JPH0662882A - Production of glucose by saccharification of starch - Google Patents

Abstract

PURPOSE:To promote saccharification reaction and efficiently produce glucose by saccharifying starch or liquefied starch in the presence of a pullulanase-like conjugated enzyme having glucoamylase activity and alpha-amylase activity. CONSTITUTION:A bacterium of the genus Bacillus which is a Bacillus subtilis TU strain (FERM P-684) is cultured to prepare a pullulanase-like conjugated enzyme having alpha-amylase activity capable of mainly decomposing starch into maltose and maltotriose. Starch or liquefied starch is subjected to saccharification reaction in the presence of glucoamylase, alpha-amylase beta-amylase, etc., and the above-mentioned pullulanase-like conjugated enzyme having the alpha-amylase activity at prescribed pH and prescribed temperature. As a result, glucose can be produced in good yield. Furthermore, since the pullulanase-like conjugated enzyme promotes saccharification reaction of starch, the glucose can usually be obtained in 0.5-3% higher final yield.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing glucose by saccharification of starch using a novel starch sugar enzyme agent.

[0002]

2. Description of the Related Art Pullulanase is α-1,6-
It is an enzyme that cleaves a glucosidic bond and ultimately produces maltotriose, and is known to exist widely in various bacteria, actinomycetes, and the like. It is known that pullulanase, when acted on amylopectin or starch containing it, cleaves its branched bond (α-1,6-glucoside bond) to produce amylose-like polysaccharide, which is associated with increased iodine reaction. However, the α-1,4-glucoside bond cannot be cleaved. On the other hand, various amylases such as α-amylase and β-amylase that cleave α-1,4-glucoside bonds cannot cleave α-1,6-glucoside bonds. With a very rare exception, glucoamylases can cleave not only α-1,4-glucoside bonds, but also α-1,6-glucoside bonds.

In addition to pullulanase, various enzymes called isoamylase, R-enzyme, and amylo-1,6-glucosidase are available as enzymes for cleaving α-1,6-glucosidic bonds, and are collectively referred to as α-1. , 6-Glucosidase or, generally, is called debranching enzyme. Debranching enzymes such as pullulanase have recently been shown to be β-
By simultaneously acting on starch in combination with amylase, maltose can be used for high-yield production, and in order to cover the branching (α-1,6-glucoside bond) cleavage ability of glucoamylase, glucoamylase When used in combination, it is a useful enzyme that is used for producing glucose from starch with high yield.

However, for example, in order to use pullulanase in combination with glucoamylase, glucoamylase should be adjusted to pH 4
〜5 、 Temperature of 55 ~ 60 ℃ has an optimum working range, so it has a long-term thermal stability of at least 55 ~ 60 ℃, and
It is required to be an enzyme capable of acting at pH 4 to 5. However, many conventionally known debranching enzymes are those of some microorganisms (Bacillus stearothermophilus (Agricultural Chemistry Society of Japan, Showa 47 abstract, page 88),
Most of them, except Bacillus acidopolyticus (Japanese Patent Laid-Open No. 57-174089, Starch, 34 , 340 (1982)), have inferior thermal stability because the optimum working temperature is around 40 to 50 ° C. Was a drawback.

[Objective] The inventors of the present invention have conducted a wide range of searches for microorganisms from the natural world for the purpose of developing a debranching enzyme which meets the above-mentioned purpose, and as a result, have a wide pH range of about 5 to about 7.5. It was found that a thermostable pullulanase-like enzyme having an optimum pH was produced by a bacterium identified as Bacillus subtilis TU. The bacterium has the mycological properties described below, and has been deposited at the Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology, as Micro Engineering Research Article No. 684.

[0006] When the enzyme acts on pullulan, it exhibits α-1,6-glucosidase activity which is almost decomposed into maltotriose. At the same time, however, when the enzyme acts on starch or the like, maltose and maltotriose are mainly present. It has a novel α-amylase activity that decomposes into aose. And
When this enzyme preparation is used in combination with glucoamylase to saccharify starch, not only the saccharification reaction is significantly promoted as compared with glucoamylase alone, but the final glucose yield can be increased by 0.5 to 3%, which is extremely useful. It is an enzyme. Further, the α-amylase activity of the enzymatic agent of the present invention produces maltose and maltotriose in a high yield of about 40 to about 50% when it is acted on amylose, amylopectin, starch, glycogen, etc. It is a novel enzyme activity. This is because this enzyme agent also retains the enzyme activity of decomposing the α-1,6-glucoside bond,
It is considered that maltose and maltotriose can be obtained in an extremely high yield even from substrates having branched bonds such as amylopectin and starch. The present invention has been made based on such findings.

[Structure] The present invention provides a starch or liquefied starch with a novel pullulanase-like enzyme agent having a α-amylase activity produced by a Bacillus bacterium, glucoamylase,
The present invention relates to a method for saccharifying starch, which is used in combination with other amylase such as α-amylase and β-amylase. The contents of the present invention will be described in more detail below.

The novel pullulanase-like enzyme having an α-amylase activity used in the present invention shows a pullulanase activity which mainly produces maltotriose when pullulan is used as a substrate, but at the same time, amylose, Α-1,4-such as amylopectin and glycogen
Α- which has glucoside bond-degrading activity and produces maltose and maltotriose in almost half amount and as the main components.
It is an interesting new enzyme agent that has amylase activity, but its pullulanase activity and α-amylase activity are characterized by ammonium sulfate fractionation, fractionation by various organic solvents, adsorption chromatography by anion exchanger, gel filtration, inorganic carrier. Sepadex G-200 (manufactured by Phermacia Fine Chemicals), which is not separated by protein purification methods such as adsorption to
Cellulofine GC700m (manufactured by Chitsso Corporation) and Biogel A 0.5m
(Bio-Rad Lab. Co., Ltd.) has a high molecular weight of 450,000 to 550,000 as measured by gel filtration method (the molecular weight of normal pullulanase is around 100,000). It is conceivable that a plurality of subunits each having γ are bound to each other fairly tightly to form a complex enzyme.
(Fig. 3 shows the elution curves of α-amylase activity, pullulanase activity and protein by Biogel A 1.5m, and the elution patterns of these three are in perfect agreement).

The enzymatic properties of the pullulan-degrading activity of the enzyme produced by the present invention are described below. (1) Action: Decomposes the α-1,6-glucoside bond of pullulan to mainly produce mantotriose. (2) Working temperature and optimum working temperature; 1% pullulan, 0.05
When reacted for 30 minutes under M Tris buffer (pH 7.0),
It works up to 80 ℃, and the optimum working temperature is 60-63 ℃ (Fig. 2). (3) Working pH and optimum working pH: When it is measured with 1% pullulan and 0.05M buffer, it works in a wide range of pH from about 4 to about 10. As shown in Fig. 1, peaks were observed near pH 5 and pH 7 to 7.5, and the optimum working pH is considered to be in a wide range of about 5 to about 7.5 (Fig. 1, citric acid-disodium phosphate buffer and phosphorus). Acid buffer, 2% pullulan, reacted at 55 ° C for 30 minutes). (4) Thermostability: After heating for 10 minutes at each temperature in 0.05 M Tris buffer (pH 7.0), residual activity was measured using pullulan as a substrate. As a result, almost no deactivation was observed until heating at 50 ° C for 10 minutes, and it was deactivated by about 30% by heating at 55 ° C for 10 minutes. Then, it was deactivated by about 80% by heating at 60 ° C for 10 minutes. (5) pH stability: After leaving it in a 0.1 M buffer at 30 ° C. for 3 hours, the residual activity was measured using pullulan as a substrate.
It was stable at a pH of about 5 to about 10. (6) Inhibitor: It was inhibited by 1 × 10 ⁻³ M HgCl ₂ and AgNO ₃ by 90% or more. Moreover, about 70% was inhibited by the same concentration of ZnSO ₄ . (7) Stabilizer: Thermal stability significantly increases in the presence of calcium ions. In the presence of 1 × 10 ^-2 M calcium chloride, the optimum working temperature was found at about 65 ° C. (1% pullulan, 30 minutes reaction). (8) Purification method: the present enzyme is obtained from the culture filtrate of a liquid culture,
After adsorbing on calcium phosphate gel and washing with distilled water,
Extract with 5 M KCl or phosphate-potassium solution, then DEAE-Sepharose column chromatography,
It can be purified chromatographically and electrophoretically to homogeneity by column chromatography using Biogel A 1.5m, re-chromatography using the same column, and the like. (9) Molecular weight; molecular weight measured by Biogel A 5.0m is about 55.
It was good. (10) Activity measuring method; pullulanase activity is measured at 0.1 M
1% pullulan solution (pH 7.0) dissolved in phosphate buffer
Add an appropriate amount of enzyme to 0.5 ml, make up to 1 ml with water, and react at 40 ° C. Under this condition, the amount of enzyme that produces a reducing power corresponding to 1 μmol of glucose per minute was defined as 1 unit.

As is clear from the above, the pullulanase activity of the present invention has an optimum working pH within a very wide pH range of about 5 to about 7.5, and the optimum working temperature is 60 to 63 ° C. Bacillus pullulanase (for example, Agric.
iol. Chem. 40 , 1523 (1976) (optimal pH 6 to 6.5, optimal temperature 50 ° C), Japanese Examined Patent Publication No. 59-39630 (optimal pH 7.0, optimal temperature 45 ° C), JP-A-57-174089 ( Optimum pH 3.5-5.5
, An optimum temperature of about 60 ° C.)], a novel pullulanase-like enzyme.

The mycological properties of the Bacillus subtilis TU strain used as an exemplary bacterium in the present invention are as follows. Has been deposited. (1) Morphological characteristics; bacillus size 0.5-0.7 x 0.8-
1.2 μ, non-motile, Gram-positive, spherical to elliptical spores. (2) Culture properties; (a) Slope culture of broth agar; Smooth surface, good growth, light yellow in the latter stage of culture.

(B) Glucose broth agar slope culture: Growth is inferior to that of broth agar culture. (C) broth liquid culture; growth is not good, but turbidity occurs,
Settle. (D) Citrate agar slope culture; grows slightly. (E) Peptone-gelatin stab culture; slow liquefaction. (F) Liquid milk culture; coagulate casein and then peptone.

(G) Potato culture; growth is not very good. (3) Biochemical properties; (a) Nitrate reduction; Negative (b) Catalase; Positive (c) Tyrosinase; Negative (d) Indole; No formation (e) Use of citric acid; Positive (f) Hydrogen sulfide Production; positive (g) urease; negative (h) hydrolysis of starch; positive (i) utilization of carbohydrates: D-glucose, D-fructose, D-mannose, D-galactose, sucrose, mantose, lactose, starch, Acids are produced from carbohydrates such as dextrin, glycogen, D-xylose, D-arabinose and L-arabinose, but no gas is observed. (4) Growth pH and growth temperature;
Grow well in weakly alkaline pH 7.5 and pH 8.5. The optimum growth temperature is 35-45 ℃, and the maximum growth temperature is about 50 ℃.

[Effect] The novel pullulanase-like oxygen agent having α-amylase activity used in the present invention is
In addition to the α-1,6-glucoside bond-degrading activity that decomposes pullulan into maltotriose, it decomposes α-1,4-glucoside bonds present in amylose, amylopectin, glycogen, etc. to produce maltose and maltotriose. Since it has an α-amylase activity that is produced, it promotes the starch saccharification reaction when used in combination with glucoamylase for saccharification of starch, compared to pullulanase, isoamylase, etc. Glucose yields can usually be obtained as high as 0.5-3%. For example, when glucoamylase alone is allowed to act on liquefied starch at a concentration of 30%, the yield of glucose is about 94%. Then, when the commercially available pullulanase was coexisted with glucoamylase, the yield of glucose was about 96.5%, but when the pullulanase-like enzyme of the present invention having the same pullulanase activity was coexisted, the glucose yield was 97.0-97.5%. was gotten. Thus, not only the yield of glucose is high, but also the time to reach the maximum saccharification rate can be significantly shortened when the enzyme preparation of the present invention is used. That is, this means that the amount of glucoamylase used can be reduced.

Further, the enzyme used in the present invention is an enzyme having extremely excellent thermostability, and it is possible to carry out a long-term reaction even at 60 ° C., which is the optimum temperature and limit temperature of glucoamylase. The glucoamylase having a pH of 4.5 to 5.0 can be suitably used even in a good working pH range. As described above, the use of the enzyme used in the present invention brings about various effects in saccharification of starch.

In order to produce the pullulanase-like enzyme agent of the present invention, as a nitrogen source, an organic nitrogen source such as peptone, meat extract, yeast extract, casein, corn steep liquor, soybean meal, and fish meal, or chloride. Either or both ammonium, ammonium sulfate, ammonium salts such as ammonium phosphate, nitrates such as sodium nitrate, potassium nitrate, or inorganic nitrogen sources such as urea are used.

As the carbon source, starch, dextrin, maltose, glucose and the like are usually used. Phosphates, magnesium salts, and water-containing manganese and iron compounds are added as supplementary nutrient sources. The culture is
It can be carried out at a pH of about 5 to about 9 and a temperature of 25 to 55 ° C., but is usually aerobically carried out at a pH of 7 to 9 and a temperature of about 30 ° C. for 2 to 4 days. Since the enzyme is mostly produced outside the cells, after culturing,
Filter or centrifuge to sterilize and collect the upper solution. Then, it is concentrated if necessary and salted out with ammonium sulfate, sodium sulfate or the like, or an organic solvent such as acetone, isopropanol, ethanol or methanol is added to recover the enzyme as a precipitate, and as a concentrated solution,
Or store as a dried product.

The reaction of saccharifying starch by using this enzyme alone or in combination with various amylases such as glucoamylase and β-amylase is usually pH 4-9 and temperature 40 ° C.
Done at ~ 70 ° C. Hereinafter, details of the present invention will be described with reference to examples.

[0019]

Example 1 Soybean meal 5%, corn steep rica
0.6%, Meat Extract 0.4%, Potassium Phosphate 0.3%, Magnesium Sulfate 0.1%, Soluble Starch 2%, Urea 0.3%, Sodium Dodecyl Sulfate 0.1%, Copper Sulfate 5 × 10 ^-5
M, manganese chloride 2.5 x 10 ^-6 M, calcium chloride 1 x 10
30 ml of a medium (pH 7.2) consisting of ^-3 M, zinc sulfate 1 × 10 ^-4 M, and iron sulfate 1 × 10 ^-5 M was placed in a 200 ml Erlenmeyer flask and sterilized by a conventional method, followed by Bacillus subtilus TU strain ( FERM B
P-684) was inoculated and cultured at 30 ° C. for 3 days with shaking. After culturing,
The pullulanase activity in the supernatant obtained by centrifugation was
It was 9.6 units per ml. The α-amylase activity was 45.2 units per ml of medium.

Here, the α-amylase activity was measured as follows. An appropriate amount of enzyme is added to 0.5 ml of 1% soluble starch solution (pH 7.0) dissolved in 0.1 M phosphate buffer, and the total volume is adjusted to 1 ml with water, followed by reaction at 40 ° C. Under this condition, the amount of enzyme that produces a reducing power corresponding to 1 μmol of glucose per minute was defined as 1 unit.

[0021]

Example 2 Bacillus subtilis TU strain (FERM BP-68) cultured in a medium having the same composition as the medium used in Example 1.
To 250 ml of the culture supernatant of 4), 150 ml each of 0.4 M disodium phosphate solution and 0.4 M calcium chloride solution was added dropwise to form a calcium phosphate gel, and the enzyme was adsorbed thereto. Then, it is filtered with a glass filter to collect the adsorbed substance, thoroughly washed with distilled water, and
The enzyme was eluted with 200 ml of 5 M monopotassium phosphate solution, dialyzed and concentrated.

Then, 2.5 × 10 ^-3 M Tris buffer (pH 7.
Treated with a DEAE-Sepharose column buffered in (0), collect the pullulanase activity fractions flowing out with the same buffer, concentrate,
After dialysis, gel filtration was performed using a Biogel A 1.5m column buffered with 2.5 × 10 ⁻³ M Tris buffer (pH 7.0) to collect active fractions, and rechromatography was repeated using the same column.
FIG. 3 shows the elution curve of the Biogel A 1.5 m column (1.5 × 87 cm). The purified enzyme has a protein curve, a pullulanase activity curve, and an α-amylase activity curve that are in perfect agreement. The final recovered enzyme preparation had a pullulanase activity of 585 units and an α-amylase activity of 10 units.
It was 70 units.

[0023]

Example 3 The enzymatic preparation prepared in Example 2 was used in combination with a commercially available glucoamylase to carry out a saccharification reaction of starch. As the substrate, DE7.7 prepared by liquefying potato starch with a commercially available liquefying enzyme was used (glucoamylase and liquefying enzyme can be obtained from Amano Pharmaceutical Co., Ltd., Novo Japan Co., Ltd., etc.).

As solid content, to each 3 g of liquefied starch, glucoamylase was added in an amount of 0.2% based on the solid content of liquefied starch and the enzyme preparation of the present invention prepared in Example 2 or a commercially available pullulanase, and 1 × 10 5 was added. -PH 4. in the presence of ^-2 M calcium chloride.
The saccharification reaction was carried out at 8 to 5.0 at 57.5 ° C (each pullulanase agent was measured under the same conditions except that acetate buffer solution was used instead of phosphate buffer solution in the above-mentioned activity measurement method at pH 5.0. The enzyme amount of 0.5 unit was added to 1 g of the substrate). The results obtained were as shown in Tables 1 and 2.

[0025]

[Table 1]

Table 1 shows DE values (reducing sugar in total sugar was expressed as glucose) at 1, 2, 3, 5 and 20 hours after the start of saccharification. Total sugar was quantified by the phenol-sulfuric acid method, and reducing sugar was quantified by the potassium ferricyanide method. As is clear from the table, it can be seen that when the enzyme of the present invention is used, saccharification at the initial stage of saccharification is promoted.
Table 2 shows the results of analyzing the sugar composition of the saccharified products 22 hours, 25 hours and 2 hours after the start of saccharification by high performance liquid chromatography. As is apparent from the table, glucose was obtained in a maximum yield of 97.1% when the enzyme of the present invention was used. Further, as is clear from the glucose yield at 22 hours after saccharification, it can be seen that the saccharification reaction is significantly promoted.

[0027]

[Table 2]

[0028]

[Example 4] In the same manner as in Example 3, 0.5 unit of the enzyme preparation of the present invention was added per g of the substrate, pH was 4.5 to 5.3, and temperature was 55 ° C.
Saccharified with. 24 hours after the start of saccharification, the yield of glucose produced every 2 hours was determined by high performance liquid chromatography. Table 3 shows the glucose yield when the maximum value was shown at each saccharified pH.

[0029]

[Table 3]

As is apparent from the table, the enzyme of the present invention has a pH
The optimum action of glucoamylase of 4.5 to 5 was effective under the pH condition, and the glucose yield could be increased by about 2% as compared with the case of no addition.

[0031]

[Example 5] In Example 3, the enzyme of the present invention used was changed to 0.2, 0.5 and 0.75 units per g of substrate, and the pH was changed.
The saccharification reaction was performed at about 5 and a temperature of 55 ° C. The results obtained are shown in Table 4.

[0032]

[Table 4]

[0033]

Example 6 Since the enzyme preparation of the present invention firmly binds to the calcium phosphate gel, this example shows an example using an enzyme adsorbed and immobilized on calcium phosphate.
Calcium phosphate is 0.5 M disodium phosphate and 0.5
It was prepared by a method of mixing equal amounts of M calcium chloride, to which the enzyme of the present invention was immobilized, and 1.2 ml per ml of gel suspension was prepared.
The unit combination was used.

The reaction solution was prepared in the same manner as in Example 3 except that the enzyme (1 unit / g substrate) immobilized on the calcium phosphate gel was used, and the reaction was carried out at pH of about 5 and at a temperature of 55 ° C. with gentle shaking. went. Then, every 24 hours, the immobilized enzyme was recovered by a centrifuge, replaced with a new reaction solution, and the immobilized enzyme was repeatedly used. The results obtained are shown in Table 5.

[0035]

[Table 5]

As is clear from the table, when the immobilized enzyme is used, the yield of glucose is 0.5 to 0.5 as compared with the free enzyme.
About 1% less, about 96%, but 9 without additive
The glucose yield was increased by about 1.5 to 2% as compared with 4.4%, indicating that the enzyme can be reused repeatedly.

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH when pullulan is used as a substrate.

FIG. 2 is a diagram showing an optimum temperature when pullulan is used as a substrate.

FIG. 3 shows elution curves of pullulanase activity, amylase activity and protein (absorption at 280 nm) on a Biogel A 1.5m column (1.5 × 87 cm).

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Claims (2)

[Claims] 1. A novel purinase having a α-amylase activity, which is produced by Bacillus bacteria and decomposes starch mainly into maltose and maltotriose when saccharifying starch or liquefied starch with glucoamylase to produce glucose. A method for producing glucose by saccharification of starch, characterized in that a complex enzyme preparation is present. 2. The Bacillus subtilis TU of Bacillus sp. Is deposited under the No. Micro Incorporated Research Institute No. 684 (FERM. BP-684).
The method for producing glucose by saccharification of starch according to claim 1, which is a strain.