JPH0763364B2 - Method for producing raw starch degrading enzyme - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for producing a novel raw starch degrading enzyme.

Background of the Invention Direct saccharification of starch granules is effective in terms of utilization of agricultural products, labor saving in brewing process, energy saving and the like. The use of raw starch degrading enzyme is considered for this purpose, but hitherto no suitable enzyme has been found.

In view of such circumstances, the present inventors have separated a large number of microorganisms from soil, flowers, fruits, etc., and have conducted extensive studies on their raw starch degrading activity. As a result, it was found that the isolated filamentous fungus produces a raw starch degrading enzyme and has a very high ratio of the raw starch degrading activity to the gelatinized starch degrading activity, and the present invention was completed. It was

DISCLOSURE OF THE INVENTION That is, the present invention relates to a novel method for producing a raw starch degrading enzyme.

The novel raw starch degrading enzyme-producing filamentous fungus of the present invention has the following mycological properties.

(1) Growing state Using Czapek's solution agar medium, plating at 28 ° C for 2 weeks, the diameter of 4
Gives ~ 5 cm colonies. In addition, most of the color is white, and a spore shape is seen in a part of the central part of the colony, and the color is pale green.

Using Malt extract agar medium, 2
By plating at 8 ° C. for 2 weeks, colonies with a diameter of about 4 cm are produced and show velvety growth. The color is green to gray-green and it spores very well. Conidia peduncle arises from aerial hyphae, with a maximum length of 200μ and width of 3μ.
The wall is smooth. In addition, the Fiaro-type conidia are made and the perfect generation is not recognized.

Czapek's solution agar The carbon source in the medium is changed to rice starch (1%) and cultivated at 30 ° C to form a clear halo.

(2) Physiological properties Hydrolyzability of starch: Positive Attitude toward enzymes: Aerobic Growth pH range: pH 3.5-12 Growth temperature range: 15-35 ℃ From these observations, this bacterium is an incomplete bacterium. Of the genus Penicillium. Penicilli are polymorphic to asymmetrical, conidia stalks are usually long, colonies become wooly, white to grayish green, and thus Penicillium comemane (P. comm.
ane) system.

Eight kinds are contained in this system, but due to the fact that sporulation occurs well on malt agar and that the surface of conidia is fine and rough, Penicillium lanosum Westling The representative Penicillium lanosum OGR-1 strain was identified as
On October 23, 2013, it was deposited in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 8491 (FERM P-8491).

This bacterium is separated and purified as follows. For separation, take an appropriate amount of sample in a Petri dish and place it on the broth agar Buillon aga.
r) It was carried out by pouring a medium in which the carbon source in the medium was changed to rice starch (1%). After incubating at 30 ° C for 3 days, a colony around which a clear rice solute-dissolved area called a halo was formed was collected and cultured in a medium similar to the above to re-establish the halo formation. The confirmed one is separated and stored (referred to as the first-pass strain).

Furthermore, for the first-pass strain, the carbon source in the broth medium was modified with rice starch (1%) in a liquid medium at 30 ° C for 5 to 7
After culturing with shaking for a day, the raw broth was used as a crude enzyme solution to measure the raw starch degrading activity and the soluble starch degrading activity, and the ratio of the raw starch degrading rate to the soluble starch degrading rate (R / S ratio)
6 strains having a ≥20 were selected, and OGR-1 was selected as the strain having the largest R / S ratio among the 6 strains. The R / S ratio is given by the following formula.

According to the present invention, the raw starch degrading enzyme is produced by inoculating the nutrient medium with the filamentous fungus and culturing it. The medium used for the culture may be any one as long as it contains epichlorohydrin-crosslinked starch as an enzyme-inducing substrate and a nutrient source utilized by the filamentous fungus, but the present inventors use it as a basic medium for enzyme production. As a result of diligent studies on 20 types of ground, 8 types of carbon sources, 8 types of nitrogen sources, and 6 types of derived substrates, it is desirable that 1% sorbitol is used as a carbon source, 1% KNO _{3 is} used as a nitrogen source, and 0.5% KH ₂ PO _{4 is used} as an inorganic component. , 0.01% FeCl ₃ ,
Comprises _{0.25% MgSO 4 · 7H 2 O} , using the unique media pH6.0 consisting of 0.5% epichlorohydrin crosslinked starch as an inducer substrate was found to be optimal.

The aeration culture method is preferable as the culture method. As a condition
1vvm is performed around 30 ℃. After culturing for 2 to 3 days, the culture solution is subjected to the following operation. Since the raw starch degrading enzyme is usually present in the culture filtrate, the cells are separated from the culture by using a means such as filtration or centrifugation, and then salting out with 80% ammonium sulfate is performed. Next, the protein precipitates deposited by salting out were collected by centrifugation, dissolved in distilled water, dialyzed,
The inner solution is treated with 80% acetone precipitation, and then dialyzed.
Lyophilize to obtain crude enzyme powder. This crude enzyme has the following physicochemical properties.

(1) Action: Used alone or in combination with a commercially available α-amylase (amylase K) to hydrolyze raw (non-steamed) starch to obtain glucose. (See FIG. 1 and FIG. 2) FIG. 1 shows the action of the novel enzyme in an aqueous dispersion of raw rice starch, wheat starch, corn starch, sweet potato starch and potato starch at 200 μg protein equivalent at pH 5. 3 is a graph showing the relationship between the incubation time and the hydrolysis rate when allowed to incubate at 37 ° C. Further, Fig. 2 shows that about 2 units of the enzyme and a commercially available α-in which the degrading activity of gelatinized starch was adjusted to 0.1U.
It is a graph when a mixture of amylase is similarly made to act on rice starch. The activity was expressed in μmol-G1c / min, and the hydrolysis rate was calculated in g-produced G1c / g-starch.

(2) Optimum pH: pH 5.0 (see FIG. 3) FIG. 3 shows the activity in the buffer solution at each pH.

(3) pH stable production: When treated in a buffer solution of each pH for 20 hours, 90% or more of residual activity is shown at pH 3 to 7. (See Fig. 4) (4) Temperature stability: 50 ℃ when treated at each temperature for 10 minutes.
Stable up to. (See FIG. 5) The residual activity is the ratio of the remaining activity when the initial activity is 100%.

The raw starch degrading enzyme thus obtained is directly used as it is, or as an enzyme agent appropriately blended with a commercially available α-amylase agent by a conventional method, in order to hydrolyze raw starch without a cooking step, it is usually used. It can be used in the same way as
This can be used for labor saving and energy saving in the brewing process, and also for energy recovery of unused starch of agricultural products.

Next, examples will be described.

Example 1 Culture for Enzyme Production One platinum loop was inoculated from a preserved slant of Penicillium lanosum OGR-1 on a plate medium of broth (pH 6.0) and cultured at 30 ° C. for 2 days.

After culturing, 10 ml of sterilized water was dropped on the formed colonies of the present bacterium to prepare a microbial cell suspension, which was filtered using a sterilized glass filter (3G-3) to obtain a spore suspension.

Then, the spore concentration was adjusted to 1 × 10 ⁶ cells / ml to 1 × 10 ⁷ cells / ml, preferably 5 × 10 ⁶ cells / ml, and this was adjusted to a broth medium containing 1% epichlorohydrin-crosslinked starch (pH 6.0). ) Inoculate 1 ml per 100 ml and culture with shaking at 30 ° C for about 3 days. (Preculture).

The pre-culture medium obtained in 1. was used as a seed, 1% sorbitol, 1% KNO ₃ , 0.5% KH ₂ PO ₄ , 0.01% FeCl ₃ , 0.25% Mg
SO ₄ 7H ₂ O, pH consisting of 0.5% epichlorohydrin crosslinked starch
Add to the main culture medium of 6.0 at a ratio of 20%,
Aeration culture is performed under the condition of 1 vvm at ℃.

Example 2 Preparation of crude enzyme powder After completion of the culture, the bacterial cells are removed by filtration or centrifugation to obtain a culture filtrate.

The culture filtrate is preferably cooled to 0 ° C to 5 ° C
While gently stirring with a stirrer under the following conditions, solid ammonium sulfate was gradually added to the culture filtrate to 80% saturation to carry out salting.

The protein precipitate deposited at the salt place was collected by centrifugation at 1000 G for 5 minutes, dissolved in distilled water, and dialyzed at 5 ° C. for 24 hours.

This dialysate was subjected to 80% acetone treatment at 5 ° C. or lower, centrifuged again at 1000 G for 5 minutes to obtain a precipitate, dissolved in distilled water, and then dialyzed under the same conditions.

Crude enzyme powder was obtained by freeze-drying the dialysate.
The enzyme powder had the physicochemical properties as described above.

[Brief description of drawings]

1 and 2 are graphs showing the hydrolysis activity of the raw starch degrading enzyme obtained in the present invention, and FIG. 3 is the optimum pH of the enzyme.
FIG. 4 is a graph showing pH stability, and FIG. 5 is a graph showing temperature stability.

Claims (2)

[Claims] 1. A raw starch-degrading enzyme-producing bacterium belonging to the genus Penicillium is treated with epichlorohydrin-crosslinked starch, sorbitol,
A raw starch degrading enzyme having the following physicochemical properties, which is characterized in that the raw starch degrading enzyme is obtained by liquid culture in a medium composed of KNO ₃ , K ₂ HPO ₄ , FeCl ₃ and MgSO _4, and obtaining the raw starch degrading enzyme from the culture filtrate. Manufacturing method. Action: Acts together with α-amylase to hydrolyze raw (non-steamed) starch to obtain glucose. Optimum pH: pH 5.0 pH stability: pH when treated in a buffer solution of each pH for 20 hours
In 3 to 7, 90% or more of residual activity is shown. Temperature stability: Stable up to 50 ° C when treated at each temperature for 10 minutes. 2. The method for producing a raw starch degrading enzyme according to claim 1, wherein the raw starch degrading enzyme producing bacterium is Penicillium lanosum OGR-1.