KR100893202B1 - Heat-resistant thermotoga maritima glucosidase and preparation method of glucose with the same - Google Patents

Abstract

The present invention relates to a heat-resistant thermomoto maritima glucosidase, and a method for preparing glucose using the same, wherein the heat-resistant thermogram, the heat-resistant enzyme of the present invention, can be used at high temperature because it is heat-resistant, By using this to prepare glucose from starch, no harmful salts are produced and glucose can be prepared safely from starch without changing pH at high temperatures.

Heat Resistant Enzyme, Starch, Glucose

Description

Heat-Resistant THERMOTOGA MARITIMA GLUCOSIDASE AND PREPARATION METHOD OF GLUCOSE WITH THE SAME}

1 is a process chart for preparing glucose from starch using an enzymatic reaction according to the prior art.

2 is a process chart for preparing glucose from starch using the heat resistant enzyme of the present invention.

3 is a result of SDS-PAGE analysis of heat-resistant thermomoto Maritrima glucosidase.

4 is a thin-film chromatography analysis showing the results of various substrate decomposition by heat-resistant thermomoto maritima glucosidase.

Figure 5 is a reaction chart showing the thermal degradation of the substrate thermolysis of maritrima glucosidase.

6 is a graph showing the stability of various buffer solutions of heat-resistant thermomoto maritima glucosidase.

7A to 7B are graphs showing the operating pH and operating temperature of the heat resistant thermomoto maritrima glucosidase.                 

8A to 8B are graphs showing the working pH and working temperature of the heat resistant pullulanase.

9A to 9B are graphs showing the working pH and working temperature of the heat resistant α-glucosidase.

Figure 10a is the HPIC results of the reaction solution treated with maltohexaose heat-resistant α- glucosidase, Figure 10b is a reaction of heat-resistant α-glucosidase and heat-resistant thermomoto treated maliti glucosidase to maltohexaose HPIC result of the solution.

11 is a diagram showing the results of analyzing the reaction solution of Example 5-1 using high performance ion chromatography.

12 is a diagram showing the results of analyzing the reaction solution obtained in Example 5-1 using thin layer chromatography.

[Industrial use]

The present invention relates to a method for producing glucose from marithima glucosidase, and starch using the same, and more particularly, because it is heat-resistant and can be used at high temperatures, thereby preparing glucose from starch. The present invention relates to a method for producing glucose from starch safely without generating harmful salts and without changing pH at high temperatures.                         

[Prior art]

Glucose production from starch includes a chemical method and a biological method using an enzyme. In the conventional biological method, as shown in FIG. 1, caustic soda (NaOH) was added to 30% of starch slurry to adjust the pH to 6.0 to 6.2, and heat resistant α-amylase was added thereto. After jetting through a jet cooker for about 5 to 10 minutes, the solution is reacted at 95 ° C. for 1 to 2 hours to complete liquefaction. At this time, hydrochloric acid was added to adjust the pH of the liquefied starch solution back to 4.2 to 4.8, followed by addition of heat-resistant pullulanase, an α-1,6 glucoside bond cleavage enzyme, and glucoamylase, a glycosylating enzyme. The method of preparing glucose by reacting at 72 DEG C for 72 hours was mainly used.

However, when glucose is prepared from starch by the above method, acid and alkali are repeatedly used during the manufacturing process, and thus, harmful salts are generated. In addition, the optimum temperature of each enzyme is different from each other to cool the solution, so there is a problem that the reaction temperature is low, there is a risk of contamination of microorganisms and a long reaction time of 72 hours or more is required.

The present invention is to solve the above-mentioned problems of the prior art, the object of the present invention is produced by Thermotoga maritima , the optimum operating pH is 6.0 to 7.0 and the optimum operating temperature is 80 to 90 ℃ To provide maritrima glucosidase, a thermostable thermogram that degrades α-1,4 glucoside bonds and α-1,6 glucoside bonds.

It is still another object of the present invention to obtain a liquefied reaction solution obtained by treating starch with α-amylase, and the liquefied reaction solution is subjected to the above-mentioned heat-resistant thermomoto glucosidase, heat-resistant fluranase and heat-resistant α-glucosidase. To provide a method for producing glucose from starch comprising the step of treating and saccharifying.

Another object of the present invention is to provide a glucose produced by the above method.

In order to achieve the above object, the present invention is produced by the Motomoto Marittima, the optimum operating pH is 6.0 to 7.0 and the optimum operating temperature is 80 to 90 ℃, α-1,4 glucoside and α-1,6 Heat-resistant thermomotors that break down glucoside bonds provide maritima glucosidase.

The present invention also obtains a liquefied reaction solution in which starch is treated with α-amylase, and the liquefied reaction solution is treated with the above-mentioned heat-resistant thermomoto maritrima glucosidase, heat-resistant fluranease and heat-resistant α-glucosidase. It provides a method for producing glucose from starch comprising the step of saccharification.

The present invention also provides glucose prepared by the above method.

Hereinafter, the present invention will be described in more detail.

FIG. 6 is a graph showing the stability of various heat buffers of thermotoga maritima glucosidase of the present invention. As shown in FIG. 6, it can be seen that the heat-resistant thermogram of the present invention maritrima glucosidase is relatively stable in a buffer solution of potassium-faspace.

7A to 7B are graphs showing the operating pH and operating temperature of the heat-resistant thermomoto of the present invention maritima glucosidase. As shown in FIG. 7A, the working pH of the heat-resistant thermomotor is maritima glucosidase is 6.0 to 8.0, the optimum working pH is 6.0 to 7.0, and as shown in FIG. The operating temperature of seades is 70 to 90 ° C. and the optimum operating temperature is 80 to 90 ° C.

Therefore, it can be used at high temperatures due to its high heat resistance and its optimum working pH is similar to that of α-amylase or heat-resistant α-glucosidase, so that the reaction conditions can be easily controlled when used with these enzymes.

Unlike the existing α-glucosidase, thermomoto maritima glucosidase does not hydrolyze maltose, and specifically recognizes substances present at the reducing end and hydrolyzes them into glucose units to finally form glucose and maltose. To produce.

In addition, Thermomoto Maritrima Glucosidase hydrolyzes α-1,4 glucosidic and α-1,6 glucosidic bonds of acarbose, a potent inhibitor of α-amylase, and glucose present at the reducing end of the substrate. can do.

The working substrates of heat-resistant thermomoto-maritimar glucosidase are maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, paranite P- nitrophenylpentaglucoside, beta-cyclodextrin, acarbose and isocarbose.

4 is a thin-film chromatography analysis showing the results of various substrate decomposition by the heat-resistant thermomoto maritima glucosidase. As shown in FIG. 4, it can be seen that the heat-resistant thermomoto maritima glucosidase can hydrolyze paranitrophenyl pentaglucoside (pNPG5), maltopentaose and cyclodextrin. The final hydrolysis products are glucose and maltose and it can be seen that maltose is not susceptible to degradation by Marittima Glucosidase. In addition, it can be seen that the heat-resistant thermomoto maritima glucosidase produces glucose and pseudo-samtan sugar (PTS) by hydrolyzing acarbose and acaocarbose.

Therefore, it can be seen that the enzyme hydrolyzes α-1,4 glucoside bond and α-1,6 glucoside bond of reducing terminal glucose.

Table 1 shows the reaction rate for the various substrates of the heat-resistant thermomoto maritima glucosidase. The rate of hydrolysis of the thermostable thermomoto on the substrate of Marittima glucosidase was in order of maltopentaose> maltotetraose> maltotriose.                     

temperament k _cat (S ^-1 ) Km (mM) k _cat / Km (S ^-1 mM ^-1 ) Malto trios 3.196 ± 0.256 20.639 ± 3.333 0.155 ± 0.028 Maltotetraos 4.336 ± 0.559 11.835 ± 1.035 0.366 ± 0.057 Maltopentaos 5.182 ± 0.232 9.947 ± 0.774 0.521 ± 0.047 Maltohexaos 4.562 ± 0.546 7.773 ± 1.617 0.587 ± 0.141 Maltoheptaos 4.090 ± 0.250 7.410 ± 0.762 0.552 ± 0.066 Paranitrophenyl pentaglucoside 16.283 ± 0.410 2.261 ± 0.164 7.201 ± 0.551 Beta-cyclodextrin 1.373 ± 0.178 14.531 ± 2.206 0.094 ± 0.019

In Figure 5, the heat-resistant thermomoto for various substrates showed the substrate degradation of Maritrima glucosidase. As shown in FIG. 5, Thermomoto Maritrima glucosidase is composed of paranitrophenylpentaglucoside ( p- nitrophenylpentaglucoside), beta-cyclodextrin, acarbose and isocarbose. Hydrolysis can be performed, whereby paranitrophenol is recognized and hydrolyzed as well as glucose present at the reducing end of each substrate.

The present invention also provides a liquefaction reaction solution obtained by treating starch with α-amylase, and the liquefaction reaction solution is treated with the above heat-resistant thermomoto maritrima glucosidase, heat-resistant fluranease and heat-resistant α-glucosidase. It provides a method for producing glucose from starch comprising the step of saccharification. Preferred pH for the reaction is 5.5 to 6.5 and the reaction temperature is 70 to 85 ℃.

When the three enzymes are added to the starch solution, the reaction mechanism for preparing starch into glucose is shown in Scheme 1 below.                     

As shown in Scheme 1, α-amylase is an enzyme that converts starch into maltodextrin, an oligosaccharide. In addition, heat-resistant thermomoto maritima glucosidase and heat-resistant α-glucosidase are enzymes that convert maltodextrin to glucose.

Among them, heat-resistant thermomoto is a high rate of decomposition of oligosaccharides (G6 ~ G7) with a relatively long glucose chain, while heat-resistant α-glucosidase is a decomposition rate for maltose with a relatively short glucose chain Is large. As described above, in the present invention, the two glycosylases can be added to the starch solution at the same time so that the reaction can be progressed to shorten the glucose production time, thereby efficiently preparing glucose.

The three enzymes used in Scheme 1, heat-resistant fluranase, heat-resistant thermomoto, and maritrima glucosidase and heat-resistant α-glucosidase are all heat-resistant enzymes and do not lower the temperature of the reaction solution to 70 to 85 ° C. or lower. You don't have to. Therefore, the reaction conditions are simple because the temperature of the reaction solution does not need to be adjusted. In addition, it is possible to maintain the temperature of the reaction solution at a high temperature by using the heat resistant enzyme, which has the advantage of preventing contamination of microorganisms during the reaction.                     

The heat resistant pullulanase used in the present invention is an enzyme that converts starch into maltodextrin. The working pH and working temperature of this heat resistant pullulanase are shown in Figs. 8A to 8B. FIG. 8a shows enzyme titers according to pH for various buffer solutions of heat resistant pullulanase. As shown in FIG. 8A, this heat resistant flulanase has a working pH of 4.0 to 7.0. In addition, it can be seen that the enzyme titer according to the pH change with respect to sodium-phosphate buffer among the various buffer solutions is weighted higher.

In addition, as shown in Figure 7b, the operating temperature is 70 to 85 ℃. This heat resistant fluranease may be prepared and used in the laboratory using the strain of Marimotoma MRS8, or commercially available Promozyme 400L (Novozymes).

In addition, the heat resistant α-glucosidase used in the present invention is an enzyme that converts starch into maltodextrin. The working pH and working temperature of this heat resistant α-glucosidase are shown in Figs. 8A to 8B. As shown in FIG. 9A, the working pH of α-glucosidase is 4.0 to 7.0, and the working temperature is 70 to 110 ° C. as shown in FIG. 8B.

The heat-resistant α-glucosidase can be prepared and used using the Sulfarobus Sulfataricus ATCC 35092 strain, or commercially available AMG E produced by Novozymes.

Hereinafter, a method of preparing glucose from starch of the present invention will be described with reference to FIG. 2.                     

The starch slurry at 30% concentration is adjusted to pH 6.0-6.2. This starch slurry is then passed through a 105 ° C. jet cooker for 5 to 10 minutes to gelatinize. Next, α-amylase is added to the cooker and reacted at 95 ° C. for 1 to 2 hours to liquefy the starch slurry.

The liquefied starch solution is then saccharified by the addition of heat-resistant thermomoto glucoma marimarima glucosidase, heat-resistant fluranase and heat-resistant α-glucosidase.

The saccharification reaction is preferably performed for 24 to 48 hours at 80 ℃ to 90 ℃, preferably 80 ℃ to 85 ℃.

The present invention relates to a heat-resistant thermomoto maritima glucosidase, and a method for preparing glucose using the same, wherein the heat-resistant thermogram, the heat-resistant enzyme of the present invention, can be used at high temperature because it is heat-resistant, By using this to prepare glucose from starch, no harmful salts are produced and glucose can be prepared safely from starch without changing pH at high temperatures.

Hereinafter, preferred examples and comparative examples of the present invention are described. The following examples and comparative examples are described for the purpose of more clearly expressing the present invention, but the contents of the present invention are not limited to the following examples.

Example 1

Example 1 Heat Resistant Thermomoto Maritrima Glucosidase

(1-1) Cloning and Expression of Heat-Resistant Thermomotor Marittima Glucosidase

After obtaining a culture medium of MARIMATIC MRS8 through the Japanese Collection of Microorganisms, the cells were collected by centrifugation and chromosomal DNA was isolated using a QIAmp Tissue Kit (QIAGEN Co., USA).

Thermostable Thermomoto prepared TMBspHI, 5-CGGTGAAATCATGAT GTACCCGATGC-3, and TMC, 5-TTCGTCGACGTCAGAATAGCGCG-3 primers to isolate genes of Marittima glucosidase, and Thermomoto polymerase chained to the chromosomal DNA of Marittima MRS8. The reaction was carried out to obtain a product of about 2.0 kb.

The amplified gene fragments were treated with BspHII and SalI and ligated with p6xHis119 vector treated with BspHII and SalI. For transformation, 5 ml of LB medium was inoculated with E. coli MC1061, a host cell, incubated at 37 ° C for 12 hours, and then 1.0 ml of the culture was inoculated in 50 ml of fresh LB medium and the absorbance was 0.5 at 600 nm. Incubated. The cells were recovered by centrifuging 1.5 ml of the culture solution at 7000 x g for 5 minutes at 4 ° C., and then, 0.75 ml of the transformed solution 1 was added thereto, and the cells were suspended for 30 minutes in ice. The cells were recovered again by centrifugation at 6000 x g for 2 minutes at 4 ° C. 0.15 ml of the transformed solution 2 was added to the recovered cells, and the cells were suspended and left in ice for 30 minutes. 0.15 ml of the suspension and 500 ng of the ligated solution were mixed and left to stand in ice for 1 hour and then subjected to thermal shock at 42 ° C. for 2 minutes. 1 ml of LB medium was added thereto, followed by incubation at 37 ° C for 1 hour, and then plated on 1.5% agar-containing LBA solid medium to select strains resistant to empicillin.

The first selected transformants were inoculated in 5 ml of LB medium and incubated at 37 ° C. for 12 hours, followed by centrifugation to obtain cells. The recovered cells were suspended in 50 mM phosphate buffer at pH 7.0, then disrupted by ultrasound and centrifuged to obtain supernatant. The strain showing the activity by measuring the enzyme titer of the supernatant was finally selected as a heat-resistant thermomotogamaritia glucosidase producing strain (Escherichia coli MC1061 / pBLTMG).

(1-2) Tablet of heat resistance thermomotoga Marittima glucosidase

Heat-resistant thermomoto was inoculated with Maritrima glucosidase producing strain (Escherichia coli MC1061 / pBLTMG) in LBA medium, incubated at 37 ° C. for 10 hours, and centrifuged to obtain cells. The recovered cells were suspended in 50 mM phosphate buffer at pH 7.0, then disrupted by ultrasound and centrifuged to obtain supernatant. The expressed dextrin glucosidase was purified by chromatography using ion exchange resin.

(1-3) Enzyme Activity Measurement

Enzyme titer was added to 0.2 ml of 50 mM phosphate buffer solution of pH 8.0 and 0.25 ml of 1% (w / v) maltotriose solution in 0.05 ml of enzyme solution, and then reacted at 85 ° C for 10 minutes and boiled for 5 minutes to stop the reaction. In addition, 0.1 ml of the reaction solution and 0.9 ml of a glucose solution (Glucose E kit, Yeongdong Pharm.) Were added and reacted for 30 minutes at 37 ° C. for color development. The absorbance was measured at 505 nm. The enzyme titer determines the amount of enzyme that produces 1 mmole of glucose per minute in 1 unit.

(1-4) Molecular Weight Measurement

Figure 3 shows the results of the analysis of the heat-resistant thermomoto prepared by using the SDS-PAGE maritimea glucosidase. As shown in FIG. 3, the molecular weight of the heat resistant thermotoga marittima glucosidase was about 55,900 to 56,300 daltons.

      Example 2: Heat Resistance α-Glucosidase

The following primers were prepared to isolate heat resistant α-glucosidase genes.

SSAgl-N (Forward Primer) 5'-ATACGGTGATAACATATGCAGACAATAAAA-3 ',

SSAgl-C (Reverse Antistatic Primer) 5'-AGATGTGAGTCGACTTGTAGGCAAGG-3 '

The primer pair was used to perform a polymerase chain reaction on the main chromosome of sulfolabus sulfataricus ATCC 35092 to obtain a product of about 2.0 kb. The amplified gene fragments were treated with NdeI and SalI and ligated with the p6xHis119 vector completely digested with the same enzyme. The strain was transformed in the same manner as in Example 1 and first selected a strain showing resistance to empicillin.

The first selected transformants were inoculated in LBA solid medium containing 1% soluble starch and incubated at 37 ° C. for about 12 hours, and 0.6% agar medium containing 3 mg of D-cycloserine was added thereto. 5 ml of the transformant was poured onto the cultured solid medium and incubated again at 37 ° C. for 12 hours. Iodine solution was poured into the solid medium, and a strain showing a transparent ring on the strain was finally selected as a heat resistant α-glucosidase producing strain (Escherichia coli MC1061 / p6xHSSAgl119).

Heat-resistant α-glucosidase producing strain (Escherichia coli MC1061 / p6xHSSAgl119) was inoculated in LBA medium, incubated at 37 ° C. for 10 hours, and centrifuged to obtain cells. The recovered cells were suspended in 50 mM Tris buffer solution at pH 7.5, then disrupted by ultrasonication, and centrifuged to obtain supernatant.

Since six histidine residues were bound to the amino terminus of amylase expressed from p6xHBTMA, the supernatant was purified by passing through a Ni-NTA column (QIAGEN Co., USA) and the enzyme titer was measured. The enzyme titer was determined in the same manner as in Example 2, except that 50 mM sodium acetate having a pH of 4.5 was used as the buffer solution.

Example 3: Heat-Resistant Plulanase

In order to isolate the heat resistant fluranease gene, a primer pair for PCR amplification was prepared.

TmPUL-N (Forward Primer) 5'-TCCGCTTTGATACATATGGAAACCACCATC-3 ',

TmPUL-C (Reverse Primer) 5'-GATCGATACGTCGACTAAAACCAGTGTCAG-3 '

In the same manner as in Example 1 using the primer pairs, Thermomoto polymerase chain reaction was carried out on the main chromosome of Maritrima MRS8 to obtain a product of about 2.0 kb. The amplified gene fragments were treated with NdeI and SalI and ligated with the p6xHis119 vector completely digested with the same enzyme. The strain was transformed in the same manner as in Example 1 and first selected a strain showing resistance to empicillin.

The primary screened transformants were inoculated in LBA solid medium containing 0.5% red flurane and incubated at 37 ° C. for about 12 hours, and 0.6% agar containing 3 mg of D-cycloserine. 5 ml of the medium was poured onto the solid medium in which the transformants were cultured, and then cultured again at 37 ° C. for 12 hours. The strain showing the yellow ring on the strain was finally selected as a heat resistant fluranease producing strain (Escherichia coli MC1061 / p6xHTMPULl119).

Heat resistant fluoranese producing strain (Escherichia coli MC1061 / p6xHTMPULl119) was purified in the same manner as in Example 2.

Enzyme titer was added to 0.2 ml of 50 mM sodium acetate buffer solution at pH 6.0 and 0.25 ml of 1% (w / v) Pulran solution at 0.05 ml of enzyme solution. After boiling for 5 minutes to develop the color was determined by measuring the absorbance at 575 nm. The absorbance obtained was compared with a maltotriose standard curve to determine the amount of enzyme that produces 1 mmole of maltotriose per minute in 1 unit.

Example 4 Preparation of Glucose from Maltohexaose

(4-1) glucose production

5% maltohexaose was added to 50 mM buffer solution (pH 6.0) to prepare a suspension, followed by addition of heat-resistant thermomoto maritrima glucosidase and heat-resistant α-glucosidase and reacted at 75 ° C. The composition of the reaction solution according to the reaction time was analyzed using high performance ion chromatography.

As a control, only heat-resistant α-glucosidase was added and reacted in the same manner as above.

(4-2) HPIC analysis of the reaction solution

The reaction solution was analyzed using a Carbopak-column (Dionex, USA) and a pad (pulsed ampherometric detector, Dionex, USA) as a detector. A 150 mM caustic soda solution was used as the eluate A. The solution was dissolved in 600 mM sodium acetate in the eluate A as a B eluent. After 40 minutes, the concentration was linearly adjusted so that the B buffer became 0 to 40%. The solution was analyzed.

10A and 10B show the results of analyzing the reaction solution and the control using high performance ion chromatography.

As shown in Figure 10a, in the control group added only heat-resistant α-glucosidase, maltohexaose (G6) remained in large amounts after 2 and 6.5 hours of reaction, and the decomposition products maltopentaose (G5) and maltotetra Os (G4), maltotriose (G3), etc. remain, and after 6 hours and 24 hours of reaction time, G6, G5, G4 and G3 remained undecomposed and were not converted to glucose.

However, as shown in FIG. 10B, in the experimental group in which 0.5 unit of heat resistant thermomoto was added maritrima glucosidase to 2 units of heat resistant α-glucosidase, G6 and G5 were almost decomposed after 2 hours of reaction, and a small amount of G4, G3 and G2 remain. After 6.5 hours, only a small amount of G2 remained, and after 14.5 hours, almost all maltooligosaccharides were decomposed and converted into glucose.

Therefore, when heat-resistant thermomoto treated Maritrima glucosidase and heat-resistant α-glucosidase at the same time, the yield of glucose was more than 95%, and it was confirmed that the manufacturing process time could be more than doubled.

10A and 10B, when only heat-resistant α-glucosidase was treated, malto-oligosaccharides such as G6, G5, and G4 remained in the reaction solution, and a small amount of a large number of products having no structure was identified. On the contrary, in the experimental group treated with both enzymes at the same time, there was no generation of foreign substances by the sugar transfer reaction, and thus it was found that there was an effect of increasing the yield of glucose. This can be presumed to be due to the heat-resistant thermomoto hydrolysis of glucose from the reducing end of maltooligosaccharides by marittima glucosidase.

Example 5 Preparation of Glucose from Glucydex 12

(5-1) glucose production

In order to perform an experiment for preparing glucose from branched oligosaccharides, a suspension was prepared by adding 5% Gludexex 12 to 50 mM buffer solution (pH 6.0), and then 1 unit of heat resistant flulanase and heat resistant α-glucose. Two units of Days and 0.5 unit of heat resistant Thermomoto were added and reacted at 75 ° C. The composition of the reaction solution according to the reaction time was analyzed using high performance ion chromatography.

As a control, 2 units of heat resistant α-glucosidase and 0.5 units of heat resistant thermomoto were added and reacted under the same conditions.

(5-2) HPIC analysis of the reaction solution

The reaction solution obtained in 5-1 was analyzed using high performance ion chromatography, and the results are shown in FIG. 11. It was found that most of the oligosaccharides were hydrolyzed and converted to glucose after 12 hours of reaction in the spheres to which heat-resistant fluoranese was added. As a result of the above experiment, the heat resistant fluranease acts on the oligosaccharide branched by the α-1,6 glucoside bond to hydrolyze the α-1,6 glucoside bond, and consists of a linear α-1,4 glucoside bond. It is assumed to act to convert to oligosaccharides.                     

The composition of the reaction solution was calculated by converting the mass ratios of the compounds present in the total solution, and the analysis showed that the glucose content of the solution in which the heat-resistant fluerase, the heat-resistant α-glucosidase, and the heat-resistant thermomoto were acted on the maritima glucosidase for 24 hours. Was 95.3%.

(5-3) TLC analysis of the reaction solution

The reaction product of 5-1 was analyzed using thin layer chromatography (Thin Layer Chromatoghy, TLC). Each sample was loaded in a Whatman K5F TLC plate (20 cm × 20 cm) by 1 mL and developed once in a developing solution with a 3: 1: 1 (v / v / v) ratio of isopropyl alcohol, ethyl acetate, and distilled water. After development, the plate was well dried, immersed in a coloring solution in which 0.3% (w / v) N- (1-naphthyl) -ethylenediamine and 5% (v / v) sulfuric acid was dissolved in methanol, and then taken out in a 110 ° C oven. 10 minutes of color development.

The reaction solution obtained in Example 5-1 was analyzed by thin film chromatography and is shown in FIG. 11.

In the control group without the addition of heat-resistant fluranease, it can be seen that oligosaccharides having a high molecular weight and expected to include α-1,6 glucoside bond after 6 and 24 hours of reaction are present at the starting line without degradation.

On the other hand, in the spheres to which heat-resistant fluoranese was added, it was found that after 12 hours of reaction, the oligosaccharide having a large molecular weight was decomposed and disappeared from the starting line. The molecular weight oligosaccharides including α-1,6 glucoside bonds are decomposed into linear oligosaccharides by the hydrolysis action of the heat-resistant fluoranese, followed by α-glucosidase and heat-resistant thermomoto by maritima glucosidase. Hydrolysis shows that glucose was produced.

The heat-resistant thermogram of the present invention, heat-resistant thermogram, maritima glucosidase, has the advantage that it can be used at high temperatures because it is heat resistant. There is an advantage to getting glucose.

Claims (8)

Produced by Thermotoga maritima , the optimum working pH is 6.0 to 7.0 and the optimum working temperature is 80 to 90 ° C, and α-1,4 glucoside bonds and α-1,6 glucosides from the reducing end The heat-resistant thermomotor maritima glucosidase which breaks a bond and does not hydrolyze maltose. 2. The heat resistant thermomoto according to claim 1, wherein the substrate of the enzyme is selected from the group consisting of maltotriose, maltotetraose, maltopentose, maltohexaose, maltoheptaose and beta-cyclodextrin. Seadays. To obtain a liquefaction reaction solution in which starch is treated with α-amylase, The method of producing glucose from starch comprising the step of saccharifying the liquefied reaction solution by the heat-resistant thermomoto of claim 1 in the treatment of maritrima glucosidase, heat resistant fluranase and heat resistant α-glucosidase. The method of claim 3, wherein the reaction conditions are pH 5.5 to 6.5 and the method for producing glucose from the starch of the reaction temperature is 70 to 85 ℃. 4. The method of claim 3, wherein the heat resistant α-glucosidase is heat resistant α-glucosidase produced by Sulfurobus sulfataricus ATCC 35092. 4. The method of claim 3, wherein the heat resistant fluranease is produced by Thermomoto Marittima. Glucose prepared according to the method of any one of claims 3 to 6. delete

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