KR100349932B1 - α-glucosidase from extreme thermophile, Thermus caldophilus GK24 and its production - Google Patents

Abstract

The present invention is a novel heat-resistant enzyme alpha-glucosidase isolated from the thermophilic bacterium Thermos Caldophilus ZK24 strain, a gene encoding the enzyme, an expression vector comprising the gene, transformed by the vector To a method for producing alpha-glucosidase comprising culturing the recombinant strain and the recombinant strain, and a method for producing a complex sugar by starch sugar hydrolysis and sugar transfer reaction using the alpha-glucosidase prepared by the above method. It is about. Recombinant alpha-glucosidase according to the present invention has an activity optimum temperature of 90 ° C., optimum pH 6.5 and catalyzes the hydrolysis and sugar transfer reaction of isomaltooligosaccharide or sugar and is very useful in the starch industry, food and biopharmaceutical industry.

Description

Novel thermostable alpha-glucosidase isolated from thermophilic thermos strain and its preparation method {α-glucosidase from extreme thermophile, Thermus caldophilus GK24 and its production}

The present invention relates to a novel alpha-glucosidase from Thermos, a thermophilic strain, and a method for preparing the same. More specifically, the present invention provides an optimal pH 6.5 isolated from the thermophilic Thermos caldophilus GK24, a heat resistant alpha-glucosidase at an optimal temperature of 90 ° C., the gene encoding the enzyme, the gene Expression vector comprising a, recombinant strain transformed with the expression vector, a method for producing the enzyme comprising culturing the recombinant strain, starch sugar hydrolysis and sugar using alpha-glucosidase prepared by the method It relates to a method for producing a complex sugar by a transfer reaction.

Enzymatic hydrolysis of starch is well known. In the hydrolysis process of starch, starch dissolved in the aqueous phase at a temperature higher than the gelling temperature is liquefied and hydrolyzed by bacterial-derived alpha-amylase at a DE value of 5 to 25 at a temperature near 95 ° C. The liquefied starch is hydrolyzed by fungal-derived glucoamylase or bacterial-derived pullulanase and isoamylase. The process is widely used in the starch industry to obtain a variety of product quality and yield. Most of the Bacillus strain-derived alpha-amylase enzymes used in the liquefaction process must be adequately stable to heat during the process and require refeeding of the enzyme. In addition to the main product as a final product, other sugars are produced. Therefore, research for improving the yield and quality of glucose is currently being conducted.

Starch is a polymeric substance in which glucose is linked in two bonds: alpha-1,4 and alpha-1,6. Most bacterial-derived alpha-amylases have alpha-1,6 hydrolysability at the terminal, but mainly act on alpha-1,4 binding. Alpha-glucosidase is known to catalyze the hydrolysis of short-length oligosaccharides, a hydrolysis product of alpha-amylase, which is mainly associated with alpha-glucosidase, which specifically hydrolyzes alpha-1,4 bonds. And oligo-1,6-glucosidase, which hydrolyzes alpha-1,6 bonds.

Alpha-glucosidase is a typical terminal starch hydrolase that cleaves alpha-glucose from the non-reducing end of a substrate oligosaccharide or polysaccharide. Many enzymes with varying substrate specificity and glucose transfer activity have been reported in microorganisms, plants, and animals (Vihinen, M. and Mantsala, P .: Crit. Rev. Biochem. Mol. Biol. , 24, 329-418 (1989) ). Most alpha-glucosidase (α-D-Glucoside glucohydrolase, EC 3.2.1.20) tends to degrade maltose (Kelly, CT et al .: Process Biochem. 18, 6-12 (1983)), but other groups, For example, dextrin 6-alpha-di-glucohydrolase (Oligo-1,6-glucosidase EC 3.2.1.10) specifically acts only on alpha-1,6-glucose binding of isomaltose (Suzuki, Y. et. al .: Biochim. Biophys. Acta. 704, 476-483 (1982). Alpha-glucosidase of Bacillus sp. SAM1606, which is reported to have the broadest substrate specificity, binds alpha-1,3, alpha-1,4 and alpha-1,6 such as nigerose, maltose and isomaltose, in particular 1- Not only O-aryl alpha-glucoside but also alpha-1, alpha-1-glucoside linkages of alpha, alpha-trehalose (Nakao, M. etal .: Appl. Microbiol. Biotechnol ., 41, 337-343 (1994)). The difference in substrate specificity and glucosyl transfer reaction is due to the difference in the structure of the enzyme active site and the binding of the enzyme to the substrate.

The present inventors found an alpha-glucosidase activity that effectively hydrolyzes isomaltose to glucose in cell extracts of the thermophilic microbial strain Summers Caldophilus ZK24 during the study of starch metabolism. Heat-resistant enzymes produced by highly thermophilic microorganisms such as the thermophilic strains are of interest in basic and applied studies, and their industrial application prospects are encouraging. In the starch bioconversion industry, processes with many of these enzymes have been developed and many studies are currently underway to explore new applications. The novel heat resistant alpha-glucosidase is distinguished from oligo-1,6-glucosidase, which exhibits similar activity against isomaltooligosaccharides in that it has an effective hydrolytic capacity against sugars, nigerose and turanose. Among the most heat-resistant alpha-glucosidases reported, the optimum pH of the enzyme activity of the enzyme according to the present invention is high enough to promote alpha-amylolysis in the liquefaction process of the starch industry.

As a result of determining the N-terminal peptide sequence of the purified Summers Caldophilus ZK24-derived alpha-glucosidase by Edman decomposition, Ser-Trp-X-Gln-Arg-Ala-Val-Ile-Tyr -Gln-Val, which was similar to the sequence of group I alpha-glucosidase (bacteria, yeast and insects).

The alpha-glucosidase of Summers Caldophilus ZK24 of the present invention exhibits unique substrate specificity. The enzyme is clearly oligo-1,6-glucosidase in terms of its action on isomaltooligosaccharides and panos, but unlike other oligo-1,6-glucosidase enzymes in sugar activity, It appears to be isomaltase-sukrase. There have been no reports that oligo-1,6-glucosidase can act on sugar. Moreover, the enzyme effectively hydrolyzes turranos and nigerose to glucose. Trehalose, maltose, pullulan and starch, however, do not hydrolyze.

The inventors of the present invention have shown that the thermophilic microorganism Somers Caldophilus ZK24 strain (Kwon ST et al .: Mole. Cells , 7, 264-271 (1997); Park, J. et al .: Eur. J. Biochem. , 214, 135-140 (1993); Taguchi, H. et al .: J. Biochem. , 93, 7-13 (1983)), the cells were recovered and disrupted, and the intracellular enzyme protein was extracted. The new heat resistant alpha-glucosidase was isolated and purified by chromatography. The enzyme was purified 80-fold and had a specific activity of 999.5 units per milligram (mg) of protein, yielding 0.83%. The molecular weight of the enzyme was also estimated at 60 kDa on SDS-polyacrylamide gels. The enzymatic activity of alpha-glucosidase was confirmed by the action of hydrolysis of para-nitrophenyl-alpha-di-glucopyranoside and isomaltose by enzyme extracts and respective chromatographic fractions of enzymes. -Accurately confirmed by activating staining of the denatured protein with 4-methylumbeliferyl-alpha-di-glucoside on polyacrylamide gels. The enzyme showed optimal activity at pH 6.5 and 90 ° C., and the purified protein was stable at pH 6.0-8.5. In addition, even after heating the purified enzyme for up to 90 hours at 90 ℃ showed more than 50% enzymatic activity. The alpha-glucosidase of Summers Caldophilus ZK24 is slightly activated by Ca ²⁺ and Ni ²⁺ ions, while the activity is severely inhibited by Hg ²⁺ ions.

The alpha-glucosidase gene of Somers Caldophilus ZK24 was cloned from total chromosomal DNA using polymerase chain reaction and Southern hybridization methods. The Alpha-glucosidase gene sequence from Somers Caldophilus ZK24 encodes 528 amino acids and has a G + C ratio of 68.4%, with a tendency for G or C to appear at the third position of the codon (93.6%). strong. Expected amino acid sequences showed high homology with sequences of alpha-glucosidase and oligo-1,6-glucosidase, yeast and insects of Bacillus. This homology indicates that Summers Caldophilus ZK24 derived alpha-glucosidase belongs to the alpha-amylase group. The catalytic activity and substrate binding sites are presumed to be Asp198, Glu265, Asp327, His101 and His326, corresponding residues already determined in many alpha-amylase family proteins.

Somers Caldophilus ZK24 derived alpha-glucosidase gene was transferred to a pET22b (+) expression carrier engineered to fit the junction of the carrier. The carrier has a powerful bacteriophage T7 transcription and translation system. Plasmid pTcAGlu was used to mass produce recombinant alpha-glucosidase in Escherichia coli BL21 (DE3). IPTG (isopropyl β-D-thiogalactopyranoside) was used to induce the expression of recombinant alpha-glucosidase, and the enzyme was simply purified by heat-treating the cell extract at 85 ° C for 30 minutes. Recombinant alpha-glucosidase was similar to alpha-glucosidase purified from Summers Caldophilus ZK24 in terms of catalyst properties, substrate specificity and electrophoretic mobility. In addition to the hydrolytic properties, the enzymes have shown that isomaltose or sugars themselves form sugar-transfer reaction products by a combination of primary molecules and acceptor molecules or other substrate acceptor molecules.

Accordingly, an object of the present invention is to provide a thermostable alpha-glucosidase derived from Thermos caldophilus ZK24, which is a thermophilic strain. Another object of the present invention is to provide a gene of the heat resistant alpha-glucosidase. Still another object of the present invention is to provide a recombinant vector containing the gene. Still another object of the present invention is to provide a recombinant strain transformed with the recombinant vector. Still another object of the present invention is to provide a method for preparing a recombinant alpha-glucosidase comprising culturing the transformed cell line. Still another object of the present invention is to provide a method for preparing complex sugars by starch sugar hydrolysis and sugar transfer reaction using alpha-glucosidase prepared by the above method.

In order to achieve the above object, the present inventors cultivated the isolates of alpha-glucosidase from the culture by cultivating the S. caldophilus ZK24 strain, and according to the molecular weight, the optimum temperature and the optimum pH of the enzyme, the addition of metal ions The activity change, the type of sugar as a substrate, and the amino acid sequence of the enzyme were determined.

In order to achieve the above another object, the present inventors have performed Southern hybridization using a labeled DNA probe having an N-terminal coding sequence after treating total chromosomal DNA of the Somers Caldophilus ZK24 strain with an appropriate restriction enzyme. DNA fragments containing alpha-glucosidase genes are recovered and linked to plasmid DNA, transformed into Escherichia coli, and only transformants containing alpha-glucosidase genes are selected through colony hybridization. The recombinant plasmid was isolated to determine the base sequence of the alpha-glucosidase gene.

In order to achieve the above another object, the present inventors produce a recombinant expression vector by linking the alpha-glucosidase gene to an appropriate expression vector, and then prepare a recombinant strain transformed with the expression vector, A method for mass production of alpha-glucosidase was established by culturing to produce alpha-glucosidase and separating and purifying the enzyme.

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

Figure 1 shows the results of SDS-polyacrylamide gel electrophoresis of alpha-glucosidase derived from Thermus caldophilus GK24 strain,

2 is a graph showing the effect of temperature on the enzymatic activity of the thermostable alpha-glucosidase derived from the Somers Caldophilus ZK24 strain,

3 is a graph showing the effect of pH on the enzymatic activity of the thermostable alpha-glucosidase derived from the Summer Caldophilus ZK24 strain,

Figure 4 shows the results of the enzyme activity analysis of thermostable alpha-glucosidase derived from the Somers Caldophilus ZK24 strain,

Figure 5 shows the results of Southern hybridization of total chromosomal DNA by probes of alpha-glucosidase derived from the Summer Caldophilus ZK24 strain,

6 shows the restriction map of the alpha-glucosidase gene and the position of the cloned DNA fragment,

Figure 7 shows the amino acid sequence of the thermostable alpha-glucosidase derived from Somers Caldophilus ZK24 strain and the base sequence encoding the same,

8 shows the results of comparing amino acid sequences between alpha-glucosidases,

FIG. 9 illustrates the construction of a recombinant expression vector comprising a thermostable alpha-glucosidase gene derived from the Somers Caldophilus ZK24 strain,

10 is a result confirming the expression of the recombinant heat-resistant alpha-glucosidase derived from the Somers Caldophilus ZK24 strain,

11 is a result analysis result of the sugar transfer reaction using the isomaltose substrate of the thermostable alpha-glucosidase derived from the Somers Caldophilus ZK24 strain,

12 is a result analysis result of the sugar transfer reaction using the sugar substrate of the recombinant heat-resistant alpha-glucosidase derived from the Somers Caldophilus ZK24 strain.

We have isolated a new heat resistant alpha-glucosidase from the thermophilic strain Thermus caldophilus GK24, as described in detail below. The scope of the present invention includes alpha-glucosidase and its amino acid sequence, and also exhibits the same or similar activity and functionally equivalent protein molecules in the form in which any amino acid residue in the amino acid sequence is substituted with another amino acid which is functionally equivalent. Even protein fragments or derivatives. The scope of the present invention also includes the base sequence encoding the alpha-glucosidase. One of ordinary skill in the art will recognize that the base sequence of the present invention also includes a base sequence by overlapping the genetic code inferred from the alpha-glucosidase amino acid sequence.

The present invention comprises the steps of centrifugation and recovery of the culture medium cultured Somers Caldophilus ZK24 strain for cryopreservation; Preparing an enzyme solution by adding ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) to the crude enzyme solution obtained by crushing and centrifuging the cultured cells in the strain culture medium, re-centrifuging, and dialysis; Separating and purifying the enzyme protein from the enzyme solution obtained through a series of column chromatography using an FPLC system; Determining the molecular weight of the purified alpha-glucosidase by SDS-polyacrylamide electrophoresis; Reacting the purified alpha-glucosidase with isomaltose to determine an optimal activity temperature; Reacting the purified alpha-glucosidase with isomaltose and various metal ions to investigate the effect of each metal ion on enzymatic activity; Reacting the purified alpha-glucosidase with various sugars to investigate a reactive sugar; Determining an N-terminal sequence of alpha-glucosidase using a peptide sequencer; Synthesizing a DNA probe based on the determined amino acid sequence; Preparing total chromosomal DNA of the strain; Reducing the total chromosome DNA by restriction enzyme treatment and performing Southern hybridization to recover a DNA fragment containing an alpha-glucosidase gene, and then linking the DNA fragment with plasmid DNA to determine the base sequence of the gene. ; Binding the determined gene sequence to a plasmid vector and transforming E. coli with the recombinant vector to prepare recombinant E. coli; Culturing the recombinant E. coli to prepare a recombinant alpha-glucosidase; And analyzing the product produced by reacting the prepared recombinant alpha-glucosidase enzyme solution with isomaltose and sugar.

Experimental methods related to such genetic manipulations can be found in Sambrook, J. et al .: Molecular Cloning. A laboratory Manual . 2nd ed., Cold Spring Harbor Laboratory. Cold Spring Harbor.New York., 1989; Ausubel, F. et. al .: Short Protocols in Molecular Biology . 3rd.John & Wiley Sons, Inc., 1995).

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples. However, the following examples are only for illustrating the present invention in detail, and the scope of the present invention is not limited only to these examples.

Example 1 Cultivation of Somers Caldophilus ZK24 Strain, Purification of Alpha-Glucosidase and Characterization of the Enzyme

Experimental Example 1: Cultivation of Somers Caldophilus ZK24 Strain

Cultivation of the Somers Caldophilus ZK24 strain was previously known (Taguchi, H. et al .: J. Biochem. , Tokyo, 91, 1343-1348 (1982); Park, JH et al .: Eur. J. Biochem ., 214, 135-140 (1993)), and cultured at 75 ° C for 24 hours in a medium containing 0.8% polypeptone, 0.4% yeast extract and basic salt (pH 7.2). , The culture was centrifuged at 9,500Xg for 30 minutes at 4 ℃, cells were recovered.

Experimental Example 2 Preparation of Enzyme Liquid

50 g of the S. caldophilus ZK24 strain cultured cells cultured and recovered in Experimental Example 1 were suspended in 40 mM potassium phosphate buffer solution (buffer A) at pH 7.0, and the cells were disrupted by an ultrasonic cell crusher. The crushed cell solution was centrifuged at 12,500 × g for 1 hour at 4 ° C. to remove unbroken cells and insoluble cell debris and to obtain a crude enzyme solution (100 ml). 50% (w / v) ammonium sulfate in a weight-to-volume ratio was slowly added to the crude enzyme solution while stirring at 4 ° C. The enzyme solution was centrifuged at 12,500 × g for 30 minutes to collect only precipitates, and the precipitates were dissolved in Buffer A and dialyzed in the same buffer. The dialysis enzyme solution was centrifuged again to remove insoluble material and then used for purification of the alpha-glucosidase enzyme.

Experimental Example 3: Determination of activity of alpha-glucosidase enzyme

Alpha-glucosidase enzyme activity was measured as the amount of para-nitrophenol liberated from p-nitrophenyl-α-D-glucopyranoside by the enzyme. 0.5 ml of the reaction, including 40 mM phosphate buffer, pH 6.8, 10 mM pienji and enzyme were reacted at 75 ° C. for 10 minutes (Suzuki, Y. et al .: Biochim. Biophys. Acta. , 445, 386-397 (1976), the reaction was stopped by heating the reaction solution in a boiling water bath for 10 minutes. The amount of para-nitrophenol produced in the reaction solution was calculated by absorbance measurement at 405 nm, and one unit of enzyme activity was an enzyme capable of catalyzing the production of 1 μmole of para-nitrophenol per minute under the above conditions. It is defined as the amount of.

Experimental Example 4 Purification of Alpha-Glucosidase

The alpha-glucosidase isolated in Experimental Example 3 was subjected to a series of column chromatography using an FPLC system to purify the enzyme protein. That is, the obtained enzyme solution was passed through a cation exchange resin column (DEAE-Sephacel column, 2.5 X 30 cm) previously equilibrated with buffer solution A, washed sufficiently with the same buffer solution, and then with a sodium chloride concentration gradient (0.1 M to 0.3 M NaCl). Protein was fractionated. Only the fractions having enzymatic activity in each of the fractionated enzyme fractions were collected, the concentrated enzyme solution (15 ml) was concentrated and the hydrophobic equilibrated with the same buffer after buffer replacement with buffer A containing 1 M ammonium sulfate. Passed through a Phenyl-Sepharose column and washed. Of the separated enzyme fractions, only enzyme-enriched fractions (6 ml) were collected, buffer exchanged with buffer A, and a strong cation exchange resin equilibrated with buffer A (MonoQ HR5 / 5 column, 5 X 50 mm). The enzyme fraction was added to and washed with the same buffer sufficiently, and the protein was fractionated with a sodium chloride concentration gradient (0.1 M to 0.35 M NaCl). In addition, only the fraction (2 ml) separated from the enzyme activity was collected, the buffer solution was replaced with buffer A containing 150 mM potassium chloride, and then passed through a gel filter resin column (Sephacryl S-300 column) equilibrated with the same buffer solution. Enzyme proteins were fractionated to yield highly pure purified alpha-glucosidase (Table 1).

Alpha-glucosidase Purification Process from Somers Caldophilus ZK24 Purification stage Total protein (mg) Total Unit Inactivity (Unit / mg) yield(%) Tablet magnification Crude protein 1875 23980 12.8 100 One 50% ammonium sulfate ppt 750 21865 29.1 91.2 2.3 DEAE-Sephacel 510 20234 39.7 84.4 3.1 Phenyl sepharose 35.5 7024 197.9 29.3 15.5 MonoQ 1.8 860 477.8 3.6 37.3 Sephacryl S-300 0.2 199.9 999.5 0.83 78.09

Purification degree of alpha-glucosidase enzyme separated and purified as described above was confirmed as a pure enzyme protein of a single composition on acrylamide gel electrophoresis as shown in FIG.

Example 2 Characterization of Alpha-glucosidase Enzyme from Somers Caldophilus ZK24

Experimental Example 1 Analysis of Molecular Weight by Electrophoresis

The molecular weight of the purified enzyme was determined by 10% SDS-polyacrylamide electrophoresis (SDS-PAGE, SDS-Polyacrylamide Gel Electrophoresis) under the same conditions as known methods (Lammli, UK: Nature , 227, 680-685 (1970)). ). The molecular weight of alpha-glucosidase measured using a molecular weight size marker (Pharmacia LKB) as the standard protein was 60 kDa.

Alpha-glucosidase activation staining is performed by the method of Vibel modified (Bibel, M. et al .: FEMS Microbiol. Lett. , 158, 9-15 (1998)) on 10% polyacrylamide gels containing SDS . It was. More specifically, the enzyme was activated by electrophoresis of the enzyme protein and washing the gel with 2% Triton X-100, and the gel was buffered with 40 mM potassium phosphate buffer containing 3 mM 4-methylumbelliferyl-alpha-di-glucoside. The solution was reacted at 65 ° C. for 15 to 20 minutes. Alpha-glucosidase activity was confirmed by fluorescence under ultraviolet (366 nm) irradiation.

Experimental Example 2: Optimal reaction temperature and thermal stability of alpha-glucosidase

The reactivity of the enzyme according to the given reaction temperature was measured in the same manner as in Experimental Example 3 of Example 1. The optimum temperature for the reaction of alpha-glucosidase was 90 ° C. and showed thermal stability below 95 ° C. as shown in FIG. 2.

Experimental Example 3 Optimum Reaction pH and pH Stability of Alpha-Glucosidase

The reactivity of alpha-glucosidase according to the given reaction pH was measured in the same manner as in Experiment 3 of Example 1. As shown in FIG. 3, the optimum pH of the enzyme was 6.5 and showed pH stability at pH 6.0-8.5.

Experimental Example 4: Effect of various metal ions on alpha-glucosidase activity

The reactivity of alpha-glucosidase with the addition of metal ions was measured in the same manner as in Experiment 3 of Example 1. As shown in Table 2 below, calcium and nickel ions slightly increased the enzyme activity when the various metal ions were added at a concentration of 1 mM, but the mercury ions were strongly inhibited. For other metal ions there was no specific change in enzyme activity.

Effect of Metal Ions on the Activities of Somers Caldophilus ZK24 Derived Alpha-Glucosidase Metal ions Concentration [mM] Relative activity None - 100.0 Aluminum Chloride (AlCl ₃ ) 1.0 99.7 Calcium chloride (CaCl ₂ ) 1.0 104.8 Cobalt Chloride (CoCl ₂ ) 1.0 99.1 Copper chloride (CuCl ₂ ) 1.0 99.4 Ferric Chloride (FeCl ₃ ) 1.0 95.3 Mercury chloride (HgCl ₂ ) 1.0 3.0 Magnesium Chloride (MgCl ₂ ) 1.0 96.4 Manganese Chloride (MnCl ₂ ) 1.0 98.3 Nickel Chloride (NiCl ₂ ) 1.0 105.1 Zinc Chloride (ZnCl ₂ ) 1.0 95.2 EDTA 1.0 99.7

Example 5 Substrate Specificity and Sugar Transfer Reaction

Substrate specificity was determined by reaction at 75 ° C. for 1 hour using alpha-glucosidase purified in 40 mM potassium phosphate buffer (pH 7.0) containing 10 mM oligosaccharide substrates. Measurement of the transfer reaction was performed by reacting 40 mM potassium phosphate buffer (pH 7.0) at 75 ° C. for 72 hours as isomaltose only or as a mixture with acceptor molecules. The reaction mixture was boiled for 10 minutes and centrifuged to stop the reaction. The sugar products of the hydrolysis and sugar transfer reaction of the substrate were Dionex high performance anion-exchange chromatography with pulsedamperometric detector and HPAEC-PAD column (Dionex CarboPac PA-1, 0.4 X 25 cm) The concentration of 60 mM sodium hydroxide and 0 to 300 mM sodium acetate concentration was analyzed using a flow rate of 1 ml / min.

Substrate specificity is shown in Table 3 below, depending on the relative degree of degradation for various substrates.

Substrate Specificity of Somers Caldophilus ZK24 Derived Alpha-Glucosidase temperament Glycoside Bonds Relative Glucose Yield (%) Isomaltose Glucose-α-1,6-glucose 100.0 Isomaltotriose Glucose-α-1,6-glucose-α-1,6-glucose 77.0 Isomaltotetraose Glucose- [α-1,6] ₃ -glucose 50.5 Panose Glucose-α-1,6-glucose-α-1,4-glucose 55.0 Gentibiose Glucose-β-1,6-glucose 0.0 Leucrose Glucose-α-1,5-fructose 0.0 Maltose Glucose-α-1,4-glucose 0.0 Lactose Galactose-β-1,4-glucose 0.0 Cellobiose Glucose-β-1,4-glucose 0.0 Nigerose Glucose-α-1,3-glucose 86.0 Turanose Glucose-α-1,3-fructose 54.8 Laminaribiose Glucose-β-1,3-glucose 0.0 Sucrose Glucose-α-1,2-fructose 65.0 Trehalose Glucose-α-1,1-glucose 0.0 Pullulan 0.0 Starch 0.0

Alpha-glucosidase produced glucose from isomaltose, panos, isomaltotriose and isomaltotetraose, but no glucose was produced from starch and pullulan. In addition, glucose was also produced from sugar, nigerose and turanose. In the case of isomaltose, the glucose production rate was the highest (FIG. 4). In more detail, the substrate specificity of the enzyme alpha-glucosidase of the present invention is best hydrolyzed to alpha-1,6 bonds such as isomaltose, isomaltoligosaccharide and panose, Alpha-1,3 linkage between glucose and alpha-1,2, alpha-1,3 linkage between glucose and fructose present in sugar, turanose, etc., are a large amount of hydrolysis, maltose, trehalose, cellobiose, gentibio and la Alpha-1,4, alpha-1,1, beta-1,4, beta-1,6, beta-1,3 bonds such as Minaribiose and alpha-1,4 bonds of Panos were not hydrolyzed. .

Experimental Example 6: N-terminal amino acid sequence determination

The purified enzyme was electrophoresed on a polyacrylamide gel, the enzyme protein on the gel was transferred to a polyvinylidine difluoride (PVDF) membrane by electroblotter, and only the enzyme protein band on the PVDF membrane was subjected to Edman degradation. N-terminal sequences were determined by analysis with an Applied Biosystems model 476A Protein / peptide sequencer. As shown in Table 4 below, the N-terminal amino acid sequence of the alpha-glucosidase of the present invention was SWXQRAVIYQV, similar to that of group I alpha-glucosidase with a molecular weight range of 50-66 kDa.

Comparison of N-terminal Amino Acid Sequences with Somers Caldophilus ZK24 Derived Alpha-Glucosidase and Group I Alpha-Glucosidase organism N-terminal amino acid sequence Somer's caldophilus GK24 ² SWXQRAVIYQV ¹² Bacillus strain SAM1606 ( Bacillus sp SAM1606) ² STALTQTSNSQQSPIRRAWWKEAVVYQI ²⁹ Bacillus sp. KP1006 ^One MERVWWKEAVVYQI ¹⁴ Bacillus cereus ^One MEKQWWKESVVYQI ¹⁴ Saccharomyces cerevisiae ^One MTISDHPETEPKWWEKEATIYQI ²² Mosquito ( Ades aegypti ) ^One MKIFVPLLSFLLAGLTTGLDWWEHGNFYQV ³¹ Worker-Bee, Apis mellifera L ^One MKAVIVFCLMALSIVDAAWKPLPENLKEDLIVYYQV ³⁵

Example 3 Cloning of Alpha-glucosidase Enzyme Gene from Somers Caldophilus ZK24 Strain

Experimental Example 1 Synthesis of DNA Brows by Polymerase Chain Reaction (PCR)

In order to synthesize probe DNA by PCR, primer 1 having the following nucleotide sequence (23mer) consisting of 23 bases was synthesized using a DNA synthesizer based on the amino acid sequence determined in Experimental Example 7 of Example 2.

Primer 1:

5'-CAGCG (G / C) GC (G / C) T (G / A / T / C) AT (A / T / C) TACCAGGT (G / A / T / C) -3 '

The amino acid sequences of the previously reported alpha-glucosidases were compared to select the well-conserved sequences therein to synthesize primer 2 having the following nucleotide sequence, the opposite oligonucleotide.

Primer 2:

5 '-(G / A / T / C) TCCCA (A / G) TT (G / A / T / C) AG (A / G) TC (G / A / T / C) GG (C / T) TG-3 '

Polymerase chain reaction was performed using the primers 1 and 2 to synthesize a 0.5 kb DNA probe. The sequence of the probe has similarities with several alpha-glucosidase, and the identification and cloning of the alpha-glucosidase gene derived from the Summers Caldophilus ZK24 strain by performing Southern hybridization using the probe. Was performed.

Experimental Example 2 Preparation of Total Chromosome DNA of Somers Caldophilus ZK24 Strain

The cells of the S. caldophyllus ZK24 strain cultured 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 0.5 mg / After adding lysozyme to ml and reacting at 37 ° C. for 30 minutes, SDS was added to 1% and RNase was added to 50 μg / ml for 1 hour at 37 ° C., followed by proteolysis to 0.2 mg / ml. Enzyme-K (Protenase K) was added and reacted at 37 ° C. for 3 hours, and then the same amount of phenol was added for 12 hours. Only the aqueous layer was recovered by centrifugation, and double alcohol was added to precipitate DNA to recover. The chromosomal DNA was washed three times with pure spirits and dissolved in a TE buffer solution to be used as total chromosomal DNA.

Experimental Example 3: Search for alpha-glucosidase gene and recombinant production

DNA fragments containing the alpha-glucosidase gene cut by restriction enzymes were selected by performing Southern hybridization on the total chromosomal DNA using the probe synthesized in Experimental Example 1. That is, the total chromosomal DNA of the Somers Caldophilus ZK24 strain as described in the general known methods and in the user manual of the Dig Labeling and Detection Kit of Boehringer Mannheim GmbH, Biochemica, Germany. Alpha-glucosidase as shown in FIG. 5 using various restriction enzymes suitable for the production of recombinant plasmids and running on agarose gel, Southern blotting, Southern hybridization method using the probe DNA fragments having a size of 3.3 kb (pAGON4) by restriction enzyme BamH I treatment and 3.1 kb (pAGON41) by Hind III treatment were isolated and purified by binding to the plasmid pUC 18, and the recombinant The plasmid was transformed into Escherichia coli to make a recombinant, and the nucleotide sequence thereof was determined.

Experimental Example 4: Determination of the base sequence of the alpha-glucosidase gene derived from the Somers Caldophilus ZK24 strain

Plasmids pAGON4 and pAGON41 were isolated from the recombinant constructed in Experimental Example 3 by a known method, and DNA fragments containing the cloned alpha-glucosidase gene were further cut and separated by restriction enzymes Bam HI, Sac I, Hind III, etc. After purification, plasmid pAGON-06BH, pAGON-08SH, pAGON-19SS and pAGON-25HB were constructed by subcloning into plasmid pUC 18 (FIG. 6). The base sequence of the alpha-glucosidase gene was determined using the plasmids pAGON4, pAGON41, pAGON-06BH, pAGON-08SH, pAGON-19SS and pAGON-25HB. The sequencing was performed using the AB1373A automatic DNA sequencer of the Biotechnology Research Institute Genome Project.

Figure 7 shows the nucleotide sequence of the alpha-glucosidase gene derived from the S. caldophilus ZK24 strain determined as a result of the experiment. According to the amino acid and DNA sequences thus determined, the alpha-glucosidase derived from the S. caldophilus ZK24 strain was composed of 529 amino acids, and the molecular weight of the enzyme was 61 kDa, and the gene of the enzyme was 1,590 bases. It is composed of 68.7% of G + C.

As shown in FIG. 8, when comparing the amino acid sequence with alpha-glucosidase isolated from other species, oligo-1,6-glucosidase of Bacillus coagulans and 36.3%, Bacillus strain SAM1606 ( . Bacillus sp SAM1606) alpha-glucosidase and 35.9%, Bacillus Thermo-glucosidase siwooseu KP1006 (raising of Bacillus thermoglucodasiu s KP1006) -1,6- glucosidase and 34.8%, Thermo North Fora larger Bata (Themonospora curvata) Homology with alpha-glucosidase of 35.9% and beta (Adult Worker Bee, Apis mellifera L) with 36.1% (Watanabe, K. et al .: Appl. Environ.Microbiol. , 62 (6), 2066-2073 (1996); Nakao, M. et al .: Eur. J. Biochem ., 220 (2), 293-300 (1994); Janda, L. et al .: J. Appl. Microbiol. , 83, 470-476 (1997); Watanabe, K. et al .: Eur. J. Biochem. , 192 (3), 609-620 (1990); Watanabe, K. et al., J . Biol Chem, 266, 24287-24294 ( 1991);.. James, AA et al .: Gene , 75, 73-83 (1989); Ohashi, K. et al .: Biochim. Biophys.Res. Commun., 221, 380-385 (1996)). Alpha-glucosidase derived from the S. caldophyllus ZK24 strain shows homology in the amino acid sequence as described above, and as shown in FIG. 8, it has six preservation zones (R1 to R6). Therefore, it can be seen that it belongs to the alpha-amylase group, and among them, it can be seen that the enzyme belongs to the alpha-amylase group I.

The alpha-glucosidase of the present invention shows a great difference in substrate specificity despite high sequence similarity with the alpha-glucosidase of various origins. By enzyme-specific action on isomaltooligosaccharides and panos, alpha-glucosidase derived from the S. caldophilus ZK24 strain is oligo-1,6-glucosidase, and sugar and alpha-1,3- as substrates. By the action of this enzyme on glucose binding, the enzyme of the present invention is sucrase-isomaltase. In addition, unlike alpha-glucosidase of Bacillus sp. SAM1606, it does not degrade maltose and trehalose. In the above, the alpha-glucosidase derived from the Summers Caldophilus strain in the present invention is a novel enzyme.

Example 4 Expression Vector Construction of Alpha-Glucosidase Gene, Expression of Recombinant Alpha-glucosidase and Trehalose Synthesis

Experimental Example 1 Preparation of Expression Vector of Alpha-Glucosidase Gene

As shown in FIG. 9, an expression vector of an alpha-glucosidase gene was constructed. DNA amplifying and recovering the DNA corresponding to the alpha-glucosidase gene (OFR, open reading frame) by polymerase chain reaction, binding to pET22b (+), a protein expression plasmid vector, and transfecting into host E. coli The conversion produced alpha-glucosidase expressing recombinants. More specifically, on the basis of the alpha-glucosidase N-terminal gene, a new PCR primer 5'-GAACATATGAGCTGGTGGCAAAGGGCG-3 'and a new C-terminus comprising a restriction enzyme Nde I cleavage site and a transcription initiation codon ATG are newly introduced. The polymerase chain reaction using PCR primer 5'-GCAAAGCTTCTAGTCTAGCCGCACCGC-3 'containing restriction enzyme Hind III cleavage site was performed to amplify and isolate the alpha-glucosidase gene DNA corresponding to 1.6 kb. The amplified alpha-glucosidase gene DNA was treated with restriction enzymes Nde I, Hind III, isolated and purified and then bound to the plasmid vector pET22b (+) for protein expression treated with the same enzyme. The alpha-glucosidase expression plasmid pTcAGlu constructed by binding to each other was transformed into expression E. coli ( E. coli BL21 (DE3)) to prepare an alpha-glucosidase expression recombinant.

E. coli BL21 (DE3) [pTcAGlu] containing the plasmid pTcAGlu has been deposited with the gene bank affiliated with the Biotechnology Research Institute as accession number KCTC 8943P (deposit date: June 1, 1999).

Experimental Example 2 Expression and Purification of Recombinant Alpha-Glucosidase

To express the cloned alpha-glucosidase gene, Escherichia coli BL21 (DE3) [pTcAGlu] was inoculated in LB liquid medium to which 50 μg / ml of ampicillin was added and cultured in large quantities. After incubation at 37 ° C. for about 3 hours, 1 mM IPTG was added at the final concentration and continued for about 12 hours to induce gene expression (FIG. 10). The expression rate was as high as 10% of the total cellular protein. The cells cultured as described above were recovered by centrifugation, suspended in 20 mM potassium phosphate buffer (pH 7.0), and the cells were disrupted by an ultrasonic cell disruptor. The crushed cell solution was centrifuged at 12,500 × g for 30 minutes at 4 ° C. to form an enzyme solution from which unbroken cells and insoluble cell debris were removed. Only the water-soluble supernatant obtained above was heat treated at 85 ° C. for 30 minutes and centrifuged to remove E. coli derived denatured protein. The characteristics of the purified alpha-glucosidase as described above are characterized by mobility, optimum temperature, pH and substrate (isomaltooligosaccharide, panose, sugar, nigerose, turanose, etc.) on SDS-polyacrylamide gel electrophoresis. In specificity, they were identical to the alpha-glucosidase derived from the Summers Caldophilus ZK24 strain.

Experimental Example 3: Sugar Transition Reaction by Recombinant Alpha-Glucosidase

As shown in Figure 11 and 12, using the recombinant alpha-glucosidase of Experimental Example 2 was confirmed the sugar transfer reaction. 10 to 500 mM isomaltose or 10 mM to 2.0 M sugar only, or in combination with 20 mM maltose, trehalose, leucross, erlos, lactose as acceptor molecules at 75 ° C. under 40 mM potassium phosphate buffer (pH 7.0) 72 hours enzymatic reaction. The reaction was boiled for 20 minutes to stop the reaction and the reaction product was analyzed by HPAEC-PAD. As a result of analyzing the reaction solution as described above (FIG. 11), as a result of enzymatic reaction with 500 mM isomaltose as a substrate, the product was partially reacted with isomaltose, which is the substrate itself, and glucose, a hydrolysis product, and glucose transfer reaction. Isomaltrios, isomaltotetraose and larger sugars with increased sugar chain length than isomaltose produced. In the case of using the sugar as a substrate (Fig. 12), the peaks of the new sugars were detected behind the sugar in addition to the remaining substrate sugar and the hydrolyzate glucose and fructose.

As described in the above Examples and Experimental Examples, the recombinant alpha-glucosidase prepared by the method of the present invention may catalyze synergistic liquefaction that can improve the glucose production yield by microbial alpha-amylase in the starch bioconversion process. In addition, the sugar transfer reaction can be used to produce new polysaccharides for food and pharmaceutical purposes, which is a very useful invention for the starch industry, food and biopharmaceutical industry.

Claims (11)

Heat-resistant alpha-glucosidase derived from the Themus caldophilus GK24 strain having the following characteristics: i) Reaction temperature: 30-100 ℃ Ii) Stable pH: pH 6.0-8.5 Substrate specificity: isomaltose, isomaltoriose, isomaltotetraose, panose, nigerose, turanose, sugar Iii) amino acid sequence: A gene comprising a nucleotide sequence encoding the alpha-glucosidase of claim 1. The gene according to claim 2, wherein the gene comprises the following nucleotide sequence. delete A recombinant gene expression vector comprising the gene of any one of claims 2 to 3. The method of claim 5, wherein the recombinant gene expression vector is pTcAGlu. E. coli transformed with the recombinant gene expression vector of any one of claims 5 to 6. The method of claim 7, wherein the transformed E. coli is BL21 (DE3) [pTcAGlu] (KCTC 8943P). E. coli BL21 (DE3) [pTcAGlu] (KCTC 8943P) transformed by a recombinant gene expression vector pTcAGlu containing a gene comprising a base sequence encoding alpha-glucosidase as described in claim 1 A method for producing alpha-glucosidase derived from Summers Caldophilus ZK 24 strain. delete A method for producing a complex sugar by starch sugar hydrolysis and reverse hydrolysis (sugar transfer reaction) using the heat resistant alpha-glucosidase of claim 1.