JPS6015313B2 - Production method of β-amylase using microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 8-amylase in high yield using Bacillus polymyxa.

Generally, B-amylase is extracted from plants such as malt, soybean, and sweet potato, and is used for industrial production of maltose.
However, the production of 8-amylase by microorganisms is
Bacillus polymyxa (armpit cilluspoIM) by Tilden et al. (J. Bact. 43 527)
Since its presence in water a) was confirmed, there have been reports on 8-amylase originating from various microorganisms, but its production has been extremely low, so industrial production has never been carried out.

As a result of intensive research into the industrial production of 8-amylase using Bacillus polymyxa, the present inventors found that it is necessary to increase the carbon source concentration of the medium in liquid deep culture in order to improve the productivity of this bacterium. It was recognized that this improved the productivity of 8-amylase.

However, in general, Bacillus polymyxa has a tendency to accumulate sticky substances as the bacteria proliferate.

The high viscosity of the culture solution caused by this mucilage significantly inhibits the aeration effect into the solution, so culturing in a tower containing a high concentration of carbon source not only makes culture efficiency even more difficult. They acknowledged that this would make it difficult to purify and recover the enzyme from the culture, and acknowledged that this would be a major obstacle to industrialization. Therefore, as a result of further research, the present inventors found that the entire culture period or up to the maximum cell growth period was 36.
It has been found that a method of culturing at a high temperature of ~40°C is extremely effective in suppressing mucilage production.

0 The present invention was completed based on this knowledge, and the present invention was completed based on this knowledge.
- In amylase production culture, this is a method of culturing at 36 to 40 qo during the entire culture period or from the start of culture to the maximum cell growth phase.

In the present invention, the pH of the culture tower is 6.0 to 7.
．． If it is adjusted to 0, 6-amylase can be produced more effectively. In the present invention, P-
To achieve high yield production of amylase, a column containing a high concentration of carbon source is used.

The higher the concentration of carbon sources such as starch and starch decomposition products in the culture medium, the better, as long as it is 6% or more, but preferably 10 to 20%.
Added to a certain extent. In addition, as nitrogen sources, commonly used organic substances such as defatted soybean powder, yeast extract, and polybeptone, inorganic salts such as ammonium sulfate and ammonium phosphorous, and essential metal ions are added after appropriately controlling the composition and concentration. The bacteria used in the present invention may be any bacteria that belongs to Bacillus polymyxa, but Bacillus polymyxa No. 5
Beat ERM-P Misaki. 3952 (hereinafter referred to as Bacterium A), Bacillus polymyxa ATCC8523 (hereinafter referred to as Bacterium B), etc. are advantageous.

Cultivation is carried out at a pH between 6.0 and 7.0 using a vent lamp.

Culture temperature is important in the present invention, and is heated to 36 to 40 qo during the entire culture period or from the initial stage of culture to the maximum bacterial growth phase. This temperature adjustment suppresses the production of mucilage and results in high-yield 8-amylase production.
In particular, it has been found that high temperature culture at the initial stage of culture not only causes suppression of mucilage but also has the effect of increasing the accumulation of 8-amylase. High-temperature culture at the early stage of culture allows bacteria to proliferate and suppresses the accumulation of 36 to 40 qo.
The effective time is the time required for bacterial growth to reach its maximum, and although the time may be longer or shorter depending on the culture conditions, 20 to 4 feeding hours is generally effective.

In order to increase the accumulation of 3-Amylase, it is more effective to lower and maintain the culture temperature after high-temperature incubation to 30-34q0, and due to these temperature crossovers, a culture solution with low viscosity and high Mu-Amylase activity is produced. Can be adjusted.

Next, test examples and examples of the present invention will be shown.

Test example 1 Bacteria A and B bacteria were used.

(Tanetoji) Phosphorus An 0.1% KC〆
○-02%MgS0
4/7 days 20 0.02% yeast extract 0.2% soluble starch
0.5% (pH=7.3) (
Main culture basic medium) Meat extract 1.0% Polybebutoso 1.0% NaC
0.3%CaC03
0.5% of each strain was inoculated into the seed soil, grown for 1 hour at 30 qo, and 50 qo of the seed culture solution was added with the soluble starch concentration varying from 1.0 to 15.0%. Add to culture basic medium 2 and add 300
The plants were cultured for 0 and 12 hours, respectively, and the amount of 8-amylase produced and viscosity (cp) were measured.

The results are shown in Table 1 below, and it can be seen that as the starch concentration in the medium increases, the yield of 8-amylase increases, but at the same time, the viscosity of the beach soil also increases. table
1 (8-amylase activity measurement method) 0.1M phosphate buffer containing 0.5% soluble starch (pH
7.0) Add 1 part of the diluted enzyme solution on top of 9, and add 3 in 4 pi 0.
Perform the reaction for 0 minutes.

Under these conditions, the amount of enzyme that produced 1 unit of maltose per reaction solution 1 was defined as 1 unit. (Viscosity measurement method) Tokyo Keiki■
The viscosity of various rotors was measured at 2° C. and 6 pm using a BL-type rotational viscometer manufactured by Kogyo Co., Ltd.

In the same manner as in the test example, two strains were used, seed cultured, and 50% of the seed nutrient solution was used.
I was added to the main culture medium supplemented with 10% soluble starch, and cultured for 120 hours at various culture temperatures.
The amount of 8-amylase produced and the concentration (cp) were measured.

The results are shown in Table 2 below, and it can be seen that the viscosity of the medium can be significantly reduced by high-temperature culture or high-temperature initial culture. Table 2 Example 1 Soluble starch 10%, meat extract 1%, polybepoton 1%,
The medium having a composition of 0.3% NaC (pH 6.5) was placed in a fermenter and sterilized under high pressure at 120°C for 30 minutes.

On medium 100, which has the composition of 0.5% soluble starch, 0.1% ammonium phosphorus, 0.02% KC, 00.02% MgS04/7 ratio, and 0.2% yeast extract (pH 7.3). Bacillus polymixa ATCC85 was sterilized under high pressure for 120 minutes in a 500' Sakaguchi flask.
Seed culture solution 2 inoculated with No. 23 and shake cultured for 30q01 hours
00' was inoculated into the above fermenter and incubated for 3 q hours at the initial stage of culture.
70°C and then cultured at 30°C for 70 hours. During the culture, the pH was adjusted to between 6.0 and 7.0 using Su-NaOH, and the pH was adjusted to between 6.0 and 7.0 using aeration rope Azusa (
25 pm, 0.5 vvm). Result 8 - amylase 89u/I, viscosity 4. You obtained a crude enzyme solution.
Example 2 Potato starch 15%, ammonium sulfate 0.75%, CSLO. 5
%, yeast extract 0.2%, K2HP040.1%, Mg
A medium having a composition of 00.05% S04/7 ratio (pH 6.5) was inoculated in the same manner as in Example 11, and cultured at 370 for 4 feeding hours at the initial stage of culture, and then for 6 field hours at 30oo.

During the culture, the pH was adjusted to between 60 and 7.0 with Su-NaOH, and the culture was carried out under aeration (25 pm, 0.5 Wm). As a result, the 8-amylase was 105 u', and the viscosity was 3. A crude enzyme solution of &p was obtained. Example 3 Dextrin 10%, ammonium sulfate 0.
75%, defatted soybean powder 1%, NaC 0.3%, phosphorus ammonium 0
．． The 500ml medium having a composition of 1% (pH 6.5) was placed in an 800ml fermenter and sterilized under high pressure at 12°C for 30 minutes.

Soluble starch 0. %, Phosphoran 0.1%, KC〆0
．． 02%, MgSQ・7QOO. 02%, yeast extract 0
．． 2% (pH 7.3) medium with a composition of 20 and 30
Bacillus polymyxa pongee was then placed in a fermentation tank and sterilized under high pressure at 120℃ for 30 minutes. 5 Snake ERM-P・Gate. 3952
was inoculated and cultured with aeration at 30°C for 20 hours (20pm, 0
．． The entire amount of the seed culture solution (5vvm) was inoculated into the above fermenter and cultured at 370℃ throughout the entire culture period. pH during culture is 6.5
±0.5 - NaO rule trowel kept for 100 hours aeration (
30pm, 0.4Wm) cultured at 11°C, viscosity 3.
A crude enzyme solution containing cells of E. p. After the culture was completed, the bacteria were removed using a Draval centrifuge, and the sterilized solution was concentrated under reduced pressure at 30°C to obtain 43 liters of concentrated crude enzyme solution containing 1050 u of 3-amylase/secretion. Example 4 Pharmacopoeia dextone 20%, phosphorus 0.75%, defatted soybean powder 1.5%, NaC 0.1%, KC 0.05%, M 04.7400.05% (pH 6.5) ) was inoculated with an inoculum solution prepared in the same manner as in Example 3, and cultured at 39° C. for 3 feeding hours, and then at 34 qo for 8 hours.

During the culture, the pH was maintained at 6.5±0.5 using a NaOH trowel and aerated with a trowel (30 pm, 0.4 wm). A bacterial cell-containing enzyme solution of &p was obtained.

Claims (1)

[Claims] 1. In β-amylase production culture of Bacillus polymyxa in a medium containing a high concentration carbon source, the entire culture period is 3.
β caused by microorganisms that are cultured at 6 to 40°C
-A method for producing amylase. 2. In β-amylase production culture of Bacillus polymyxa in a medium containing a high-concentration carbon source, the culture is carried out at 36 to 40°C from the start of culture to the maximum cell growth phase, and then at 30°C.
β- by a microorganism characterized by culturing at ~34°C
Production method of amylase. 3. The method according to claim 1 or 2, wherein the pH of the culture medium is adjusted to 6.0 to 7.0.