JPH042600B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel amylase inhibitor T-76 obtained from Streptomyces bacteria and a method for producing the same. (Prior Art) Research on amylase inhibitors has been conducted for a long time, and several peptide-based and oligosaccharide-based amylase inhibitors derived from plants, microorganisms, etc. are known. As a plant-derived amylase inhibitor, a heat-stable basic protein obtained from wheat is particularly famous. As amylase inhibitors derived from microorganisms, Ueda et al.'s peptide-like substances (Agric.Biol.
Chem., 37 , 2025 (1973)), Haim of Murao et al.
(Agric.Biol.Chem., 44 , 1679 (1980)) and
Paim (Agric.Biol.Chem., 47 , 453 (1983)), Goto et al.'s X-2 (Special Publication No. 54-11395), Harada et al.'s AI-B (Special Publication No. 59-10193) , I-1001C by Miyagawa et al. (Japanese Unexamined Patent Publication No. 61-74587) and Volker Eeding et al. (Japanese Patent Publication No. 62-36040). By administering an amylase inhibitor, amylase activity is inhibited in the body, such as in the digestive tract and oral cavity. Therefore, it can be effectively used as a therapeutic agent for hyperlipidemia, diabetes, etc., or as a preventive agent for dental caries. It is also useful as a reagent for various analyzes for clinical diagnosis. For example, body fluid components in human blood or urine are usually measured using amylase derived from microorganisms, or at this time, human amylase originally contained in the body fluid acts to correct the measurement results. gives an error of Therefore, it is used to prevent such errors by inhibiting the target activity of human-derived amylase. The amylase inhibitor used in this way is an inhibitor that selectively deactivates amylase derived from humans and other higher animals, but does not deactivate amylase derived from bacteria or fungi. Among conventionally known inhibitors, inhibitors that selectively inactivate amylases derived from humans and other higher animals, but do not inactivate amylases derived from bacteria and fungi, are reported by Murao et al. (cited above), Goto et al. (supra), Miyagawa et al. (supra), and Volker-Edeing et al. (supra). However, the inhibitors of Murao et al. (supra) and Goto et al. (supra) have low productivity by microorganisms and are not practical. Miyagawa et al. (cited above) also found Streptomyces bacteria that produced inhibitory substances at high rates.
amylase inhibitor I-1001C, which contains pepsin, trypsin, alpha
- Inactivated by the following proteases: chymotrypsin, thermolysin, papain and fuicin. On the other hand, the amylase inhibitors of Volker Eeding et al. (cited above) are relatively less susceptible to the action of proteases, but they are also gradually inactivated by proteolysis. Thus, known amylase inhibitors have problems in their productivity and stability. Amylase inhibitors that have the desired specificity of action, are active and stable over a wide temperature and PH range, and are not inactivated by prothiases and can therefore be used for various prophylactic or analytical purposes; It is hoped that a method for effectively manufacturing the product will be developed. (Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and its purpose is to selectively inactivate amylase derived from higher animals and to inactivate amylase derived from bacteria. The object of the present invention is to provide an amylase inhibitor that has the specificity of action of not causing oxidation, is active and stable in a wide temperature range and PH range, and is not inactivated by various proteases. Another object of the present invention is to provide a method for effectively producing the above-mentioned excellent amylase inhibitor. (Means for Solving the Problems) The amylase inhibitor T-76 of the present invention has the following properties. Action: Inhibits human pancreatic amylase, human salivary amylase, and porcine pancreatic amylase;
Stable pH that does not inhibit α-amylase from Bacillus, β-amylase from soybeans, β-amylase from barley, glucoamylase from Rhizopus, isoamylase from Cydomonas, pepsin, trypsin, and α-chymotrypsin. Range and thermal stability: 30℃ at PH3-12
Stable for 24 hours at pH 3-9
Molecular weight: Molecular weight determined by SDS-polyacrylamide gel electrophoresis is approximately 8000. Isoelectric point: The isoelectric point using ampholine as a carrier is 3.8. Maximum absorption is 278nm
and has the following amino acid composition, with minimum absorption at 250 nm:

【table】

[Table] The method for producing the amylase inhibitor T-76 of the present invention includes the steps of culturing a microorganism belonging to the genus Streptomyces that can produce the above-mentioned inhibitor, and collecting the inhibitor from the resulting culture. The above object is achieved. The amylase inhibitor T-76 of the present invention is produced by Streptomyces, particularly Streptomyces nitrosporeus strain T-76 (FERMP-9882). produced. This strain is a new strain isolated from soil by the inventors. The mycological properties of this strain are shown below. The culture temperature was 30°C unless otherwise specified. 1. Morphology The branches of spore-forming hyphae are straight or curved simple branches, forming spores with 10 or more spores chained together. The spore surface is smooth, the size of the spore is (0.6-0.8) x (0.7-1.0) μm, and the formation of flagellated spores and sporangia is not observed. 2 Growth status on various media (1) Growth on seuucrose/nitrate agar medium: poor, colorless aerial hyphae: abundant, gray soluble pigment: not observed (2) Growth on glucose/asparagine agar medium: poor, orange-yellow to grayish Aerial hyphae: Abundant, white to grayish soluble pigment: Not observed (3) Growth on glycerin/asparagine agar medium: Poor, dark orange-yellow Aerial hyphae: Abundant, gray soluble pigment: Not observed (4) Growth on starch/inorganic salt agar medium: poor, dark orange-yellow to brown aerial hyphae: abundant, light gray soluble pigment: not observed (5) Growth on tyrosine agar medium: poor, brown aerial hyphae: thick, light gray soluble pigment: observed (6) Growth on nutrient agar medium: poor, grayish yellow aerial hyphae: abundant, white soluble pigment: not observed (7) Growth on yeast/malt agar medium: good, orange-yellow aerial hyphae: few, white; Wrinkled soluble pigment: Not observed (8) Oatmeal agar medium Growth: Good, orange-yellow to brown Aerial mycelia: Abundant, white to gray Soluble pigment: Not observed 3 Physiological properties (1) Growth temperature range: 16℃ It grows at ~40℃, and the optimum growth temperature is around 30℃. (2) Liquefaction of gelatin: Positive (glucose-peptone-gelatin medium and simple gelatin medium) (3) Hydrolysis of starch: Positive (starch agar medium) (4) Coagulation of skim milk: Negative (5) Peptone in skim milk Formation: Positive (6) Melanin-like pigment production: Negative (Peptone,
Yeast-iron agar, tryptone-yeast broth agar, and tyrosine agar) (7) Nitrate reduction: Positive 4 Carbon source assimilation (Pridham-Gottlieb agar) D-glucose, D-xylose, L-arabinose , and D-mannose; L-rhamnose, D-fructose, D-
Galactose, and salicin are utilized sparingly; raffinose, D-mannitol, inositol, and sucrose are not utilized. Identification of bacterial strain The strain that produces the amylase inhibitor T-76 of the present invention was identified based on the above-mentioned mycological properties as described in Bergey's Manual of Determinative Bacteriology, 8th edition (1974).
of Determinative Bacteriology, 8th, Edd.
(1974)]; International Streptomyces progiect [International
Report of the Strepomyces Project (ISP) [International Journal of Systematic Bacteriology]
of Systematic Bacteriology) Vol. 18, pp. 69-189 (1968), Vol. 18, pp. 279-392 (1968), Vol. 19
391-512 (1969) and Vol. 22, pp. 265-394 (1972)]; and Waksman, "The Actinomycetes", Volume 2, this strain is as follows: Similar to Streptomyces nitrosporeus except that Streptomyces nitrosporeus (1) coagulates skimmed milk, (2) assimilates L-rhamnose well, and ( 3) Do not assimilate D-fructose. However, since it matches Streptomyces nitrosporeus in other respects, the strain of the present invention is considered to be a new strain belonging to Streptomyces nitrosporeus.
The present inventors named this strain Streptomyces nitrosporeus T-76 strain. Culture Conditions The medium for the above bacterial strain does not need to be special, and a normal medium can be used. As the carbon source, starch, glycerin, glucose, sucrose, etc. are used. As nitrogen sources, polypeptone, meat extract, soybean protein, general amino acids, etc. are used. Inorganic salts include common salt (NaCl), monopotassium phosphate, dipotassium phosphate,
Magnesium sulfate and the like are used. In addition, micronutrients such as vitamins may be added as necessary. For example, a medium having the following composition is preferably used. Medium composition

[Table] Culture pH is 6-8, preferably 7.0, culture temperature is
The culture is carried out at 25-35°C, preferably 28°C, for about 2-7 days, preferably about 4 days, with aerobic stirring or shaking. The amylase inhibitor of the present invention is secreted outside the bacterial cells and can be recovered from the medium. Method for collecting inhibitory substance In order to collect and purify the amylase inhibitory substance of the present invention from the above-mentioned culture solution, known purification methods can be used alone or in combination. For example, the culture solution is filtered or centrifuged to remove bacterial cells to obtain a filtrate or supernatant. This filtrate or supernatant is concentrated and subjected to ultrafiltration or dialysis, if necessary. This is further salted out with ammonium sulfate, etc., and then dialyzed, and then various chromatographies such as ion exchange chromatography using anion exchange cellulose, anion exchange gel, and hydrophobic chromatography (for example, TEAE cellulose) are performed. , DEAE Toyopearl, Octylsepharose CL-4B, etc.) alone or in combination. An example of a purification method is shown below. (1) After removing the bacterial cells from the culture solution, salt out with ammonium sulfate (80% saturation), dissolve the resulting precipitate, and perform dialysis. This is subjected to (2) TEAE cellulose column chromatography (PH6.0, 0-1.25M NaCl concentration gradient), followed by dialysis. Next, (3) dialysis is performed after subjecting it to Octyl Sepharose CL-4B column chromatography ((PH6.0; 1.5M NaCl)). (4) The dialyzed solution is subjected to DEAE Toyopearl column chromatography (PH6.0; 1.5M NaCl). (2) 0-0.3M NaCl concentration gradient) and dialysis. (5) Freeze-dry to obtain a purified sample. The amylase inhibitor purified by this method is subjected to disk electrophoresis and SDS-polyacrylamide gel analysis. Gel electrophoresis shows a single band.The properties of the amylase inhibitors shown below were investigated using the purified preparations obtained in this way.Amylase inhibitory activity measurement methodThe amylase activity used as a control was as follows. It is measured as follows: 0.2 ml of purified water is added to 0.2 ml of an enzyme solution containing approximately 2 units of porcine pancreatic amylase, and pre-incubation is performed at 37°C for 5 minutes.Then, this solution contains 1.5% soluble Add 0.8 ml of starch solution (0.1M Tris-HCl buffer solution; PH7.0) and react at 37°C for 10 minutes.To this reaction solution, add 0.5N acetic acid (CH ₃ COOH) and 0.5N hydrochloric acid (HCl). The amylase reaction is stopped by adding 2 ml of a 5:1 mixture of
Add 5 ml of a solution containing 0.005% iodine and 0.05% potassium iodide, shake and leave at room temperature for 20 minutes. The absorbance of this solution at 660 nm is measured, and this absorbance is designated as C. Separately, a reaction system was prepared as a blank using purified water instead of the above amylase enzyme station, and the absorbance obtained by performing the same operation was designated as B. From the absorbances C and B thus obtained, amylase activity A is calculated using the following formula (1). When the calculated value of A is 0.35, it is defined as one unit of amylase activity. A=(B-C)/B...() Therefore, if the amylase activity in the absence of any inhibitor is _A0 , then _A0 is measured in the same manner as the above amylase activity measurement, and the obtained absorbance is
When T ₀ , it is calculated using the following formula (). _A0 =(B- _T0 )/B...() The inhibitory activity of the amylase inhibitor is measured as follows. A reaction system is prepared using 0.2 ml of the inhibitor solution instead of 0.2 ml of purified water in the reaction system for amylase activity measurement, and the same operation is performed. Let the absorbance obtained by this operation be Ti. Assuming that the amylase activity in the presence of an inhibitor is Ai, Ai is calculated using the following formula (). Ai=(B-Ti)/B...() When the inhibition rate (%) when an inhibitor is present is I, I is calculated using the following formula (). I = (A ₀ - Ai) × 100 / A ₀ ... () If the amount of amylase inhibitor that inhibits 50% of 2 units of the above porcine pancreatic amylase activity is 1 inhibition unit (1U), the above formula ( )~()from,
The inhibitory activity of the amylase inhibitor is calculated using the following formula (). Inhibitory activity = (A ₀ /0.7) x (I/50) x dilution factor of inhibitor () Properties of inhibitor The physicochemical properties of the amylase inhibitor T-76 of the present invention are shown below. Effect Various amylases and proteases were used in place of porcine pancreatic amylase, and measurements were performed according to the amylase inhibitory activity assay method. The reaction conditions were conditions suitable for each amylase and protease reaction. As a result, it inhibited human pancreatic amylase, human salivary amylase, porcine pancreatic amylase, feline pancreatic amylase, horse pancreatic amylase, rat pancreatic amylase, sheep pancreatic amylase, goat pancreatic amylase, and carp pancreatic amylase, snake pancreatic amylase, rabbit pancreatic amylase. , dog pancreatic amylase, guinea pig pancreatic amylase, α-amylase derived from Bacillus spp., β-amylase from soybean
It was found that it did not inhibit amylase, barley β-amylase, Rhizopus-derived glucoamylase, Pseudomonas-derived isoamylase, pepsin, trypsin, and α-chymotrypsin. From this, the inhibitor T-76 of the present invention is an inhibitor that selectively inhibits amylases derived from many higher animals such as humans and pigs, and does not inhibit amylases derived from plants, bacteria, and fungi. Furthermore, it is clear that it does not act on proteases. Stable PH range and thermostability This inhibitor T-76 was tested in 13 types with a PH range of 1 to 13.
After dissolving each in PH buffer and keeping at 30°C for 24 hours, PH was dissolved in 1M Tris-HCl buffer (PH
7.0) to adjust to a neutral range, and residual inhibitory activity was measured according to the above measurement method. The results are shown in FIG. Separately, after holding each pH at 100°C for 15 minutes, the pH was similarly adjusted to the neutral range, and the residual inhibitory activity was measured. The results are shown in FIG. 1 by broken lines. The buffer used was 50% at pH 1-3.
mM hydrochloric acid-sodium acetate buffer (○ mark in the figure),
For pH 4 to 6, 50mM acetate buffer (● mark in the figure),
50mM Tris-acetate buffer (marked with an x in the figure) for pH 7-8, 50mM glycine-sodium hydroxide buffer (marked △ in the figure) for pH9-11, and 50mM for pH12.
M sodium hydroxide-disodium hydrogen phosphate buffer (marked ▲ in the figure), and 250mM sodium hydroxide solution (marked □ in the figure) for PH13. From Figure 1, it can be seen that the stable PH range is PH 3 to 12 (100% activity remaining) at 30°C, and PH 3 to 9 (approximately 90% or more activity remaining) at 100°C. Inhibitor T-76 is stable even under heating conditions over a wide pH range, especially between pH 3 and 9.
It is extremely stable. Molecular weight Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gives a single band. The molecular weight determined by SDS-polyacrylamide gel electrophoresis is approximately 8000. Isoelectric point The isoelectric point measured using ampholine as a carrier is 3.8. Ultraviolet absorption spectrum Maximum absorption is 273nm, minimum absorption is 250n
It is m. A chart of this ultraviolet absorption spectrum is shown in FIG. Color reaction: Appears blue using the copper phorin method. Inhibitor Add the compound shown below to a solution (PH7.0) containing 100 units of this inhibitor T-76, and
After treatment for 60 minutes, residual inhibitory activity was measured according to the amylase inhibitory activity measurement method. As a result, the present inhibitor T-76 was inhibited by disodium ethylenediaminetetraacetate (10mM), p-chloromercurybenzoate (5mM), monoiodoacetic acid (10mM), o-phenanthroline (10mM), or 8-quinolinol (1mM).
It was found that the activity was not lost with M), and more than 70% of the activity remained with sodium dodecyl sulfate [1%, (treated at 100°C, 10 minutes)] or guanidine hydrochloride (4M). Thus, the present inhibitor T-76 is stable against various inhibitors. Inactivation conditions This inhibitor T-76 was added to 1N hydrochloric acid at 37℃ for 15 minutes.
When treated for 1 minute, the residual activity is 50%, and when treated for 15 minutes at 100°C with 1N hydrochloric acid or at a pH of 10 or higher, it is completely inactivated. 1%2-
At 100℃ in the presence of mercaptoethanol
It is completely inactivated after 15 minutes of treatment. Action of phlotease Add 20 PU of each of the following proteases to a solution containing 130 units of this inhibitor T-76, and add the optimal pH of each protease (pepsin has a pH of
2.0, trypsin and α-chymotrypsin
PH8.5) and 37°C for 210 minutes. this
Inactivate the protease by treating at 100℃ for 5 minutes, adjust the pH to around neutrality using 1M Tris-HCl buffer (PH7.0), and proceed according to the amylase inhibitory activity assay method. Amylase activity inhibition was measured. Inhibitor T-76, which was not subjected to the above protease treatment, was similarly measured and used as a standard. As a result, this inhibitor T-76 does not inactivate any of the proteases pepsin, trypsin, and α-chymotrypsin when acting alone, and sequentially inhibits these proteases (pepsin then trypsin; pepsin then trypsin; It was found that the enzyme was not inactivated even when it was acted on by α-chymotrypsin; and pepsin, then trypsin, and α-chymotrypsin (in that order). Thus, the present inhibitor T-76 is stable against various proteases. Amino acid composition The amino acid composition of the amylase inhibitor T-76 was determined by Spackman et al. (Anal.Chem., 30 , 1190).
(1958)) using a Hitachi 835 high-speed amino acid analyzer (Hitachi). Pretreatment of samples for the fast amino acid analyzer was performed as follows: the inhibitor T-76 was placed in an ampoule with redistilled 5.7N HCl, vacuum-sealed, and incubated at 110°C for 24, 48 days. and hydrolyzed by heating for 72 hours. Cystine, methionine and tryptophan are not measured by this method, so another method was adopted. That is,
Methionine, cystine (and cysteine) are
Schram's method (Biochem.J., 57 33 (1954)
as well as Moore's method (J.Biol.Chem., 238 , 235
(1963)), the mixture was oxidized with performic acid and then hydrolyzed, and quantified as methionine sulfonic acid and cysteic acid. Cysteine can also be measured using Ellman's colorimetric method (Arch.Biochem.Biophy., 82 70 (1959)).
It was quantified according to the following. For tryptophan, the method of Edelhoch et al. (Biochemistry, 6 ,
1948 (1967)). The results are shown in the table below.

[Table] (Examples) The present invention will be explained below with reference to Examples. Example 1 100 ml of a medium (PH7.0) containing 3% corn starch, 1.0% meat extract, 1.0% polypeptone, 0.2% yeast extract, 0.5% dipotassium phosphate, and 0.05% magnesium sulfate was placed in a 500 ml Sakaguchi flask. , Streptomyces nitrosporeus T.
-76 strain was inoculated and cultured with shaking at 28°C for 4 days. The amylase inhibitory activity obtained by filtering this culture solution was about 2000 U/ml. The culture filtrate thus obtained was collected to a volume of 2000 ml, and the filtrate was then subjected to ammonium sulfate fractionation. First, ammonium sulfate powder was added to the culture filtrate until it reached 80% saturation, and the deposited precipitate was collected by centrifugation. The precipitate was dialyzed overnight against 0.01M phosphate buffer (PH6.0) to perform desalting. The obtained dialyzed fluid was applied to a TEAE cellulose column (2.8 x 34 cm: Serva) equilibrated with 0.01M phosphate buffer (PH6.0) to adsorb amylase inhibitors. Gradient elution was then performed by linearly increasing the saline (NaCl) concentration in phosphate buffer from 0 to 1.25M. Fraction showing amylase inhibitory activity (NaCl concentration 0.9~
1.2M) and 0.02M containing 1.5M NaCl.
Dialysis was performed overnight against phosphate buffer (PH6.0).
This dialysate was applied to an Octyl Sepharose CL-4B column (2.8 x 34 cm; Pharmacia
The colored substance and the inhibitory substance were separated by applying the same buffer solution and developing with the same buffer. (Although both colored substances and inhibitors are not adsorbed to this resin, inhibitors have a higher affinity. Therefore, inhibitors are eluted later than colored substances.) Most colored substances The active fraction from which was removed was collected and dialyzed against 0.01M phosphate buffer (PH6.2) overnight. This dialyzed solution was added to a DEAE Toyopearl column (50 ml; Toyo Soda Kogyo Co., Ltd.) equilibrated with 0.01M phosphate buffer (PH6.2), and the inhibitor was adsorbed in the same manner as above. Non-adsorbed substances were washed and removed. Elution was then performed by linearly increasing the NaCl concentration in the phosphate buffer from 0 to 0.3M. FIG. 3 shows the relationship between the fractions obtained by elution and absorbance, and the relationship between the obtained fractions and amylase inhibitory activity. In FIG. 3, the ● mark indicates absorbance at OD ₂₈₀ nm (absorption by protein), and the circle mark indicates amylase inhibitory activity (x10 ^-4 U/ml).
Amylase inhibitory activity appeared as a single peak in the same fraction as the protein fraction. This active fraction
NaCl concentration 0.2-0.24M) was collected and extensively dialyzed against water. This dialyzed fluid was freeze-dried to obtain 33 mg of a purified sample of amylase inhibitor T-76. The amylase inhibitory activity of this purified sample is 26000U/mg
It was hot. This purified sample showed a single band when subjected to SDS-polyacrylamide gel electrophoresis and disk electrophoresis, and was electrophoretically shown to be a single substance. (Effects of the Invention) According to the present invention, the novel amylase inhibitor T-76 is thus provided. This inhibitor T
-76 is effectively produced by Streptomyces sp. This inhibitor T-76 selectively inhibits amylase derived from higher animals, is not inactivated by proteases such as pepsin and trypsin contained in digestive juices, and is stable over a wide temperature and PH range. . Therefore, it can be used extremely effectively as a therapeutic or preventive drug administered to inhibit amylase activity in the digestive tract, oral cavity, and other parts of the body. Since it does not act on amylase derived from microorganisms, it is also useful as a specific inhibitor of amylase derived from microorganisms, which causes errors when measuring biological components of higher animals using amylase derived from microorganisms.

[Brief explanation of drawings]

Figure 1 shows the inhibitory substance T-76 at 30°C and 100°C.
Figure 2 is a chart showing the ultraviolet absorption spectrum of this inhibitor T-76, Figure 3 is the elution curve of DEAE-Toyopearl column chromatography of this inhibitor T-76. It is a graph.

Claims (1)

[Claims] 1. An amylase inhibitor T- having the following properties:
76: Action: Inhibits human pancreatic amylase, human salivary amylase, and porcine pancreatic amylase; α-amylase from Bacillus, β from soybean
- Does not inhibit amylase, barley β-amylase, glucoamylase derived from Rhizopus, isoamylase derived from Pseudomonas, pepsin, trypsin and α-chymotrypsin, stable PH range and thermostability: 30℃ at PH3-12
Stable for 24 hours at pH 3-9
Molecular weight: Molecular weight determined by SDS-polyacrylamide gel electrophoresis is approximately 8000. Isoelectric point: The isoelectric point using ampholine as a carrier is 3.8. Maximum absorption is 278nm
, the minimum absorption is at 250 nm, and has the following amino acid composition: [Table] [Table] * Two disulfide bonds
Equivalent to individual parts (cystine is
2 exist).
2. The amylase inhibitor T-76 according to claim 1, which is obtained from Streptomyces bacteria. 3. The amylase inhibitor T-76 according to claim 2, obtained from Streptomyces nitrosporeus T-76 strain. 4. A method for producing the amylase inhibitor T-76 according to claim 1, comprising: culturing a microorganism belonging to the genus Streptomyces capable of producing the inhibitor, and the obtained product. A method for producing amylase inhibitor T-76, comprising the step of collecting the inhibitor from a culture. 5 Streptomyces
5. The production method according to claim 4, wherein the microorganism belonging to the genus Streptomyces nitrosporeus.