JPS62126992A - Production of maltose with amylase g2 - Google Patents

Abstract

PURPOSE:To obtain maltose in high yield, by reacting starch, amylopectin or glycogen with amylase G2 in the presence of alpha-1,6-glycosidase. CONSTITUTION:In treating starch, amylopectin, glycogen or a derivative thereof with amylase G2 to produce maltose from starch, the reaction is carried out in the presence of alpha-2,6-glucosidase to give the aimed maltose. A bacterium which is separated from soil and identified as Bacillus megaterium is recognized to be novel and to produce a thermostable maltose forming enzyme and this enzyme is named amylase G2. To saccharify starch with amylase G2, firstly starch is liquefied with an acid or a starch liquefying enzyme, alpha-amylase. Degree of liquefaction has a great influence upon yield of maltose and maltose is obtained in higher yield by using liquefied starch having lower degree of liquefaction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for producing maltose using amylase G2 produced by Bacillus bacteria.

[Prior art]

The enzyme that breaks down starch into maltose is β-amylase produced by plants and Bacillus bacteria, or Aspergillus oryzae.
) Various # elements are known, such as α-amylase produced by filamentous fungi.

β-Amylase is an enzyme that regularly releases β-maltose from the non-reducing end of amylose and amylopectin. On the other hand, α-amylase produced by Aspergillus oryzae and the conventionally well-known bacteria of the genus Bacillus and Streptomyces degrades amylose and amylopectin in the endo-type, so in addition to maltose, it also degrades glucose,
Maltotriose and other oligosaccharides are also produced in large amounts as by-products.

Recently, H, Out, t, rup and F3. E, No
rn+an is Bacillus stearothermophilus (Ba
cillus 5jearot, hermo-ρhi
It has been reported that a novel maltogenic enzyme produced by A. lus) produces α-maltose in exo form from the non-reducing end of starch. This enzyme does not hydrolyze strictly 5 maltose units like β-amylase,
At the beginning of the reaction, in addition to maltotetraose (G4), maltotriose (Ni3), and maltose (G2), small amounts of maltopentaose (G5) and maltohexaose (
G6) is also generated. Also, Schardinger (Shar
dinger) Dex-1-phosphorus is decomposed into maltose and glucose, and maltotriose is decomposed into maltose and glucose. Therefore, the starch decomposition product produced by this enzyme contains 6 to 8% glucose.

[Purpose and effect]

The present inventor has extensively searched for microorganisms in search of amylase-producing bacteria that specifically produce maltose from starch, and as a result, isolated from soil, Bacillus, Megaterium (
A bacterium identified as Bacillus megaterium was found to produce a novel and heat-stable maltogenic enzyme. This enzyme is thought to hydrolyze starch (from its non-reducing end to maltose units).
It was confirmed by barbaric light and gas chromatographic analysis of the product that the sugar produced was the α-type. This enzyme cannot break down the α-1,6-glucoside bond of amylopectin, so it leaves limit dextrin.
The decomposition rate of amylopectin to maltose is about 2 to 3% higher than that of β-amylase, and it is thought that it can be decomposed closer to branched bonds (α-1,6-glucosidic bonds). In addition, in the early stage of the reaction, no products other than flu-1--se are detected, and because the affinity for maltotriose is small, there is almost no glucose in the final reaction product. thought to be an enzyme. In the juice of the present invention, this enzyme was named amylase G2. Details of the properties of this enzyme are described below.

Details of the enzymatic properties of amylase G2 are as follows.

(1) Action: Hydrolyzes α-1,4-glucoside bonds of amylose, amylopectin, and glycogen into maltose units from their non-reducing ends. Since the sugar produced is α-maltose, it is considered to be a type of α-amylase, but it does not show endo-type degrading action and leaves limit dextrin from amylopectin and glycogen. It does not decompose cyclodextrin, and its ability to decompose maltotriose is weak. Except that the sugar produced is α-type.

It is similar to β-amylase in plants and bacteria, but its degradation limit for amylopectin and starches containing it is 2-3% higher.

(2) Working temperature range and optimum temperature; 1% soluble starch;
When working under 0.05M phosphate buffer.

It works up to about 75°C, and the optimum temperature is about 60°C (first
Figure (a)).

(3) Effect Pi (range and optimum pH; about 3 to about 11
It acts on a wide pl+ range. Q: Appropriate pH is approximately 7-7.5
(Works under 0.05M phosphate buffer, 1% soluble starch, (Figure 1(b)).

(4) Thermal stability; 0.05M Tris buffer (pH 7
, 0), no deactivation was observed until heating at 50°C for 110 minutes. Approximately 10% of the activity was inactivated by heating at 60°C for 10 minutes, approximately 70% was inactivated by heating at 65°C for 10 minutes, and more than 90% was inactivated by heating at 60°C for 10 minutes (Figure 1 (C) ).

(5) pH stability; under 0.1M buffer at room temperature (3
After standing at 0° C. for 3 hours, residual activity was measured. As a result, it was stable in the range of pl+4.5 to 8 (Fig. 1(d)
)).

(6) Stabilization: No increase in thermal stability due to calcium ions was observed.

(7) Inhibitor; This enzyme is 1X10-3M llgc
It was inhibited by about 85%, about 75%, about 70%, and about 60% by l2, AgN03, CuS04, and N15O4, respectively. It was also inhibited by about 70% by 1X10-3M of ρ-chloromercuribenzoate.

(8) Purification method: The enzyme is purified from the supernatant of the liquid culture solution.
It can be purified to homogeneity by disk electrophoresis by ammonium sulfate fractionation, DEAE-Sepharose column chromatography, rechromatography using the same column, and column chromatography using Biogel A0.5m.

(9) Molecular weight: The molecular weight measured by Sephadex G-200 was about 60,000.

(10) Titer measurement method i Add an appropriate amount of enzyme to 0.5 mQ of 2% soluble starch dissolved in 0.1M phosphate buffer, make a total i1mu with water, and react at 40°C.

Under these conditions, the plant-derived β-amylase known before the present invention, Bacillus The results of comparison with β-amylase of bacteria of the genus Bacillus stearothermophilus and maltose-producing amylase of Bacillus stearothermophilus are shown in Table 1.

That is, plant β-amylase and Bacillus flexi β-amylase
While amylase only produces β-maltose,
The enzyme of the present invention produces only alpha-maltose. Like the enzyme of the present invention, Bacillus stearothermophilus amylase produces α-maltose, but in the early stage of the reaction it produces maltotetraose, flu-1-triose, and trace amounts of maltohexaose and maltopentaose. It is reported that it generates. Therefore, these enzymes are essentially different from the enzymes of the present invention. In addition, optimal pH 1 optimal temperature,
Since differences are also observed in enzymatic properties such as molecular weight, amylase G2 can be said to be a new enzyme.

Since amylase G2 cannot break down the α-1,6-glucoside bond in amylopectin, the yield of maltose from amylopectin and starch containing it is approximately 60%.
and the rest remains as dextrin. However, it breaks down the α-1,6-glucoside bond of amylopectin,
When the reaction is carried out in the presence of α-1,6-glucosidase such as isoamylase or pullulanase, 80
It was found that maltose was obtained in high yields of ~90%. The present invention has been made based on this knowledge.

〔composition〕

A novel amylase G2 that produces maltose from starch is reacted with α-1,6-glucosidase in the presence of α-1,6-glucosidase when acting on starch, amylopectin, glycogen, or derivatives thereof. This invention relates to a method for producing maltose using 1,6-glucosidase.

An example of a bacterium that produces the enzyme of the present invention is Bacillus megum.

The mycological properties of this bacterium are as shown below, and it has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7978.

(1) Form; Width 0.7-1.3x2.4-5.0μ,
Usually, two or more are connected and formed in a short chain. Durham negative.

(2) Spores; 2 near each end. Swelling of cells containing spores is hardly observed.

(3) Growth: Growing aerobically. Almost no growth is observed under anaerobic conditions.

(4) Meat juice; cloudy and sediment.

(5) Meat juice agar: Good growth, smooth surface, light brown color, but no pigment formation.

(6) Glucose broth agar: Grows better than broth medium.

(7) Glucose nitrate agar; grows well. Smooth surface. Shows light brown color.

(8) Tyrosine agar; produces brown pigment.

(9) Glucose-asparagine agar; Grows quite well.

(10) Potato: Good growth 1 The surface is smooth and light brown in color. Produces a brown pigment in the medium.

(11) Use of citric acid) Positive.

(12) Starch hydrolysis; positive.

(13) Acetylmethylcarbinol: Not produced.

(14) Gelatin; decomposes.

(15) Milk; coagulates and peptonizes.

(16) Nitrate reduction; positive.

(17) Catalase; positive.

(18) Utilization of carbohydrates; D-glucose, D-fructose, D-galactose, D-mannose, D-ribose, di-rhamnose, di-arabinose, D-xylose, D-mannitol, D-sorbitol, maltose, sucrose These methods are used to produce gas without producing any gas.

(19)! Suitable growing temperature: around 40℃.

(20) Maximum growth temperature; approximately 50°C.

(21) Killing temperature: Not killed even by heating at 100°C for 20 minutes.

Regarding the above mycological properties, please refer to Bergcy's Manual of Determinative Bacteriology.
s Marr'ntryl of Determina
7th and 8th editions of tive [1 act, eriology) (The Williams and Wilkins Company)
Company, 1957 and 1974), and this bacterium was classified as
cgaterium) G-2.

In the present invention, α used simultaneously with amylase G2
As the -1,6-glucosidase (α-1,6-glucoside bond degrading enzyme), for example, Aerobacter aerogenes
, Streptomyces, Bacillus, and Psundomonas amylodermosa.
Isoamylase from bacteria belonging to the genus Pseudomonas, such as Pseudomonas odaramosa), is used.

When producing maltose by saccharifying liquefied starch with amylase G2, if these are treated in the presence of α-1,6-glucosidase, the yield of maltose increases by 20 to 30% compared to the case without treatment. Maltose can be obtained with a yield of 80-90%.

By culturing Bacillus megaterium G2, amylase G2
The general culture for producing is carried out as follows.

Cultivation is usually carried out by aeration and agitation in a liquid medium. Nitrogen sources include meat extract, peptone, yeast extract, casein, cornstap liquor, soybean meal, and other organic nitrogen sources that are commonly used when culturing microorganisms, or supplementary nitrogen sources such as ammonium chloride, Inorganic nitrogen sources such as ammonium phosphate and nitrates are used.

As the carbon source, starch, dextrin, maltose, glucose, sucrose, etc. are used.

Cultivation is carried out aerobically at p) 15-9 and at a temperature of 20-60°C.

Since amylase G2 is an enzyme produced outside the bacterial cells, after the culture is completed, bacteria are removed by filtration or centrifugation, and the supernatant is collected.

The culture supernatant is concentrated if necessary, and ammonium sulfate,
Is it due to salting out with sodium sulfate, etc.?

Or acetone, inpropertool, ethanol,
A π-organic solvent such as methanol is added to obtain the enzyme as a precipitate, which is then dried and stored.

The reaction of saccharifying starch using amylase G2 is carried out as follows.

Starch is first liquefied with acid or the starch liquefaction enzyme α-amylase. The degree of liquefaction (DE) of starch significantly affects the yield of maltose, and maltose can be obtained in higher yields by using liquefied starch with a lower degree of liquefaction. (DE is the percentage of reducing power expressed as glucose in the solid content). The substrate concentration is usually 5 to 40%, reaction P11 is 5 to 8, and the temperature is 50 to 60°C.

α-1,6-glucosidase is usually amylase G2
Although used simultaneously, it may be treated with α-1,6-glucosidase in advance and then treated with amylase G2.

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

Example 1 Soybean protein 2%, rice bran 2%, K2HPO40.3%
, Mg5O4-7H□00.1%, soluble starch 2%, C
aC121X10-3M, MnCl21XIO-'M
,CuSO42,5X10-'M,FeC122,
5X10-'M, sodium uryl sulfate 2.5
Medium consisting of X10-”% (PH7,0) 30+lIQ
was placed in a 200+n Q Erlenmeyer flask and heated to 120°C.
After 5 minutes of sterilization.

The amylase G2 activity produced in the supernatant obtained by centrifugation after inoculation of Bacillus megaterium (Feikoken Bacteria No. 7978) and shaking culture (160 rpm) at 30°C for 3 days was determined. As a result of the measurement, the medium was 1ml! The hit was 9.0 units.

1 g of liquefied starch of DEI, 43, 5 units of amylase 2G, and Klebsiella pneumonia.
1.2 or 3 units of pullulanase (manufactured by Nagase Biochemical Co., Ltd.) produced by P. pneumoniae was added, and the pH was adjusted to 7.
, the reaction was carried out at 55°C for 3 days. After the reaction, the sugar composition of the saccharified solution was analyzed and the results are shown in Table 2.

Table 2

[Brief explanation of drawings]

Figure 1 (a), (b), (c) and (d) show the optimum temperature, optimum pH1 thermostability and pH of amylase G2, respectively.
It shows stability. Ya 113a

Claims (1)

[Claims] A novel amylase G2 that produces maltose from starch is reacted with α-1,6-glucosidase in the presence of α-1,6-glucosidase when acting on starch, amylopectin, glycogen, or derivatives thereof. Method for producing maltose using 1,6-glucosidase.