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New proteinaceous α-amylase inhibitor (T-76) from Streptomyces nitrosporeus
JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 68, No. l, 56-57. 1989
New Proteinaceous a-Amylase Inhibitor (T-76) from
Streptomyces nitrosporeus MOTOO ARAI, 1. TAKAHISA NISHIMURA, 1 KOUKI TSUKAO, 1 TAKASHI KAWAGUCHI, 1 HIDEO HAYASHI, 1 YASUHIRO SHIMIZU, 2 AND SAWAO MURAO l §
Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, l and Daiwa Kasei K. K., Kouga, Shiga 520-32, 2 Japan Received 14 March 1989/Accepted 11 May 1989 An actinomycete, strain no. T-76, which a produced new proteinaceous a-amylase inhibitor, was isolated from soil. The strain was identified as Streptomyces nitrosporeus from its taxonomical characteristics. The inhibitor, tentatively named T-76 was purified from the culture filtrate by ammonium sulfate precipitation, DEAE and TEAE cellulose column chromatography, acid precipitation, and Octyl Sepharose hydrophobie chromatography. The molecular weight and isoelectric point of the inhibitor were estimated to be 8,000 and 4.0, respectively. The amino acid composition of the inhibitor was different from that of proteinaceous inhibitors of other strains. The inhibition spectrum of the inhibitor was also different from that of other origins.
Sepharose CL-4B equilibrated with P-buffer containing 1.5 M NaCI and eluted with a linear gradient from 1.5 to 0 M NaC1. The colored fractions without inhibitory activities were eluted first and then the active fraction was eluted. The final inhibitor preparation was studied by polyacrylamide gel electrophoresis at pH 9.5. A single band was obtained. A summary of purification is shown in Table 1. The yield of the inhibitor was 54.1% and its specific activity was 24.2 times higher than that of the culture filtrate. A total of 2.5 g of the inhibitor was obtained from 47 1 of the culture filtrate. The relative activity of T-76 on various amylases is shown in Table 2. T-76 was clearly different from Haim or Paim in its inhibition spectra. The molecular weight of T76 was estimated to be 8,000 by SDS gel electrophoresis and gel filtration on a column of Sephadex G-100. The isoelectric point of T-76 was found to be pH 4.0 by isoelectric focusing. The amino acid composition of T-76 was as follows: Asxg, Thrll, Ser6, GlxT, ProT, GIy7, Ala12, 1/2Cys4, Vals, Met0, Ilel, Leu0, Tyro, Phe2, Lysl, H i s , Argo, and Trp2. T-76 contained a large amount of alanine but no leucine. Comparison of amino acid composition of aamylase inhibitors is given in Table 3. T-76 was clearly different from other inhibitors. The primary structure of T-76 is now under investigation.
There are many proteinaceous inhibitors of animal aamylases (1-9). They are useful for the investigation of amylases, and of differences between amylases of various origins. We have isolated amylase inhibitors named Haim and Paim (1--4). The two inhibitors are similar in their inhibition of animal a-amylases, but different in their inhibition of human and pig a-amylases. Paim does not inhibit human a-amylase. This difference is probably due to the difference in their primary structure of the inhibitors. So these primary structures were elucidated (3, 4), as were those of the amylase inhibitors Hoe-467A, Z-2685, and AI-3688 (5-9). To expand the study of this line, we have isolated several new proteinaceous inhibitors from the culture filtrate of microorganisms. This paper describes some properties of an inhibitor tentatively named T-76. The assay system for the inhibitor was similar to that reported previously (1, 2). The microorganism, strain no. T-76, that produced T-76 was identified as Streptomyces nitrosporeus from its taxonomical characteristics. Strain no. T-76 was grown aerobically at 30°C for 30h in a medium consisting of 3% corn starch, 1% Polypepton, 1% meat extract, 0.2% yeast extract, 0.5% K2HPO4, and 0.05% MgSO4.7H20, pH 7.0. To purify T-76 from the culture filtrate, it was brought to 80% saturation with ammonium sulfate and the resulting precipitate was collected by filtration and dissolved in water. The solution was dialyzed against 10mM phosphate buffer, pH 6.0 (Pbuffer). The dialyzed solution was put onto a column on DEAE cellulose equilibrated with P-buffer and eluted with a linear gradient of NaC1 concentrations from 0 to 0.5 M. The active fractions were collected and dialyzed against Pbuffer. The dialyzed solution was put on a column of TEAE cellulose equilibrated with P-buffer and eluted with a linear gradient of NaC1 from 0 to 1 M. The active fractions were collected and their pH was adjusted to 4.0 with acetic acid. The resultant precipitate was collected by centrifugation and dissolved in P-buffer containing 1,5 M NaC1. The solution was put on a column of Octyl * Corresponding author. Present address: Kumamoto Institute Kumamoto 860, Japan.
of
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan. This work was also partly supported by a grant from the Fujisawa Foundation. TABLE 1. Summary of purification of the inhibitor Step Culture filtrate Ammonium sulfate ppt DEAE cellulose TEAE cellulose Acid treatment Octyl Sepharose
Technology, 56
Total Total Specific Yield activity protein activity (u × 10 4) (A2s0 × ml) (u/A2so) (°~°) 1,680 184,000 91 100 1,480 38.900 380 88.1 1,450 16,600 873 86.3 1,090 6,700 1,630 64.9 1,070 5,590 1,910 63.7 909 4,140 2,200 54,1
Vol., 68, 1989
NOTES TABLE 3.
TABLE 2.
Effects of amylase inbibitors on various pancreatic aanfylases '
T,76
Halm
Paim
Snake Cat Horse
++ ~+ ++
~ ~ +~
~+
Rai Sheep
+
+
-
~-
Rabbit Guinea pig Goat Cow Dog Pig Chicken Human Human'
+++ ~+ +H+ ~ +
~ ~ -~ +¢-¢+~+ ~ ~ ~
+ + --~ +~¢4+ -
+
Each enzyme sOlution was incubated with 0.5 ml of inhibitor solution at 37°C for 10 min and the residual activity was assayed. In the experimental condition, the degree of inhibition of each inhibitor on pig pancreatic amylase was 80~0. Degree of inhibition: ~ , 80-100~; ~r, 50-80°~; -e, 20-50~o; - ,
0--20oA.
Amino acid compositions (mol%) of a-amylase inhibitors
Z-2685 Hoe-467A
r
Amylase '
Asx Thr Ser Olx Pro Gly Ala 1/2Cys Val Met Ile Leu Tyr Phe Lys His Arg Trp
57
7,9 11.8 5.3 7.9 6.6 11.8 10.5 5,3 10.5 0 1.3 1.3 6.6 1.6 0 3.9 2.6 3.9
8.1 10.8 6.8 9.5 4.1 9.5 9.5 5.4 10,8 0 2.7 5.4 8.1 0 1.4 2.7 4.1 2.4
AI-3688 i1.I 8.3 11.1 11.1 5.6 5.6 8.3 5.6 16.7 0 0 0 5.6 2.8 0 0 5,6 2.8
Haim II Palm II 14.3 10.4 5.2 1.3 6.5 10.4 11.7 5,2 10.4 0 3.9 5.2 5.2 3.9 0 1.3 3.9 1.3
7.9 9.5 9.5 7.1 4.8 9.5 14.3 4.8 9.5 2.4 0 4.8 4.8 O 0 4.8 2,4 2.4
T-76 10.0 13.8 6.3 8.8 6.3 10.0 13.8 5.0 10.0 0 1.3 0 5.0 3.8 t .3 1.3 1.3 2.5
trometry. Biochemistry, 26, 6483-6488 (1987).
5. Asehsuer, H., Ve~tesy, L., and Braaalt=er, G.: The sequence of
a From saliva.
REFERENCES 1. Murao, S., Goto, A., Matsui, Y., Ohyama, K., and Aral, M.: Isolation and identification of a hog pancreatic a-amylase inhibitor (Haim) producing gtreptomyces. Agri~; Biol. Chem., ,15, 2599-2604 (1981). 2. Murao, S., OOuehi, N., Goto, A., and Arai, M.: New proteinaceous a-amylase inhibitor (Palm) from Streptomyces corchorshii, Agric. Biol. Chem., 47, 453-454 (1983). 3. Mural, H., Ham, S., lkenaka, T., Goto, A., Anti, M., and Murao, S.: Amino acid sequence of protein a-amylase inhibitor from Streptomyces griseosporeus YM-25. J. Biochem,, 97, 11291133 (1985).
4. Hlrayama, K., Tnkahashl, R., Akashl, S., Fukuhara, K., Oouehl, N., Mural, A., Anti, M., Murao, S., Tanaka, K., and No|iron, I.: Primary structure of Palm I, an a-amylase inhibitor from Streptomyces corchorshii, determined by the combination of Edman degradation and fast atom bombardment mass spec-
the a-amylase inhibitor Hoe-467A (a-amylase inactivator Hoe467A) from Streptomyces tendae 4158. Hoppe-Seyler's Z. Physiol. Chem., 362, 465--467 (1981).
6. Aschaner, H., Verte~y, L., Naemann, G., and Braun/tzer, G.: Die prim~struktur des a-amylasein_hibitors Hoe 467A aus gtreptomyces tendae 4158. eine neue klasse von inhibitoren. HoppeSeyler's Z. Physiol. Chem., 364, 1347-1356 (1983).
7. Vestesy, L., Oeding, V., Bender, R,, Zepf, K., and N e s e m g n , G.: Tendamistat (HOE 467), a tight-binding a-amylase inhibitor from gtreptomyces tendae 4158. Eur. J. Biochem., 141, 505-512 (1984). 8. Hofmann, O., Vertesy, L., and Braunltzer, G.: The primary structure of a-amylase inhibitor Z-2685 from Streptomycea parvullus FH-1461. Biol. Chem. Hoppe-Seyler, 366, I161-I168 (1985). 9. Vertesy, L. and Tripler, D.: Isolation and structure elucidation of an a-amylase inhibitor, AI-3688, from Streptomyces aureofaciens. FEBS Lett., 185, 187-190 (1985).
Vol. 68, No. l, 56-57. 1989
New Proteinaceous a-Amylase Inhibitor (T-76) from
Streptomyces nitrosporeus MOTOO ARAI, 1. TAKAHISA NISHIMURA, 1 KOUKI TSUKAO, 1 TAKASHI KAWAGUCHI, 1 HIDEO HAYASHI, 1 YASUHIRO SHIMIZU, 2 AND SAWAO MURAO l §
Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, l and Daiwa Kasei K. K., Kouga, Shiga 520-32, 2 Japan Received 14 March 1989/Accepted 11 May 1989 An actinomycete, strain no. T-76, which a produced new proteinaceous a-amylase inhibitor, was isolated from soil. The strain was identified as Streptomyces nitrosporeus from its taxonomical characteristics. The inhibitor, tentatively named T-76 was purified from the culture filtrate by ammonium sulfate precipitation, DEAE and TEAE cellulose column chromatography, acid precipitation, and Octyl Sepharose hydrophobie chromatography. The molecular weight and isoelectric point of the inhibitor were estimated to be 8,000 and 4.0, respectively. The amino acid composition of the inhibitor was different from that of proteinaceous inhibitors of other strains. The inhibition spectrum of the inhibitor was also different from that of other origins.
Sepharose CL-4B equilibrated with P-buffer containing 1.5 M NaCI and eluted with a linear gradient from 1.5 to 0 M NaC1. The colored fractions without inhibitory activities were eluted first and then the active fraction was eluted. The final inhibitor preparation was studied by polyacrylamide gel electrophoresis at pH 9.5. A single band was obtained. A summary of purification is shown in Table 1. The yield of the inhibitor was 54.1% and its specific activity was 24.2 times higher than that of the culture filtrate. A total of 2.5 g of the inhibitor was obtained from 47 1 of the culture filtrate. The relative activity of T-76 on various amylases is shown in Table 2. T-76 was clearly different from Haim or Paim in its inhibition spectra. The molecular weight of T76 was estimated to be 8,000 by SDS gel electrophoresis and gel filtration on a column of Sephadex G-100. The isoelectric point of T-76 was found to be pH 4.0 by isoelectric focusing. The amino acid composition of T-76 was as follows: Asxg, Thrll, Ser6, GlxT, ProT, GIy7, Ala12, 1/2Cys4, Vals, Met0, Ilel, Leu0, Tyro, Phe2, Lysl, H i s , Argo, and Trp2. T-76 contained a large amount of alanine but no leucine. Comparison of amino acid composition of aamylase inhibitors is given in Table 3. T-76 was clearly different from other inhibitors. The primary structure of T-76 is now under investigation.
There are many proteinaceous inhibitors of animal aamylases (1-9). They are useful for the investigation of amylases, and of differences between amylases of various origins. We have isolated amylase inhibitors named Haim and Paim (1--4). The two inhibitors are similar in their inhibition of animal a-amylases, but different in their inhibition of human and pig a-amylases. Paim does not inhibit human a-amylase. This difference is probably due to the difference in their primary structure of the inhibitors. So these primary structures were elucidated (3, 4), as were those of the amylase inhibitors Hoe-467A, Z-2685, and AI-3688 (5-9). To expand the study of this line, we have isolated several new proteinaceous inhibitors from the culture filtrate of microorganisms. This paper describes some properties of an inhibitor tentatively named T-76. The assay system for the inhibitor was similar to that reported previously (1, 2). The microorganism, strain no. T-76, that produced T-76 was identified as Streptomyces nitrosporeus from its taxonomical characteristics. Strain no. T-76 was grown aerobically at 30°C for 30h in a medium consisting of 3% corn starch, 1% Polypepton, 1% meat extract, 0.2% yeast extract, 0.5% K2HPO4, and 0.05% MgSO4.7H20, pH 7.0. To purify T-76 from the culture filtrate, it was brought to 80% saturation with ammonium sulfate and the resulting precipitate was collected by filtration and dissolved in water. The solution was dialyzed against 10mM phosphate buffer, pH 6.0 (Pbuffer). The dialyzed solution was put onto a column on DEAE cellulose equilibrated with P-buffer and eluted with a linear gradient of NaC1 concentrations from 0 to 0.5 M. The active fractions were collected and dialyzed against Pbuffer. The dialyzed solution was put on a column of TEAE cellulose equilibrated with P-buffer and eluted with a linear gradient of NaC1 from 0 to 1 M. The active fractions were collected and their pH was adjusted to 4.0 with acetic acid. The resultant precipitate was collected by centrifugation and dissolved in P-buffer containing 1,5 M NaC1. The solution was put on a column of Octyl * Corresponding author. Present address: Kumamoto Institute Kumamoto 860, Japan.
of
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan. This work was also partly supported by a grant from the Fujisawa Foundation. TABLE 1. Summary of purification of the inhibitor Step Culture filtrate Ammonium sulfate ppt DEAE cellulose TEAE cellulose Acid treatment Octyl Sepharose
Technology, 56
Total Total Specific Yield activity protein activity (u × 10 4) (A2s0 × ml) (u/A2so) (°~°) 1,680 184,000 91 100 1,480 38.900 380 88.1 1,450 16,600 873 86.3 1,090 6,700 1,630 64.9 1,070 5,590 1,910 63.7 909 4,140 2,200 54,1
Vol., 68, 1989
NOTES TABLE 3.
TABLE 2.
Effects of amylase inbibitors on various pancreatic aanfylases '
T,76
Halm
Paim
Snake Cat Horse
++ ~+ ++
~ ~ +~
~+
Rai Sheep
+
+
-
~-
Rabbit Guinea pig Goat Cow Dog Pig Chicken Human Human'
+++ ~+ +H+ ~ +
~ ~ -~ +¢-¢+~+ ~ ~ ~
+ + --~ +~¢4+ -
+
Each enzyme sOlution was incubated with 0.5 ml of inhibitor solution at 37°C for 10 min and the residual activity was assayed. In the experimental condition, the degree of inhibition of each inhibitor on pig pancreatic amylase was 80~0. Degree of inhibition: ~ , 80-100~; ~r, 50-80°~; -e, 20-50~o; - ,
0--20oA.
Amino acid compositions (mol%) of a-amylase inhibitors
Z-2685 Hoe-467A
r
Amylase '
Asx Thr Ser Olx Pro Gly Ala 1/2Cys Val Met Ile Leu Tyr Phe Lys His Arg Trp
57
7,9 11.8 5.3 7.9 6.6 11.8 10.5 5,3 10.5 0 1.3 1.3 6.6 1.6 0 3.9 2.6 3.9
8.1 10.8 6.8 9.5 4.1 9.5 9.5 5.4 10,8 0 2.7 5.4 8.1 0 1.4 2.7 4.1 2.4
AI-3688 i1.I 8.3 11.1 11.1 5.6 5.6 8.3 5.6 16.7 0 0 0 5.6 2.8 0 0 5,6 2.8
Haim II Palm II 14.3 10.4 5.2 1.3 6.5 10.4 11.7 5,2 10.4 0 3.9 5.2 5.2 3.9 0 1.3 3.9 1.3
7.9 9.5 9.5 7.1 4.8 9.5 14.3 4.8 9.5 2.4 0 4.8 4.8 O 0 4.8 2,4 2.4
T-76 10.0 13.8 6.3 8.8 6.3 10.0 13.8 5.0 10.0 0 1.3 0 5.0 3.8 t .3 1.3 1.3 2.5
trometry. Biochemistry, 26, 6483-6488 (1987).
5. Asehsuer, H., Ve~tesy, L., and Braaalt=er, G.: The sequence of
a From saliva.
REFERENCES 1. Murao, S., Goto, A., Matsui, Y., Ohyama, K., and Aral, M.: Isolation and identification of a hog pancreatic a-amylase inhibitor (Haim) producing gtreptomyces. Agri~; Biol. Chem., ,15, 2599-2604 (1981). 2. Murao, S., OOuehi, N., Goto, A., and Arai, M.: New proteinaceous a-amylase inhibitor (Palm) from Streptomyces corchorshii, Agric. Biol. Chem., 47, 453-454 (1983). 3. Mural, H., Ham, S., lkenaka, T., Goto, A., Anti, M., and Murao, S.: Amino acid sequence of protein a-amylase inhibitor from Streptomyces griseosporeus YM-25. J. Biochem,, 97, 11291133 (1985).
4. Hlrayama, K., Tnkahashl, R., Akashl, S., Fukuhara, K., Oouehl, N., Mural, A., Anti, M., Murao, S., Tanaka, K., and No|iron, I.: Primary structure of Palm I, an a-amylase inhibitor from Streptomyces corchorshii, determined by the combination of Edman degradation and fast atom bombardment mass spec-
the a-amylase inhibitor Hoe-467A (a-amylase inactivator Hoe467A) from Streptomyces tendae 4158. Hoppe-Seyler's Z. Physiol. Chem., 362, 465--467 (1981).
6. Aschaner, H., Verte~y, L., Naemann, G., and Braun/tzer, G.: Die prim~struktur des a-amylasein_hibitors Hoe 467A aus gtreptomyces tendae 4158. eine neue klasse von inhibitoren. HoppeSeyler's Z. Physiol. Chem., 364, 1347-1356 (1983).
7. Vestesy, L., Oeding, V., Bender, R,, Zepf, K., and N e s e m g n , G.: Tendamistat (HOE 467), a tight-binding a-amylase inhibitor from gtreptomyces tendae 4158. Eur. J. Biochem., 141, 505-512 (1984). 8. Hofmann, O., Vertesy, L., and Braunltzer, G.: The primary structure of a-amylase inhibitor Z-2685 from Streptomycea parvullus FH-1461. Biol. Chem. Hoppe-Seyler, 366, I161-I168 (1985). 9. Vertesy, L. and Tripler, D.: Isolation and structure elucidation of an a-amylase inhibitor, AI-3688, from Streptomyces aureofaciens. FEBS Lett., 185, 187-190 (1985).