JPH0795950B2 - Method for producing carboxypeptidase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing carboxypeptidase from a filamentous fungus belonging to the genus Paecilomyces that has carboxypeptidase-producing ability. Carboxypeptidase is an enzyme that has the property of releasing peptides and proteins sequentially from the carboxyl terminal. This enzyme, alone or in combination with proteinase, is used for pharmaceuticals such as foods, digestive agents, industrially for the production of amino acid mixtures, and as a biochemical reagent for the determination of the amino acid sequence of proteins. It has become an increasingly important enzyme for use in the removal of bitter peptides.

(Prior Art) Conventionally, as carboxypeptidase, for example, carboxypeptidase A (Methods in Enzymology 19 extracted from bovine pancreas)
Vol. 475), Carboxypeptidase B extracted from pig pancreas (Methods in Enzymology Methods)
in Enzymology 19: 504), and in plants, carboxypeptidase C (Nature Na) of citrus peels.
ture (London) 201 613 1964), citrus leaf carboxypeptidase (Hopepe Zylers Zite Shrift Fur Physiologish Chemistry Hoppe-Seylers Z.Physiol. Chem. 352, 1524, 1971
Enzymes of kidney bean leaves (Journal of Biological Chemistry J. Biol. Chem. 247, 5573, 1)
972), Enzymes of sprouted barley (European Journal of Biochemistry Eur.J.Biochem. 7: 193,
1969), enzymes of sprouted wheat (plant physiology)
Plant Physiol. 58, 516, 1976), Enzymes of germinated cottonseed (Journal of Biological Chemistry J.
Biol. Chem. 247, 5034, 5041, 1972), tomato enzyme (agricultural, biological, chemistry, Agric Biol. Chem. 38, 1901, 1974), watermelon enzyme (agricultural).・ Biological chemistry Agric.Biol.Chem. 38, 1891, 1974) and enzymes in bromelain powder (J. Biochemistry 75, 881, 1974) are known. .

However, the carboxypeptidase described above is not suitable for industrial scale production as a raw material, and thus carboxypeptidase has been obtained from a filamentous fungus having no resource problem. For example, the enzymes of Aspergillus saitoi (Biochimica, Biophysica, Acta Biochim.
S.Acta 258, 274, 1972), Enzyme of Aspergillus oryzae (Agricultural Biological Chemistry Agric.
Biol. Chem. 36, 1343, 1474 Hyachi, 1481, 1972, 37,
1237, 1973), Aspergillus sp.
ergillus) enzyme (JP-A-47-25382) and Yokoyama et al., Penicillium enzyme (JP-A-4-25)
8-35084), Kumagai et al. (As)
pergillus) (Japanese Patent Laid-Open No. 51-95182), H. Hoffmann et al.
m janthinellum) penicillocarboxypeptidase S
-1 and penicillo carboxypeptidase S-2 (Methods in Enzymology 45
Vol., P. 587) is known.

(Object of the Invention) An object of the present invention is to provide a method for producing carboxypeptidase from a filamentous fungus belonging to the total of Paecilomyces having carboxypeptidase-producing ability.

(Means for Achieving the Purpose) As a result of screening various filamentous fungi, the present applicant found that filamentous fungi belonging to the genus Paecilomyces produce a novel carboxypeptidase, and finally completed the present invention. It is a thing.

That is, the gist of the present invention is a method for producing a carboxypeptidase from a filamentous fungus belonging to the genus Paecilomyces that has a carboxypeptidase-producing ability.

The details will be described below.

Examples of filamentous fungi belonging to the genus Paecilomyces that can be used in the present invention include Paecilomyces carneus (Pa
ecilomyces carneus), Paecilomyces elegans, Paecilomyces farinosus, Paecilomyces fumosoroseus,
Paecilomyces isa
rioides), Paecilomyces japonicas (Paecilo)
myces javanicus), Paecilomyces marquandii, paecilomyces
Palio (Paecilomyces variotii), Paecilomyces bacillisporu (Paecilomyces bacillisporu)
s), Paecilomyces canadensis (Paecilomyces)
canadensis), Paecilomyces clavispolis (Pa
ecilomyces clavisporis), Paecilomyces cremeo-rosei, Paecilomyces dactyl echilomorphis (Paecilomyces d)
actylethromorphis), Paecilomyces fusisporus, Paecilomyces
Paecilomyces griseoviridis,
Paecilomyces humicol
a), Paecilomyces infratus
ces inflatus), Paecilomyces vario
ti var.antibioticus), Paecilomyces varioti variati brunnerorumu (Paecilomyces v)
arioti var.brrunneolum), Paecilomyces viridis, Paecilomyces lilacinus, Paecilomyces ochraceus, Paecilomyces perochinus
s), Paecilomyces ro
seolus), Paecilomyces striatisporus, Paecilomyces
Subglobosus (Paecilomyces subglobosus), Paecilomyces tenuipes (Paecilomyces tenuipes),
Paecilomyces terricol
a), Paecilomyces
variabilis), Pacilomyces mandshuricum, etc., but as long as it belongs to the genus Paecilomyces of the filamentous fungus, it can be used without limitation to its variants and mutants, and Paecilomyces cerevisiae in terms of carboxypeptidase productivity. Carneus (Pa
ecilomyces carneus) is preferred.

In addition, carboxypeptidase is obtained by culturing the filamentous fungus in order to obtain a large amount, and the culturing method may be solid culture or liquid culture. A raw material having an appropriate solid culture value, for example, wheat malt, defatted soybeans, rice bran, rapeseed meal, wheat, rice, etc. may be appropriately selected singly or in combination, and an appropriate nutrient source is added as necessary. You can do it. Water is added to this raw material, steam pressure sterilization is performed, and the mixture is allowed to cool, and then filamentous fungi are inoculated into it for culturing. The culture conditions are 15 to 35 ° C, preferably around 20 to 25 ° C, at a temperature at which filamentous fungi can grow, and the number of days of culture is 3 to 10 days, and the culture is terminated when the carboxypeptidase-producing ability reaches the maximum. To do. In addition, when liquid culture is performed, appropriate carbon source, nitrogen source, if cold,
Contains wheat, defatted soybeans, defatted soybeans, rice bran, rapeseed meal, starch, glucose, etc., selected singly or in combination, and further necessary components for the growth of filamentous fungi, such as phosphate when cold Water is added to the medium containing the inorganic salt and the medical metal salt, steam pressure sterilization is performed, and the mixture is allowed to cool, and the filamentous fungus is inoculated into the medium for culturing. The culture conditions are adjusted to maximize the carboxypeptidase-producing ability depending on the bacteria and medium used. Generally, the pH of the medium is 2.0 to 6.0 and the culture temperature is 15
Culturing is preferably carried out at ~ 35 ° C for 3 to 10 days, and the cultivation is terminated when the carboxypeptidase-producing ability reaches the maximum. For the liquid culture, any culture method such as stationary culture, shaking, stirring, and aeration culture may be used.

Further, in the case of solid culture, if the culture is cooled, water or an appropriate salt solution, a buffer solution or the like is added to the culture and extracted, and then the solution is treated as a crude enzyme solution. In the case of liquid culture, a solution obtained by treating the culture due to excess or the like may be used as a crude enzyme solution. The crude enzyme solution can be made into a precipitate form by adding an organic solvent or the like, and can also be made into a crude enzyme preparation form by freeze-drying or the like. The crude enzyme solution or crude enzyme preparation thus obtained is concentrated by ultrafiltration, dialysis using a dialysis membrane, etc., salting out with ammonium sulfate, sodium chloride, etc., adsorption and elution with various ion exchange substances, molecular weight It can be purified by gel filtration or the like. The purification methods may be used alone or in combination.

Next, the properties of the thus obtained carboxypeptidase (hereinafter referred to as the present enzyme) will be described. In addition, it was revealed by purification that there are two kinds of enzymes, a low molecular type which is the main part and a high molecular type which is the minor part. The following properties describe low molecular weight enzymes.

(1) Measurement of enzyme activity Carbobenzoxy-glutamyl-tyrosine as a substrate is added to 0.5 ml of the enzyme solution appropriately diluted according to the measurement conditions to make 10 ^-3 M in 1/20 M sodium acetate-hydrochloric acid buffer solution (pH 3.0). Add 0.5 ml of the thus-dissolved substrate solution and react at 30 ° C for 20 minutes.
Then, 1.0 ml of ninhydrin reagent is added to stop the reaction. Furthermore, 1 / 2M sodium citrate-citrate buffer (pH 5.
3.0 ml of 0) was added, heated at 100 ° C for 15 minutes to develop color, and then rapidly cooled in ice water, and then the absorbance was measured at a wavelength of 570 nm using a spectrophotometer. As a control, the ninhydrin reagent was added to the enzyme solution, the substrate was then added, and the same operation was performed thereafter to measure the absorbance. Ninhydrin reagent is methyl cellosolve
1 ml of 1 / 100M potassium cyanide was added to 118 ml, and 1 g of ninhydrin was added.
Was prepared by dissolving. As a standard for calculating the amount of amino acid released from the substrate, 1.0 ml of ninhydrin reagent was added to 1.0 ml of 10 ⁻⁴ M tyrosine solution for each measurement, and the same procedure was followed to measure the absorbance. Regarding the enzyme activity unit, one enzyme capable of purifying 1 mol of free tyrosine per second under the above-mentioned conditions was defined as one enzyme activity unit (1 kat). In addition, 1
Nano Qatar (1 nkat) is 10 ^-9 Qatar.

(2) Action Hydrolyzes the peptide bonds at the carboxyl terminals of proteins and peptides under acidic conditions to release amino acids sequentially.

(3) Substrate specificity Table 1 shows the relative enzyme activities of carbobenzoxydipeptides and benzoyl-glycyl-lysine against carbobenzoxy-glutamyl-tyrosine.

(4) Optimal action pH As shown in Fig. 1 and Fig. 2, when the enzyme was used in the range of pH 2 to 7 with carbobenzoxy-glutamyl-tyrosine and carbobenzoxy-phenylalanyl-alanine as substrates. The optimum working pH is 4.0 for both substrates.
Met.

(5) pH stability As shown in Fig. 3, this enzyme was treated at 30 ° C for 120 minutes.
It was stable between pH 2 and 8.

(6) Effects of various inhibitors and metal salts Table 2 shows the effects of various inhibitors and metal salts. It was found to be a serine carboxypeptidase since it was 100% inhibited by phenylmethylfluorosulfonic acid (PMSF) and not inhibited by pepstatin A. In addition, serine carboxypeptidase is named by the nomenclature committee of the International Union of Biochemistry, and is known as Enzyme Nomenclature, ed. By Nomenclature Com
mittee of the International Union of Biochemistry,
Academic Press, New York, 1984, p300). In addition, this enzyme is 100% inhibited by parachloromercury benzoic acid, suggesting that the thiol group is involved in the enzyme activity.

(7) Molecular weight This enzyme has a molecular weight of about 45,000 as determined by the gel permeation method of Sephadex G-100 (Pharmacia Fine Chemical Co., Ltd.) according to the method of Andrius.
(Low molecular type). In addition, TSK-GEL G-3000SW (manufactured by Toyo Soda Kogyo Co., Ltd.) by high performance liquid chromatogram
It was about 47,000 as determined by the gel filtration method using a column. By the way, the polymer type which is the minor part is about 93,00 when it is obtained by the gel filtration method of Sephadex G-100.
It was 0.

(8) Isoelectric point The isoelectric point of this enzyme was pI3.9 by isoelectric focusing using Pharmalite 2.5 to 5.0 (Pharmacia Fine Chemical Co., Sweden).

(9) Absorption coefficient The enzyme was freeze-dried, the weight was measured, and the absorbance at 280 nm was measured to obtain the absorption coefficient. ▲ A ^1% _{1 cm} ▼ is 14.8
Met.

(10) Kinetic analysis Table 3 shows carbobenzoxydipeptide and benzoyl-
The reaction rate constant of this enzyme when glycyl-lysine was used as a substrate is shown.

(11) Disc electrophoresis Polyacrylamide gel pH 4.3, gel concentration 7.5%
The gel was subjected to electrophoresis at 5 mA per gel for 4 hours at 4 ° C, and then stained with Coomassie Brilliant Blue R250. As a result, as shown in Fig. 5, a single band was observed about 0.7 cm from the origin (anode end) to the cathode side, demonstrating that this enzyme is an electrophoretic homogeneous sample. .

(Example) Example 1 (Production by solid-state culture) 2.1 g of water was added to 3 g of wheat flour in a 100 ml Erlenmeyer flask and kneaded well, sterilized under pressure at 120 ° C for 20 minutes, allowed to cool, and then pre-cultured in advance. One platinum loop was inoculated from a slant culture of various Paecilomyces spp. This was shaken twice a day at 24 ° C. to thoroughly loosen the filamentous fungi in the flask and aerated, followed by static culture for 7 days. After culturing, 30 ml of 1/20 M sodium acetate-hydrochloric acid buffer solution (pH 3.0) was added to the solid culture and shaken vigorously.
After leaving it at 2 ° C. for 2 hours, an excess of this solution was used as a crude enzyme solution. The results of measuring the enzyme activity of the obtained crude enzyme solution are shown in Table 4.

Example 2 (Production by liquid culture) To a Sakaguchi flask of 500 ml, 3 g of wheat bran and 1 g of defatted soybeans and 3 ml of tap water containing 0.2 g of potassium phosphate 1 were added and kneaded well, and 93 ml of tap water was added. After adjusting the pH to 3.0 with 3.5 ml of 1M tartaric acid, it was sterilized under pressure at 120 ° C. for 20 minutes, allowed to cool, and 1 platinum loop was inoculated from sucrose culture of various Paecilomyces sp. This at 24 ℃, 120 (amplitude 10 cm)
The culture was carried out with shaking at a reciprocating rate of 7 minutes. The enzyme was extracted with No. 2 Toyo Paper (manufactured by Toyo Roshi Co., Ltd.), and this solution was used as a crude enzyme solution. The results of measuring the enzyme activity of the obtained crude enzyme solution are shown in Table 5.

Example 3 (Purification of carboxypeptidase) 90 g of wheat bran and 63 ml of tap water were well kneaded and then dispensed into 3 Erlenmeyer flasks and sterilized at 120 ° C for 20 minutes to prepare a medium.
Paecilomyces carneus
s) About 1 cm ^{2 of} IFO7012 was cut off, then aseptically crushed into fine pieces, inoculated into the medium, and static culture was carried out at 24 ° C. for 7 days. During the culture, the filamentous fungi in the flask were thoroughly shaken and aerated by shaking twice a day. After culturing, 900 ml of 1/20 M sodium acetate-acetic acid buffer (pH 4.0) was added to the culture, and the mixture was vigorously shaken and then left standing for 2 hours for extraction. The culture and the extract were separated with No.2 Toyo Paper (manufactured by Toyo Roshi Kaisha, Ltd.). Ammonium sulfate was added to 407 g of this solution and dissolved (80% saturation)
Then, it was left standing overnight for salting out (80% ammonium sulfate salting out). Next, this enzyme was precipitated as a precipitate by centrifugation (14,000 xg / 10 min) for a few times.
2 g was obtained. This precipitate was dissolved in 1 / 20M sodium acetate-acetic acid buffer (pH4.0), put in a dialysis tube and dialyzed overnight with 1 / 20M sodium acetate-acetic acid buffer (pH4.0), and the dialysate was centrifuged. (25,000 × g / 10 min) to obtain 8.2 ml of supernatant. The enzyme recovery rate here was 72.7% of the culture extract solution. This supernatant was applied to Sephadex G-100 column (1.8φ x 72c
When gel filtration was performed with m, 1/20 M sodium acetate-acetic acid buffer (pH 4.0), as shown in Fig. 6, the minor portion was a high molecular type with a molecular weight of about 93.000, and the main portion had a molecular weight of 4
It was divided into 5,000 low molecular weight carboxypeptidase fractions. The low-molecular-weight enzyme activity, which is the main part, was measured and found to be 109 nkat / ml and a specific activity of 74.7 nkat / ml. 51 ml of this low-molecular-weight fraction was obtained, and the recovery rate was 45.2%. Next, the obtained low-molecular-weight fraction was dialyzed against 1/20 M sodium acetate-acetate buffer (pH 4.5), and the dialysate was treated with DEAE-cellulose (DE3
2) (Whatman, England) column (1.8φ x 41c
m, 1 / 20M sodium acetate-acetic acid buffer (pH 4.5)), and then ion-exchange chromatogram was performed with the above buffer pH 4.5 at a concentration gradient from 1 / 20M to 1 / 2M. It was (7th
The figure shows the elution pattern of the ion-exchange chromatogram with DEAE-cellulose (DE32). ) The main fraction obtained was 55 ml, the enzyme activity was 53.7 nkat / ml, and the specific activity was 245 n.
Kat / A280, recovery rate was 24.0%. In addition, a rechromatogram was performed using a DEAE-cellulose (DE32) column. The column was equilibrated with 1.8φ x 41 cm, 1/20 M sodium acetate-acetic acid buffer (pH 4.5) in advance, and elution with a concentration gradient from 1/5 M to 1/2 M with the above buffer (pH 4.5) was performed. The main fraction having carboxypeptidase activity was obtained as a purified enzyme preparation. From this operation, the enzyme activity was 46.7nk
50 ml of enzyme solution with at / ml and specific activity of 313 nkat / A280 was obtained. (Eighth
The figure shows the elution pattern of the ion-exchange rechromatogram with DEAE-cellulose (DE32). ) All the work after the extraction process was performed at 4 ° C.

(Effects of the Invention) As described above, according to the present invention, since filamentous fungi are used as a raw material, culturing for mass production is easy and suitable for industrial scale production.

[Brief description of drawings]

Figure 1 shows the action pH range of this enzyme when using carbobenzoxy-glutamyl-tyrosine as a substrate, and Figure 2 shows this enzyme when using carbobenzoxy-phenylalanyl-alanine as the substrate. Fig. 3 shows the stable pH range of this enzyme, Fig. 4 shows the molecular weight of this enzyme, Fig. 5 shows the disk electrophoresis of this enzyme, Figure 6 shows the elution pattern of this enzyme on Sephadex G-100, and Figure 7 shows the elution pattern of this enzyme.
The figure which shows the elution pattern of the ion exchange chromatography by DEAE-cellulose, and FIG. 8 are the figures which show the elution pattern of the ion exchange rechromatography of this enzyme by DEAE-cellulose.

Claims (1)

[Claims] 1. A method for producing carboxypeptidase from a filamentous fungus belonging to the genus Paecilomyces which has the ability to produce carboxypeptidase.