JPS63219376A - Production of protease - Google Patents

Abstract

PURPOSE:To efficiently produce protease having high titer, by maintaining >=2.0mg/l dissolved oxygen concentration of culture solution after end stage of mold multiplication, adding a liquid medium containing soybean flour to the mold and cultivating the mold. CONSTITUTION:In cultivating a mold capable of producing protease in a liquid medium, dissolved oxygen concentration of the culture solution is maintained at >=2.0mg/l at the end stage of the multiplication, a liquid medium containing soybean flour is continuously or intermittently added to the mold and the mold is cultivated to produce protease. The dissolved oxygen concentration of the culture solution in and after the end stage of the multiplication is preferably maintained at >=2.0mg/l by the use of pure oxygen or oxygen enriched air. In and after the end stage of the multiplication, the mold is preferably cultivated while keeping nitrogen concentration in the culture solution at 0.05-0.30% (W/V) and saccharide concentration at <=0.10% (W/V).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for efficiently producing high-titer protease using filamentous fungi capable of producing protease.

[Conventional technology]

Protease is a hydrolase that acts on proteins or partially hydrolyzed products thereof to break down peptide bonds, and is widely used in medicines, brewed foods, detergents, etc.

Conventionally, research on the production of protease by microorganisms has focused on screening methods for microorganisms with high protease-producing ability or methods for breeding them. In addition, many of the studies on culture conditions have focused on the composition of the culture medium, especially those that add inorganic salts to the culture medium (for example, Japanese Patent Publication No. 50-22111), and the type of nitrogen source in the culture medium (for example, Japanese Patent Publication No. 50-22111).
95180, etc.), the type of carbon source (for example, Japanese Patent Publication No. 5
2-12797, etc.), culture methods, especially those that control the oxygen transfer rate (Japanese Patent Publication No. 52-43916),
These methods are limited to those that control the dissolved oxygen partial pressure to 0.20 atm or higher (Japanese Patent Publication No. 53-27789), and all of these methods are limited to batch production methods.

[Problem that the invention seeks to solve]

In the conventional methods proposed for protease production, mainly due to the batch production method, the protease production period is limited to the stationary phase after the bacterial cells have proliferated, and moreover, during the stationary phase, there is no Since it is extremely difficult to control the carbon source and nitrogen source, there are problems in that the industrial harvest period for protease is short and the protease titer obtained is relatively low.

Further, in the method of controlling the oxygen transfer rate,
The problem is that the bacterial respiration rate (oxygen absorption rate) per unit bacterial cell increases and exceeds the oxygen supply rate of the culture conditions, and bacterial cell growth is limited by the oxygen supply rate, resulting in a decrease in protease productivity. there were. Furthermore, even in the method of controlling only the dissolved oxygen partial pressure, there is a problem that the protease activity remains at a certain point and it is not possible to obtain even higher activity.

[Means for solving problems]

The present invention was completed as a result of research conducted to overcome the problems of these prior applications and conventional methods.

When producing protease by culturing filamentous fungi capable of producing protease in a liquid medium, the present invention maintains the dissolved oxygen concentration of the culture solution at 2.0x71 or higher after the final stage of growth, and contains soybean flour. This is a method for producing protease, which is characterized by culturing by continuously or intermittently adding a liquid medium.

The microorganism used in the present invention is a filamentous fungus having protease-producing ability belonging to the genus Aspergillus, Penicillium, Mucor, Rhizopus, etc., and specifically, Aspergillus sojae (I AM 2703).
, Aspergillus soya (IAM 2631), Aspergillus oryzae (IAM2609), Aspergillus oryzae (I F O4176), Aspergillus tamari (T AM 2156), Penicillium
Chrysogenum (HU T 4019), Penicillium
Luteum (A HU 8022), Mucor racemosus (A HU 6002), Mucor hiemalis (H
UT 1131), Rhizopus formosaensis (
IF O4732), Rhizopus javanicus (IF
O5441) ), etc.

It is desirable that the filamentous fungi having protease-producing ability used in the present invention have salt tolerance from the viewpoint of preventing bacterial contamination (a rough guideline for salt tolerance is 5% or more salt, preferably 10% or more salt). .

In the present invention, the culture up to the addition of the liquid medium containing soybean flour, that is, the culture for simply multiplying the bacterial cells, is hereinafter simply referred to as preculture.

As the medium used for pre-culture, any liquid medium conventionally used in the liquid culture method of filamentous fungi having protease-producing ability may be used. Starch, sucrose, dextrin, cellulose, glycerin, etc. are used as nitrogen sources, soybean flour, peptone, meat extract, yeast extract, bran, casein, polypeptone, gluten, etc. are used as nitrogen sources, and inorganic salts include various types of phosphorus. Acid salts, sulfates, hydrochlorides, etc. are used, and if necessary, a liquid medium to which vitamins, nucleic acids, etc. are appropriately added is used.

Among these, soybean flour, which has the effect of significantly increasing protease activity, is preferred as a nitrogen source. In addition, the above-mentioned nitrogen source may be used in combination with soybean flour.

The above liquid medium is inoculated with filamentous fungal cells capable of producing protease, followed by liquid culture. The culture conditions at this time, such as the culture temperature, pH 1 of the medium, and aeration rate, vary depending on the strain used, medium composition, etc., but the culture temperature is usually 25
~40°C, medium pH 6-8, aeration rate 0.1-2
It is about V, V, M.

Thus, after passing through the lag phase at the initial stage of culture and entering the growth phase, the bacterial cells proliferate significantly. Normally, proliferation stops approximately 1 to 4 days after the start of culture, and after the final stage of proliferation, the cell enters an almost stationary phase.

In the present invention, the dissolved oxygen concentration of the culture medium is reduced to 2. Otgl 1 or more, preferably 2.5
Maintain at ~15 liters. As a maintenance method, aeration gas may be stirred and mixed into the culture solution. However, when the stirring speed becomes high, there is a possibility that physical effects such as shearing of bacterial cells by the stirring blades may appear, so the amount of aeration gas, the type of aeration gas, and the shape of the stirring blades should be adjusted to prevent this effect from occurring. It is better to decide etc. From the viewpoint of dissolution efficiency, etc., it is preferable to use pure oxygen gas or oxygen-enriched air as the ventilation gas.

Furthermore, in the present invention, a culture solution containing soybean flour is added continuously or intermittently after the end of the growth phase, that is, during the enzyme production phase.

At this time, in addition to soybean flour, other protein raw materials may be added as needed.
For example, isolated soy protein, milk casein, egg albumin,
One or more of bovine serum albumin, wheat gluten, peptone, soytone, etc. may be used in combination.

This liquid medium containing soybean flour contains carbohydrate raw materials such as glucose, sucrose, lactose, galactose, soluble starch, dextrin, cellulose, and wheat, as well as inorganic salts such as various phosphates, sulfates, and hydrochlorides. , vitamins, nucleic acids, etc. may be added.

In the present invention, the reason why a liquid medium containing soybean flour is added to the culture solution after the end of the growth phase is as follows.

In the culture solution, proteins that are inducers of protease production contained in the early stage of culture are degraded into amino acids by proteases, peptidases, etc. that are produced in small amounts as the bacterial cells multiply, so there is little or no protein present, and proteases are not present. Since it is difficult to induce production, production of highly active protease cannot be achieved after the final stage of proliferation.

Therefore, in order to increase protease production, a liquid medium containing soybean flour can be added to the culture solution after the final stage of growth.

In addition, when adding a culture solution containing soybean flour after the end of the growth phase, the nitrogen concentration in the culture solution should be adjusted to 0.05-0°30% (
(W/V) and maintain the sugar concentration below 0.10% (W/V) (hereinafter sometimes simply referred to as carbon rate control), or reduce the nitrogen concentration in the culture solution to 0.05%. (W/V) (hereinafter sometimes simply referred to as nitrogen rate limiting).

) is preferable because the protease activity can be further enhanced.

First, as a means of adding a liquid medium containing soybean flour after the end of the growth phase so that the carbon rate is controlled, the composition of the medium,
Although the addition rate, addition ratio, etc. can be adjusted and other appropriate means can be selected, one example of preferred means is as follows.

First, the sugar source is broken down by filamentous fungi into monosaccharides,
Since this acts as a suppressor for protease production, it is necessary to consider the carbon, nitrogen, and phosphorus composition of the bacterial cell itself and lower the content of sugar sources in the medium compared to proteins and phosphorus. Therefore, when determining the composition of a liquid medium containing soybean flour to be added to the culture medium after the final stage of growth, we experimentally cultivate filamentous fungi with protease-producing ability to be used in this liquid medium in advance to ensure that the sugar source is completely absorbed. The blending ratio of sugar sources, proteins, and other carbon sources is determined so that the nitrogen concentration remaining when consumed is 0.06 to 0.3% (W/V), and soybeans with the blending ratio determined in this way A sugar concentration of 0.10% (W/V
) or less, preferably 0.05% (W/V) or less, the nitrogen concentration in the culture solution will be 0.06-0.
It is maintained within a range of 30% (W/V).

In addition, when the liquid medium containing soybean flour does not contain sugar, the nitrogen concentration in the culture medium is 0.06 to 0.30.
% (W/V) of the medium containing soybean flour may be added.

On the other hand, as a method of adding a liquid medium after the final stage of growth so that nitrogen becomes rate-limiting, the composition of the liquid medium containing soybean flour to be added is compared to the C, N, and P composition of the bacterial cells themselves. N
There are ways to reduce this.

First, the N source is decomposed by the filamentous fungus into amino acids, which act as inhibitors of protease production. Therefore, the content of Niff1 in the medium should be adjusted by considering the carbon, nitrogen, and phosphorus composition of the fungus itself. It needs to be lower than phosphorus. Therefore, in determining the composition of a liquid medium containing soybean flour to be added to the culture medium after the final stage of growth, we experimentally cultivated filamentous fungi capable of producing protease to be used in the liquid medium, and Compared to other nutrient sources, the blending ratio of sugar source, other nutrient sources, and protein was determined so that the N source was completely consumed, and the liquid medium containing soybean flour with the blending ratio determined in this manner was prepared as described above. If added to the culture solution after the end of growth so that the nitrogen concentration is less than 0.05% (W/V), the nitrogen concentration in the culture solution will be 0.05% (W/V)
is kept within the range below.

In either method, the liquid medium containing soybean flour can be added continuously or intermittently to the culture solution after the final stage of growth.

When culturing is carried out by adding a liquid medium containing soybean flour after the final stage of growth, the liquid medium is continuously added and the culture solution is continuously taken out for continuous culture, or the liquid medium is added intermittently. The culture is carried out by intermittently removing the culture medium, or by adding the liquid medium continuously or intermittently to perform fed-batch culture.

The continuous culture method and the fed-batch culture method can be carried out according to conventional methods except as described above. In addition, in culture in which a liquid medium containing soybean flour is intermittently added and the culture solution is intermittently removed, the addition amount and removal amount may be the same or different, and the addition time and removal time may be at the same time. It may be different or different.

In addition, when culturing by continuously or intermittently adding a liquid medium containing soybean flour, in any of the above-mentioned cultures,
The normal temperature is 25 to 35°C, and the pH of the medium is about 6 to 8.

As a means of recovering protease from the culture solution obtained as described above, for example, the culture solution is filtered by a conventional method to separate the bacterial cells, and if necessary, dialysis, salting out, ion exchange resin, gel filtration, etc. Examples include a method of purification by, etc.

[For production]

In the present invention, by continuously or intermittently adding a liquid medium containing soybean flour while maintaining the dissolved oxygen concentration of the culture solution at 2.0 mg/l or more after the final stage of growth, the bacterial cell concentration after the final stage of growth can be reduced. In addition, by preferably controlling the rate of carbon or nitrogen, monosaccharides or amino acids, etc. that inhibit protease production are kept in extremely small amounts in the culture solution, so protease activity can be maintained at a high level. This will significantly accelerate production.

〔Effect of the invention〕

According to the present invention, it is possible to efficiently obtain a protease with extremely high titer, so the present invention is extremely significant industrially.

The present invention will be further explained below with reference to Examples.

〔Example〕

Example 1 Soybean flour 1.6χ (W/V), soluble starch 5.0χ (W/V)
V) 1MgSO4・7HzOO, 5X (W/V), K
HzPO40.5X (W/V), yeast extract 0.03χ
(W/V), ZnC1z O,01χ(W/V),
CaC1z 0.02% (W/V) and NaC110
, 0χ (W/V) was charged into a 21 mini jar at 1.51 liters of a liquid medium (pH 6,5), then heat sterilized in an autoclave, and then spores of Aspergillus oryzae (I A M 2609) were added to the medium. When inoculating the suspension, the number of spores is 10 spores/d), and the stirring speed is 250-400 rpm.
Cultured in l. 5 so that the pH during culture is 6.5.
Control was performed with N-HtSO and 5N-NaOH.

After 80 hours, the growth reached the final stage, so continuous culture was started. That is, first, pure oxygen is blown into the culture medium, and the stirring speed is set to 400 to 700 rpm, so that the dissolved oxygen concentration in the culture solution is always between 0 and less than 2 ■/β, around 2.5 ■/β, and 15 ■
It was controlled to be around /l.

At the same time, use the same liquid medium as above at a dilution rate of 0.02V/V.
-hr was continuously supplied from the supply port of the mini-jar, and the same amount of culture solution as the supplied amount was continuously collected from the discharge port. The temperature during continuous culture was 30°C, and the medium pH was 6.5.
maintained.

The nitrogen concentration in the culture solution after the start of liquid culture is 0.025 to 0.
．． It was maintained at 0.3% (W/V).

Protease titer (P, U, /d) in the collected culture solution
was measured by the modified Anson-Hagiwara method. The results are shown in Figure 1.

From Figure 1 above, in the method of the present invention, the protease activity is maintained high, while the dissolved oxygen concentration is 2■/l.
If it is less than that, it can be seen that the protease activity is inferior to that of the present invention.

Example 2 Soybean flour 2.0χ (W/V), soluble starch 1.0χ (W/V)
V) 9MgSO4・7H, OO, 5χ (W/V), K
HgPO4o, sχ (W/V), yeast extract 0.03
χ(W/V), ZnC1z O,01χ(W/V),
CaC1t O,02X (W/V) and NaC110
, 0X (W/V) was heated and sterilized in an autoclave at normal pressure, and charged into a 21 mini jar at 1.51 μl, and the spores of Aspergillus oryzae (I AM 2609) were added to the medium. When inoculating the suspension, the number of spores was 106/mZ), 002/Il, and the stirring speed was 40.
Culture was performed at 0 rpm. The pH during the culture was controlled to 6.5 using 5N 1hsO4 and 5N-N'aOH.

After 48 hours, the cells reached the final stage of growth, so continuous culture was started. That is, first, pure oxygen is blown into the culture medium, and the stirring speed is set to 500 to 700 rpm, so that the dissolved oxygen concentration in the culture solution is always O2/l. Three types of continuous culture were carried out while controlling the concentration to be 3 .mu./l and 15 .mu./l.

At the same time, protein raw material [1,0χ (W/V) starch, 2.
0X (W/V) soybean flour, 0.5χ (W/V) KH2PO
4,0,05χ(W/V) Mg5o,.

0.01(W/V) CaC1z, 0.03χ(W/V
) A liquid medium containing yeast extract with a pH of 6.5 that was sterilized by heating at normal pressure in an autoclave] was diluted at a dilution rate of 0.02V.
/V-hr was continuously supplied from the supply port of the mini-jar, and the same amount of culture solution as the supply amount was continuously collected from the discharge port. The temperature during continuous culture was 30°C, and the medium pH was 6.
．． It was kept at 5.

11M98 degrees in the culture solution after the start of liquid culture is 0.1% (
-/V), the nitrogen concentration is 0.05 to 0.20% (W/V) or less.
V) D was maintained.

Protease titer (P, U, /-) in collected culture solution
was measured by the modified Anson-Hagiwara method. Table 1 shows the activity on the 10th day after the start of continuous culture.

(This page, below in the margins) As is clear from the upper table of Table 1, it was observed that the protease activity significantly increased in the method of the present invention.

[Brief explanation of the drawing]

The drawings show the results of Examples and Comparative Examples of the present invention.

Claims (1)

[Scope of Claims] 1) When producing protease by culturing a filamentous fungus capable of producing protease in a liquid medium, the dissolved oxygen concentration of the culture solution is maintained at 2.0 mg/l or more after the final stage of growth, and 1. A method for producing protease, which comprises culturing by continuously or intermittently adding a liquid medium containing soybean flour. 2) After the final stage of growth, reduce the nitrogen concentration in the culture solution to 0.
05-0.30% (W/V), sugar concentration 0.10% (W/V)
/V) The production method according to claim 1, which comprises culturing while maintaining the following conditions. 3) After the final stage of growth, reduce the nitrogen concentration in the culture solution to 0.
2. The production method according to claim 1, wherein the production method is performed by culturing while maintaining the concentration below 0.5% (W/V). 4) The production method according to any one of claims 1 to 3, wherein the filamentous fungus having protease-producing ability is a salt-tolerant bacterium. 5) The manufacturing method according to any one of claims 1 to 4, wherein the dissolved oxygen concentration is maintained at 2.0 mg/l or more using pure oxygen gas or oxygen-enriched air.