JP4902140B2 - Protein having α-amylase inhibitory activity - Google Patents

Description

  The present invention relates to a novel protein derived from rye bran and having α-amylase inhibitory activity.

  In recent years, diabetes and obesity have been increasing steadily in Japan due to westernization and satiety of eating habits. Furthermore, the significant postprandial blood glucose level seen in diabetics or their preparatory group is associated with serious complications such as hyperlipidemia, arteriosclerosis and myocardial infarction. In view of these circumstances, the importance of blood glucose control has been recognized, but strict dietary restrictions and continuous exercise are not always easy in modern day-to-day life. A highly safe substance is desired.

  α-Amylase is an enzyme that breaks down starch into sugar and is contained in saliva and pancreatic juice in humans. It is known that the increase in blood glucose level after ingestion of starch can be delayed by inhibiting the activity of this α-amylase (Patent Document 1, Patent Document 2). So far, α-amylase inhibitors have been isolated from many plants including wheat, barley, rye, rice and kidney beans (Non-patent Document 1).

JP 07-41499 A JP 09-194392 A Eur. J. et al. Biochem. 269, 397-412, (2002)

  However, conventional α-amylase inhibitors have different specificities for various α-amylases such as bacteria, insects and animals, and α-amylase having strong inhibitory activity against α-amylase derived from human saliva and human pancreas. There is a need for amylase inhibitors.

  In this situation, an object of the present invention is to provide a substance having an inhibitory activity against α-amylase derived from human saliva and human pancreas.

  As a result of earnest search for α-amylase inhibitors that can be used as safe materials derived from natural products, the present inventors have searched for α-amylase inhibitors from rye bran, and have proteinaceous inhibitors clearly different from conventional ones. Was isolated. The N-terminal amino acid sequence of this protein was determined, and it was confirmed that the protein was novel by analysis using a database. The present invention was completed by naming it RBAI.

That is, the protein having α-amylase inhibitory activity according to the present invention is characterized by the following properties.
1) It shows a molecular weight of 12.0 kDa by SDS polyacrylamide electrophoresis and 23.2 kDa by gel filtration chromatography.
2) The subunit has the following N-terminal amino acid sequence:
SGPWMCYPGQAFQVPALPNM
(Wherein S is serine, G is glycine, P is proline, W is tryptophan, M is methionine, C is cysteine, Y is tyrosine, Q is glutamine, A is alanine, F is phenylalanine, V is valine, L is leucine and N is asparagine.)
3) 1 mol of the dimer of the subunit reacts with 2 mol of α-amylase, and competitively inhibits the activities of porcine pancreas, human saliva and α-amylase derived from human pancreas.

  In addition, the method for producing a protein having an α-amylase inhibitory activity according to the present invention obtains a rye bran water extract from the supernatant of rye bran suspension water, fractionates this rye bran water extract and recovers the recovered precipitate. After obtaining an ammonium sulfate precipitate fraction solution from the supernatant of the aqueous solution of the product, the active fraction solution of the protein having α-amylase inhibitory activity is separated from the ammonium sulfate precipitate fraction solution by ion exchange chromatography, A protein having an α-amylase inhibitory activity is purified by fractionating an active fraction solution of a protein having an α-amylase inhibitory activity from the active fraction solution by action chromatography.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited by these.

Purification of RBAI The protein amount in each purification step was determined by the Lowry method using bovine serum albumin as a standard substance. Moreover, the inhibitory activity of each chromatographic fraction was determined according to the method of Oneda et al. (J. Biochem. 135, 421-427 (2004)), porcine pancreatic α-purified from pancreatin (manufactured by Wako Pure Chemical Industries, Ltd.). Evaluation by inhibition amount of degradation activity of amylase isomer I (hereinafter referred to as PPA) by p-nitrophenyl-α-D-maltoside (p-nitrophenyl-α-D-maltoside; manufactured by Wako Pure Chemical Industries, Ltd., hereinafter referred to as pNP-G2) did.

The enzyme reaction was performed by adding 50 μL of PPA solution and 50 μL of chromatographic fraction solution to 50 mM Tris-HCl buffer (pH 6.9) containing 750 μL of 1.0 mM CaCl ₂ and 30 mM NaCl. After incubation at ° C for 3 minutes, 50 μL of pNP-G2 was added to start. The amount of p-nitrophenol (hereinafter referred to as pNP) produced by the PNP-G2 hydrolysis reaction with PPA was continuously monitored using the increase in absorbance at 400 nm as an index, and the molecular extinction coefficient of pNP at 400 nm was 9, The increase rate was calculated using 470M ⁻¹ cm ⁻¹ (J. Biochem. 110, 605-607 (1991)), and this was used as the enzyme activity. The measurement conditions were an initial concentration of PPA of 1.17 μM (FIG. 1) and 0.513 μM (FIG. 2), an initial concentration of pNP-G2 of 1.67 mM, and the enzyme reaction was performed at 30 ° C. using a thermostatic cell holder. .

  35 g of rye bran was suspended in 300 mL of distilled water and stirred at room temperature for 1 hour. Subsequently, the precipitate was removed by centrifugation at 3,500 g × 10 minutes, and distilled water was added to the resulting supernatant to 350 mL, which was used as a rye bran water extract (854 mg protein). The fraction of ammonium sulfate saturation 20-40% of the rye bran water extract was collected by centrifugation at 3,500 g × 10 minutes and dissolved in 35 mL of distilled water. This solution was dialyzed against 2 L of distilled water for 12 hours, and further dialyzed against 50 mM acetate buffer (pH 5.0) for 12 hours. The dialyzed internal solution was centrifuged at 10,000 g × 5 minutes, and the same buffer was added to the resulting supernatant to make 40 mL, which was used as a 20-40% ammonium sulfate precipitation fraction solution (240 mg protein).

  The 20-40% ammonium sulfate precipitation fraction solution was fractionated using a cation exchange HPLC column, TSKgel SP-5PW [7.5 mm (ID) × 7.5 cm; manufactured by Tosoh Corporation]. The HPLC conditions are as follows: eluent 50 mM acetate buffer (pH 5.0), flow rate 1.0 mL / min, sample injection amount 3.0 mL, detection wavelength 280 nm, preparative amount 2.0 mL / fraction. Thus, the adsorbed protein was separated with a linear gradient in which the NaCl concentration increased from 0 to 0.5M. Inhibitory activity was found in the fraction eluted at 22-26 minutes, and this fraction was collected (FIG. 1). This operation was repeated to obtain 52 mL of active fraction solution from 40 mL of 20-40% ammonium sulfate precipitation fraction solution. This was designated as an SP fraction solution (32.1 mg protein).

  Subsequently, the SP fraction solution was fractionated using a hydrophobic interaction HPLC column, TKSgel Phenyl-5PW [7.5 mm (ID) × 7.5 cm; manufactured by Tosoh Corporation]. A sample solution was prepared by mixing 52 mL of the SP fraction solution with an equal amount of 1M ammonium sulfate. The HPLC conditions were 50 mM acetate buffer (pH 5.0) containing 0.5 M ammonium sulfate as eluent, flow rate 1.0 mL / min, sample injection amount 3.0 mL, detection wavelength 280 nm, fractionation amount 2.0 mL / fraction. Yes, as shown in the figure, the adsorbed protein was separated with a linear gradient in which the ammonium sulfate concentration decreased from 0.5 to 0M. Activity inhibition was observed in the 38-41 minute elution fraction, and the 39-40 minute elution fraction was collected (FIG. 2). This operation was repeated to obtain 35 ml of active fraction solution from 52 ml of SP fraction solution. This was used as the final purified sample (6.65 mg protein).

Properties of RBAI Inject 500 μL of this purified sample into a gel filtration HPLC column, TSKgel G2000SW _XL [7.8 mm (ID) × 30 cm; manufactured by Tosoh Corporation] previously equilibrated with 50 mM acetate buffer (pH 5.0). When analyzed at a flow rate of 1.0 mL, this sample eluted at a molecular weight of 23.2 kDa (FIG. 3), and the molecular weight was confirmed to be 23.2 kDa by gel filtration chromatography.

  Further, the RBAI purified sample together with the sample at each purification stage was analyzed by SDS polyacrylamide electrophoresis (SDS-PAGE) (PAG Mini “Daiichi” 15/25; manufactured by Daiichi Chemicals) under non-reducing conditions. . Electrophoresis was performed at a constant current of 40 mA, and the purified sample showed a single band at a molecular weight of 12.0 kDa (FIG. 4). From these results, it was found that RBAI exists as a dimer composed of subunits having a molecular weight of about 12.0 kDa.

  RBAI was reduced S-alkylated, and the N-terminal amino acid sequence was analyzed using a protein sequencer (G1000A; manufactured by Hewlett Packard). , M is methionine, C is cysteine, Y is tyrosine, Q is glutamine, A is alanine, F is phenylalanine, V is valine, L is leucine, and N is asparagine. It was. When this sequence was searched for homology with a known protein using SWISS-PLOT, which is a protein amino acid sequence database, it was found that RBAI was a novel substance.

Inhibitory properties of RBAI α-amylase activity (1)
The inhibitory effect of the purified RBAI preparation on PPA and human salivary α-amylase (hereinafter referred to as HSA) activity was examined. Since HSA contains a small amount of isozyme, HSA (Type XIII-A crude) manufactured by SIGMA is used as a hydrophobic interaction HPLC column, TSKgel Ether-5PW [7.5 mm (ID) × 7.5 cm; manufactured by Tosoh Corporation ] Was used for the experiment.

  The HPLC conditions were 50 mM 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid {2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic containing 0.5 M ammonium sulfate as an eluent. acid; (HEPES)} buffer (pH 6.9), flow rate 1.0 mL / min, 1.0 mg / mL crude HSA (manufactured by SIGMA) containing 0.5 M sample ammonium sulfate, sample injection amount 500 μL, detection wavelength At 280 nm, 10 to 35 minutes after sample injection, the adsorbed protein was separated with a linear gradient in which the ammonium sulfate concentration decreased from 0.5 M to 0 M. Under this separation condition, the main component of HSA was eluted from 17.3 minutes to 18.0 minutes, and this fraction was fractionated to obtain a purified HSA sample.

The effect of RBAI on pNP-G2 degradation activity of PPA and HSA was examined kinetically under conditions of initial enzyme concentration ([E] _O ) >> inhibitor constant (K _i ).

Enzymatic reaction was performed by adding 10 μL of RBAI solution and 50 μL of PPA or HSA solution to 50 mM Tris-HCl buffer (pH 6.9) containing 700 μL of 1.0 mM CaCl ₂ and 30 mM NaCl. After incubating at C for 3 minutes, 40 μL of pNP-G2 solution was added and started. Measurement conditions were an initial concentration of PPA and HSA of 179 nM, an initial concentration of pNP-G2 of 4.0 mM, and the enzyme reaction was performed at 30 ° C. using a thermostatic cell holder.

This increase in A ₄₀₀ with increasing pNP produced by the reaction was calculated increase initial rate of continuously tracked to pNP was the enzyme activity was investigated the effect of adding RBAI on the enzymatic activity.

Calculating a K _i for each α- amylase RBAI approximates the RBAI concentration dependence of the data of PPA and HSA activity formula given in Equation 1, to evaluate the inhibitory activity with a K _i value (Fig. 5).

v: enzyme activity in the presence of RBAI, v _O : enzyme activity in the absence of RBAI, [E] _O : initial enzyme concentration, [S] _O : initial substrate concentration, [I ₂ ] _O : RBAI (dimer) Initial concentration, K _m : Michaelis constant.

As a result, the equivalence point of α-amylase and RBAI is α-amylase: RBAI (dimer) = 2: 1 (mole), and two amylases bind to one molecule of RBAI dimer. Indicated. pH6.9,30 K _i for PPA of RBAI in ° C is 17.7 ± 1.1nM, K _i for HSA is sought respectively 893 ± 228pM, RBAI is very strong inhibition against these α- amylase It was shown to have activity.

Inhibitory properties of RBAI α-amylase activity (2)
P-nitrophenyl-α-D-maltopentaoside of PPA, HSA and human pancreatic α-amylase (CALZYME Laboratories, hereinafter referred to as HPA) (Wako Pure Chemical Industries, Ltd.) (Hereinafter referred to as pNP-G5) The effect of RBAI on the degradation activity was examined kinetically under the condition [E] _O << K _i . An example of measuring the pNP-G5 degradation activity of α-amylase is shown below. 10 μL of α-amylase solution and 10 μL of RBAI solution were mixed with 50 μM Tris-HCl (Tris-HCl) buffer (pH 6.9) containing 2000 μL of 1.0 mM CaCl ₂ and 30 mM NaCl, and set to 30 ° C. After treatment for 30 minutes in a constant temperature water bath, 100 μL of a pNP-G5 solution was added to initiate the reaction.

  After a certain time, 200 μL of the reaction solution was mixed with 50 μL of 5% trifluoroacetic acid (hereinafter referred to as TFA) to stop the reaction, and reverse phase HPLC column, CAPCELL PAK C8UG 120 [4.6 mm (ID) × The reaction product was separated and quantified using 25 cm; The HPLC conditions were as follows: eluent 0.1% TFA, flow rate 1.0 mL / min, sample injection volume 200 μL, detection wavelength 313 nm, linear gradient in which acetonitrile concentration increased from 5% to 50% 5 to 20 minutes after sample injection. The sample adsorbed with was separated. The degradation products of pNP-G5 by the α-amylase examined are mainly p-nitrophenyl-α-D-maltoside and p-nitrophenyl-α-D-maltotrioside ( p-nitrophenyl-α-D-maltotrioside), and the initial rate of increase was defined as the enzyme activity.

Substrate concentration dependence of pNP-G5 degradation activity of PPA was examined in the presence of RBAI and analyzed using Hanes-Woolf plot. As a result, inhibition of α-amylase activity by RBAI was found to be an antagonistic type caused by an increase in Michaelis constant. It was discriminated (FIG. 6). In addition, the dependency of RBAI concentration was studied with constant PPA and substrate concentrations. Was calculated a K _i for the PPA RBAI measurement data by approximating the equation represented by the number 2 (Fig. 7).

v: enzyme activity in the presence of RBAI, v _O : enzyme activity in the absence of RBAI, [S] _O : substrate initial concentration, [I ₂ ] _O : RBAI (dimer) initial concentration, K _m : Michaelis constant, It is.

As a result, K _i for PPA of RBAI in pH6.9,30 ° C was determined to 17.4 ± 0.5 nM. Similarly, K _i for HSA was 1.01 ± 0.06 nM (FIG. 8), and K _i for HPA was 18.2 ± 0.8 nM (FIG. 9).

  The novel protein RBAI of the present invention has a strong inhibitory activity against the activity of porcine pancreas, human saliva, and α-amylase derived from human pancreas. By containing RBAI as an amylase inhibitor, an inhibitor of blood glucose level, and an active ingredient in foods, prevention and treatment of diseases considered to be caused by excessive nutrition such as diabetes, obesity, hyperglycemia, and arteriosclerosis It is effective for.

In Example 1, it is a figure which shows the fraction of RBAI by a cation exchange column. In Example 1, it is a figure which shows the fraction of RBAI by a hydrophobic interaction column. In Example 1, it is a figure which shows the elution pattern of the RBAI refinement | purification sample by a gel filtration column. In Example 1, it is a figure which shows the migration pattern in SDS-PAGE of each refinement | purification stage sample and an RBAI refinement | purification sample. In Example 2, it is a figure which shows the addition effect of RBAI with respect to pNP-G2 degradation activity of PPA and HSA. In Example 3, it is a figure which shows the addition effect of RBAI with respect to the substrate concentration dependence of pNP-G5 degradation activity of PPA. In Example 3, it is a figure which shows the addition effect of RBAI with respect to pNP-G5 degradation activity of PPA. In Example 3, it is a figure which shows the addition effect of RBAI with respect to the pNP-G5 degradation activity of HSA. In Example 3, it is a figure which shows the addition effect of RBAI with respect to pNP-G5 degradation activity of HPA.

Claims (2)

A protein derived from rye bran having α-amylase inhibitory activity, characterized by exhibiting the following properties.
1) It shows a molecular weight of 12.0 kDa by SDS polyacrylamide electrophoresis and 23.2 kDa by gel filtration chromatography.
2) The subunit has the following N-terminal amino acid sequence:
SGPWMCYPGQAFQVPALPNM
(Wherein S is serine, G is glycine, P is proline, W is tryptophan, M is methionine, C is cysteine, Y is tyrosine, Q is glutamine, A is alanine, F is phenylalanine, V is valine, L is leucine and N is asparagine.)
3) 1 mol of the dimer of the subunit reacts with 2 mol of α-amylase, and antagonistically inhibits the activities of porcine pancreas, human saliva and α-amylase derived from human pancreas. Rye bran water extract is obtained from the supernatant of rye bran suspension water, this rye bran water extract is subjected to ammonium sulfate fractionation, and ammonium sulfate precipitation fraction solution is obtained from the supernatant of the recovered precipitate aqueous solution, followed by ion exchange. Chromatographic separation of the active fraction solution of the protein having α-amylase inhibitory activity from the ammonium sulfate precipitate fraction solution, and then the activity of the protein having α-amylase inhibitory activity from the active fraction solution by hydrophobic interaction chromatography The method for producing a protein derived from rye bran having an α-amylase inhibitory activity according to claim 1, wherein a protein having an α-amylase inhibitory activity is purified by fractionating a fraction solution. .

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