JPS6025113B2 - Enzyme extraction method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for efficiently extracting enzymes from solid-state cultured malt of Rhizobus bacteria.

In general, Rhizopus bacteria are currently used to produce various useful enzymes, and most of the production methods involve extraction from solid cultured koji (hereinafter referred to as koji).

However, since Rhizobus koji contains sticky substances and the extract is highly viscous, the cumulative leaching method (
High-concentration extraction methods such as immersion (hereinafter referred to as cumulative immersion), countercurrent extraction, and semi-countercurrent extraction cannot be applied; The drawback is that the yield of the enzyme decreases because it is difficult to carry out.

In other words, generally to obtain enzymes and other target substances from a solid culture of microorganisms, the culture is immersed in a suitable yuzu agent for extraction. For the purpose of increasing as much as possible, it is common to obtain immersion liquid by, for example, the cumulative leaching method (hereinafter abbreviated as cumulative immersion liquid). However, when applying multiple immersion to obtain enzymes such as lipase and protease from Rhizobus koji, the viscosity of the immersion liquid is abnormally high, making it impossible to accumulate. It is necessary to use a small amount and make the koji layer in the extraction tank extremely thin. This means that in order to process a certain amount of koji within a certain amount of time, it is necessary to install several times as many extraction tanks, or if it is not possible to increase the number of extraction tanks, the amount of koji to be processed must be reduced to a fraction. However, such measures only enable the detection of accumulated liquid. In addition, when handling enzymes, various operations must be carried out at as low a temperature as possible to prevent heat inactivation, but keeping the enzyme at low temperatures naturally leads to an increase in viscosity, so the cumulative soaking temperature must be set high, and therefore heat inactivation is necessary. In order to prevent this, the extraction time has to be shortened, resulting in poor extraction efficiency and large enzyme deactivation during extraction. Moreover, even in the post-process operation of separating the enzyme from the accumulated liquid, there are many process losses due to the high viscosity, and the workability in furnace filtration and purification is very poor, so the low temperature conditions have to be relaxed, resulting in heat loss. Problems such as oxidation occurring everywhere and yield decline becoming increasingly severe remain unresolved. Although the koji of Rhizobus bacteria has many problems, the enzymes obtained from it, such as protease and pase, are excellent enzymes with properties suitable for use in medicine, food industry, feed, etc. In order to solve these problems, the present inventors conducted extensive research and found that a very large amount of crude enzymes derived from filamentous fungi and bacteria was pre-dissolved in the koji extract (
Hereinafter referred to as crude enzyme yuzu agent.

), by extracting koji, it is possible to thicken the koji layer in the extraction tank, and furthermore, it is possible to perform cumulative immersion with high extraction efficiency in a short time even under low temperature conditions that are sufficient to prevent heat deactivation of enzymes during extraction. The present invention was completed based on the discovery that the various steps after extraction can be carried out under suitable low temperature conditions. The present invention provides a method for extracting enzymes from a solid culture of several koji of Rhizobus spp., which is characterized in that a crude enzyme agent derived from filamentous fungi or bacteria is added to the extraction agent. be.

Numerical culturing is carried out by conventional methods.

Further, as the citrus agent, a solution of water and an acid, an alkali, a salt, etc. or a mixture of water and a water-soluble organic solvent is used. The enzyme agent derived from filamentous fungi or bacteria added to these extractants may be highly purified or not sufficiently purified.

Examples include extracts of enzyme-producing cultures, simple purified products, and commercially available enzyme preparations. The amount of the crude enzyme agent added to the yuzu agent is very small, preferably about 0.1 to 0.001%.

Extraction is preferably carried out by cumulative immersion, but according to the method of the present invention, a cumulative immersion solution can be obtained in a much shorter time than in the case where no enzyme is added. Next, test examples and examples of the present invention will be shown, and the koji used in the various tests was prepared in the same manner as in Example 1.

The viscosity of the immersion liquid and the cumulative immersion liquid was measured using an Ostwald viscometer, and the viscosity (SV) was expressed as a relative value with the value of water as 1 at a temperature of 5°C, and the falling time (seconds) was also measured at 5°C. Lipase activity was determined by the Machida method (Nichino Kagaku Shi, Vol. 36, p. 8, 1962), and protease activity was determined by the Kageyama method (Chikou Shi, Vol. 33, p. 28, 1953) using milk casein at pH 3.0. Measured according to King). Test example 1 15k9 of koji was packed in a 20-ton column (koji layer thickness: 100 layers), poured 25 liters of 5℃ water, and left to stand for 2 hours while keeping at 5℃. The liquid and the washing liquid obtained by the same operation after collecting the soaking liquid once are used to make a new koji 15.
Table 1 shows the relative values of the viscosity and enzyme activity of the cumulative immersion liquid obtained by cumulative immersion, which was used as an extractant in the next column packed with k9.

(Table 1) From these results, it can be seen that although the enzyme concentration is higher in cumulative immersion, the viscosity is extremely high, so cumulative immersion cannot be performed on an industrial mass production scale.

In order to compare the liquid permeability at each temperature for the cumulative immersion liquid obtained in the same manner, the falling time (seconds) was measured and shown in FIG.

These results show that the permeability of the cumulative immersion liquid deteriorates significantly at low temperatures. Table 2 shows the relative values of the enzyme activities obtained by extracting the single immersion solution obtained in the same manner at various temperatures.

(Table 2) From this result, protease is good, but reverse is 10qo.
It can be seen that the following low temperature maintenance is essential.

Test Example 2 Using the same method as Test Example 1, 0.1% of various enzyme preparations were added to the cumulative immersion liquid, and after being left at 20°C for 1 hour, the temperature was increased to 5°C.
When the viscosity and relative enzyme activity values were compared, the third
The results shown in the table were obtained.

Table 3 (Note) Items 1 to 13 are Amano Pharmaceutical KK products, item 14 is a Daiwa Kasei KK product, item 15 is a Kaken Chemical KK product.

From this result, we learned that various crude enzyme preparations have the effect of reducing viscosity.

Test Example 3 Regarding Cellulase Amano A, which was found to be effective in Test Example 2, the relationship between the added amount, action temperature, and viscosity reduction was tested by allowing it to act on the cumulative immersion liquid obtained in the same manner as Test Example 2 for 2 hours. The results are shown in Figure 2.

From this result, it is possible to achieve the most ideal temperature of 5°C with the addition of the most characteristic amount.
It was found that it was effective in reducing viscosity both before and after. Test example
4 Using the cumulative immersion liquid obtained in the same manner as in Test Example 2, we compared the viscosity and enzyme activity relative value after changing the amount of each paper enzyme agent and leaving it at 5℃ for 2 hours. Table 4 shows the results. The following results were obtained.

From the results in Table 4, we can see that some substances are effective in reducing viscosity but also cause lipase to be lost, and that there is a difference in the effect when added at the same amount, but in any case, the effect is less than 0.1%. 4.0, which allows the viscosity to be maintained at a low temperature of around 5°C without deactivating the enzyme in the cumulative immersion liquid by adding a certain amount, and without causing heat inactivation of the enzyme.
It has been found that it is possible to reduce the

Test Example 5 200kg of koji was packed in two columns of 100K9 and 5Koji (Koji layer thickness: 90K), 180kg of yuzu was poured from above the first column for each yuzu agent, and the columns were left to stand for 1 hour. The double immersion liquid is detected from below, then poured from above the second column, and 1 hour later, the cumulative immersion liquid is detected from the bottom of the column.The immersion liquid and cumulative immersion liquid are collected using the gravity flow method and extracted. The temperature was 5°C.

A 0.001% aqueous solution of Cellulase Amano A and water were used as the yuzu agent for cumulative immersion, and the viscosity of each cumulative immersion liquid and the time required to collect the immersion liquid were compared and the results are shown in Table 5.

Table 5 Even from these results, cumulative immersion using the water extraction method is not possible on an industrial scale due to the high viscosity of the cumulative immersion liquid and the time required to collect the immersion liquid. It turns out that you can only find things that correspond to liquid.

Therefore, in the method of the present invention, the viscosity of the immersion liquid is significantly reduced, so that it is possible to carry out cumulative leaching on an industrial scale. It increases markedly as if it were Similar results were obtained with Cellulase Amano T, Gluczyme 600, Cellulase Amano N, Lipase Amano/A, and Biodiastase 1000YL-5. The viscosity of the cumulative immersion liquid is 4.
The fact that it is less than 0 means that operations in subsequent steps can be carried out very efficiently.

In addition, since the amount added is extremely small, there is no indication that the components of the crude enzyme agent used will be transferred to the product. Crude enzyme preparations that can be used in the present invention include those derived from microorganisms such as filamentous fungi belonging to the genus Asbergillus, Trichoderma, and Rhizorbus, and bacteria belonging to the genus Aeromonas and Achromobacter. The amount of addition is required (preferably 0.1 or less)
A crude enzyme agent that has the effect of lowering the viscosity of the cumulative immersion liquid (preferably SV at 500 is 4.0 or less) and does not deactivate the target enzyme during extraction (preferably has a deactivation rate of 15% or less). It's good to have.

Example 1 Well water of the same weight as the number of wheat was sprinkled, steamed at 12,000 for 40 minutes, left to cool, and then inoculated with seed koji of Rhizobus deremar lAM6038 crab strain, plated on chamber lids, and grown in a culture chamber of approximately 3000℃. Pack 180k9 of koji that has been cultured for 40 hours into a 50-arc stainless steel column (koji layer thickness: 15), and pour it from the top of the column.
300 grams of a 0.001% aqueous solution of cellulase Amano A cooled to 5° C. was sprayed and injected, and the immersion liquid was collected from the bottom of the column immediately after leaving it for 2 hours.

Next, the same koji 180 [9] as above was added to another column similar to the above.
The collected long liquid was sprayed into the column and left to stand for 2 hours, after which the accumulated liquid was collected from the bottom of the column. The base was 180 long. The viscosity of this cumulative immersion liquid was 2.8, and the total time required to collect the immersion liquid once was 2.8. It was bug time. Extracting only water required a lot of time to collect even one immersion liquid, so repeated immersion work was not possible. Example 2 A cumulative infusion solution obtained in the same manner as in Example 1 using koji obtained in the same manner as in Example 1 using Rhizobus niveus M6031 strain and using a 0.01% solution of Cellulase Amano N as a yuzu agent. The base was 180 long.

Its viscosity was 3.6, and the total time required to collect the single immersion liquid and the multiple immersion liquid was 4 hours. On the other hand, 40 kg of koji was packed in a column (koji layer thickness: 35 mm), and 60 kg of water cooled to 5°C was sprayed and injected from the top of the column. The total time required for collecting the one-time immersion liquid and the multiple immersion liquid in Step 5 was 3 hours. Furthermore, the relative values of the enzyme activities of both cumulative immersion solutions are 100 for the water extraction method, and 170 for reverse and 150 for protease for the enzyme liquid extraction method.
Met. Example 3 The bacterial strain used was Rhizobus delemer M6040 strain, and the combined enzyme agent used and the amount added were 0 and 0 Cellulase Amano T.
．． 001% and carried out in the same manner as in Example 1. As a result, the cumulative amount of immersion liquid was 180 mm, the viscosity was 3.0, and the time required to collect the immersion liquid was 3 hours.

Example 4 The results were carried out in the same manner as in Example 1, using the Rhizobus niveus M6035 strain as the bacterial strain, and using the crude enzyme agent used and the amount of gluczyme 6000 added at 0.001%.
The cumulative amount of immersion liquid was 180 mm, the viscosity was 3.3, and the time required to collect the immersion liquid was 3. It was immediate.

[Brief explanation of drawings]

FIG. 1 shows the liquid permeability of the cumulative immersion liquid at various temperatures in Test Example 1. FIG. 2 shows the effects of the amount of crude enzyme added to the extraction agent and the temperature at the time of action on the viscosity in Test Example 3. a...cumulative immersion liquid, b...water, c...2℃, d...
...1 pi○, e...20℃. Figure 1 Figure 2

Claims (1)

[Claims] 1. A method for extracting enzymes from solid-cultured wrinkled koji of Rhizobus spp., which comprises adding a crude enzyme agent derived from filamentous fungi or bacteria to the extraction agent.