JPH0789925B2 - Strong thermostable pullulanase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a pullulanase having excellent thermostability.

(Prior art) α such as amylopectin, glycogen, branched oligosaccharides
As an enzyme (α-1,6-glucosidase) that hydrolyzes only -1,6-bond, pullulanase, isoamylase and the like are known. These are used, for example, in combination with glucoamylase to produce glucose from a starchy substrate, and in combination with β-amylase or maltotriose or higher malto-oligosaccharide-forming enzyme from maltose, maltotriose or maltotetraose from a starchy substrate. In the production of maltooligosaccharides such as aus, maltopentaose and maltohexaose, it has been recognized that they are effective in increasing the yield of these sugars. It is desirable that the reaction temperature during the production of the above-mentioned sugar from a starchy substrate is high in terms of reaction rate, substrate solubility and prevention of microbial contamination. However, the optimum temperature and stable temperature range of α-1,6-glucosidase known so far are low, usually around 40 ° C to 50 ° C. There are only a few of these enzymes that act at high temperatures, Bacillus acidopullulytic.
us) and Clostridium thermohydrosulfurium-derived pullulanases are only known (Japanese Patent Publication No. 62-
25037 Publication; and Applied and Environmental Micro
biology (1985) 49, 1168-1173). The optimum temperature of the former enzyme is about 65 to 70 ℃, and the optimum temperature of the latter is about 85 ℃.
Is known to be. In addition to these, there is a demand for α-1,6-glucosidase that works well at high temperatures and has excellent heat resistance.

(Object of the Invention) An object of the present invention is to provide an α-1,6-glucosidase that has a high optimum temperature of reaction and excellent heat resistance. Another object of the present invention is to provide a pullulanase having the above-mentioned excellent properties and effective for producing maltooligosaccharides.

(Summary of the Invention) The highly thermostable pullulanase of the present invention has the following properties.

Action and substrate specificity: It acts on pullulan and mainly produces maltotriose. Also, amylopectin,
It cleaves the α-1,6-glucoside bonds of β-limit dextrin and soluble starch.

Optimum pH range: 6.2 to 6.5 Stable pH range: 3.2 to 9.5 at 35 ° C.

Range of optimum temperature of action: The optimum temperature is 75-80 ° C when 5% pullulan is used as a substrate; and the optimum temperature is 80-85 ° C when 5% soluble starch is used as a substrate.

Thermal stability: Stable up to 90 ° C when kept at pH 6.8 for 10 minutes.

Molecular weight: The molecular weight by sodium dodecyl sulphate electrophoresis is 55,000.

Michaelis constant (Km) for pullulan: 5.1 mg / ml.

The pullulanase of the present invention is a bacterium of the genus Bacillus,
Especially highly thermophilic bacterium Bacillus flavocaldarius (Bacill
us flavocaldarius) KP1228 strain (Microtech Lab. No. 9542; FE
It is produced by RM P-9542). This strain is a new strain isolated and collected from the Izumine self-spraying spring by the present inventors (Abstracts of the Abstracts of the Japan Agricultural Chemistry Conference 1984, p. 542). It has been clarified by the present inventors that this strain produces a thermostable exo-oligo-1,6-glucosidase in addition to the pullulanase of the present invention (Abstracts of the Annual Meeting of the Japan Agricultural Chemistry in 1985, p. 375). ). The mycological properties of this strain are shown in Table 1. The culture temperature is 60 ° C. unless otherwise specified in Table 1.

Culture conditions The culture medium of the above strains does not have to be special, and the usual culture medium is used. Soluble starch, amylopectin, amylose, dextrin, etc. are used as carbon sources. Corn, potatoes, honeydew, etc. can also be used. As the nitrogen source, peptone, yeast extract, meat extract, soybean powder, corn distillers solubles, etc. are used. Inorganic salts include K ₂ HPO ₄ , KH ₂ PO ₄ , CaCl ₂ , MgSO ₄ , Na ₂ HPO ₄ ,
(NH ₄ ) ₂ HPO ₄ or the like is used. For example, the following medium is preferably used.

Culture pH is 6.3-8.7, preferably 6.8-8.0, culture temperature is 51-
The temperature is 82 ° C, preferably around 76 ° C, and the culture is performed for about 18 hours while aerobically stirring or shaking. The pullulanase of the present invention is mainly accumulated in cells.

Enzyme Collection Method In order to collect and purify the enzyme of the present invention from the above culture solution, known purification methods can be used alone or in combination. For example,
The cells in the culture solution are crushed, extracted, and then salted out with ammonium sulfate or the like. This is applied to a column of CM-Sephadex, hydroxyapatite, phenyl sepharose, etc. for purification. An example of the purification method is shown below.

(1) Cells are collected from the culture solution by centrifugation, pulverized with alumina, and extracted with an appropriate buffer solution. (2) Salt out the obtained extract with ammonium sulfate (0.2 to 0.5M). (3) The obtained crude enzyme was added to DEAE-Sephadex A
Subject to -50 column chromatography (pH 8.0).
(4) Next, it is subjected to CM-Sephadex C-50 column chromatography (pH 6.0; 0-0.8M NaCl concentration gradient).
(5) Hydroxyapatite (biogel HTP) chromatography (pH 6.8; phosphate buffer 5 to 300m
M concentration gradient). (6) Subject to phenyl sepharose CL-4B column chromatography (pH 6.8 to 7.5; ethylene glycol 0 to 50%, (NH ₄ ) ₂ SO ₄ 1 to 0 M concentration gradient).
(7) CM-Sephadex C-50 column chromatography (pH 6.0; 0-0.3M NaCl concentration gradient). Table 2 shows the degree of purification of the enzyme in each step when purified by such a method. The activity in Table 2 is a value measured by the activity measuring method described below. The value of step 1 is bacterial
732 g (wet weight) was pulverized with alumina, extracted, and centrifuged to obtain a supernatant solution, which was concentrated and used as a sample. In this way, step 7 yields 11.5 units / mg protein of purified enzyme (1040-fold slower compared to the original enzyme supernatant). The properties of the enzyme described below were examined using this purified enzyme.

Activity measurement method Contains 5 mM ethylenediaminetetraacetic acid (EDTA) 4
Pullulan is added as a substrate to 0 mM K-phosphate buffer (pH 6.8) at a concentration of 1%, and 0.1 ml (0.02-0.05 unit) of enzyme solution of unknown activity is added to make the total volume 0.25 ml. . This is incubated at 75 ° C for 20 minutes and cooled to stop the enzymatic reaction. The increase in reducing power is measured by the Somogyi Nelson method. One unit of enzyme is the amount of enzyme that produces 1 μmol of formyl group per minute.

Properties of enzyme The physicochemical properties of the enzyme of the present invention are shown below.

Action and Substrate Specificity -1 Substrate Specificity Instead of pullulan, this enzyme was added to each of the phosphate buffers containing the following substrates, and the enzyme reaction was carried out according to the activity measurement method. It was measured. However, α-limit dextrin (1) and (2), dextrin is 3,5-dinitrosalicylic acid method, panose and isomaltose are glucose B test reagents (Wako Pure Chemical Industries), and other substrates are The number of moles of the formed formyl group was measured by the Moggy Nelson method. The results of experiments conducted using various substrates are shown in Table 3.

In Table 3, α-limit dextrin (1) uses human sleep gland α-amylase, and α-limit dextrin (2) indicates Bacillus subtilis.
Were prepared using the α-amylase produced by E. coli.

-2 Mechanism of action The enzyme reaction was performed under the same conditions using pullulan as a substrate.
After 0 minutes, 40 minutes, and 60 minutes, the reaction solutions were collected and subjected to paper chromatography. As the developing solvent, n- butanol - pyridine _{-H 2 O (6: 4:} 3) was used. The results are shown in FIG. In FIG. 1, G _{1 to} G ₆ indicate the developed positions of maltooligosaccharides having a glucose number of 1 to 6, respectively. IG ₂ indicates the deployment position of isomaltose, and PA indicates the development position of panose. From FIG. 1, it can be seen that this enzyme acts on pullulan and mainly produces maltotriose, but not panose and isomaltose.

Next, using pullulan as a substrate, another enzymatic reaction was carried out under the same conditions, and after 10 minutes, 60 minutes, and 120 minutes, 10 μl of the reaction solution was collected and subjected to high performance liquid chromatography. Ja as a column for high performance liquid chromatography
A sco Finepak SIL NH ₂ column was used, and acetonitrile-H ₂ O (6: 4) was used as the eluent. The eluent was run at a rate of 1 ml / min and detected with a Jasco RID-300 R1 detector. The obtained charts are shown in FIGS. 2 (a), (b) and (c). In Figure 2, 1 is the peak of maltotriose, and 2 denotes a peak of 6 ^{3-.alpha.-maltotriosyl} maltotriose.

From the experimental results of -1 (substrate specificity) and -2 (mechanism of action), the present enzyme was identified as pullulan, amylopectin
It can be seen that it acts on starch-like α-glucans such as β-limit dextrin and soluble starch. Good substrates are pullulan and β-limit dextrin. The pullulan to produce maltose and a small amount of 6 ^{3-.alpha.-maltotriosyl} maltotriose. Amylose, glycogen, α
-Limited dextrins, panose, isomaltose and dextrins do not decompose. From the characteristics of the above-mentioned substrate and product, this enzyme is a typical pullulanase, unlike isopullulanase. As a product,
Since the presence of 6 ^{3-.alpha.-maltotriosyl} maltotriose is a dimer of maltotriose has been confirmed, the enzyme is seen to hydrolyze the substrate to the end type.

Optimum pH range and stable pH range Using this enzyme, enzymatic reaction was carried out under the pH condition of pH 3.0 to 9.2 according to the method of activity measurement. However, the buffer used was McClvain's buffer in the range of pH 3.0 to 7.2, and 50 mM glycine-NaCl-NaOH buffer in the range of pH 6.5 to 9.2. The relative activity is shown in FIG. From Fig. 3, it can be seen that the optimum pH is about 6.2 to 6.5 at 75 ° C.

The enzyme was dissolved in a buffer having a predetermined pH and kept at 35 ° C for 15 hours, and then the residual activity was measured. The pH was set to 12 types shown in FIG. The results are shown in FIG. The buffer used was McCurvein buffer at pH 2.5-7.2, pH 7.5.
At ~ 9.5 is 50 mM glycine-NaCl-NaOH buffer. The measured value in the reaction system of McClvain buffer is
The values are shown by ● in the figure and the measured values in the reaction system of glycine-NaCl-NaOH buffer are shown by ▲ in Fig. 7. Stable pH range is 3.2 at 35 ° C
It turns out to be ~ 9.5.

Optimum temperature range of action Using this enzyme, pullulan (5%) was used as a substrate, and the enzyme reaction was carried out in the range of 35 to 100 ℃ under 14 kinds of temperature conditions according to the activity measurement method. The relative activity is shown by the solid line in FIG. It can be seen from FIG. 5 that the optimum temperature is 75-80 ° C. when pullulan (5%) is used as the substrate.

Next, using soluble starch (5%) as a substrate, the same enzymatic reaction was carried out in the range of 45 to 100 ° C under 11 kinds of temperature conditions. The relative activity is shown by the broken line in FIG. From Fig. 5, the optimum temperature is 80-85 when soluble starch (5%) is used as a substrate.
It can be seen that the temperature is ° C.

At 94 ° C, 67% of the optimum activity is obtained when pullulan is used as the substrate, and 55% of the optimum activity is obtained when soluble starch is used as the substrate. Even when the reaction temperature is close to 100 ° C., the activity is about 50% of the optimum activity in any case.

Conditions for inactivation due to pH, temperature, etc. From Fig. 4, it is clear that this enzyme is inactivated at pH 3.0 or lower at 35 ° C in 15 hours.

This enzyme was dissolved in a phosphate buffer of pH 6.8 and kept at a predetermined temperature for 10 minutes, and its residual activity was measured. The holding temperature was set to 6 types shown in FIG. The residual activity of each is shown in FIG. It can be seen from FIG. 6 that this enzyme is stable up to 90 ° C. under the above conditions. It can be seen that about 50% of the activity remains even near 100 ° C.

Molecular weight Sodium dodecyl sulfate (SDS) electrophoresis detects a single protein band. The molecular weight by SDS electrophoresis is about 55,000.

Km value The Km value when using pullulan as a substrate is 5.1 mg / ml. However, the enzyme reaction was performed according to the activity measuring method.

Comparison with known enzyme This enzyme was compared with the previously reported pullulanase.

Table 4 shows the characteristics (molecular weight, optimum pH, optimum temperature and stable temperature) of this enzyme and various pullulases.

It can be seen from Table 4 that this enzyme is different from the six enzymes below pullulanase derived from Bacillus acid pullulitics because the optimum reaction temperature is as high as 75 to 85 ° C. This enzyme is similar to the pullulanase derived from Clostridium thermohydrosulfurium at the optimum temperature, but its thermostability is 90 ℃, which is 10 ℃ higher than 80 ℃ of the pullulanase, when kept at pH6.8 for 10 minutes. Furthermore, the optimum pH is 6.2 to 6.5
And is more neutral than the pullulanase derived from Clostridium thermohydrosulfurium.
As described above, it was revealed that the pullulanase of the present invention is a novel enzyme that is different from any known pullulanase, and that it has the highest thermostability and functions at high temperature.

Example (A) Culture of Bacillus flavocaldarius KP1228 strain: dextrin 2.0%, peptone 1.2%, yeast extract 0.3%, meat extract 0.05%, K ₂ HPO ₄ 0.3%, KH ₂ PO ₄ 0.1% and CaCl ₂ 0.0.
Using a solution containing 1 mM (pH 7.5) as the medium, Bacillus flav
The ocaldarius KP1228 strain was cultured. Culture is about 1 at 76 ℃
The culture was performed for 8 hours by the rotary shaking culture method.

(B) Collection and preparation of enzyme: The culture broth obtained in (A) was centrifuged to obtain 732 g of bacterial cells (wet weight). This was pulverized with alumina and then extracted with a phosphate buffer (pH 7.0). The extract was concentrated, and the resulting crude enzyme solution was subjected to salting out with ammonium sulfate (0.2 to 0.5M). The crude enzyme obtained was first subjected to DEAE-Sephadex A-50 column chromatography (pH 8.0) and then to CM-Sephadex C-50 column chromatography (pH 6.0; 0).
~ 0.8M NaCl concentration gradient). Furthermore, hydroxyapatite (biogel HTP) chromatography (pH 6.8; phosphate buffer solution 5 to 300 mM concentration gradient), phenyl sepharose CL-4B column chromatography (pH 6.8 to 7.5; ethylene glycol 0 to 50%, (NH ₄ ) ₂ SO ₄ 1-0 M concentration gradient) and CM-Sephadex C-50 column chromatography (pH 6.0-0.3 M NaCl concentration gradient) were sequentially applied to obtain a purified enzyme. This enzyme was found to be 11.5 units / mg protein when measured using the above-mentioned activity measuring method. The method of Lowry et al. Was adopted for protein quantification.

(C) Preparation of maltotriose: 0.25 ml of 40 mM phosphate buffer (pH 6.8) containing (C) -1 pullulan at a ratio of 1% was added to the enzyme solution obtained in (B) (0.05 unit of enzyme). Was added) and the enzyme reaction was carried out for 2 hours according to the activity measurement method. It was subjected to the reaction solution to HPLC, the presence of 6 ^{3-.alpha.-maltotriosyl} maltotriose maltotriose and traces were observed.

(C) -2 To 0.25 ml of 40 mM phosphate buffer (pH 6.8) containing 1% pullulan, the enzyme solution (having 0.05 unit of enzyme) obtained in (B) was added, and the mixture was heated at 75 ° C. for 2 hours. The reaction was carried out over time. After deactivating the enzyme in the reaction solution, it was desalted and concentrated to obtain a colorless transparent starch sugar solution. When the product composition of this starch sugar solution was confirmed by high performance liquid chromatography,
The content of maltotriose was 88% by weight based on the total carbohydrate content.

(Effect of the Invention) According to the present invention, a novel pullulanase is thus obtained from Bacillus flavocaldarius KP1228 strain.
This enzyme has a very high thermostability of 90 ° C and is the most thermostable enzyme among pullulanases known to date. Since the optimum pH of this enzyme is near neutral,
For example, it is advantageous to produce a maltooligosaccharide (used for foods, reagents, pharmaceuticals, etc.) from a starch-based substrate by combining an amylase that works appropriately near neutrality. Since it works effectively at high temperature, it is possible to effectively produce maltooligosaccharides by using high temperature as the reaction temperature.

[Brief description of drawings]

Fig. 1 shows the results of paper chromatography of the reaction solution when pullulan was used as a substrate and the enzyme reaction was performed using this enzyme. Fig. 2 is the enzyme reaction using this enzyme with pullulan as the substrate. Chart of high performance liquid chromatography showing the reaction product with time, Fig. 3 is a graph showing the optimum pH of the enzyme of the present invention, and Fig. 4 is the stable pH of the enzyme.
A graph showing the range, FIG. 5 is a graph showing the optimum temperature of this enzyme, and FIG. 6 is a graph showing the stable temperature range of this enzyme.

Claims (3)

[Claims] 1. A pullulanase having the following properties and excellent heat resistance. Action and substrate specificity: It acts on pullulan and mainly produces maltotriose. Also, amylopectin,
It cleaves the α-1,6-glucoside bonds of β-limit dextrin and soluble starch. Optimum pH range: 6.2 to 6.5 Stable pH range: 3.2 to 9.5 at 35 ° C. Range of optimum temperature of action: The optimum temperature is 75-80 ° C when 5% pullulan is used as a substrate; and the optimum temperature is 80-85 ° C when 5% soluble starch is used as a substrate. Thermal stability: Stable up to 90 ° C when kept at pH 6.8 for 10 minutes. Molecular weight: The molecular weight by sodium dodecyl sulphate electrophoresis is 55,000. Michaelis constant (Km) for pullulan: 5.1 mg / ml. 2. The pullulanase according to claim 1, which is obtained from a bacterium of the genus Bacillus. 3. Bacillus flavocaldarius
flavocaldarius) KP1228 strain, The pullulanase according to claim 1 or 2.