JPH037356B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying acidic carboxypeptidase collected from a culture obtained by culturing acidic carboxypeptidase-producing bacteria belonging to the genus Penicillium. Traditionally, unique microorganisms have been used to produce fermented foods such as miso, soy sauce, and cheese, and many of these microorganisms have strong proteolytic activity. Generally, the degradative enzymes of these microorganisms are a mixture of exopeptidase and endopeptidase.
It is known to play an important role in the flavor and tissue formation of fermented foods. On the other hand, it is also known that when proteins are degraded, they sometimes exhibit an unpleasant bitter taste due to the peptides produced. Therefore, if an attempt is made to actively utilize proteolytic enzymes to promote decomposition, bitterness may develop, making the product unsuitable as a food. However, traditional protein-rich fermented foods
It provides a unique taste due to the delicate balance of endopeptidase and various exopeptidases that can degrade bitter peptides, and almost never exhibits bitterness. This reminds us how important the role of exopeptidase is. Considering these functional characteristics of exopeptidases, if acidic carboxypeptidase, a type of exopeptidase, can be obtained without any endopeptidase, it could be used to remove bitter peptides from foods or enhance the flavor of foods. Furthermore, by adjusting the activity ratio of carboxypeptidase and endopeptidase, it is thought that new foods using these enzymes will be developed. Conventionally, carboxypeptidases have been isolated and purified from citrus peels, germinated wheat and barley, animal pancreas, etc. (carboxypeptidases A and B), and have also been isolated and purified from yeast and actinomycetes (yeast The product isolated from the enzyme is called carboxypeptidase Y). In addition, Aspergillus saitoi, Aspergillus oryzae,
Penicillium
The isolation and purification of carboxypeptidase from filamentous fungi such as P. janthinellum has also been reported. However, in the conventional purification of carboxypeptidase, in order to obtain carboxypeptidase that does not contain endopeptidase, a salting-out method using ammonium sulfate or Glauber's salt, an organic solvent precipitation method using alcohol, acetone, dioxane, etc.
Currently, multi-step purification methods are being carried out using a combination of gel perchromatography such as Sephadex or biogel. Therefore, such a purification method is not suitable for industrial mass production because of complicated operations and high costs. In view of the above-mentioned background, the present inventors conducted studies to provide a purification method suitable for large-scale purification of acidic carboxypeptidase on an industrial scale. We have discovered a purification method suitable for advantageous mass production of acidic carboxypeptidase obtained by culturing acidic carboxypeptidase-producing bacteria, leading to the present invention. The present invention will be explained in detail below. The present invention involves passing a culture solution obtained by culturing acidic carboxypeptidase-producing bacteria belonging to the genus Penicillium through a carboxymethyl-sephadex, and adsorbing the acidic carboxypeptidase fraction in the culture solution onto the column; The column is then washed and the above fraction is eluted. Carboxymethyl-cephadex (hereinafter referred to as CM-cephadex) used in the present invention to adsorb the acidic carboxypeptidase fraction is known to have properties as a weakly acidic cation exchanger. The present inventor discovered for the first time that the adsorption amount of cation exchange resin is significantly larger than that of other cation exchange resins. In the present invention, the culture solution obtained by culturing acidic carboxypeptidase-producing bacteria belonging to the genus Penicillium is obtained as follows. Examples of the acidic carboxypeptidase-producing bacteria include Penicillium caseicolum No. 6009, or Penicillium roqueforti ATCC38099.
At a temperature of about 20 to 25°C, the cells were grown for 8 to 10 days in a wheat-based medium, which is commonly used for culturing filamentous fungi.
After culturing for 1 day, add 7 to 10 times the volume of the resulting culture to 5
Add ~10mM acid buffer (PH3.0) to the mixture.
5℃ while adjusting the pH to 3.0-4.0, preferably 3.5
The enzyme in the culture is extracted for about 5 hours. Next, this extraction mixture is subjected to solid-liquid separation by centrifugation or over-operation to obtain a culture solution containing the crude enzyme (ie, a crude enzyme extract). Next, the culture solution obtained in this way (PH
3.0 to 4.0, preferably 3.5) is passed through a CM-Sephadex column equilibrated in advance with 10 mM acetate buffer (PH 3.5) to adsorb the acidic carboxypeptidase fraction in the above solution. Here, the enzyme
Since the CM-Sephadex adsorption capacity is approximately 25 units/ml resin, the column volume used should be at least twice the exchange capacity calculated from this. In addition, the column should be made as thick as possible to ensure the flow rate.
It is preferable to use a short shape, particularly one with a diameter to length ratio of 1:3 to 10. When the acidic carboxypeptidase fraction adsorbed on the CM-Sephadex column is eluted as described above, the endopeptidase activity in the resulting eluate is approximately 1/10 of the activity in the crude enzyme extract (culture solution). decreases to In other words, CM
- It can be confirmed that acidic carboxypeptidase in the crude enzyme extract is selectively adsorbed by passing the solution through the Sephadex column. In the present invention, in order to obtain high-purity acidic carboxypeptidase that is substantially completely free of endopeptidase, the column is washed prior to the elution process to remove a small amount of the acidic carboxypeptidase adsorbed on the column. Elute and remove the endopeptidase present. This washing is carried out using a 10 mM acetate buffer (PH3.5) containing 0.2 M sodium chloride at a flow rate of at least 2 BV (vehicle volume; resin volume). Note that no elution of acidic carboxypeptidase is observed by this washing. After the endopeptidase is removed by washing the column, the adsorbed acidic carboxypeptidase is eluted at an ionic strength of 0.5M or higher and a pH of 3.0 to 4.0, using, for example, a 10mM acetate buffer (PH3.5) containing 0.5M sodium chloride. Let's do it. This elution can be completed in 1 to 2 BV, and no endopeptidase is detected in the elution fraction obtained. In addition, it is preferable that the liquid flow rate in the above adsorption and elution operations in the CM-Sephadex column be carried out at an SV (flow rate per unit hour divided by resin capacity) of 2 to 3. is 0~
Care should be taken to perform the test at a low temperature of 5°C. As described above, according to the present invention, highly pure acidic carboxypeptidase free from endopeptidase can be obtained by simple operations of adsorption onto a CM-Sephadex column, washing and elution. In other words, the present invention can be said to have made it possible to efficiently and completely separate endo-type and exo-type peptidases, which had been considered difficult in the past. EXAMPLES The present invention will be specifically described below with reference to Examples. In addition, the enzyme activity in the examples is shown as a value determined by the following measurement method. 1 Acidic carboxypeptidase activity: 1mM, benzyloxycarbonylglutamyltyrosine (Z-Glu-Tyr) solution 0.5ml, 50ml
M acetate buffer 0.45ml (PH3.0) and enzyme solution 0.05
After reacting ml of the mixture at 30℃ for 20 minutes,
Add 0.2 ml of 0.3M sodium hydroxide solution to stop the reaction, and leave it for 20 minutes. Then do this
After neutralizing with 0.2 ml of 2.5% acetic acid solution, perform a ninhydrin reaction and measure the absorbance at 570 nm. The amount of enzyme that releases 1 μM tyrosine in 1 minute at 30°C by this reaction is expressed as one unit. 2. Acid protease activity: Add 0.1 ml of enzyme solution to 1.9 ml of substrate mixture (0.32% casein, 50 mM lactic acid solution, PH3.0), react at 30°C, and stop the reaction by adding 2.0 ml of 10% trichloroacetic acid solution. Hold at 30 °C for 30 minutes. The resulting precipitate is then filtered and the increase in absorbance of the liquid at 280 nm is measured. One enzyme unit was defined as the amount of enzyme that increased the absorbance at 280 nm by 1 per minute under the above measurement conditions. Example 1 Wheat = 3 kg of ion-exchanged water containing 10.5 g of dibasic potassium phosphate and 1.05 g of magnesium sulfate were mixed, kneaded well, and then dispensed into 3-volume Erlenmeyer flasks and sterilized at 121°C for 1 hour. . This was previously cultured on potato dextrose agar slants.
After inoculation with Penicillium caseicolum SBT7010 (Feikoken Bacteria No. 6009), the cells were cultured at 20°C for 10 days. Add 20 mL of 5mM acetate buffer (PH3.0) to the resulting culture.
was added and extracted by immersion for 5 hours. During this time, adjust the pH with hydrochloric acid.
Adjusted to 3.5 and held constant. Next, this was passed to obtain about 17 ml of clear crude enzyme extract. This was passed through a CM-Sephadex column (column capacity: 200 ml, diameter: 4 cm x length: 16 cm) that had been equilibrated with 10 mM acetate buffer (PH 3.5) to adsorb the acidic carboxypeptidase fraction. Note that the flow rate at this time was SV2. Further this
The mixture was washed with 400 ml of acetate buffer containing 0.2M sodium chloride to completely elute the contaminating endopeptidase. next,
Acidic carboxypeptidase was eluted with 400 ml of the same buffer containing 0.5M sodium chloride, and the resulting active fractions were collected. Through the one-step purification process described above, endo- and exo-type peptidases could be completely separated, and 300 ml of an acidic carboxypeptidase active fraction containing no endopeptidase activity was obtained. The results of each enzyme activity during the purification process are shown in Table 1, and the recovery rate of enzyme activity was 91%. In addition, the acidic carboxypeptidase obtained above has a capacity of approximately
55 times, and the specific activity (activity/protein amount) is approximately 22
concentrated twice.

[Table] Next, the acidic carboxypeptidase obtained as described above was used to examine its ability to decompose bitter peptides. Casein bitter peptide preparation
% casein solution was added to 0.01% crystalline trypsin and decomposed at 25°C for 24 hours at pH 7.5. After extracting the bitter fraction in the decomposition solution with n-butanol, acid treatment (PH
Protein removal was performed according to 5.4), and the bitter peptide was obtained by freeze-drying. To 1 ml of the 1% solution of the bitter peptide obtained here, 0.3 units of this enzyme preparation was added and reacted at 25° C. and PH 3.0, and the reduction in bitterness was examined by sensory evaluation. In the sensory evaluation, five panelists were used to compare the intensity of bitterness between the enzyme solution and the bitter peptide and enzyme mixture before and after the reaction. As shown in Table 2, the present enzyme clearly has bitter peptide degrading activity and was judged to be effective.

[Table] Example 2 10 kg of wheat and 8 kg of ion-exchanged water were mixed, and this mixture was placed in a toilet dedicated to culturing koji and sterilized at 121°C for 30 minutes. Penicillium, which was previously cultured on potato dextrose agar medium,
After inoculating with roqueforti ATCC No.38099, 25℃, humidity
The cells were cultured in a 100% chamber for 8 days. The culture was collected and diluted with 5mM acetate buffer (PH
3.0), and while adjusting the pH to 3.5, 10℃,
A 4 hour immersion extraction was performed. After immersion extraction, first remove large solid matter using a sieve, then completely remove the precipitate using a continuous high-speed centrifuge (manufactured by Shinmaru Enterprises) to obtain a clear crude enzyme solution 65. . separately
Prepare a CM-Sephadex column (10 columns with a diameter of 3 cm and a length of 20 cm, each column capacity is 100 ml) equilibrated with 10 mM acetate buffer (PH3.5). Then, simultaneously flow the crude enzyme extract at a rate of 6.5 times per column.
Acidic carboxypeptidase was adsorbed. Next, 10mM containing 0.2M salt as in Example 1.
Washing with acetic acid buffer (2BV) was followed by elution of acidic carboxypeptidase with the same buffer (2BV) containing 0.5M NaCl. The purification results are shown in Table 3. As seen in Table 3, carboxypeptidase and endopeptidase could be completely separated. In addition, the active fraction can be obtained at 1.2, and the volume is
The specific activity was 54 times more concentrated, and the specific activity was 21 times more concentrated.

【table】

Claims (1)

[Scope of Claims] 1. A filtrate of a culture obtained by culturing an acidic carboxypeptidase-producing bacterium belonging to the genus Penicillium is passed through a column of carboxymethyl-sephadex, and the acidic carboxypeptidase fraction in the culture filtrate is collected in the column. 1. A method for purifying acidic carboxypeptidase, which comprises adsorbing it to a column, washing the column, and eluting the fraction. 2 The filtrate of the culture was adjusted to an ionic strength of 0.2M or less and a pH of 3.0.
-4.0 through the column and the acidic carboxypeptidase fraction adsorbed on the column at ionic strength
The purification method according to claim 1, which elutes at 0.5M or more and pH 3.0 to 4.0.