JPS594118B2 - Method for producing oligosaccharides using amylase G4,5 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a saccharide containing maltotetraose and maltopentaose as main components from starch.

Conventionally, various amylases such as α-amylase, β-amylase, and glucoamylase, which have different decomposition modes for starch, have been known and have been used in the production of glucose and maltose.

Recently, molecules with larger molecular weights such as maltotriose (G3), maltotetraose (G4), maltopentaose (G5), and maltohexaose (G6) have been introduced.
There is a demand for the development of oligosaccharide manufacturing technology such as

It is thought that these sugars can be widely used in foods, drugs, and various industrial products as sweeteners, bulking agents, excipients, and inclusion agents, but a method for producing them has not yet been established.

The present inventor has extensively searched for microorganisms in soil in search of microorganisms with a strong ability to produce these oligosaccharides.
) and maltopentaose (G5) as its main components into saccharified products were found to be produced in large amounts outside the bacterial cells.

The enzyme that produces maltotetraose is Pseu.
It is known that amylase produced by Domonas 5tuzeri generates maltotetraose from the non-reducing end of cutin to amylose and amiD- (Ar
Chive of Biochemistry and
Biophyaics Vol. 145, pp. 105-114 (
However, this enzyme does not produce maltopendaose.

On the other hand, amylase that produces maltopentaose is Bacillus licheniformis.
It has been reported that thermostable α-amylase produced by starch produces glycated products containing G5 as the main component.
The ability of this enzyme to generate maltotetraose is significantly lower than that of maltopentaose, and is about 1/4.

In addition, this enzyme is an enzyme that simultaneously produces each component of Gl to G12 (Archive of Biochem
istry and Bio-physics Part 1
55, 290-298 (1973)).

In addition, α-amylase of Bacillus 5ubtilis also produces a mixture of various sugars including maltotetraose and maltopentaose from starch, but since maltotetraose and maltopentaose are not the main components, these It is not used in sugar production.

However, the sugar composition of the starch decomposition product produced by the amylase of the present invention varies depending on the DE of the starch used and the reaction conditions during saccharification (initial starch concentration, pH 1 enzyme concentration), but normally maltotetraose is 15-35%, maltopentaose accounts for 115-30%, and the total of these sugars reaches 30-60%.

As described above, the amylase produced by the present invention is an amylase that generates a saccharide whose main components are maltotetraose and maltopentaose from starch, and no enzyme with such characteristics has been known so far. This is a completely new enzyme, and the present inventor named this enzyme amylase G4,5.

The properties of this enzyme are described below.

(1) Action: Decomposes starch, amylose, amylopectin, and dextrin into maltotetraose and maltopentaose units to produce saccharified products containing these sugars as main components.

(2) Working pH range and optimum working pH; pH 4,5-1
1, with an optimum pH of 6.5-7.5 (Figure 1b).

(3) Action temperature range and optimum action temperature: It works up to about 70°C, and the optimum action temperature is 50-55°C (reaction for 30 minutes at an initial concentration of 1% starch, Figure 1a).

(4) Thermal stability; 0.05M Tris buffer (pH 7.0
), when heated at 50°C for 10 minutes,
0-90% inactivation.

(5) pH stability: After standing in a 0.05M buffer for 3 hours, residual activity was measured, and the result was that it was stable in the pH range of 6 to 10 (Figure 1C).

(6) Stabilization: Increased thermal stability was observed due to the presence of calcium ions (Fig. 1d).

(Structural harming agent; This enzyme contains 5X10-3M HgCl2゜ZnSO4, CuSO4, Fe, SO4, CoCl2.
Approximately 95% or more of the activity was inactivated by AgNO3 and the like.

(8) Purification method and molecular weight: This enzyme was purified from tear fluid of liquid culture by ammonium sulfate fractionation, DEAE-Sepharose column chromatography, Sephadex G-200 and Sephadex G-100 column chromatography, etc. It is refined to a single state.

Sephadex G-200 column chromatography separates it into two isomers with molecular weights of approximately 40,000 and 110,000, but the main component is the component with a lower molecular weight.
There is no significant difference in the enzymatic properties of the two.

(9) Titer measurement method; 0. Add an appropriate amount of enzyme to 0.5 ml of 1% soluble starch solution (pH 7.0) dissolved in IMIJ acid buffer, make up to 1 rnl with water, and react at 40°C.

Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μg of glucose per hour was defined as 1 unit.

Bacillus circulans G45 is an example of a bacterium of the genus Bacillus that produces this enzyme.

The mycological properties of this strain are as follows, and this strain has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 6237.

(1) Form: Bacillus, size, width 0.7-0.8μ x length 2.5-5μ, 2. There are many cases where there are ~3 pieces in a row.

Nonmotile, Durham negative (2) spores; prespore cells swell to form spherical to oval spores.

(3) Gelatin; liquefies.

(4) Meat juice agar; good growth, pale yellowish brown (5)
Glucose broth agar: Poor growth, pale yellow (6) Glucose nitrate agar: Slight growth, translucent (7) Meat juice: Slightly turbid, cloudy, precipitated (8) Salt juice: Grows even at 1-10% salt concentration, Growth is promoted at 1-5% (9) Milk; decomposition is not strong, but coagulates and then peptonizes, lidomas reduction 00) Potato; growth is normal, no pigment is produced aυ Tyrosine agar; growth is quite good, pale yellow, tyrosinase 02 ) Glucose-asparagine agar; almost no growth.

α3) Indole; not produced.

α(a) Acetylmethylcarbinol; not produced σ■
Hydrogen sulfide: generated although weak.

α6) Nitrate reduction; positive aη urease; not produced (18) catalase; positive α9) starch hydrolysis; positive (20) utilization of carbohydrates; glucose, fructose,
Utilizes and produces mannose, galactose, D-xylose, L-arabinose, sucrose, maltose, L-sorbose, mannitol, and starch, but does not generate gas.

Production from lactose, raffinose, sorbitol, and inulin is weak, but almost non-existent.

Cυ methylene blue; reduces.

(221 citric acid; not used.

(23) Growth temperature: Optimum growth temperature is about 30°C, maximum growth temperature is 50-57°C (24) Death temperature: Will not die even if heated at 100°C for 10 minutes.

(251 optimum growth pH; for mycological properties of 7.5 to 8.5 or higher, see Bergey's Ma-nnual
of Determinative Bacteria
7th and 8th editions of Logic (The Willia
ms & Wi Ikins Company 1957
(1974), this microorganism is Bacillus
Circulans (Ba-cillus C1rcula)
It was identified as a microorganism closely related to ns).

This strain produces pullulanase at the same time as amylase G4,5, and both enzymes work together to yield maltotetraose and maltopentaose from substrates with branched bonds (α-1,6-glucosidic bonds) such as starch and amylopectin. It plays an important role in producing well.

The enzymatic properties of pullulanase produced by Honkan are as shown below.

(1) Action: Decomposes α-1,6-glucoside bonds present in pullulan to produce maltotriose.

It breaks down α-1,6-glucosidic bonds in starch, amylopectin, glycogen or its derivatives.

(2) Working temperature range and optimum working temperature: works up to about 70°C, and the optimum working temperature is 50 to 55°C (1% pullulan, 0
．． 05 MI-IJ buffer solution and reaction for 30 minutes).

(3) Action pH range and optimum action pH: It acts in the range of pH 5 to about 9, and the optimum action pH is around 7 (1%
Pullulan, 40 °C under 0.05 M ToIJS buffer
reaction).

(4) Thermal stability; 0.05M 1-Lis buffer (pH 7,
0), the residual activity was measured after heating at each temperature for 10 minutes.

As a result, approximately 73% of the activity was inactivated by heating at 50°C for 10 minutes, and 5
Approximately 97% of the activity was inactivated by heating at 5° C. 10 minutes.

(5) pH stability; stable at pH about 6 to about 9 (0,1
At room temperature (25°C) under M acetate or phosphate buffer
Measure the residual activity after leaving it for a while).

(6) Inhibitor: This enzyme contains Cu”, Zn2+, Ag
``*kg'''' is strongly inhibited by 5Fe2+, etc.

(7) Stabilization: Calcium ions increase the thermostability of this enzyme.

(8) Purification method: This enzyme is extracted from cultured tear fluid by ammonium sulfate fraction, D
EAE-Sepharose column chromatography (KC
It can be separated from amylase G4,5 by gradient elution with Io of 2 to 0.5M, and then purified to chromatographic homogeneity by Sephadex G-200 column chromatography.

(9) Molecular weight: The molecular weight determined by Sephadex G-200 gel filtration method was approximately 70,000.

(10) Titer measurement method; 0. Add an appropriate amount of enzyme to 0.5 rIL1 of 1% pullulan solution (pH 7,0) dissolved in IMIJ acid buffer, make up to 1 volume with water, and react at 40°C.

Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 mg of maltotriose per hour was defined as 1 unit.

In addition to pullulanase produced by this bacterium, for example, Aerobacteraerogenas
enes) and Streptomyces (Strepto
- pullulanase produced by the genus Pseudomonas amyces and Pseudomonas amy
α-1,6-glucosidase such as isoamylase produced by L. lo-deramosa) can be used similarly.

α-1,6-glucosidase is usually amylase G
4,5, but the substrate may be treated with α-1,6-glucosidase prior to saccharification with amylase G4.5.

When producing maltotetraose and maltopentaose by saccharifying liquefied starch with amylase G4,5,
When treated with these α-1,6-glucosidases, the yield increases by at least 5 to 10% compared to the case without treatment.

The culture for enzyme production according to the present invention is usually carried out using a solid medium or a liquid medium, and nitrogen sources for the liquid culture medium include peptone, meat extract, yeast extract, casein, and cornstarch liquor. Starch, texturin, maltose, glucose sucrose, etc. are used as carbon sources, and as supplementary nutritional sources, a medium containing inorganic nitrogen sources, phosphates, magnesium salts, and metal salts is used. used.

Cultivation is carried out at pH 6-9 and temperature 25-55° C. by aerated cultivation.

Since amylase G4,5 is an enzyme produced outside the bacterial cells, after the culture is completed, bacteria are removed by filtering 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 acetic acid, ethanol, methanol, isopropanol, etc. is added to obtain the enzyme as a precipitate, dried, save.

The saccharification of starch using this enzyme is usually carried out by adding the enzyme to 5-40% liquefied starch at a pH of 6-9 and a temperature of 40-60°C.

Starch, amylose and amylopectin are liquefied during saccharification.

Liquefaction is carried out by conventional methods using acids or bacterial α-amylase, and since the degree of liquefaction (degree of partial decomposition) is related to the yield of maltotetraose and maltopentaose, it is desirable for the production of these oligosaccharides. Starch liquefaction degree DE
(the percentage of reducing sugars expressed as quercose in the solid content) is less than 15, and when such liquefied starch is used, the total yield of maltotetraose and maltopentaose is about 30 to about 60%. .

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

Example 1 In a 2t Erlenmeyer flask, Horipeptone S (made from soybean, sold by Wako Pure Chemical Industries, Ltd.) 4%, K2HPO40.3%,
Liquid medium (pH 7.0) consisting of MgSO4.7H200.1%, soluble starch 1% and ammonium sulfate 0.1%.
), and after sterilization by a conventional method, Bacillus circulans G45 (Feikoken Bacterial Serial No. 6237) was inoculated and cultured with shaking at 30°C for 2 days.

After culturing, bacteria were removed using a centrifuge, and amylase G4 and G5 produced in the resulting supernatant were measured.
It was 6.1 units per unit.

Since pullulanase is produced at the same time in the main culture solution, collect the fraction that precipitates with 90% ammonium sulfate from the culture p solution.
After dialysis with distilled water, amylase free of pullulanase was obtained by adsorbing it on a DEAE-Sepharose column buffered with 0.025M ToIJ buffer containing 0.2M KCl and gradient elution from 0.2M KCl to 0.5M KCl. Obtained classification (67 units/CD280mμ)
.

Add 20 units of the enzyme solution and 5XIO-3M amount of calcium chloride to 1 g of liquefied starch with DE4.2, making a total volume of 10 ml.
pl(s, o) and reacted for 24 hours.

The sugar composition in the obtained starch saccharide was analyzed using a liquid chromatograph (column Showa Denko NHpack J-4).
11. As a result of separation and quantitative analysis using an eluent (acetonitrile 6.5%; water 35%), the following results were obtained: crucose 1.3%, maltose 10.9%, maltotriose 8.5%, maltotetraose 178%, maltopenta The sugar composition consisted of 24.5% ose, 32% maltohexaose, and 33.8% others.

On the other hand, when the same reaction was carried out in the presence of 30 units of pullulanase produced by Honkan, the sugar composition of the glycated product was 2.3% glucose, 12.4% maltose, 10.9% maltotriose, The sugar composition consisted of 22.8% tetraose, 280% maltopentaose, 5.7% maltohexaose, and 17.9% other sugars.

[Brief explanation of drawings]

Figure 1 a, b, c, and d are the optimum temperature, optimum pH, thermostability, and p for starch decomposition reaction by amylase G4.5, respectively.
It shows H stability.

Claims (1)

[Claims] 1. In the presence of α-16-gelcoditase, when the Parsis amylase G4,5, which produces maltotetraose and maltopentaose from starch or a partial decomposition product thereof, is allowed to act on starch, amylose, amylopectin, or a partial decomposition product thereof, 1. A method for producing a sugar solution containing maltotetraose and maltopentaose using Bacillus amylase G4.5, which is characterized by reacting with the following: