JPS61162197A - Method of saccharifying starch - Google Patents

Abstract

PURPOSE:In saccharifying starch or liquefied starch, to increase yield of glucose, by using a novel pullulanase-like complex enzyme agent having alpha-amylase activity, produced by a bacterium belonging to the genus Bacillus. CONSTITUTION:A bacterium such as Bacillus subtilis Tu(FERM P-684), etc. belonging to the genus Bacillus is cultivated in a medium containing a nitrogen source, a carbon source, an iron compound, etc., and an enzyme produced outside the mold is collected. The aimed pullulanase-like complex enzyme gent having alpha-amylase activity is obtained from the culture solution after removal of the mold by concentration, salting-out, etc. Starch or liquefied starch is treated with the prepared enzyme agent or it and various amylases such as glucoamylase, beta-amylase, etc., so starch is saccharified to produce glucose.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for saccharifying starch using a novel starch sugar enzyme agent.

[Prior art]

Pullulanase is an enzyme that cleaves the α-1,6-glucoside bond of pullulan and ultimately produces maltotriose, and is known to be widely present in various bacteria, actinomycetes, and the like. When pullulanase acts on amylopectin or starch containing it, its branched bonds (α-
It is known that α-1,4-glucosidic bonds cannot be cleaved, but α-1,4-glucosidic bonds cannot be cleaved to produce amylose-like polysaccharides. Various amylases such as α-amylase and β-amylase, which cleave α-1,4-glucosidic bonds, cannot cleave α-1,6-glucosidic bonds. As a very rare exception, glucoamylase has not only α-1,4-glucosidic bonds but also α-1
, 6-glucosidic bonds can also be cleaved.

In addition to pullulanase, enzymes that cleave α-1,6-glucosidic bonds include isoamylase, R-enzyme, and amyl-1
There are various enzymes called α-1,6-glucosidase or generally,
debranching enzyme
It is called.

Recently, debranching enzymes such as pullulanase have recently been used in combination with β-amylase to simultaneously act on starch to produce maltose in good yields, ) It is a useful enzyme used to produce glucose from starch in good yield by using it in combination with glucoamylase to enhance its cleavage ability.

However, for example, in order to use pullulanase in combination with glucoamylase, glucoamylase must be at a pH of 4 to 5. In order to have an optimal working area at a temperature of 55-60°C, at least 5
It has thermal stability that can be used for a long time at 5-60℃, and
An enzyme that can act at H4-5 is required.

However, many of the branch-cutting enzymes known so far are those of some microorganisms [Bacillus stearothermophilus (1971 lecture abstract of the Japanese Society of Agricultural Chemistry, p. 88), Bacillus acidopurus, Lyticus (JP-A-57-1
74089.5earch, 34,340 (1982)
)), most of them had an optimum operating temperature around 40 to 50°C, and had a disadvantage of poor thermal stability.

〔the purpose〕

With the aim of developing a debranching enzyme that meets the above purpose, the present inventors have extensively searched for microorganisms in the natural world and found that the optimum pH is within a wide pH range of about 5 to about 7.5. A thermostable pullulanase-like enzyme is produced by Bacillus subtilis TO
It was approved that it could be produced by a bacterium identified as

When this enzyme acts on pullulan, it exhibits α-1,6-glucosidase activity that decomposes it mostly into maltotriose, but at the same time, when the enzyme acts on starch, etc., it mainly degrades maltose and maltotriose. It has a novel α-amylase activity that decomposes it into When this enzyme agent is used in combination with glucoamylase to saccharify starch, not only is the saccharification reaction significantly accelerated compared to the case of glucoamylase alone, but the final glucose yield is 0.5~
It is an extremely useful enzyme that can increase the amount by 3%. Furthermore, the α-amylase activity of the enzyme agent of the present invention is such that when it acts on amylose, amylopectin, starch, glycogen, etc., maltose and maltotriose are
Nire is an interesting new enzymatic activity that is produced in a high yield of about 50%.Since this enzymatic agent also retains the enzymatic activity that degrades α-1,6-glucoside bonds, it has a high yield of about 50%. It is thought that maltose and maltotriose can be obtained in extremely high yields even from substrates with branched bonds such as . The present invention has been made based on this knowledge.

〔composition〕

The present invention provides starch or liquefied starch with a pullulanase-like enzyme agent having a novel α-amylase activity produced by Bacillus bacteria alone or with other amylases such as glucoamylase, α-amylase, and β-amylase. The content of the present invention will be explained in more detail below in 6, which relates to a method for saccharifying starch, which involves the use of starch in combination with.

The novel pullulanase-like enzyme agent with α-amylase activity used in the present invention exhibits pullulanase activity that ultimately produces mainly maltotriose when pullulan is used as a substrate, but at the same time it exhibits pullulanase activity that mainly produces maltotriose, but at the same time it also produces amylose, amylopectin, and glycogen. It has α-1,4-glucoside bond decomposition activity such as, and reduces the amount of maltose and maltotriose by almost half.

and has α-amylase activity produced as a main component,
Although it is an interesting new enzyme agent, the pullulanase activity and α-amylase activity can be measured by ammonium sulfate fractionation, fractionation with a variety of organic solvents, adsorption chromatography using an anion exchanger, gel filtration, and transfer to an inorganic carrier. SephadexG-
200 (Pharmacia Fine Chemical
ls), Ce1lulofine GC700m (made by Chisso ■), Biogel A 0.5m (Bio-R
The molecular weight measured by the gel filtration method using ad Lab, ■) etc. is high, with a molecular weight of 450,000 to 550,000 (the molecular weight of normal pullulanase is around 100,000). It is thought that the subunits combine quite tightly to form a composite enzyme (
Figure 3 shows the elution curves of α-amylase activity, pullulanase activity, and protein using RoHel A 1.5m, and the elution patterns of these kings are completely consistent).

The enzymatic properties of the pullulan-degrading activity of the enzyme produced according to the present invention are described below.

r[) action: decomposes the α-1,6-glucoside bond of pullulan and mainly produces maltotriose.

O) Working temperature and optimum working temperature; 1% pullulan, 0.0
When reacted for 30 minutes under 5M Tris buffer (pH 7,0), it acts up to about 80℃, and the optimum temperature is 60~
63°C (Figure 2).

(3) Action pH and optimal action pH; 1% pullulan, 0
．． It acts over a wide range of pH from about 4 to about 10 when measured in 0.05M buffer. As shown in Figure 1, peaks are observed around pH 5 and pH 7 to 7.5, and the optimum pH for action is thought to be in a wide range of about 5 to about 7.5 (Figure 1, citric acid-phosphoric acid disodium buffer and phosphate buffer, 2% pullulan, react at 55°C for 30 minutes).

(4) Thermal stability: 0.05M Tris buffer (pH 7
, 0) for 10 minutes at each temperature, the residual activity was measured using pullulan as a substrate. As a result, 50℃, 10
Almost no deactivation was observed until heating for 55°C, 1 min.
Approximately 30% of the activity was inactivated by heating for 0 minutes. And 60℃, 1
Approximately 80% of the activity was inactivated by heating for 0 minutes.

(5) pi (stability; 30 under 0.1M buffer)
After being left for 3 hours at °C, residual activity was measured using pullulan as a substrate, and the results showed that it was stable at a pH of about 5 to about 10.

46) Inhibitor i1X10-3M HgC12,AgN0
3 inhibited by more than 90%. In addition, the same concentration of Zn5O4 inhibited about 70%.

(7) Stabilizer; thermal stability increases significantly in the presence of calcium ions. In the presence of I x 10-''2M calcium chloride, the optimum working temperature was found at about 65°C (1% pullulan, 30 min reaction).

(8) Purification method: This enzyme is extracted from the culture filtrate of a liquid culture.
After adsorbing on calcium phosphate gel and washing with distilled water, 0
．． Extract with 5M CQ or phosphoric acid-hydrium solution, then DIEAE-Sepharose column chromatography, column chromatography with I3iogel A1.5m, rechromatography with the same column, etc. until chromatographically and electrophoretically homogeneous. It can be refined into

Molecular weight: The molecular weight measured with Biogel Ao, 5m was about 550,000.

Technique 4) Activity measurement method; measurement of pullulanase activity is 0.1
1% pullulan solution dissolved in M phosphate buffer (pH 7)
,0)0.5+m Add an appropriate amount of enzyme to Q, and dilute the total amount with water to 1
mg, and reacted at 40°C. 1 per minute under these conditions
The amount of enzyme that generates the reducing power equivalent to μmoff of glucose was set at about 1%.

As is clear from the above, the pullulanase activity of the present invention has an optimum action pH in an extremely wide range of about pH 5 to about 7.5, and an extremely thermostable action temperature with an optimum action temperature in the range of 60 to 63°C. Bacillus pullulanase, which is an excellent enzyme and was known before the present invention [for example, Agric
, Biol, Che+m, 40.1523 (1
976) (optimal pH 6-6.5, optimum temperature 50°C),
JP 59-39630 (optimum pH 7.0, optimum temperature 45°C), JP 57-174089 (optimum PH 3.5)
~5.5. It can be said that it is a novel pullulanase-like enzyme, which is different from the one with an optimal temperature of about 60°C).

The mycological properties of the Bacillus subtilis Tu strain used as an exemplary bacterium in the present invention are as follows. There is.

(1) Morphological properties: Bacillus, size 0.5-0.7 x 0.8-1.2μ, non-motile, Durham positive, spores spherical to oval.

Cultural properties; C, &) Meat juice agar slant culture; smooth surface and good growth;
In the late stage of culture, the color is pale yellow.

(b) Glucose broth agar slant culture; growth is inferior to broth agar culture.

(c) Broth liquid culture: Growth is not good, but it causes turbidity.
Sediment.

(d) Citric acid agar slant culture: Slight growth.

(e) Peptone-gelatin puncture culture; slowly liquefy.

(f) Milk liquid culture; casein is coagulated and then peptonized.

(g) Potato culture; growth is not very good.

(3) Biochemical properties; (a) Reduction of nitrate; Negative catalase; Positive tyrosinase; Negative ty indole; Not produced (use of citric acid; Positive (f) Production of hydrogen sulfide ; Positive (g) Urease; Negative (b) Hydrolysis of F2 flour; Positive (i) Utilization of carbohydrates; D-glucose, D-fructose, D-mannose, D-galactose, sucrose, mandose, lactose, starch, Acid is produced from carbohydrates such as dextrin, glycogen, D-xylose, O-arabinose, and L-arabinose, but no gas generation is observed.

(4) Growth pl+ and growth temperature; In this case, the pH is slightly alkaline, 7.5, rather than near neutral.
Grows well at pH 8.5. Optimum growth temperature is 35-4
5°C, and the maximum growth temperature is about 50°C.

〔effect〕

The novel pullulanase-like oxygen agent with α-amylase activity used in the present invention has an α-1,6-glucoside bond decomposition activity that decomposes pullulan into maltotriose, and also exists in amylose, amylopectin, glycogen, etc. α-1,4-/It) II # α that decomposes cytobonds to produce maltose and maltotriose
- Since it has amylase activity, compared to pullulanase and isoamylase, which do not have this activity, when used in combination with glucoamylase to saccharify starch, it accelerates the saccharification reaction of starch and produces the final glucose. Yields can typically be obtained 0.5-3% higher. For example, when glucoamylase alone acts on liquefied starch at a concentration of 30%, the yield of glucose is about 94%. When commercially available pullulanase was allowed to coexist 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 made to coexist, the yield of glucose was 97.0-97%. Glucose was obtained with a yield of 5%. As described above, not only the yield of glucose is high, but also the time to reach the maximum saccharification rate can be significantly shortened when using the enzyme preparation of the present invention. This also means that the amount of glucoamylase used can be reduced.

However, when the enzyme of the present invention acts on amylose, amylopectin, starch, glycogen, etc., it mainly produces maltose and maltotriose. , maltose and maltotriose can be obtained in extremely high yields. For example, when this enzyme agent is applied to liquefied starch, both maltose and maltotriose can be obtained in high yields of about 40 to about 50%. An amylase that produces maltose and maltotriose with such a sugar composition is not yet known. Such a saccharified product obtained using the enzyme agent of the present invention has the characteristics of both maltose and maltotriose, and can be used as a sweetener, sweetness regulator, food filler, etc., and as an additive for various foods. It can be used as a. In addition, the enzyme used in the present invention is an enzyme with extremely excellent thermostability, and can not only carry out long-term reactions even at 60°C, which is the optimum and limit temperature for glucoamylase, but also at pH 4,
Good action of glucoamylase of 5-5.0 p)l can be suitably utilized.

Using the enzyme used in the present invention as φ, t
It has various effects on starch saccharification.

In order to produce the pullulanase-like enzyme agent of the present invention, peptone, meat extract, yeast extract, casein, and corn steep liquor are used as nitrogen sources.

Use organic nitrogen sources such as soybean meal or fish meal, and/or inorganic nitrogen sources such as ammonium salts such as ammonium chloride, ammonium sulfate, ammonium phosphate, nitrates such as sodium nitrate, potassium nitrate, or urea. do.

As the carbon source, starch, dextrin, maltose, glucose, etc. are usually used. Phosphates, magnesium salts, and water containing manganese and iron compounds are added as supplementary nutritional sources. Cultivation can be carried out at a pH of about 5 to about 9 and a temperature of 25 to 55°C, but usually
p) 17-9. Temperature around 30'C? It is carried out aerobically for 2-4 days. Since most of the enzyme is produced by bacteria in the wild, after culturing, bacteria are removed by filtration or centrifugation, and the supernatant is collected. Then, if necessary, the enzyme is concentrated and salted out with ammonium sulfate, sodium sulfate, etc., or an organic solvent such as acetone, isopropanol, ethanol, methanol, etc. is added to collect the enzyme as a precipitate.

Store as a concentrated solution or as a dry product.

Use this enzyme alone or with glucoamylase.

The reaction for saccharifying starch using various amylases such as β-amylase is usually carried out at a pH of 4 to 9. Temperature 40℃~7
Performed at 0°C.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Soybean meal 5%, corn steep liquor 0.6%, meat extract 0.4%, dipotassium phosphate 0.3%, magnesium sulfate 0.1%, soluble starch 2%, urea 0.3 %, Sodium Dodecyl Sulfate 0.1%, Copper Sulfate 5X10
5M, manganese chloride 2.5X10-'M, calcium chloride 1X10-3M, zinc chloride 1Xto-'M
, m-acid iron txto-' medium (pH 7,2) 3
0 m Q was placed in a 200 + s Q volume Erlenmeyer flask, and after sterilization by a conventional method, Bacillus, Zubuchis TU strain (FER
N. jug) was inoculated and cultured at 30°C for 3 days. After culturing, the supernatant obtained by centrifugation had an IF@pullulanase activity of 9.6 units per 1 sQ of medium. Also α−
Amylase activity was 45.2 units per 1+m of medium.

Here, α-amylase activity was measured as follows.

1% soluble starch solution dissolved in 0.1M phosphate buffer (
Add an appropriate amount of enzyme to pH 7,0) 0.5+s 11,
The total volume was made up to 1 mfl with water, and the reaction was carried out at 40°C. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit.

Example 2 Bacillus subtilis Tt1 strain (FERN BP-1) was cultured in a medium with the same composition as that used in Example 1.
684) culture supernatant (250 wr Q), add 1 each of 0.4 M disodium phosphate solution and 0.4 M calcium chloride solution.
50mQ was added dropwise to form a calcium phosphate gel and the enzyme was adsorbed onto it. Next, the adsorbate was collected by filtration with a glass filter, and after thorough washing with steamed water, 0.5 M phosphate-potassium solution 20
The enzyme was eluted with G+F1, dialyzed, and concentrated.

Then, it was treated with a DEAE-Sepharose column buffered with 2.5×10″3M Tris buffer (pH 7°O), and the pullulanase active fraction flowing out was collected with the same buffer.
After concentration and dialysis, 2,5X10-3M Tris buffer (pH
Gel filtration was performed on a Biogel A1.5n+ column buffered with 7,0), the active fraction was collected, and rechromatography was repeated on the same column. Figure 3 shows Biogel
The elution curve with A1.5m column (1,5X87c+m) is shown. The protein curve, pullulanase activity curve, and α-amylase activity curve of the purified enzyme completely match each other. The pullulanase activity of the finally recovered enzyme preparation was 585 units, and the α-amylase activity was 1070 units.

Example 3 The enzyme preparation prepared in Example 2 was used in combination with commercially available glucoaminase to perform a starch saccharification reaction.

The substrate was D, which was made by liquefying potato starch with a commercially available liquefying enzyme.
H7,7 was used (glucoamylase).

Denkazo is available from Amano Pharmaceutical ■, Novo Japan ■, etc.).

As a solid content, glucoamylase was added in an amount of 0.2% based on the solid content of the liquefied starch, and the enzyme agent of the present invention prepared in Example 2 or commercially available pullulanase was added to 3 g of each liquefied starch, and 1X10-''M In the presence of calcium chloride, PH4,
8 to 5.0, and the saccharification reaction was carried out at 57.5°C. The same conditions were used except for the measurement, and 0.5 units of enzyme was added per 1 g of substrate). The results obtained are shown in Tables 1 and 2.
It was as shown in the table.

Table 1 Table 1 shows the DB values (reducing sugars in total sugars were expressed as glucose) at 1.2.3.5 and 20 hours after the start of saccharification. Total sugars were determined by the phenol-sulfuric acid method, and reducing sugars were determined by the potassium ferricyanide method. As is clear from the table, when the enzyme of the present invention is used, saccharification is promoted at the initial stage of saccharification initiation.

Table 4 shows the results of analyzing the sugar composition of the glycated products by high performance liquid chromatography at 22 hours, 25 hours, and 28 hours after the start of saccharification. As is clear from the table,
Using the enzyme of the invention, glucose was obtained with a yield of up to 97.1%. In addition, as is clear from the glucose yield after 22 hours of saccharification, it can be seen that the saccharification reaction is significantly accelerated.

Table 4 Example 4 In the same manner as in Example 3, the enzyme agent of the present invention was added to 0 per substrate ε.
．． Add 5 units, pH 4.5-5.3. Saccharification was carried out at a temperature of 55°C. After 24 hours from the start of saccharification, the yield of glucose produced was determined every 2 hours by high performance liquid chromatography. Table 5 shows the maximum glucose yield at each saccharification pH.

As is clear from Table 4, the enzyme of the present invention is P old, 54-5
Optimum effect of glucoamylase The present invention used in Example 3 acted effectively under pH conditions and was able to increase the relative yield of beglucose by about 2% when no additive was used. The enzyme was changed to 0.2, 0.5 and 0.75 units per g of substrate, and the pH was about 5.
The saccharification reaction was carried out at a temperature of 55°C. The obtained results are shown in the sixth
Shown in the table.

Table 6 Example 6 Since the enzyme preparation of the present invention binds strongly to calcium phosphate gel, this example shows an example using an enzyme adsorbed and immobilized on calcium phosphate.

Calcium phosphate is 0.5M disodium phosphate and 0
．． The enzyme was prepared by mixing an equal amount of 5M calcium chloride, and the enzyme of the present invention was immobilized thereon in a bound amount of 1.2 units per mQ of gel suspension.

The reaction solution composition was the same as in Example 3, except that the enzyme immobilized on calcium phosphate gel (1 unit/g substrate) was used, and the reaction was carried out at a pH of approximately 5 and a temperature of 55°C with gentle shaking. . Then, the immobilized enzyme was collected every 24 hours using a centrifuge, replaced with a new reaction solution, and used repeatedly. The obtained results are shown in the seventh
Shown in the table.

As is clear from Table 7, when using an immobilized enzyme, the yield of glucose is about 96%, which is about 0.5 to 1% lower than when using a free enzyme, whereas when no additive is used, the yield of glucose is about 94%. 4%
An increase in glucose yield of approximately 1.5 to 2% was observed compared to the above, indicating that the enzyme can be repeatedly reused.

Example 7 Enzyme prepared in Example 2 (11 as pullulanase activity)
of liquefied starch with a DH of 4.2 (I as solid content)
In addition to g), the content was adjusted to 10+uQ, and the reaction was carried out at 50°C for 2 days while maintaining the pH around 7. After the reaction was completed, the sugar composition of the saccharide was measured by high performance liquid chromatography, and the results are shown in Table 8.

As is clear from Table 8, the main sugar composition of the obtained saccharide was maltose and maltotriose, each of which accounted for about 40%.

Example 8 To 1 g of soluble starch, 0.5 units of the culture solution of Bacillus subshinorus ru strain (FERM BP-684) prepared in Example 1 was added as pullulanase activity, and 10 n of raw nails was added with water.
+ Q and reacted for 2 days at pH 7 and temperature 50°C.

After the reaction, the sugar composition of the saccharified solution was analyzed by high performance liquid chromatography, and the results are shown in Table 9.

Table 9 Example 9 To 2 g of liquefied starch with a DE of 4.2, 500 units of commercially available vegetable layer β-amylase was added, and the total amount was made up to 10+w12 with water.
The reaction was carried out at 5°C for 20 hours. The sugar composition of the obtained saccharified liquid was 1.1% glucose, 52.5% maltose, 4.9% maltotriose, and 41.5% other sugars.

One portion of the enzyme solution prepared in Example 1 was added to a portion of this saccharified solution (solid content: Ig) for pullulanase activity, and the mixture was reacted at 50° C. for 24 hours. The sugar composition of the obtained saccharified liquid was 4.6% glucose, 60.5% maltose, 30.9% maltotriose, and 4.0% other sugars.

[Brief explanation of the drawing]

FIG. 1 shows the optimum pH when pullulan is used as a substrate. Figure 2 shows the optimum temperature when pullulan is used as a substrate. Figure 3; BiogelA 1.5m column (1,5X
87c+m) shows elution curves of pullulanase activity, amylase activity, and protein (absorption at 280 nm). Patent Applicant: Director of the Agency of Industrial Science and Technology, etc., 7 Rakun 1 N Office Application Procedures Specialist, Fu Chungsho (Spontaneous) August 2, 1985 1, Display of Case 1986 Patent Application No. 58
6 No. 2, Name of the invention Method for saccharification of starch 3, Person making the amendment 4, Designated representative 6, Number of inventions increased by the amendment None 7, Subject of the amendment ``Detailed description of the invention'' column of the description 8. Contents of the amendment As shown in the attached sheet, "half amount" in Attachment (1), page 6, line 6 of the specification is corrected to "equal amount." (2) "Mandose" on page 11, line 16 of the specification is replaced with "
Corrected to "maltose". (3) rp+rr, s, P on page 12, line 3 of the specification
Correct H8,5J to r9H1,5~plI8.5. (4) "Ontoku" in the third line of page 12 of the specification is corrected to "temperature". (t> Correct "amount of water" in line 5 of page 15 of the specification to "a small amount". (7) "While maintaining, from..." in lines 5 to 6 on page 24 of the specification should be corrected to "While maintaining...". (8) In the first line from page 12, page 3 of the specification, "Zubshinorusu" is corrected to "Zubutilus."

Claims (2)

[Claims] (1) Starch or liquefied starch is produced by Bacillus bacteria,
A method for saccharification of starch, characterized by applying a pullulanase-like complex enzyme agent having a novel α-amylase activity that decomposes starch mainly into maltose and maltotriose. (2) When starch or liquefied starch is saccharified with glucoamylase to produce glucose, a prunase-like complex is produced by Bacillus bacteria and has a novel α-amylase activity that decomposes starch mainly into maltose and maltotriose. A method for increasing glucose yield, characterized by the presence of an enzyme agent.