose synthase with maltose or hydrolytic product of starch.

Description

Production of trehalose using a novel recombinant trehalose synthase, an expression vector into which a gene encoding the same, and a recombinant enzyme are inserted.

The present invention relates to a novel recombinant trehalose synthase, an expression vector into which a gene encoding the enzyme is inserted, and mass production of trehalose using the recombinant enzyme. More specifically, the present invention relates to the heat-resistant trehalose synthase that produces alpha, alpha-trehalose and alpha, beta-trehalose from maltose. After determining the gene sequence, preparing an expression vector into which the gene is inserted, culturing the transformed strain with the vector to produce a large amount of recombinant trehalose synthase, and reacting the recombinant enzyme with maltose to prepare trehalose. It is about.

Trehalose (D-glucopyranosyl- (1↔1) -D-glucopyranoside, see Manaught, AD, (1997) Adv. Carbohydr. Chem. Biochem., 52, 43-177) is shown to have two glucoses as shown in FIG. Each of these carbon positions is a non-reducing disaccharide having three kinds of isomers bonded to each other. The most common isomers in nature are alpha and alpha-trehalose, which are a type of storage carbohydrate for many types of bacteria, fungi, yeasts, insects, animals and plants, and act as a power source for flying insects. , Yeasts and Escherichia coli, etc., as a resistance to external physical effects (Elbein, AD (1974) Adv. Carbohydr. Chem. Biochem., 30, 227-256; Yoshida, M. et al. (1995) Oyo Toshitsu Kagaku, 42, 19-25; Van Laere, A. (1989) FEMS Microbiol. Rev., 63, 201-210; Kassen, I. et al. (1994) Gene, 145, 9-15).

Trehalose is a sweetener with about half the sweetness of sugars of the same concentration, as well as a variety of applications for the stabilizing effects of biomolecules in fields such as basic science, medicine, agriculture and bio-electronics. (Colaco, C. et al. (1992) Bio / Technology., 10, 1007-1011). Trehalose can be used as a stabilizer in dry or frozen foods and as a diagnostic reagent and pharmaceutical pharmaceutical additives, as a stabilizer to prevent thermal coagulation and thermal denaturation of bioactive proteins, including enzymes, as well as moisturizers in people's cosmetics. (1994) Eur. J. Biochem., 219, 187-193; Schick, I. et al. (1991) Biochem. Eng.-Stuttgart, Proc. Int. Symp.], 2nd, 126-129; Carninci, P. et al. (1998) Proc. Natl. Acad. USA, 95, 520-524). The production method of alpha, alpha-trehalose is obtained from yeast cells, Plesiomonas, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium ( Separation from microbial culture media such as Microbacterium, Nocardia, and Filobasidium can be performed by using starch converting enzymes of Sulfobus solfataricus or aspergillus or Methods using enzymes such as glucoamylase of Aspergillus niger and beta-glucanase of penicillium emersonii (see Lama, L. et al. (1991) Biotech.Forum Eur., 8, 201-203 .; Vuorio, OE et al. (1993) Eur. J. Biochem., 216, 849-861).

The present inventors found that the thermophilic microorganism Thermus caldophilus GK24 strain (Kwon S.-T. et al. (1997) Mol. Cells, 7, 264-271; Park, J. et al. (1993) Eur. J. Biochem., 214 (1983) J. Biochem. 93, 7-13), the cells were recovered and crushed, and extracted from the heat-resistant trehalose synthase and cloned from the extracted enzyme gene. Next, the strain was transformed and cultured with this gene to mass produce recombinant trehalose synthase, and the produced enzyme was reacted with starch hydrolyzate and maltose to prepare trehalose.

Accordingly, an object of the present invention is to determine and provide a gene sequence of the heat resistant trehalose synthase. Another object of the present invention is to produce a recombinant vector based on the gene, transform the strain with the vector and then culture to produce a large amount of recombinant trehalose synthase. It is another object of the present invention to provide a method for preparing trehalose by reacting the recombinant trehalose synthase produced in large quantities with maltose or starch hydrolysis products.

The above object of the present invention is to cultivate the Thermus caldophilus GK24 strain and to purify the trehalose synthase from the culture medium, the molecular weight of the enzyme, the optimum activity temperature, the optimum activity pH, the buffer solution concentration and the type of metal ion added during the enzyme reaction Plaque hybridization using a labeled DNA probe having an amino terminal sequence after determining the type of sugar reacting with the enzyme and the amino-terminal amino acid sequence of the enzyme and making a lambda library from the chromosomal DNA of Thermus caldophilus GK24 strain Lambda recombinants containing trehalose synthase genes were selected by plaque hybridization. Subsequently, the DNA of the lambda recombinant was isolated and treated with an appropriate restriction enzyme. Through Southern hybridization, a DNA fragment containing the trehalose synthase gene was recovered, ligated with plasmid DNA, and transformed into E. coli. After colony hybridization method, only transformants including trehalose synthase gene were selected from the transformed Escherichia coli, and the plasmid DNA was isolated to determine the trehalose synthase gene sequence, and then the determined trehalose synthase gene was determined appropriately. This is achieved by connecting to an expression vector capable of expressing an enzyme protein, making a recombinant Escherichia coli recombinant, culturing it to produce a recombinant trehalose synthase, isolating and purifying the enzyme, and reacting with maltose to produce trehalose. It was.

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

FIG. 1 is a structural formula showing three isomers of maltose and trehalose, alpha, alpha-trehalose, alpha, beta-trehalose, beta, and beta-trehalose.

2 is a graph showing the effect of temperature on the conversion reaction by the heat-resistant trehalose synthase produced from Thermus caldophilus GK24 strain.

Figure 3 is a graph showing the effect of pH on the conversion reaction by the heat-resistant trehalose synthase produced from Thermus caldophilus GK24 strain.

Figure 4 shows the restriction fragment map of the heat-resistant trehalose synthase gene and the position of the cloned DNA fragment from Thermus caldophilus GK24 strain.

Figures 5a, 5b, 5c shows the nucleotide sequence and amino acid sequence of the heat-resistant trehalose synthase isolated from Thermus caldophilus GK24 strain.

6a and 6b show the results of comparing amino acid sequences between trehalose synthase enzymes.

Figure 7a, 7b 7c, 7d shows the results of the base sequence comparison between trehalose synthase.

8 is a recombinant expression vector construct of a heat resistant trehalose synthase gene derived from Thermus caldophilus GK24 strain.

Figures 9a, 9b, 9c are gene sequences and amino acid sequences of the heat-resistant trehalose synthase expressed in the recombinant E. coli MV1184 [pTSEHS3].

10 is a detection diagram of alpha, alpha-trehalose, glucose, alpha, beta-trehalose by high base cation exchange chromatography.

The present invention comprises the steps of centrifuging the culture medium cultured Thermus caldophilus GK24 strain to recover the cells after cryopreservation; Preparing an enzyme solution by pulverizing the cultured cells in the Thermus caldophilus GK24 strain culture medium, adding the emulsion to the crude enzyme solution obtained by centrifugation, and centrifuging again and dialysis; Reacting the enzyme liquid obtained in the step with maltose to generate trehalose; Purifying the resulting trehalose reactant, separating only trehalose, dissolving in heavy water, and analyzing the structure with a nuclear magnetic resonance spectrometer; Separating the enzyme protein by purifying the enzyme by performing a series of column chromatography using the FPLC system on the enzyme obtained by culturing the Thermus caldophilus GK24 strain; Analyzing the molecular weight of the purified trehalose synthase by SDS-polyacrylamide electrophoresis; Measuring the temperature exhibiting maximum activity while reacting the purified trehalose synthase with maltose; Measuring a pH exhibiting maximum activity while reacting the purified trehalose synthase with maltose; Measuring the concentration of the buffer solution exhibiting maximum activity by varying the concentration of the buffer solution while reacting the purified trehalose synthase with maltose; Irradiating the purified trehalose synthase with maltose while adding metal ions to investigate metal ions that do not inhibit the activity of the enzyme; Reacting various kinds of sugars with trehalose synthase to examine a reactive sugar; Amino-terminal sequencing of trehalose synthase using a gaseous peptide sequencer equipped with a PTH-Xaa analyzer; Synthesizing an oligo DNA probe based on the determined amino acid sequence; Recovering the DNA of the cultured Thermus caldophilus GK24 strain cells, and infecting and amplifying E. coli XL1-Blue MRA to prepare a Thermus caldophilus GK24 strain lambda library; Selecting a lambda recombinant comprising a trehalose synthase gene by plaque hybridization using an amino terminal probe from the prepared lambda library; Recovering DNA fragments containing trehalose synthase gene by separating the DNA of the lambda recombinant and subjecting to restriction enzyme treatment, and then linking these DNA fragments with plasmid DNA to determine nucleotide sequences; Preparing the recombinant E. coli by transforming E. coli with the vector Thermus caldophilus GK24 strain trehalose synthase gene sequence into one plasmid vector; Culturing the recombinant E. coli to produce a recombinant trehalose synthase; The recombinant trehalose synthase solution is reacted with maltose to produce trehalose and to analyze the type of trehalose produced.

General molecular biology experiments related to the above genetic manipulations are known methods (see Sambrook, J. et al. (1989) Molecular Cloning. A laboratory Manual.2nd ed., Cold Spring Harbor Laboratory.Cold Spring Harbor.New York. Ausubel, F. et al. (1995) Short Protocols in Molecular Biology.3rd.John & Wiley Sons, Inc.

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

Example 1 Culture of Strain and Purification of Enzyme for Isolation of Trehalose Synthase from Thermus caldophilus GK24 Strain

Experimental Example 1: Cultivation of Thermus caldophilus GK24 strain

The Thermus caldophilus GK24 strain was cultured for 24 hours at 75 ° C. under medium conditions as shown in Table 1 using known methods (Brock, TD (1978) Thermophilic microorganisms and life at high temperature.Spring-Verlag, Heidelberg .; Williams, RAD (1992) 3745-3753.In: A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications.vol. 4.Springer-Verlag, Heidelberg.), Cultures 9,500 xg at 4 ° C. Cells were harvested by centrifugation for 30 minutes. The cultured and recovered cultured cells were washed once with 10/10 volume of 10 mM potassium phosphate buffer pH 7.0 and then stored frozen or suspended in the same buffer solution.

Culture medium of Thermus caldophilus GK24 strain Polypeptone 8.0g East ext 4.0g Nitrilotriacetic acid 100mg CaSO ₄ 2H ₂ O 150 mg MgSO _{4 7} H ₂ O 100mg NaCl 8mg KNO ₃ 103mg NaNO ₃ 689 mg FeCl ₃ 28mg H ₂ SO ₄ 0.05mL MnSO ₄ H ₂ O 220 mg ZnSO ₄ · 7H ₂ O 50 mg H ₃ BO ₃ 50 mg CuSO ₄ 1.6mg Na ₂ MoO ₄ 2H ₂ O 2.5mg CoCl ₂ · 6H ₂ O 4.6 mg PH 7.5 with NaOH Total volume 1.0L

Experimental Example 2: Preparation of Enzyme Liquid

100 g of the Thermus caldophilus GK24 strain cultured cells cultured and recovered in Experimental Example 1 were suspended in 10 mM potassium phosphate buffer solution pH 7.0 and crushed by an ultrasonic cell crusher. The crushed cell solution was centrifuged at 17,000 × g for 30 minutes at 4 ° C. to form a crude enzyme solution from which unbroken cells and insoluble cell debris were removed. The crude enzyme solution (NH ₄ ) ₂ SO ₄ ) which is 30% by weight to volume ratio was added at 4 ° C. and stirred well for 4 hours. The enzyme solution was centrifuged to collect only the solution, and a solution having a final concentration of 60% by weight to volume ratio was added at 4 ° C. and stirred well for 4 hours. After collecting the precipitate by centrifugation of the enzyme solution, the precipitate was dissolved in 10 mM potassium phosphate buffer pH 7.5 and dialyzed overnight in the same buffer solution. The enzyme solution was centrifuged again to remove insoluble matter and then used as an enzyme source for trehalose synthesis or for enzyme purification.

Experimental Example 3: Measurement of Trehalose Synthase Activity

Trehalose synthase reaction was carried out from maltose using partially or purely purified enzyme solution. The final concentration in the reaction solution was 1-100 mM maltose, 20 mM potassium phosphate buffer pH 6.3, and the enzyme solution were mixed and reacted at 30-90 ° C. for 1 hour. The reaction solution was heated in a boiling water bath for 10 minutes. The reaction was terminated. The unit activity of trehalose synthase was determined by the ability to synthesize 1 μmole of trehalose per minute. Enzyme reactants were analyzed for the formation of trehalose using high pH Anion Exchange Chromatography with Pulsed Amperometric Detector (HPAEC-PAD). In other words, a ion chromatography system (Dionex, USA) was used, and CarboPac PA1 (4 × 250 mm) was used as a column, and a wave with gold electrode was used as a detector. The reaction product was detected and analyzed using a pulsed amperometric detector. At this time, a sodium hydroxide (NaOH) solution was used as the developing solution, and the sodium hydroxide solution of various concentrations was tested at a flow rate of 1 mL per minute, and the 140 mM condition was the best in each mixture.

Experimental Example 4 Separation and Structure Analysis of Trehalose

The trehalose produced in Experimental Example 3 was separated from the reaction. That is, the reaction solution is extracted with phenol, only the aqueous layer is obtained, and then extracted with ethylether to obtain only the aqueous layer, and the remaining organic solution is transferred to Sep-Pak C18 Cartridge (Waters). Removed by passage. Some of the purified reaction solution was concentrated by lyophilization, dissolved in an appropriate amount of demineralized water, and then trehalose only was used in an ion chromatography system from Dionex, and used for semi-preparative CarboPac PA1, 9 × 250. mm), a detector using a wave current whose electrode is gold as a detector was separated by flowing 2.5 mL of a 140 mM sodium hydroxide solution per minute as a developing solution. The eluate containing trehalose having a pH of 13-14 that passed through the detector was passed through an anion neutralizing device, a cationic micromembrane suppressor (AMMS-1), and neutralized and fractionated. At this time, 0.3 N sulfuric acid solution was used as the neutralizing solution of the neutralizer. This process was repeated several times to recover a large amount of trehalose fraction. The recovered trehalose fraction was replaced with heavy water and its structure was confirmed by nuclear magnetic resonance spectroscopy. In other words, neutralized by freeze-drying the collected trehalose to remove water and the process repeated three times to dry only the re-collected was dissolved in heavy water and then dissolved in heavy water for building a replacement of an -OH group trehalose -OD then 500 ㎕ ¹ H, The structure was analyzed by ¹³ C nuclear magnetic resonance spectroscopy. The structure of the synthesized trehalose was analyzed and compared with the ¹³ C- and ¹ H-nuclear magnetic resonance data of the trehalose standard material.

Experimental Example 5 Purification of Trehalose Synthase

In order to purer trehalose synthase more purely, the enzyme protein was separated by performing a series of column chromatography using the FPLC system. That is, the obtained enzyme solution was added to a cation exchange resin column (DEAE-Sephacel column, 5 x 50 cm) previously equilibrated with 10 mM potassium phosphate buffer pH 7.0, washed sufficiently with the same buffer, and then potassium chloride from 0 M to 1 M. Proteins were fractionated by concentration gradient. Of the separated enzyme fractions, only the fractions with trehalose synthase were collected and applied to the next column chromatography. The collected enzyme solution was concentrated, buffer replacement was completed with 10 mM potassium phosphate buffer pH 7.0 containing 1 M oil solution, and then added to a hydrophobic resin column (Phenyl-Toyopearl 650M column, 5 x 20 cm) equilibrated with the same buffer and washed. . The proteins were then separated with a lean concentration gradient from 1 M to 0 M. Only the separated fractions were identified and the buffer solution was replaced with 10 mM potassium phosphate buffer pH 7.0 containing 150 mM potassium chloride, pH 7.0, and the gel filtration resin column equilibrated with the same buffer solution (Superose 6 HR10 / 30 column, 1 x 30). cm), and the enzyme protein was fractionated, and only the fraction with trehalose synthase was isolated from each of the separated enzyme fractions, and the buffer solution was replaced with 10 mM potassium phosphate buffer pH 7.0. In addition, the enzyme fraction was added to a strong cation exchange resin (MonoQ HR5 / 5 column, 5 x 50 mm) equilibrated to the same 10 mM potassium phosphate buffer pH 7.0, washed with the same buffer, and then washed at 0 M to 1 M. Proteins were fractionated with a potassium chloride concentration gradient of up to very purified trehalose synthase. Purification degree of the trehalose synthase separated and purified as described above was confirmed as a pure enzyme protein of a single composition on acrylamide gel electrophoresis, the yield of the enzyme was 40.1 active units in 100 g of wet cells, the specific activity 33.42 active units / mg It was.

Example 2: Characterization of trehalose synthase from Thermus caldophilus GK24 strain

Experimental Example 1: Determination of the molecular weight of trehalose synthase

The molecular weight of trehalose synthase is 372 ± 5 kD on Size Exclusion Chromatography equilibrated with 10 mM potassium phosphate buffer pH 7.0 with undenatured conditions, 150 mM potassium chloride, and known methods (see Lammli, Analysis was carried out using denaturing conditions such as UK (1970) Nature 227, 680-685) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). As a result, the single molecular weight of trehalose synthase was 105 ± 5 kD.

Experimental Example 2: investigation of optimal reaction temperature with trehalose synthase

The reactivity of trehalose synthase according to the given reaction temperature was measured in the same manner as in Experiment 3 of Example 1. As shown in FIG. 2, the titration temperature of the enzyme was 30-80 ° C. and the optimum reaction temperature was 63 ° C. when reacted in 20 mM potassium phosphate buffer pH 6.3 for 1 hour. After 1 hour of heat treatment at a given temperature, more than 50% of the enzyme activity showed live thermal stability and the temperature reached 83 ° C.

Experimental Example 3: investigation of optimal pH with trehalose synthase

The reactivity of trehalose synthase according to the given reaction pH was measured in the same manner as in Experimental Example 3 of Example 1. As shown in FIG. 3, the reaction titration pH of the enzyme was 5.2-7.3 and the optimum reaction pH was 6.3 when reacted at 63 ° C. for 1 hour. 50% or more enzyme activity from pretreatment for 1 hour at a given pH was 4.4-9.5 in terms of live pH stability.

Experimental Example 4: Concentration of buffer solution for optimal reaction with trehalose synthase

The reactivity of trehalose synthase according to the concentration of the buffer solution was measured in the same manner as in Experimental Example 3 of Example 1. When the concentration of potassium phosphate buffer solution of pH 6.3 was changed from 0 mM to 200 mM, trehalose production was not significantly changed, but it was most responsive between 20 mM and 50 mM.

Experimental Example 5: Investigation of metal ions for optimal reaction with trehalose synthase

The reactivity of trehalose synthase according to the addition of metal ions was measured in the same manner as in Experiment 3 of Example 1. The metal ion effect during the reaction showed that the reactivity of trehalose synthase was inhibited at the concentration of 1 mM and at least 70% for AlCl ₃ , CuCl ₂ , FeCl _{3 and} at least 50% of CaCl ₂ , CoCl ₂ , ZnCl ₂ Was inhibited by more than 90%, but the activity was maintained in the case of MgCl ₂ .

Experimental Example 6: Investigation of the reactivity of trehalose synthase with various sugars

The reactivity of trehalose synthase to the maltose and trehalose described as reactants and products, as well as structurally similar to maltose and trehalose or derivatives thereof, was determined in the same manner as in Experiment 3 of Example 1 The concentration was 10 mM, the reaction was carried out at 63 ° C. and pH 6.3 for 1 hour, and confirmed by measuring the degree of reduction of a given sugar. Experimental results showed cellobiose, chitobio, alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, decyl-beta-di-maltoside, dodecyl-di-maltoside, erlos, fructose, gentibio, glucose , Glucose-1-phosphate, isomaltose, isomaltotriose, isomaltotetraose, lactose, laminaribiose, leucross, maltitol, maltotriose, maltotetraose, maltopentaose, maltohexaos, maltohepta Oslo, maltose-1-phosphate, maltulose, water soluble starch, raffinose, betabeta-trehalose, trehalose-6-phosphate, turanoose, xylobiose, etc., were not reactive with trehalose synthase. It showed less than 10% reactivity with os, panos, sugar, trehalosamine, alpha, beta-trehalose, and less than 30% with alpha, alpha-trehalose, and 8 with maltose. Reactivity was greater than 0%.

Experimental Example 7: Determination of the amino-terminal amino acid of trehalose synthase

The amino-terminal sequence of trehalose synthase was determined using a gaseous peptide sequencer (Applied Biosystem model 477A) equipped with a PTH-Xaa analyzer (Applied Biosystems model 120A). In other words, the purified trehalose synthase was electrophoresed on a polyacrylamide gel, the enzyme protein on the gel was transferred to an electroblotter on a FVD membrane (MSI, Westboro, MA 01581) and then trehalose on the FVD membrane. Only the amino acid sequence was determined by the above device for the synthetase protein band, and the determined amino acid sequence is as follows.

1 10 20

MDPLWYKDAV IYQLHVRSFF

Example 3: Cloning of Trehalose Synthase Gene from Thermus caldopilus GK24 Strain

Experimental Example 1: Amino Terminal Oligo DNA Probe Synthesis

Based on the amino acid sequence determined in Experiment 7 of Example 2 26 oligo DNA probe # 1 was synthesized as follows.

1 10 20 26

5'-TGGTA (C / T) AA (A / G) GA (C / T) GCNGTNAT (A / C / T) TA (C / T) CA-3 '

Southern hybridization, plaque hybridization, colony hybridization, and the like were performed using the # 1 probe to identify and clone the trehalose synthase gene derived from Thermus caldophilus GK24 strain.

Experimental Example 2: Preparation of lambda library of Thermus caldophilus GK24 strain

Cells of Thermus caldophilus GK24 strain grown as in Experiment 1 of Example 1 were suspended in lysis buffer (0.1 M NaCl, 10 mM EDTA, 20 mM Tris, pH 7.5), and lysozyme was added to 0.5 mg / mL. After reacting at 37 ° C. for 30 minutes, 1% SDS and RNase were added at 50 μg / mL, and then reacted at 37 ° C. for 1 hour, followed by reaction at 37 ° C. for 3 hours with 0.2 mg of protease. Was added for 12 hours. Only the aqueous layer was recovered and twice the alcohol was added to precipitate the DNA and recovered. The chromosomal DNA was partially digested with restriction enzyme Sau3A I, ultracentrifugation by sugar concentration gradient, 9-23 kb in size DNA fragments were recovered and linked to the λ EMBL3 vector and infected with E. coli XL1-Blue MRA (P2), Amplification produced a lambda library of Thermus caldophilus GK24 strain.

Experimental Example 3: Screening of Trehalose Synthase Gene and Recombinant Production in Lambda Library

In the lambda library of Thermus caldophilus prepared as in Experimental Example 2, a lambda recombinant containing trehalose synthase gene was selected by performing plaque hybridization using the amino terminal probe obtained in Experimental Example 1 of this example. . That is, the lambda library is described as NZCYM (general known method) and as described in the user manual of the Dig Labeling and Detection Kit of Boehringer Mannheim GmbH, Biochemica (Germany). 10 g NZ amine, 5 g NaCl, 5 g Bacto yeast extract, 1 g Casamino acids, 2 g MgSO ₄ · 7H ₂ O, pH 7.0) Escherichia coli XL1-Blue MRA (P2) on agar plate medium to form plaque The DNA was eluted and fixed by transfer to a nylon membrane of (+) polarity. Plaque hybridization was performed using the amino terminal probe of Experiment 1 of Example 3 labeled with digoxigenin-11-ddUTP (DIG) by terminal transferase, and the chemical luminescence CSP ( 5 lambda recombinants containing trehalose synthase genes were selected by photosensitive X-ray film using CSPD). Selected lambda recombinants were infected with Escherichia coli in ENC YM medium, and amplified and purified by treatment with chloroform, DNase I, RNase, 1 M sodium chloride and 10% sebum (PEG, polyethylene glycol).

Experimental Example 4: Determination of Trehalose Synthase Gene Sequence of Thermus caldophilus GK24 Strain

EDTA (final conc. 20 mM), proteinase K (final conc. 50 μg / mL), SDS (final conc. 0.5%) from the lambda recombinant purified in Experimental Example 3 ), Phenol / chloroform (1: 1 v / v) and alcohol were used to isolate pure DNA of selected lambda recombinants. The isolated lambda recombinant DNA was treated with various kinds of restriction enzymes suitable for making a recombinant plasmid and developed on an agarose gel, Southern blotting, and Southern hybridization method using the above probe. DNA fragments of 3.0 kb (pTSBm30) by restriction enzyme BamH I, 2.9 kb (pTSH29) by Hind III and 3.0 kb (pTSBg30) by Bgl II were isolated and purified. The nucleotide sequence was determined by binding to plasmid pUC 18. As shown in FIG. 4, since the above three DNA fragments contain only the amino terminal portion of the entire gene of trehalose synthase, the nucleotide sequence of the carboxy terminal direction of the DNA obtained by Bgl II is used to secure the carboxy terminus. The same oligo DNA probe # 2 was prepared.

1 10 20

5'-CCTGAGGAACCAGCACGCCA-3 '

Southern hybridization was performed using the oligo DNA probe # 2 to isolate and purify DNA fragments of size 2.2 kb (pTSBm22) by restriction enzyme BamH I including the carboxy terminus, and 7.0 kb (pTSH70) by Hind III. The nucleotide sequence was determined by binding to plasmid pUC 18. As a result, the nucleotide sequences of the trehalose synthase gene of the Thermus caldophilus GK24 strain were as shown in Figs. 5A, 5B and 5C. According to the amino acids and DNA sequences thus determined, the trehalose synthase of Thermus caldophilus GK24 strain consists of 965 amino acids. The molecular weight of the enzyme protein is 110 kDa, the calculated pI is 5.73, and the gene of this enzyme is composed of 2,898 bases. The composition of G + C is 69.3%. Comparing the amino acid sequence with trehalose synthase isolated from other species, as shown in Figures 6a, 6b and 98.2% of the enzyme of Summers aquaticus ATCC33923, Pimelobacter sp. 50.3% with the enzyme of R48 (Tsusaki, K. et al. (1997) Biochim. Biophys. Acta., 1334, 28-32; Tsusaki, K. et al. (1996) Biochim. Biophys. Acta., 1290, 1- 3) showed homology. In comparison between enzyme genes, as shown in FIGS. 7A, 7B, 7C, and 7D, 98.9% of Summer Aquaticus ATCC33923, Pimelobacter sp. It has a 53.4% homology with R48. Compared more closely with the trehalose synthase of the most similar Thermos aquaticus ATCC33923, the trehalose synthase of the Thermus caldophilus GK24 strain of the present invention has two more amino acids in terms of enzyme protein and thus 10 different amino acids and thus molecular weight Calculated by 246 Da was larger and pI value was 0.1 higher. In terms of enzyme genes, 6 bases were more, 26 bases were different, and the composition ratio of G + C was 0.1% higher.

Experimental Example 5: Expression vector construction of trehalose synthase gene

In order to construct the expression vector of the trehalose synthase gene, the first step was to collect the structural genes of the enzyme into one plasmid vector. That is, as shown in FIG. 8, the plasmid pTSBm30 was treated with restriction enzymes Bgl II and Hind III to elute 1.8 kb of DNA and the plasmid pTSH70 with restriction enzymes Hind III and BamH I. The plasmid pTSBgBm was constructed by linking with each other. This plasmid contains all of the structural genes of trehalose synthase. In the second step, the amino terminus of the enzyme gene containing the ATG start codon and the EcoR I site for cloning into the expression vector was obtained. That is, as shown in FIG. 8, the plasmid pTSBgBm was used as a template, and a polymerase chain reaction (PCR) was performed using primers ExEN and ExTRev as described below to obtain 160 bp of DNA, thereby connecting to plasmid pUC18. The plasmid pEXENTR was constructed comprising the gene of the amino terminal portion.

1 21

5'-GAATTCATGGACCCCCTCTGGTACAAG-3 '

134 154

5'-TCAACACCCTCTGGCTCATGC-3 '

In the third step, plasmids pTSBgBm and pEXENTR were treated with restriction enzymes Hind III, Sal I, EcoR I and Hind III to elute DNAs of 3.1 kb and 0.1 kb, respectively. And the expression vector pJR treated with restriction enzymes EcoR I and Sal I (see Korean Patent Application No. 92-6370; Kwon S.-T. et al. (1997) Mol. Cells, 7, 264-271). Trehalose synthase expression vector pTSEHS3 was constructed and transformed into E. coli MV1184 to construct a recombinant strain, E. coli MV1184 [pTSEHS3] (FIGS. 9A, 9B and 9C).

The present inventors deposited the E. coli MV1184 [pTSEHS3] containing the plasmid pTSEHS3 with the gene number KCTC 0551BP to the Gene Bank in the Biotechnology Research Institute of Korea Institute of Science and Technology as of November 20, 1998.

Example 4 Expression and Trehalose Synthesis of Recombinant Trehalose Synthase

Experimental Example 1 Expression and Purification of Recombinant Trehalose Synthase

To express the cloned trehalose synthase gene, Escherichia coli MV1184 [pTSEHS3] was inoculated in an Elbi liquid medium to which 50 μg / mL of ampicillin was added and cultured in large quantities. After incubation at 37 ° C. for about 2 hours, 0.2 mM ipitage (IPTG, isopropyl β-D-thiogalactopyranoside) was added and the culture was continued for about 10 hours to induce gene expression. The cells were recovered by centrifugation, then suspended in 10 mM potassium phosphate buffer pH 7.0 as in Example 1, the cells were disrupted with an ultrasonic cell crusher, and the crushed cell solution was centrifuged at 17,000 xg for 30 minutes at 4 ° C. Enzyme solution was removed from unbroken cells and insoluble cell debris. Subsequently, only the obtained aqueous supernatant enzyme solution was heat-treated at 75-85 ° C. for 30 minutes and centrifuged to remove denatured protein derived from E. coli. Add this enzyme to a cation exchange resin column (DEAE-Sephacel column, 1 x 10 cm) equilibrated to 10 mM potassium phosphate buffer pH 7.0, wash thoroughly with the same buffer, and then gradient the potassium chloride concentration from 0 M to 1 M. Proteins were fractionated. Only active fractions were then collected and concentrated using an ultrafilteration apparatus. Trehalose synthase activity was measured in the same manner as in Experiment 3 of Example 1 using the enzyme solution thus treated, and showed 39.44 active units per mg of partially purified protein. The amino terminus of the recombinase was the same as that of the enzyme protein purified from the Thermus caldophilus GK24 strain. It was also similar to the intrinsic properties of the enzyme investigated above. Trehalose synthase produced from E. coli MV1184 [pTSEHS3] by the above induction of expression is produced up to 528 active units per g of wet cells, which is a 78-fold increase in the amount of 6.8 active units per g of wet cells from the Thermus caldopilus GK24 strain. to be.

Experimental Example 2 Production of Trehalose from Maltose by Recombinant Trehalose Synthase

When trehalose was produced from maltose using the recombinant enzyme of Experimental Example 1, 86% of alpha, alpha-trehalose and 3.5% of alpha, beta-trehalose were produced. That is, trehalose synthase was added to 20 mM potassium phosphate buffer pH 6.3, 9 active units / mL containing 10 mM maltose, and trehalose production reaction was carried out for 72 hours at a reaction temperature of 40 ° C to 90 ° C. As a result of measuring the degree of trehalose production by graphigraphy, the production rate of alpha, alpha-trehalose reached 86% at 40 ° C., and the production rate of alpha, beta-trehalose reached 3.5% at 80 ° C.

The present invention is a recombinant trehalose synthase mass-produced by E. coli recombined with trehalose synthase gene as described through the examples and experimental examples to stabilize the biomolecules and act as a stabilizer for dried or frozen foods by reacting with maltose Alpha, alpha-trehalose, which is not only used as an additive in diagnostic reagents and pharmaceuticals, but also as a stabilizer to prevent thermal coagulation and thermal denaturation of bioactive proteins including enzymes and as a moisturizing agent in cosmetics Alpha, beta-trehalose (α, β-trehalose) is a very useful invention in the biopharmaceutical, food and cosmetic industry because it has an excellent effect of producing.

Claims (8)

Gene encoding the heat resistant trehalose synthase from Thermus caldophilus GK24 strain. The gene encoding trehalose synthase according to claim 1, wherein the gene has a base sequence or a form in which a part thereof is removed or modified. The gene encoding trehalose synthase according to claim 1, wherein the gene has a form in which the following amino acid sequence is removed or a part thereof is removed or modified. Recombinant heat-resistant trehalose synthase derived from Escherichia coli transformed with a recombinant plasmid vector into which the gene of claim 2 is inserted. Recombinant plasmid vector pTSEHS3 into which a heat-resistant trehalose synthase gene was isolated from the strain with Thermus caldophilus GK24. E. coli MV1184 [pTSEHS3] strain transformed with the recombinant plasmid pTSEHS3 inserted with the trehalose synthase gene isolated from Thermus caldophilus GK24 strain (Accession No. KCTC 0551BP) Method for producing recombinant trehalose synthase by culturing E. coli MV1184 [pTSEHS3] strain (Accession No. KCTC 0551BP) transformed with recombinant plasmid pTSEHS3 inserted with trehalose synthase gene isolated from Thermus caldophilus GK24 strain A method for producing alpha, alpha-trehalose or alpha, beta-trehalose or derivatives thereof by reacting the recombinant heat resistant trehalose synthase according to claim 4 with maltose or starch hydrolysis products.