JPS6083595A - Direct enzymatic saccharification of starch - Google Patents

Abstract

PURPOSE:To produce efficiently a saccharide, by reacting an amylolytic enzyme produced by a new strain Aspergillus K27 with starch. CONSTITUTION:Aspergillus K27 (FERM-P No.7158) is novelly separated. The resultant bacterium resembles Aspergillus fumigatus, but differs in that the hue of the conidiophores is colorless and the growth temperature range is within 10-55 deg.C. The bacterium is then cultivated at 10-55 deg.C and 3-8pH, particularly 4-7pH for 3-7 days by the solid or liquid culture method to produce a remarkable amount of amylolytic enzyme. The resultant culture fluid is directly used as an enzymic solution or filtered to give a filtrate which is used as the enzymic solution. The enzymic solution is reacted with starch at 3-8pH and 45-60 deg.C to produce a remarkable amount of glucose.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for direct enzymatic saccharification of starch, and more particularly to a method for direct enzymatic saccharification of starch using an amylolytic enzyme produced by a new bacterial species, Aspergillus K27.

Traditionally, to produce glucose using starch as a raw material,
A method has been adopted in which starch milk suspended in water is heated (85 to 120'C), gelatinized, liquefied with α-amylase, and then saccharified by adding glucoamylase. However, gelatinization and liquefaction of highly concentrated starch milk require a large amount of thermal energy and, due to its extremely high viscosity9, require special equipment.

On the other hand, when starch is directly saccharified in one step at a temperature of 25 to 70'C, the resulting liquid has a lower viscosity than the conventional method, which simplifies the process and is economically advantageous. .

The present invention was completed in view of the above circumstances, and provides a method for directly saccharifying various starches in one step using a specific amylolytic enzyme with high starch degrading ability. The gist of this article is that Aspergillus 7mi is a mycological species that belongs to the genus Aspergillus and is characterized by (1) colorless conidiophores, and (2) growth temperature range of 10 to 55°C. Starch content 1'l produced by Aspergillus K27, a new bacterial species with different properties? It consists in a patented direct enzymatic saccharification method characterized by the enzyme acting on whole starch.

Aspergillus I (27) used in the present invention is a new strain belonging to the genus Aspergillus, and one of the strains, Aspergillus K2? Details of this strain are described in Japanese Patent Application No. 144544/1982, and its bacteriological properties are shown below.

■ Growth status on each medium (1) Malt extract agar medium Growth is good, with about 5 Onon at 30th day at 37°C.
reaches a diameter of

The basal hyphal layer is thin and flat. The colony surface is velvety to woolly. Colonies are initially white in color and turn green to dark green when a large number of conidia are formed.

The underside of the colony is initially colorless and later becomes pale yellow.

(2) Growth on Czapek agar medium was good, with approximately 451,111 cells growing on the 3rd day at 37°C.
It reaches a diameter of 11.

The basal hyphal layer is relatively thin and flat. The colony surface is velvety to woolly. The color of the colony is initially white, and turns green to green when many conidia are formed.The underside of the colony is initially colorless and later becomes pale yellow.

++, Physiological properties (1) Growth range (using malt extract medium) pH: 3-
8 Temperature: 10-55°C (2) Optimum growth conditions (malt extract medium J old pH: 4
~7 Temperature: 35-45°C II+, Morphological properties Conidial head: cylindrical, length 120-2 () Oμ.

Diameter 30-60μ, pale green.

Conidiophore: length 150-30 nμ, diameter 2°5-8μ
. It branches off and stands up from the basal hyphae or buyer hyphae.

Smooth surface, colorless.

Common sac: 15-28μ in diameter, 7×-y shape, pale green, forming a phialide from about 1 in the upper 2 momme.

Metre: Metre is not formed.

Fearride II 6.5-9. SX2-2.5μ, pale green.

Conidia: diameter 2.5-3.0 μ, spherical to sub-spherical, rough surface,
The agglomerates are dark green.

In order to produce an amylolytic enzyme using the bacterial strain of the present invention, a culture medium usually used for amylase production is required.
℃, preferably 35 to 45℃, +) I (3 to 8, preferably 4 to 7, may be solid culture or liquid culture, 3 to
A significant amount of amylolytic enzyme accumulates in 7 days.

Production of starch degrading enzymes [Carbon sources for the culture medium used in this process include, for example, various starches, starch hydrolysates,
Cornmeal, flour, and molasses are used. As the nitrogen source, for example, peptone cottonseed residue, meat extract, fermented rice extract, casein, cornstarch liquor, malt extract, soybean oil, skim milk powder, inorganic ammonium salt, inorganic nitrate, etc. are used. The pond, KH2PO4, Fe50
．． ,M8SO,,KCI,CnC12,CoCl2,M
n5O, inorganic salts, and, if necessary, some trace nutrients can be added to the medium.

The culture solution obtained in this manner can be used as it is as an enzyme source, but both the bacterial cells isolated from the obtained culture solution and the culture solution can also be used as crude enzymes. Matoko, 60% ammonium sulfate fraction of cultured nine-head solution,
Further, a 55% inpropatol fraction thereof can also be used as an amylolytic enzyme.

The activity of the above-described amylolytic enzyme is measured as follows: The enzyme solution is allowed to act on a 1% soluble starch solution dissolved in a mild 1ΦJ solution at pH 4.5 at 45°C for 15 minutes. The reducing sugars produced afterwards were treated with Somogyi-Nelson (S 0111 (1-
Hyi Nelson) method. Under these conditions, the power to generate 1 μmole of glucose per minute 1
+lli is 1 unit.

Decomposition of starch using the amylolytic enzyme produced in this way is usually carried out at 25-70°C, preferably at 45-60°C.
It can be carried out at a temperature of 0° C. and a pH of 3 to 8, preferably 4 to 6. The decomposition reaction may be carried out by standing still, but it is preferably carried out by stirring gently to the extent that the starch milk is uniformly suspended. The appropriate amount of enzyme to be added is 0.05 to 1.0 units per m8 of starch, but the reaction time may be increased by reducing the amount of enzyme. The type of starch that can be decomposed is not limited, and examples include cornstarch, potato, sweet potato, rice, wheat, tapioca, and mackerel.

In the production method of the present invention, if a microorganism capable of producing alcohol from glucose is used in combination, alcohol can be produced from starch in one step.

Examples and reference examples are shown below, and the separation of non-inventive symptomatic organisms from soil, production of starch-decomposing oxygen, and starch saccharification will be specifically explained.

Reference Example 1 Soil collected at Korimoto 1-chome, Kagoshima City was diluted 1,000 times with sterile physiological saline, 1 m, g of it was mixed with 9J of the following separation agar medium (I), placed in a sterile petri dish, and
The cells were cultured at ℃ for 2 days.

Isolation agar medium (I) % N1-1. NO3' 0.1 Mg5 O-・7 N20 (1,02KH2P Oh
+), i 4 Yeast extract o, tB a-R31) (J, 5 Agar 1.5 (Sil. 16.1-6.3) Note 1) Collect insoluble starch obtained when wheat starch is liquefied and freeze it. dried.

Colonies generated in the above culture were transferred using a platinum loop to a slanted agar medium (11) with the following composition and cultured for 2L1 at 45' (:).

Slanted agar medium (II) % Peptone O, S Yeast extract 0.3 Malt extract 0.3 Glucose 0.2 Agar 1.5 (pH 7,0) One platinum loop of bacteria generated on the medium by the above culture was added to physiological saline. Diluted 10,000 times with water, mixed the IJ with the above separation agar medium (■) 9-, and placed it in a sterile petri dish for 45 minutes.
The cells were cultured at ℃ for 2 days, and it was confirmed visually and microscopically that the multiple colonies that emerged were not different from each other.

Ten of the above colonies were cultured on slanted agar plates (II) for 28 hours at 45°C, and it was confirmed macroscopically and microscopically that the bacteria on the 10 slanted agar plates were the same bacteria. confirmed. Furthermore, it was confirmed that the properties and physiological properties on each medium of these 10 cultured bacteria were the same and as described above.

From this result, it can be seen that all 10 cultured bacteria are monobacteria that have been purely isolated from nature.

Next, a protective agent (10% skim milk and 1% sodium glutamate droplet solution) was added to the pure cultured bacteria on the malt extract slant agar medium to prepare a spore suspension as described above. Add this spore suspension to an ampoule with 0.2J
The solution was divided into portions and freeze-dried.

The drying method involves slow freezing to -30 to -40°C, then
(It was dried at room temperature for 18 to 20 hours at 11.03 Lorr. Then, it was vacuum sealed with a gas burner and stored at 1.1°C.

The freeze-dried bacteria thus obtained were reconstituted after 3 months. At this time, sterile physiological saline was used as the condensate, and malt extract medium was used as the culture medium. The properties and physiological properties of the reconstituted bacteria in each medium were the same as in Freezing 11;i.

Reference Example 2 Aspergillus K27AC-1 was grown on a slanted agar medium (II
) for 2 days at 45°C, and one platinum loop of the bacterial cells grown on the slant was taken and inoculated into 100+n4 medium of the following composition in a 500J flask. After culturing with shaking at 45°C for 5 days, the culture solution was filtered and the starch decomposition activity of the resulting solution was measured and found to be 12 units/■A.

Level % Wheat starch 2.0 NH4NO30.1 Yeast extract 0.01 Corn stay liquor 0.08 KH2P0. 0.14 F eS 04 ・7 H2O0,O(11M g S
O4? H2O0.05KCI, 0.05 (pH 6.1 to 6.3) Example 1 The culture furnace solution obtained in Reference Example 2 (amount equivalent to 25 units of starch decomposition activity) was suspended in 5 J of buffer solution (pH 4.5). The cloudy cornstarch 25+ag was allowed to act at 55°C. The produced reducing sugars were measured over time using the Somogyi-Nerlan method, the decomposition rate (produced reducing sugars/total sugars) was determined, and a decomposition curve was created. The decomposition curve for corn starch is shown in FIG.

Under these conditions, the decomposition rate of cornstarch is approximately 1 in 7 hours.
Reached 00%.

Example 2 25 units of the amylolytic enzyme obtained in Reference Example 2 were added to a buffer solution (
pH 4,5) Cornstarch 250 suspended in 5'J
+nε at 55°C, and saccharification was performed by standing still. The saccharification rate was determined by quantifying the produced glucose by the glucose oxyglucose/peroxidase method. Under this condition, 2
The saccharification rate was 85% in 4 hours and 95% in 48 hours.

Example 3 The same procedure as in Example 2 was repeated except that the liquid was stirred at 1001 m. The saccharification rate is 98% in 7 hours.
11) l),% in 24 hours. This result is the second
As shown in the figure.

[Brief explanation of drawings]

FIG. 1 is a graph of the decomposition rate curve in Example 1, 12
The figure is a graph of the saccharification rate curve in Example 3. Patent applicant Daikin Industries Co., Ltd. Agent Patent attorney Aoyama Ao (2 others)

Claims (2)

[Claims] 1. What is Aspergillus 7 migatos, which belongs to the genus Supergillus? (1) The color of the conidiophore is colorless; (2) The growth temperature range is 10 to 55°C. A method for direct enzymatic saccharification of starch, which involves the action of a starch-degrading enzyme produced by Aspergillus K27, a new bacterial species with different mycological properties, on starch.