KR20050027239A - Process for obtaining serine proteinase inhibitor from canavalia gladiata - Google Patents

Abstract

A process for preparing serine proteinase inhibitors from Canavalia gladiata is provided, which serine proteinase inhibitors have high inhibiting activity for trypsin and chymotrypsin. The process for preparing serine proteinase inhibitors from Canavalia gladiata comprises the steps of: pulverizing Canavalia gladiata seeds, removing fat from the pulverized Canavalia gladiata seeds, adding deionized water into the Canavalia gladiata seeds and stirring the mixture at 2 to 5 deg. C for 10 to 30 hours to obtain the total proteins from Canavalia gladiata; concentrating the total proteins of Canavalia gladiata with a saturated ammonium sulfate solution; and subjecting the concentrated proteins of Canavalia gladiata to gel filtration, anion exchange resin or reverse-phase column chromatography, wherein the serine proteinase inhibitors have high inhibiting activity for trypsin and chymotrypsin, and have molecular weights of 7806.5 Da, 7919.9 Da, 8163.4 Da.

Description

PROCESS FOR OBTAINING SERINE PROTEINASE INHIBITOR FROM CANAVALIA GLADIATA}

The present invention relates to a method for obtaining a serine protease inhibitor from canavalia gladiata, and more particularly, a serine protease having a molecular weight of 7806.5, 7919.9, and 8163.4 having trypsin and chymotrypsin inhibitory activity from seeds of soybeans. The present invention relates to a method of separating an inhibitor.

In general, proteinases are widely present in tissues, cells, and microorganisms of animals and plants. Most are simple proteins with tens of thousands of molecular weights, sometimes complex proteins with sugars or metal ions. By the decomposition mode, it is largely divided into exopeptidase and endopeptidase. Exopeptidase acts only at the ends of the peptide chain to release amino acids in turn. Leucine aminopeptidase or carboxypeptidase are typical. Endopeptidase acts internally rather than terminally, releasing peptides of various sizes. There are numerous examples, including pepsin, trypsin, chymotrypsin. Protease is required when ingesting amino acids, which are indispensable materials of proteins by other proteolysis, and plays an important role in determining the amino acid sequence of proteins.

Protease inhibitors are known to be widely distributed in animal and plant systems. For example, it is present in the liver and epidermis of rats, in white blood cells and urine of humans, in colostrum of cows, etc., and in soybeans and cucumbers in plant systems. These protease inhibitors function as control factors for physiological activity such as blood coagulation and are used for the treatment and prevention of thrombotic diseases such as cerebral infarction and myocardial infarction. Among these protease inhibitors, trypsin inhibitors with high trypsin inhibitors have been known so far, such as deodorant basic trypsin inhibitors, soy trypsin inhibitors, bovine colostrum trypsin inhibitors, and the like. It is known to have high inhibitory activity against trypsin (see Japanese Patent Laid-Open No. 7-82296). In addition to the soybean-derived inhibitors described above, other plant-derived trypsin inhibitors have been reported to have powders or granules obtained by heating and drying hot water extracts contained in cucumbers and seeds thereof (Japanese Patent Laid-Open No. 8-143467).

The present inventors have conducted numerous studies and experiments to obtain novel trypsin and chymotrypsin inhibitors derived from plants. As a result, three proteins having a molecular weight of 7806.5, 7919.8, and 8163.4 obtained from seed extracts of canavalia gladiata were trypsin and chymotrypsin. It has been found that the inhibitory action against is excellent and the present invention has been completed.

The soybean bean is named Canavalia gladiata, which is cultivated in the southern part of Korea, and in the north of central part. Bean beans are beans as big as small beans, which is the largest soybean pod of about 20-30cm in length, and its nutrition consists of protein, starch, lipids, various minerals and vitamins. In particular, hemagglutinin, which has been disadvantaged in soybeans, contains a large amount of protein called Concanavalin A, which helps in detoxification and anticancer activity, and has a strong inhibitory effect on transformed cells produced by various types of carcinogens. In addition, it has a double-sided effect of suppressing toxicity to normal cells, and is known to be effective in purulent diseases such as sinusitis, otitis media and boils.

According to the present invention, a serine protease inhibitor having a molecular weight of 7806.5, 7919.8, and 8163.4 is degreased by grinding soybean seeds, and then extracted by stirring with deionized pure water, extracting total protein, and then concentrated by saturated ammonium sulfate solution, followed by gel filtration. It can be obtained by separating by anion exchange resin or reverse phase column chromatography the part which shows trypsin and chymotrypsin inhibitory activity. At this time, the deionized water is kept stirring for 10 to 30 hours while maintaining the temperature of 2 to 5 ℃ under weakly acidic conditions to maintain the extraction conditions. Separation by gel filtration, anion exchange resin, and reverse phase chromatography yields a fraction with high inhibitory activity against trypsin and chymotrypsin.

Hereinafter, the present invention will be described in more detail based on Examples and Experimental Examples, but the technical scope of the present invention is not limited to these Examples.

In the examples, trypsin and chymotrypsin activity measurements were similar to each other and were performed in the following manner. A suitable amount of trypsin and chymotrypsin is dissolved in 20 mM calcium chloride-containing 1 mM hydrochloric acid to prepare an enzyme solution having a concentration of 1 mg / ml. Separately, 0.5 mM benzoyl-L-tyrosine-p-nitroanilide solution for trypsin and chymotrypsin inhibition experiments was prepared using 50 mM-tris hydrochloric acid buffer (pH 7.5) and used as a substrate solution. 15 µl of the above enzyme solution is added to 10 µl of the sample aqueous solution, followed by reaction at 37 ° C for 10 minutes. In addition, after adding 150 µl of the substrate solution, the reaction was performed under the same conditions for 30 minutes, and then 50 µl of 30% acetic acid was added to stop the reaction. All reactions were performed in 96well microplates and finally absorbance was measured using a microplate reader at 415 nm.

Example

The soybean seed was ground, degreased with petroleum ether and dried to obtain a skim powder. 500 ml of deionized pure water was added to 100 g of the powder, adjusted to pH 4.0 with hydrochloric acid, and stirred at 4 ° C. overnight to extract total protein. 450 g of ammonium sulfate powder was added to 500 ml of the extract, and the mixture was stirred well to precipitate and recover total protein.

The precipitate of total protein obtained by the above method was dissolved in a small amount of deionized pure water, and treated with deionized pure water and phosphate buffer (pH 7.1) containing 0.2M sodium chloride for 24 hours, respectively, and then sent to Centriplus. Plus Amicon Co., Ltd.). This concentrate was passed through the prepared HiLoad 26/60 Superdex 200 column (Amasham, Palmacia, 26 x 600 mm) at a rate of 2 ml / min and again the phosphate buffer indicated above in the elution buffer. Gel filtration chromatography was performed by eluting with. The dissolution pattern obtained by this method is shown in FIG. 1. Trypsin and chymotrypsin activity were measured for each of the obtained fractions, and activity was recognized in the 60-67th fraction (in vitro), and the fraction was recovered.

This fraction was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) for 24 hours and concentrated to 10 ml using Centri Plus. 5 ml of the concentrate was passed through a UNO-Q1 column (7 x 35 mm), leveled with Tris-HCl buffer at a flow rate of 1 ml / min, followed by elution of 50 mM Tris-HCl buffer containing 50 mM and 0.5 M sodium chloride. Anion exchange chromatography was performed with buffer. As a result, an elution pattern as shown in Fig. 2 was obtained. At this time, the elution was performed by linearly increasing the concentration of 0.5 mM sodium chloride-containing 50 mM Tris-HCl buffer as indicated by the dotted line (gradient concentration method, gradient method). For each fraction, trypsin and chymotrypsin activity was measured to recognize activity in fractions 45-48. These fractions were recovered and purified again using reverse phase high performance liquid chromatography (reverse phase HPLC).

These fractions were dried with a centrifugal evaporator (vacuum concentrator), dissolved in acetic acid (TFA) with 0.1% triflow, and then 10 µl each of which was leveled with acetic acid with 0.1% triflow, respectively. A solution of 80% acetonitrile mixed with 20% acetic acid in 0.1% triflow and 20% acetic acid in 0.1% triflow was passed through a 120-mm column (4.6x150mm, manufactured by Waters). Reverse phase high performance liquid chromatography was performed. The result is shown in FIG. At this time, elution was performed by linearly increasing the concentration of the mixture of trifluoroacetic acid and acetonitrile as indicated by the dotted line in FIG. 3. Thus, as shown by an asterisk in FIG. 3, A was obtained from the mixed fraction of 45 and 46 fractions, B was 47 from the fraction, and C was trypsin and the chymotrypsin inhibitor from the 48 fractions. The molecular weight of these trypsin and chymotrypsin inhibitors was measured by TOF-MS, and A was 7806.5, B was 7919.8, and C was 8163.4.

Experimental Example

Trypsin and chymotrypsin inhibitory activity was evaluated using l-BAPA and l-BTPA as substrates, respectively. The molecular weight of the inhibitor was measured using the Voyager-KP matrix assisted laser desorption and ionization time of the flight mass spectrometer (MALDI-TOF-MS, PerSeptive Biosystemes). In this analysis, α-cyano-4-hydroxyninnamic acid was used as the matrix. Pyridylethylation of inhibitors with 4-vinylpyridine was performed by the methods of Cavins and Friedman (Cavins, JF and Friedman, M., Anal. Biochem., 35, 489-493 (1970)), pyridylethyl Fire inhibitors were purified by RP-HPLC using a μBondasphere C4 column (3.9 x 300 mm, water). The protein was eluted with a linear gradient of acetonitrile in aqueous trifluoroacetic acid (TFA), and the eluate was retrieved by ultraviolet absorption at 220 nm. Proteolysis limited to trypsin and chymotrypsin was performed for 24 hours under acidic conditions with formate buffer (pH 3.5), where the enzyme to substrate weight ratio was 1:50. Lyylene dopeptidase digestion was performed as follows. The protein was reduced, alkylated and then digested with 0.2 M N-methylmorpholine acetate buffer (pH 8.1) at 37 ° C. for 6 hours. To define disulfide bonds, proteins were dissolved in 0.2M N-methyl-morpholine acetate buffer (pH 8.1) containing 2M urea and digested with trypsin (enzyme / substrate, 1:50) for 12 hours at 37 ° C. . Peptides derived from enzymatic digestion were separated by RP-HPLC on a Puresyl C18 column (4.6x150 nm, water) using an acetonitrile gradient in aqueous TFA as described above.

The amino acid composition was analyzed as follows. Proteins and peptides were hydrolyzed in a vacuum sealed tube at 5.7 N HCl containing 0.02% 2-mercaptoethanol for 24 hours at 110 ° C. Amino acids were analyzed by L8500A system amino acid analyzer. Amino acid sequences were analyzed by model 476A protein / peptide sequencer (Applied Biosystms). The C-terminal amino acid sequence was analyzed by carboxypeptidase Y (CPase Y) using Secuazime C-peptide sequencing kit (Perseptive Biosystems) followed by peptide analysis by MALDI-TOF-MS.

All experimental procedures were performed at 4 ° C. The total protein was extracted from seed (200 g) of the migraine with vigorous stirring for 24 hours at different pHs (4, 7 and 11) and the supernatant was separated by centrifugation for 20 minutes at 13,000 rpm. Proteins were concentrated for 24 hours by salting technique using ammonium sulfate. The precipitate was collected by centrifugation and then dialyzed against 10 nM phosphate buffer (pH 7.5). The protein solution was concentrated and placed in a Hirod 26/60 column (Pharmacia Biotech) equilibrated with the same buffer on Biologic Fast Protein Liquid Chromatography (FPLC, BioRad). Fractions containing trypsin inhibitory activity were further purified by anion cross chromatography on an Uno Q-1 column (BioRad) previously equilibrated with 10 mM Tris-HCl buffer (pH 8.0) and then programmed in FPLC in buffer. Elution was by linear gradient of NaCl from 0 to 0.3 M. Final purification was done by model 600E reverse phase high pressure liquid chromatography using Puresil C18 column (4.6 × 150 mm, water). The major forms of serine protease inhibitors extracted from peas at different pHs (4, 7 and 11) were homogeneously purified using a series of chromatographic steps. Purification proceeded with a gradient of trypsin and chymotrypsin in inhibitory activity. As a result, at least three types of inhibitors were found in the soybeans by anion exchange chromatography of the inhibitor fraction. That is, three inhibitors SBI-1, -2 and -3 having molecular weights of 7806.5, 7919.8 and 8163.4, respectively, were obtained by RP-HPLC (see FIG. 4). Inhibitors SBI-1, -2 and -3 analyzed by MALDI-TOF-MS in Figure 4 showed a single peak under reducing and non-reducing conditions, indicating that the purified protease inhibitor is present as monomer. In addition, the amino acid sequence is determined by the amino acid sequencer from Figure 4 it can be seen that A is composed of 71 amino acids, 73 B, 75 C. The entire amino acid sequence of SBI-1, -2 and -3 was obtained by automated sequencing analysis of the enzymatically cleaved protein (see Figure 5). These results confirm the amino acid composition of protein or peptide and C-terminal sequence analysis by Cpase Y digestion.

According to the method of the present invention, a serine protease inhibitor having a high molecular weight of 7806.5, 7919.9, and 8163.4 having a trypsin and chymotrypsin inhibitory activity can be obtained from the soybean seed.

Figure 1 shows the elution pattern of gel bean seed extract by gel filtration chromatography.

2 shows an anion exchange chromatogram of active fractions obtained from gel filtration chromatography.

Figure 3 shows a reverse phase HPLC elution pattern of the active fractions obtained by anion exchange chromatography.

Figure 4 shows the molecular weight range measured by TOF-MS of inhibitors obtained from reversed phase HPLC.

Figure 5 shows the amino acid sequence of protease inhibitors obtained from seeds of soybeans.

Claims (2)

Grind and degreas the seeds of Canavalia gladiata, try to extract the total protein by stirring with deionized water, concentrate with saturated ammonium sulfate solution, isolate by gel filtration, anion exchange resin or reverse phase column chromatography, trypsin And a serine protease inhibitor having a molecular weight of 7806.5, 7919.9, and 8163.4 having chymotrypsin inhibitory activity. The method of claim 1, The deionized water is a method for obtaining a serine-based protease inhibitor from soybeans, characterized in that the stirring is maintained for 10 to 30 hours while maintaining the temperature of 2 to 5 ℃ under weakly acidic conditions and maintaining the extraction conditions.