KR100186901B1 - Pullulanase produced from thermus caldophilus gk-24 - Google Patents

Abstract

The present invention relates to a pullulanase derived from the extremely thermophilic strain, Thermus caldophilus GK-24, a preparation method thereof, and a use as a carbohydrate hydrolase.

Description

Enzyme pullulanase produced from Thermus caldophilus GK-24

1 is a chromatogram showing the purification process of pullulanase.

a is Sephacryl S-200 gel filtration chromatography.

b is DEAE-Sephacel ion exchange chromatography.

c is a high performance liquid chromatography of DEAE Toyopearl 5PW.

d is Mono-Q chromatography using FPLC.

■; Enzyme activity, .......; Protein (OD _{280 nm} )

―; NaCl concentration gradient

Figure 2 is a SDS-modified polyacrylamide gel electrophoresis of purified pullulanase. Large arrows indicate the location of purified pullulanase and small arrows indicate standard proteins.

Lane 1; Purified pullulanase (3 μg), lane 2; Purified pullulanase (4 μg), Jane 3; Purified pullulanase (5 µg), standard protein; Myosin (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), ovoalbumin (43 kDa), carbonic anhydrase (29 kDa) ), β-lactoglobulin (β-lactoglobulin, 18.4 kDa).

3 is a photograph of the resulting IEF identifying the isoelectric point.

Lane 1; Purified pullulanase (5 μg), lane 2; Isoelectric point (pI) calculation standard protein.

4 is a diagram showing the optimum temperature.

■; Comparative activity (%)

5 is a diagram showing the optimum pH.

■; Activity of the enzyme (OD _{540 nm} )

FIG. 6 is a comparative analysis of the amino acid sequence of the amino terminus of the purified enzyme and the amino terminus sequence of the enzyme present in other prokaryotes. The same amino acids were painted black.

(a) T. caldophilus GK24, (b) T. saccharolyticum B6A-R1, (c) T. thermousulfurigens EM1, (d) Thermoanaerobacter ethanolicus 39E, (e) K. pneumoniae ATCC 1505, (f) B. flavocaldarius.

7 is a TLC analysis of the product produced by the purified pullulanase.

Lane 1; Standard sample comprising maltooligosaccharides, lane 2; Panose, lane 3; Lanes 4 without pullulan; Reaction for 5 minutes, lane 5; Reaction for 10 minutes, lane 6; Reaction for 30 minutes, Lane 7; 60 minutes reaction.

The present invention relates to a pullulanase derived from the pyrogenic strain Thermus caldophilus GK-24.

More specifically, the present invention relates to pullulanase derived from the thermophilic strain T. caldophilus GK-24, a preparation method thereof, and a use as a carbohydrate hydrolase.

In recent years, the use of sugar, especially syrups, has increased in the food industry or pharmaceutical industry, and the demand is increasing rapidly. Various carbohydrate hydrolases are used for saccharification of starch. Amylase is mainly used as an enzyme for hydrolyzing starch, but in order to enhance the saccharification of starch, combination of enzymes capable of hydrolyzing various starches is required. Pullulanase hydrolyzes α-1,6-glucosidic linkages of pullulan, amylopectin, glycogen and the like, thereby promoting saccharification of starch along with various amylases, resulting in glucose, maltose or high glucose syrups. Used to make

Pullulanase (pullulanase: pullulan-glucanohydrolase, EC 3.2.1.41) was first found in Klebsiella pneumoniae and described by Bender, H. et al. (1961) Enzym. Biochem. Z. 334: 79-94] Enzymes involved in the hydrolysis of α-1,6-glucosidic linkages in pullulan to form maltotriose as a final product [Abdullah, M. et al., (1966) Nature 210, 200], but also cuts α-1,6-glucosidic linkages from polysaccharides with eggplants such as amylopectin and glycogen as well as pullulan.

The enzyme that cleaves α-1,6-glucosidic linkage also includes isoamylase, while isoamylase hydrolyzes α-1,6-glucosidic linkage of α-D-glucans. One does not hydrolyze the α-1,6-glucosidic linkage of pullulan.

There are two types of pullulanase.

Type I pullulase hydrolyzes only the α-1,6-glucosidic linkage of pullulan, and K. pneumoniae, Bacteroides thetaiotaomicron 96-1, alkaline alkaline Bacillus sp. KSM-1876, Alkaline Thermus aquaticus YT-1, Alkaline Bacillus sp. S-1, alkalescent Micrococcus sp. Y-1 and Bacillus acidopullulyticus.

Type II pullulanase hydrolyzes not only the α-1,6-glucosidic linkage of pullulan but also the α-1,4-linkage of other polysaccharides and is present in most thermophilic bacteria [Bender, H. et al. , (1961) Enzym. Biochem. Z. 334: 79-942; Kim, C. H. et al., (1993) J. Industrial Microbiol, 12: 48-57; Kim, C. H. et al., (1993) Biosci. Biotechnol. Biochem. 57: 1632-1637.

After the discovery of pullulanase in K. pneumoniae, the enzyme has been purified and characterized in a number of low temperature or thermophilic microorganisms, etc. [Spreinat, A. et al., (1990) Appl. Microbiol. Biotechnol. 33: 511-518; Kim, C. H. et al. (1995) Eur. J. Biochem. 227: 687-693], for the extremely thermophilic bacteria, Thermus aquaticus and Thermus sp. Except for AMD33, no biochemical or substrate specificity of pullulanase has been studied [Plant, A. R. et al. (1986) Enz. Microb. Technol. 8: 668-672; Nakamura, N. et al., (1989) Starch / Starke 41: 112-117].

The reaction of hydrolyzing and saccharifying carbohydrates is preferably carried out at high temperature if possible. When reacting at high temperature, the solubility of the carbohydrate used as a substrate is increased first, the substrate is decomposed well, and other bacteria can be prevented from working on the carbohydrate.

All liquefaction and saccharification require simultaneous reaction of the branched cleavage enzyme pullulanase (α-1,6-glycolysis cleavage) and amylase (α-amylase and glucoamylase), but heat resistant flulan Yesterday has not been studied in detail.

The present inventors routinely grow at a temperature of 70-75 ° C., and several species have tried to find carbohydrate-related enzymes in extremely thermophilic bacteria that also grow at 85 ° C., resulting in maximum temperature at a high temperature from the extremely thermophilic strain Thermus caldophilus GK-24. Pullulanase showing the activity of was isolated to complete the present invention.

Accordingly, an object of the present invention is to provide a carbohydrate hydrolase, pullulanase, isolated from bacteria of the genus Thermus.

The present invention is described in more detail as follows.

After culturing T. caldophilus GK-24 by a known method, the cells are recovered and crushed, and the pullulanase enzyme activity is measured. Starch was hydrolyzed using the cell extract at high temperature (75 ° C.) to confirm the presence of the enzyme and then purified using conventional column chromatography methods [Taguchi, H., Hamaoki, M., Mastsuzawa, H. And Ohta, T., (1983) Heat stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK-24, an extremely thermophilic bacterium. J. Biochem. 3: 7-13.

The amino acid sequence of the heat resistant pullulanase purified from T. caldophlius GK-24 as described above is conventional amino acid sequencing method, for example, Edman degradation method [Edman, P., (1949) Arch. Biochem. Biophys. 22: 475-476], and amino acid sequencing by automated digestion and cleavage of fragments by enzymes such as trypsin.

Enzyme activity measuring method and protein quantification method used in the present invention is shown as a reference example.

Reference Example 1 Enzyme Activity Measurement Method

The activity of pullulanase or amylase can be determined by measuring the amount of reducing sugars freed from pullulan or soluble starch [Kim, C. H. et al. (1992) Biochim. Biophys. Acta 1122: 243-250. The reaction mixture (1.0 ml) is made by adding pullulan or soluble starch (1%, w / v) to 50 ml Tris-HCl (pH 7.0) buffer solution, and adding enzyme solution (5-10 µg) for 30 minutes at 73 ° C. Reacted.

The resulting reducing sugar was measured using the known method of dinitrosalicylic acid (DNS) method [Roybyt, J. K. et al., (1972) Anal. Biochem. 45: 510-516; Bernfeld, P., (1955) Methods Enzymol. 1: 149-150. One unit of enzyme was defined as 1 μM of reducing sugar (glucose) produced in units of minutes under the conditions mentioned above.

Reference Example 2 Protein Assay

Protein was quantified using a quantitative curve using bovine serumalbumin as a standard, according to the well-known method of Lowry et al.

Hereinafter, the method for producing and separating pullulanase from Thermus caldophilus GK-24, the properties of the enzyme and the amino acid sequence will be described in detail with reference to Examples, but the Examples are merely illustrative of the present invention. It is not limited by.

Example 1 Culture of T. caldophilus GK-24 Strain and Preparation of Crude Extract

The strain of T. caldophilus GK-24 was inoculated with the strain in 10 liters of culture, incubated at 75 ° C for 2 hours, and centrifuged to recover the strain.

Dissolve 15g of T. caldophilus Gk-24 in Buffer A [50 mM Tris-HCl (pH 7.5), 1 mM β-mercaptoethanol, 0.5 M EDTA] Cells were completely disrupted in succession. The precipitate was centrifuged (Sorvall SS-34 rotor, 15000 × g, 20 min, 4 ° C.) in 100 ml of the supernatant obtained by fractional precipitation between 30% and 70% using ammonium sulfate [(NH ₄ ) ₂ SO ₄ ]. After centrifugation (Sorvall SS-34 rotor, 15000 × g, 20 min, 4 ° C.), the precipitate was recovered to a minimum volume and dialyzed three times for 4 hours against 100 times buffer A. This was again centrifuged to remove the insoluble material and used as a crude enzyme solution.

Example 2 Purulanase Purification from T. caldophilus GK-24

All experiments were performed at 4 ° C using column chromatography, which is commonly used for purification of common enzymes, and the purification process was completed by the following four steps of chromatography.

[Step 1] Sephacryl S-200 Gel Filtration Chromatography

Sephacryl S-200 gel (2.5 × 120 cm) was equilibrated with Buffer A [50 mM Tris-HCl (pH 7.5), 1 mM β-mercaptoethanol, 0.5 M EDTA). Next, a co-enzyme solution of dialyzed ammonium sulphate precipitate was mounted to perform gel filtration. Elution was carried out at a rate of 0.4 ml per minute, divided into 8 ml fractions, and the pullulanase active portion was collected (enzyme solution 1) (see FIG. 1 a).

[Step 2] DEAE-Sephacel Ion Exchange Chromatography

After equilibrating the DEAE-Sephacel column (1.5 × 20 cm) with buffer A [50 mM Tris-HCl (pH 7.5), 1 mM β-mercaptoethanol, 0.5 M EDTA] used in Example 1, the enzyme solution 1 of step 1 was adsorbed. After washing with the same buffer solution at a rate of 30 ml per hour until the absorbance was 0.01 at 280 nm, NaCl lineargradient from 0M to 1M concentration was performed in an amount of 300 ml. The active portion of ADP-glucose synthase (0.17-0.25 M KCl) was collected (50 ml) and dialyzed in buffer A (enzyme solution 2) (see b in FIG. 1).

[Step 3] DEAE Toyopearl 5PW Ion Exchange Fast Liquid Chromatography

After absorbing the enzyme solution (5 ml) concentrated in DEAE Toyopear l 5PW (10 × 100 mm; Tosoh Co., Tokyo, Japan) of high-performance liquid chromatography equilibrated with buffer solution A, the absorbance was 0.01 at 280 nm with the same buffer solution A. After washing at a rate of 30ml per hour until the NaCl linear gradient of concentration from 0M to 0.5M concentration was carried out in an amount of 10ml. The active portion of the pullulanase enzyme (0.17 to 0.19 M NaCl) was collected (5 ml) and dialyzed in buffer solution A (enzyme solution 3) (see FIG. 1C).

[Step 4] Mono-Q FPLC Chromatography

After absorbing the concentrated enzyme solution 3 (5ml) on Mono-Q HR 10/10 (10 × 100mm) already equilibrated with buffer A, wash it with the same buffer solution A at a rate of 30ml per hour, and then concentration from 0M to 0.5M. A concentration gradient of NaCl linear up to was performed in an amount of 100 ml. The active portion of pullulanase (0.11-0.13 M NaCl) was collected (2 ml) to obtain a finally purified enzyme (see FIG. 1 d).

Table 1 summarizes the purification from the crude enzyme solution through four steps of column chromatography.

Example 3 Determination of Molecular Weight of Purified Pullulanase

By using a well-known gel filtration method, about 10 μg of protein was added to each column and developed on Sepharose 4B gel to confirm that the molecular weight of the enzyme was 63 kDa.

In addition, electrophoresis and analysis on modified polyacrylamide gels by known methods [Laemmli, UK, (1970) Nature 227: 680-685] showed that the standard protein myosin (200 kDa), phosphorylase b (phosphorylase b). , 97.4 kDa), bovine serum albumin (BSA, 68 kDa),

In view of ovoalbumin (43 kDa), carbonicanhydrase (29 kDa) and lysozyme (18.4 kDa), pullulanase has a molecular weight of 64 kDa, which means that B. stearothermophilus (70 kDa) or T similar to the case of aquaticus YT-1 (70 kDa) (see Figure 2).

Example 4 Isoelectric Point of Purified Pullulanase

In order to identify the isoelectric point of pullulanase, known isoelectric focusing (IEF) was used. PhastGels (Phamacia LKB Biotechnology Inc.), which formed a gradient of pH from 3.5 to 9.0, trypsinogen (pI = 9.0), lentil lectin-basic band (pI = 8.65), Lentil lectin-middle band (pI = 8.45), lentil lectin-acidic band (pI = 8.15), myoglobin-basic band (pI = 7.35), myoglobin acid band (pI = 8.45) myoglobin-acidic band (pI = 6.85), human carbonic anhydrase B (pI = 6.56), bovine carbonic anhydrase B (pI = 5.86), β-lactoglobulin Analysis of A (β-lactoglobulin A, pI = 5.20) and soybean trypsin inhibitor (pI = 4.60) showed that the isoelectric point of this pullulanase was 6.1 (see FIG. 3).

Example 5 Optimal Temperature of Purified Pullulanase

In order to find the temperature showing the maximum activity, 2% pullulan and 0.087 unit (U) enzyme were added from 20 ° C to 100 ° C, which was confirmed by the above-mentioned activity measuring method. Showed. In addition, it was confirmed that the activity was lost after 30 minutes at 95 ℃ as a result of reacting in a buffer solution of pH 6.0 after leaving for 60 minutes at various temperatures to see the stability at a specific temperature (see Figure 4).

Example 6 Optimum pH of Purulanase

To see the effect of pH on activity, reaction solution [30 mM sodium acetate buffer (pH 3.0-6.0), 30 mM sodium phosphate buffer (pH 6.0-8.0), 30 mM Tris-HCl buffer (pH 8.0-10.0), 30 mM Glycine buffer (pH 9.0-12.0)] was added 0.023 unit (U) pullulanase was investigated for activity. Maximum activity was shown at pH 5.5 (see Figure 5).

Example 7 Effects of Various Reagents on the Activity of Purified Pullulanase Enzyme

As a result of examining the effect of various cations on enzyme activity, , Mg And Mn The cation enhances the enzyme activity. , Cu And Fe The cation showed an inhibitory effect on the enzyme activity. In addition, EDTA coordinating with metal had no effect on enzyme activity.

Example 8 Confirmation of Substrate Specificity of Purified Pullulanase

As a result of confirming the decomposition reaction with carbohydrate, pullulan was best hydrolyzed, α, β-limited dextrin, β-limited dextrin, soluble starch, amylopectin , Potato starch and glycogen were degraded to some extent. In the case of dextran, α-, β-, or γ-cyclic dextrin (α-, β-, γ-cyclodextrin), the enzyme was virtually not degraded. Table 3 shows the relative activity measured based on the pullulan degradation reaction of this enzyme.

Example 9 Amino Acid Sequence (NH-terminal) of Purulanase

To determine the amino acid sequence of the amino terminus of pullulanase prepared through the purification process from the cells of T. caldophius GK-24, it was electrophoresed on a modified polyacrylamide gel and then adsorbed on PVDF membrane with electricity and then pullulanase. After confirming the band through the dyeing and desalination, the amino acid sequence of the amino terminal (NH-terminal) was determined by a known method.

The amino terminal sequence determined was Ala-Pro-Gln-Asp-Asn-Ser-Leu-Xaa-Ile-Gly-Ala- (Ser). Comparison with the amino acid sequence of the amino terminus of pullulanase present in other prokaryotes revealed that the amino acid sequence was almost different (see FIG. 6).

Example 10 The product fished by purified pullulanase was analyzed by TLC method

Purified pullulanase was reacted with 2% pullulan at 50 ° C. (0.5 ml). 10 μl each of the reaction solution was placed on a TLC, and the sample was prepared by using malto-oligosaccharide and the sample containing panose, and without the sample, and then reacted for 5 minutes, 10 minutes, 30 minutes, and 60 minutes. (See Figure 7.) As a result, purified pullulanase was identified as an enzyme that cleaves maltotriose units from the non-reducing end from the beginning of the reaction, and was also identified as a type I pullulanase that cleaves α-1 and 6-sugar bonds.

As can be seen in the above examples, the pullulanase of the present invention is a novel pullulanase first isolated from T caldophilus, exhibiting maximum activity at high temperature, and best hydrolyzed pullulan, and α, β-limiting. Hydrolytic activity was also demonstrated for dextrin (α, β-limited dextrin), β-limited dextrin, soluble starch, amylopectin, potato starch and glycogen.

Therefore, the pullulanase of the present invention is capable of cleaving the α-1, 6 defects of starch at a high temperature, which is newly isolated from T. caldopilus, which is a very thermophilic strain, so that the high temperature α-amylase is used during the liquefaction process. It is a new heat-resistant branched enzyme that can be used simultaneously. This can proceed the hydrolysis reaction at a high temperature of 70 ~ 80 ℃, solubility of the carbohydrate used as a substrate increases, the substrate is in a good state to hydrolyze, the yield of the hydrolysis product is high, the reaction proceeds at high temperature Therefore, it exhibits an excellent effect of preventing contamination of various bacteria by inhibiting the activity of other bacteria.

Claims (6)

A pyrolytic enzyme pullulanase having an amino terminus derived from Thermus genus having the sequence Ala-Pro-Gln-Asp-Asn-Ser-Leu-Xaa-Ile-Gly-Ala- (Ser). The enzyme pullulanase of claim 1, wherein the enzyme pullulanase is produced from Dermus caldophilus GK-24. The method according to claim 1 or 2, wherein the furfuran, α, β-limited dextrin, β-limited dextrin, soluble starch, amylopectin, potato starch and glycogen Enzyme pullulanase, characterized in that the decomposition. The enzyme pullulanase according to claim 1 or 2, wherein the molecular weight is 64 kDa. Enzyme pullulanase according to claim 1 or 2, which exhibits maximum activity at pH 5.0 to 60. The flulanase according to claim 1 or 2, which exhibits maximum activity at 70 to 80 ° C.