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A novel pyrazine compound produced from chitin by the activity of the enzyme from Vibrio alginolyticus TK-24
JOURNAL OFFERMENTATION ANDBIOENGINEERING Vol. 80, No. 4, 406-407. 1995
A Novel Pyrazine Compound Produced from Chitin by the Activity of the Enzyme from Vibrio aZginolyticus TK-24 TAKAAKI
YANAI,‘*
ATSUSHI
MATSUDA,’ KAZUHIKO AND SAWAO MURA02
OKAMURA,’
TAKASHI
SHIN,2
Central Research Laboratories, Mercian Corp., 9-I Johnan I-chome, Fujisawa-shi, Kanagawa 251’ and Department of Applied Microbial Technology, The Kumamoto Institute of Technology, 4-22-l Ikeda, Kumamoto 860,= Japan Received 15 May 1995/Accepted 26 June 1995 Au enzyme from Vibrio alginolyticus TK-24 which converted chitin to a novel pyrazine compound, VAPY was purified to electrophoretic homogeneity and is suggested to be a novel enzyme as determined from examination of its ten N-terminal amino acid residue sequence and its characteristic activity. The structure of VAPY was elucidated by mass spectroscopy, ‘I-I nuclear-magnetic-resonance (‘II NMR) and 13C nuclearmagnetic-resonance (1% NMR) spectroscopy, and from the spectroscopic data it was determined to be a novel pyrazine compound. [Key words:
Vibrio alginolyticus,chitin, pyrazine compound] Erlenmeyer flask. The enzyme was adsorbed on chitin and recovered by the following method. After centrifugation to remove cells and residual chitin, the culture supernatant containing VAPY-producing enzyme was lyophilized. Equal amounts (15 g) of chitin and the lyophilizate containing VAPY-producing enzyme were suspended in 50mM borax-HCl buffer (pH9.0). The mixture was incubated for 1 h on ice, and centrifuged at 3,000 X g for 15 min. The pellet was washed twice with distilled water, prior to resuspension in the buffer and incubated for 1 h at 30°C with agitation. Remaining particulate materials were removed by centrifugation and the supernatant was dialyzed using dialysis tubing against distilled water. The inner dialysate was loaded onto a DEAE-Toyopearl 650M (Tosoh Co. Ltd.; 2.6x 8.5 cm) column that had been previously equilibrated with 50 mM borax-HCl buffer (pH 9.0). Elution was conducted with a linear gradient of NaCl (O-O.5 M) in the same buffer. Fractions with VAPY-producing enzyme activity were combined and concentrated by pressure filtration using a PM-10 Amicon ultrafilter at 4°C. The dialysate was loaded onto a Butyl-Toyopearl 650s (Tosoh Co. Ltd.; 2.6~ 6 cm) column that had been previously equilibrated with 100mM sodium phosphate (pH 7.0) containing 0.3 M (NH4)$04. The enzyme was eluted with a linear gradient of (NH&SO4 (0.3-O M) in the same buffer. The active fractions were combined and concentrated. The dialysate was loaded onto a TSK gel Phenyl-5PW (Tosoh Co. Ltd.; 2.1 x 15 cm) column. The enzyme was eluted with a linear gradient of (NH&SO4 (1.0-O M) in the same buffer. The active fractions were combined, concentrated, dialyzed against distilled water and lyophilized as previously specified. Finally, 6.5 mg of VAPY-producing enzyme was obtained in the form of a powder. The purified enzyme was revealed to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be approximately 98,000 by both native-polyacrylamide gel and SDS-polyacrylamide gel electrophoresis with standard protein markers (Pharmacia). These findings indicated that the enzyme was composed of a single polypeptide chain. The
Chitin, an insoluble polysaccharide consisting of p(1,4)-linked N-acetyl-D-glucosamine units, is one of the most abundant organic compounds in nature. In recent years, a significant amount of research has been directed toward the use of chitin and related materials in fields as diverse as the wastewater treatment, drug delivery, wound healing, soil amendment, and dietary fiber (l-3). Chemical derivatives of chitin may be complemented and extended by the use of chitin-degrading and -modifying enzymes. There is a considerable amount of literature concerning the chitinolytic enzymes, namely chitinase [EC 3.2. 1.141 and /3-N-acetyl-D-glucosaminidase [EC 3.2.1301, but only a few reports on the enzymes that produce a useful chitooligosaccharide from chitin (4, 5; Usui, T. and Hayashi, Y., Abstr. Annu. Meet. Jpn. Sot. Biotechnol. Agrochem., p. 651, 1987). Murao et al. (6) have reported that a chitinase from Vibrio alginolyticus TK-24 had a novel activity yielding N,N’-diacetylchitobiose and N,N,N”,N’“,iV”“-pentaacetylchitopentaose from colloidal chitin. This study was undertaken with the intent of investigating enzymes other than the chitinase reported by Murao et al. (6) which are produced by V. alginolyticus TK-24. In this paper, we report the characterization of a new pyrazine compound (VAPY)-producing enzyme isolated from V. alginofyticus TK-24, the production of VAPY from chitin as a result of enzyme activity and the structure of VAPY. VAPY-producing enzyme activity was determined in a 0.1 ml reaction mixture containing 1 mg flake chitin (Nacalai Tesque Inc., Kyoto) in 50 mM borax-HCl buffer (pH 9.0) and the enzyme. The reaction was allowed to proceed for 4 h at 37°C and the amount of VAPY formed was measured by high performance liquid chromatography (HPLC), using a TSK gel Amide-80 (Tosoh Co. Ltd., Tokyo; 4.6 x 25 cm) column. V. alginolyticus TK-24 was cultivated in a medium (100 ml, pH 7.5) containing 0.7% meat extract, 1% Polypepton, 0.3% flake chitin, and 2% NaCl in a 500ml * Corresponding author. 406
NOTES
VOL. 80, 1995 Physicochemical properties of VAPY
TABLE 1. Appearance: Molecular formula: HREI-MS (m/z) Calcd: Found: UV A,, (nm): IR KBr (cm I): TABLE 2. Position
727.2885 727.2874 275 3385, 1649, 1559, 1076, 1038
13Cchemical shifts” [DDIlll
\..
6 2’ 3’ 4’ 5’ 6’
102.0 (d) 56.5 (d) 74.4 (d) 70.4 (d) 76.2 idj 61.3 (t)
NAc
White powder CzsH&sN4
FIG. 1.
-
‘H and 13CNMR chemical shifts of VAPY
143.2 (d) 155.1 (s) 71.9 (d) 82.3 cd) 71.9 idj 62.8 (t)
2 3 4
23.0 (9) 175.6 (s)
‘H chemical shiftsb (ppm)
I
8.66 (s) 5.14 4.09 3.78 3.51 3.72 4.39 3.57 3.39 3.22 3.08 3.32 3.44 2.01
(d, J=3.3 Hz) cdd. J=3.7. 7.0 Hz) idt,‘J=2.9,‘6.6Hzj (dd, J=6.6, 11.7 Hz) (dd, J=2.9, 11.7 Hz) (d, J=8,4 Hz) (dd, J=8.1, 10.3 Hz) (dd, J=8.8, 10.3 Hzj ct. J=9.2 Hz) (ddd, J=2.2; 5.1, 9.9 Hz) (dd, J=5.1, 12.1 Hz) (dd, J=2.2, 12.1 Hz) (s)
a The sample was dissolved in DtO. Chemical shifts are shown with reference to 1,4-dioxane as 67.4 ppm at ambient temperature. b Chemical shifts are shown with reference to 1,4-dioxane as 3.70 ppm at ambient temperature. point was 3.6. The enzyme showed maximum activity at pH 9.0 in borax buffer. When the enzyme was kept at 28°C for 46 h with buffers of various pHs, almost no loss of activity was between pH 8.0 and pH 9.0. The optimum temperature for enzyme activity was at 37”C, and the enzyme activity was stable up to 40°C for a 24 h incubation period at pH 9.0. Furthermore, amino acid sequence analysis was carried out using an Applied Biosystems protein sequencer model 477A. The N-terminal amino acid sequence of the enzyme was Ala-Pro-Thr-Ala-Pro-Ser-Ile-Asp-Met-Tyr-. Enzymatic production of VAPY was conducted in a reaction mixture of 50mM borax-HCl buffer (PH 9.0), containing 3 g of flake chitin and 0.1 mg of the purified enzyme in a total volume of 25 ml. The reaction was allowed to proceed at 37°C for 4 h with agitation. VAPY formed in the reaction mixture was isolated by preparative HPLC. VAPY was then lyophilized to give a yield of 30 mg. The physicochemical properties of VAPY are summarized in Table 1 and the ‘H and 13C NMR spectroscopic data for VAPY are shown in Table 2. From these results, the structure of VAPY was determined to be as 2,5-Bis[2-O-(2-acetamido-2-deoxy-j-Dglucopyranosyl) - 1,3,4 - trihydroxybutyllpyrazine (Fig. 1). The product other than VAPY produced by this enzyme was identified as N-acetylglucosamine by HPLC. The enzyme specificity was examined with some chitin related compounds. The relative production rates of VAPY from N-acetylglucosamine, N,N’-diacetylchitobiose, N,N,N”-triacetylchitotriose, and N,N’,N’,N”:N”“isoelectric
407
Structure of VAPY,
pentaacetylchitopentaose were 0, 14.1, 2.0, and 35.1% of that of N,hr’,N’:N”-tetraacetylchitotetraose, respectively. Of the chitooligosaccharides tested, N,N’,N’,N”‘-tetraacetylchitotetraose was the best substrate, achieving the highest production rate of VAPY by the enzyme. The VAPY formation process may be involved in at least two enzymatic and chemical reaction steps. At first, the enzyme hydrolyzes chitin and N-acetylchitooligosaccharides to the corresponding oligomers and dimers. Then, the enzyme deacetylates one of the N-acetyl groups in the dimer. The resulting two compounds may soon be condensed to VAPY via dehydration and deprotonation. We examined some characteristics of the physiological activity of VAPY and found that VAPY had the activity of preventing collagen-induced platelet aggregation and concanavalin A-induced hemagglutination in vitro (data not shown). VAPY may be of medical use for treating arterial embolism, cerebral infarction and diabetes mellitus due to the above physiological activities. A comparison of the N-terminal amino acid sequence of the VAPY-producing enzyme with amino acid sequences listed in databases using GENETYX-Mac software (Software Development Co. Ltd., Tokyo) showed no significant degree of homology with other known chitinase sequences. Though high homology (80%) with aldose Iepimerase [EC 5.1.3.31 of Acinetobacter calcoaceticus (7) was detected, aldose 1-epimerase has not been reported to show activity toward chitin and related compounds. Thus it is conceivable that the VAPY-producing enzyme isolated in this study is a new enzyme and efforts to clarify its characteristics in detail are underway. REFERENCES 1. Brine, C. J.: Chitin; accomplishments and perspectives, p. 1824. In Zikakis, J. P. (ed.), Chitin, chitosan and related enzymes. Academic Press Inc., New York (1984). 2. Adachi, K., Kobayasbi, M., and Takahashi, E.: Effect of the application of lignin and/or chitin to soil inoculated with Fusarium oxysporum on the variation of soil microflora and plant growth. Soil Sci. Plant Nutr., 33, 245-259 (1987). 3. Watanabe, K., Saiki, I., Urakl, Y., Tokura, S., and Azuma, I.: 6-0-Carboxymethyl-chitin (CM-chitin) as a drug carrier. Chem. Pharm. Bull., 38, 506-509 (1990). 4. Ohtakara, A., Mitsutomi, M., and Uchida, Y.: Purification and some properties of chitinase from Vibrio sp. J. Ferment. Technol., 57, 169-177 (1979). 5. Usui, T., Hayashi, Y., Nanjo, F., Sakai, K., and Isbido, Y.: Transglycosylation reaction of chitinase purified from Nocardia orientakis. Biochim. Biophys. Acta, 923, 302-309 (1987). 6. Murao, S., Kawada, T., Itoh, H., Oyama, H., and Shin, T.: Purification and characterization of a novel type of chitinase from Vibrio alginolyticus TK-22. Biosci. Biotech. Biochem., 56, 368-369 (1992). 7. Gatz, C., Altschmied, J., and Hillen, W.: Cloning and expression of the Acinetobacter calcoaceticus Mutarotase gene in Escherichia coli. J. Bacterial., 168, 31-38 (1986).
A Novel Pyrazine Compound Produced from Chitin by the Activity of the Enzyme from Vibrio aZginolyticus TK-24 TAKAAKI
YANAI,‘*
ATSUSHI
MATSUDA,’ KAZUHIKO AND SAWAO MURA02
OKAMURA,’
TAKASHI
SHIN,2
Central Research Laboratories, Mercian Corp., 9-I Johnan I-chome, Fujisawa-shi, Kanagawa 251’ and Department of Applied Microbial Technology, The Kumamoto Institute of Technology, 4-22-l Ikeda, Kumamoto 860,= Japan Received 15 May 1995/Accepted 26 June 1995 Au enzyme from Vibrio alginolyticus TK-24 which converted chitin to a novel pyrazine compound, VAPY was purified to electrophoretic homogeneity and is suggested to be a novel enzyme as determined from examination of its ten N-terminal amino acid residue sequence and its characteristic activity. The structure of VAPY was elucidated by mass spectroscopy, ‘I-I nuclear-magnetic-resonance (‘II NMR) and 13C nuclearmagnetic-resonance (1% NMR) spectroscopy, and from the spectroscopic data it was determined to be a novel pyrazine compound. [Key words:
Vibrio alginolyticus,chitin, pyrazine compound] Erlenmeyer flask. The enzyme was adsorbed on chitin and recovered by the following method. After centrifugation to remove cells and residual chitin, the culture supernatant containing VAPY-producing enzyme was lyophilized. Equal amounts (15 g) of chitin and the lyophilizate containing VAPY-producing enzyme were suspended in 50mM borax-HCl buffer (pH9.0). The mixture was incubated for 1 h on ice, and centrifuged at 3,000 X g for 15 min. The pellet was washed twice with distilled water, prior to resuspension in the buffer and incubated for 1 h at 30°C with agitation. Remaining particulate materials were removed by centrifugation and the supernatant was dialyzed using dialysis tubing against distilled water. The inner dialysate was loaded onto a DEAE-Toyopearl 650M (Tosoh Co. Ltd.; 2.6x 8.5 cm) column that had been previously equilibrated with 50 mM borax-HCl buffer (pH 9.0). Elution was conducted with a linear gradient of NaCl (O-O.5 M) in the same buffer. Fractions with VAPY-producing enzyme activity were combined and concentrated by pressure filtration using a PM-10 Amicon ultrafilter at 4°C. The dialysate was loaded onto a Butyl-Toyopearl 650s (Tosoh Co. Ltd.; 2.6~ 6 cm) column that had been previously equilibrated with 100mM sodium phosphate (pH 7.0) containing 0.3 M (NH4)$04. The enzyme was eluted with a linear gradient of (NH&SO4 (0.3-O M) in the same buffer. The active fractions were combined and concentrated. The dialysate was loaded onto a TSK gel Phenyl-5PW (Tosoh Co. Ltd.; 2.1 x 15 cm) column. The enzyme was eluted with a linear gradient of (NH&SO4 (1.0-O M) in the same buffer. The active fractions were combined, concentrated, dialyzed against distilled water and lyophilized as previously specified. Finally, 6.5 mg of VAPY-producing enzyme was obtained in the form of a powder. The purified enzyme was revealed to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be approximately 98,000 by both native-polyacrylamide gel and SDS-polyacrylamide gel electrophoresis with standard protein markers (Pharmacia). These findings indicated that the enzyme was composed of a single polypeptide chain. The
Chitin, an insoluble polysaccharide consisting of p(1,4)-linked N-acetyl-D-glucosamine units, is one of the most abundant organic compounds in nature. In recent years, a significant amount of research has been directed toward the use of chitin and related materials in fields as diverse as the wastewater treatment, drug delivery, wound healing, soil amendment, and dietary fiber (l-3). Chemical derivatives of chitin may be complemented and extended by the use of chitin-degrading and -modifying enzymes. There is a considerable amount of literature concerning the chitinolytic enzymes, namely chitinase [EC 3.2. 1.141 and /3-N-acetyl-D-glucosaminidase [EC 3.2.1301, but only a few reports on the enzymes that produce a useful chitooligosaccharide from chitin (4, 5; Usui, T. and Hayashi, Y., Abstr. Annu. Meet. Jpn. Sot. Biotechnol. Agrochem., p. 651, 1987). Murao et al. (6) have reported that a chitinase from Vibrio alginolyticus TK-24 had a novel activity yielding N,N’-diacetylchitobiose and N,N,N”,N’“,iV”“-pentaacetylchitopentaose from colloidal chitin. This study was undertaken with the intent of investigating enzymes other than the chitinase reported by Murao et al. (6) which are produced by V. alginolyticus TK-24. In this paper, we report the characterization of a new pyrazine compound (VAPY)-producing enzyme isolated from V. alginofyticus TK-24, the production of VAPY from chitin as a result of enzyme activity and the structure of VAPY. VAPY-producing enzyme activity was determined in a 0.1 ml reaction mixture containing 1 mg flake chitin (Nacalai Tesque Inc., Kyoto) in 50 mM borax-HCl buffer (pH 9.0) and the enzyme. The reaction was allowed to proceed for 4 h at 37°C and the amount of VAPY formed was measured by high performance liquid chromatography (HPLC), using a TSK gel Amide-80 (Tosoh Co. Ltd., Tokyo; 4.6 x 25 cm) column. V. alginolyticus TK-24 was cultivated in a medium (100 ml, pH 7.5) containing 0.7% meat extract, 1% Polypepton, 0.3% flake chitin, and 2% NaCl in a 500ml * Corresponding author. 406
NOTES
VOL. 80, 1995 Physicochemical properties of VAPY
TABLE 1. Appearance: Molecular formula: HREI-MS (m/z) Calcd: Found: UV A,, (nm): IR KBr (cm I): TABLE 2. Position
727.2885 727.2874 275 3385, 1649, 1559, 1076, 1038
13Cchemical shifts” [DDIlll
\..
6 2’ 3’ 4’ 5’ 6’
102.0 (d) 56.5 (d) 74.4 (d) 70.4 (d) 76.2 idj 61.3 (t)
NAc
White powder CzsH&sN4
FIG. 1.
-
‘H and 13CNMR chemical shifts of VAPY
143.2 (d) 155.1 (s) 71.9 (d) 82.3 cd) 71.9 idj 62.8 (t)
2 3 4
23.0 (9) 175.6 (s)
‘H chemical shiftsb (ppm)
I
8.66 (s) 5.14 4.09 3.78 3.51 3.72 4.39 3.57 3.39 3.22 3.08 3.32 3.44 2.01
(d, J=3.3 Hz) cdd. J=3.7. 7.0 Hz) idt,‘J=2.9,‘6.6Hzj (dd, J=6.6, 11.7 Hz) (dd, J=2.9, 11.7 Hz) (d, J=8,4 Hz) (dd, J=8.1, 10.3 Hz) (dd, J=8.8, 10.3 Hzj ct. J=9.2 Hz) (ddd, J=2.2; 5.1, 9.9 Hz) (dd, J=5.1, 12.1 Hz) (dd, J=2.2, 12.1 Hz) (s)
a The sample was dissolved in DtO. Chemical shifts are shown with reference to 1,4-dioxane as 67.4 ppm at ambient temperature. b Chemical shifts are shown with reference to 1,4-dioxane as 3.70 ppm at ambient temperature. point was 3.6. The enzyme showed maximum activity at pH 9.0 in borax buffer. When the enzyme was kept at 28°C for 46 h with buffers of various pHs, almost no loss of activity was between pH 8.0 and pH 9.0. The optimum temperature for enzyme activity was at 37”C, and the enzyme activity was stable up to 40°C for a 24 h incubation period at pH 9.0. Furthermore, amino acid sequence analysis was carried out using an Applied Biosystems protein sequencer model 477A. The N-terminal amino acid sequence of the enzyme was Ala-Pro-Thr-Ala-Pro-Ser-Ile-Asp-Met-Tyr-. Enzymatic production of VAPY was conducted in a reaction mixture of 50mM borax-HCl buffer (PH 9.0), containing 3 g of flake chitin and 0.1 mg of the purified enzyme in a total volume of 25 ml. The reaction was allowed to proceed at 37°C for 4 h with agitation. VAPY formed in the reaction mixture was isolated by preparative HPLC. VAPY was then lyophilized to give a yield of 30 mg. The physicochemical properties of VAPY are summarized in Table 1 and the ‘H and 13C NMR spectroscopic data for VAPY are shown in Table 2. From these results, the structure of VAPY was determined to be as 2,5-Bis[2-O-(2-acetamido-2-deoxy-j-Dglucopyranosyl) - 1,3,4 - trihydroxybutyllpyrazine (Fig. 1). The product other than VAPY produced by this enzyme was identified as N-acetylglucosamine by HPLC. The enzyme specificity was examined with some chitin related compounds. The relative production rates of VAPY from N-acetylglucosamine, N,N’-diacetylchitobiose, N,N,N”-triacetylchitotriose, and N,N’,N’,N”:N”“isoelectric
407
Structure of VAPY,
pentaacetylchitopentaose were 0, 14.1, 2.0, and 35.1% of that of N,hr’,N’:N”-tetraacetylchitotetraose, respectively. Of the chitooligosaccharides tested, N,N’,N’,N”‘-tetraacetylchitotetraose was the best substrate, achieving the highest production rate of VAPY by the enzyme. The VAPY formation process may be involved in at least two enzymatic and chemical reaction steps. At first, the enzyme hydrolyzes chitin and N-acetylchitooligosaccharides to the corresponding oligomers and dimers. Then, the enzyme deacetylates one of the N-acetyl groups in the dimer. The resulting two compounds may soon be condensed to VAPY via dehydration and deprotonation. We examined some characteristics of the physiological activity of VAPY and found that VAPY had the activity of preventing collagen-induced platelet aggregation and concanavalin A-induced hemagglutination in vitro (data not shown). VAPY may be of medical use for treating arterial embolism, cerebral infarction and diabetes mellitus due to the above physiological activities. A comparison of the N-terminal amino acid sequence of the VAPY-producing enzyme with amino acid sequences listed in databases using GENETYX-Mac software (Software Development Co. Ltd., Tokyo) showed no significant degree of homology with other known chitinase sequences. Though high homology (80%) with aldose Iepimerase [EC 5.1.3.31 of Acinetobacter calcoaceticus (7) was detected, aldose 1-epimerase has not been reported to show activity toward chitin and related compounds. Thus it is conceivable that the VAPY-producing enzyme isolated in this study is a new enzyme and efforts to clarify its characteristics in detail are underway. REFERENCES 1. Brine, C. J.: Chitin; accomplishments and perspectives, p. 1824. In Zikakis, J. P. (ed.), Chitin, chitosan and related enzymes. Academic Press Inc., New York (1984). 2. Adachi, K., Kobayasbi, M., and Takahashi, E.: Effect of the application of lignin and/or chitin to soil inoculated with Fusarium oxysporum on the variation of soil microflora and plant growth. Soil Sci. Plant Nutr., 33, 245-259 (1987). 3. Watanabe, K., Saiki, I., Urakl, Y., Tokura, S., and Azuma, I.: 6-0-Carboxymethyl-chitin (CM-chitin) as a drug carrier. Chem. Pharm. Bull., 38, 506-509 (1990). 4. Ohtakara, A., Mitsutomi, M., and Uchida, Y.: Purification and some properties of chitinase from Vibrio sp. J. Ferment. Technol., 57, 169-177 (1979). 5. Usui, T., Hayashi, Y., Nanjo, F., Sakai, K., and Isbido, Y.: Transglycosylation reaction of chitinase purified from Nocardia orientakis. Biochim. Biophys. Acta, 923, 302-309 (1987). 6. Murao, S., Kawada, T., Itoh, H., Oyama, H., and Shin, T.: Purification and characterization of a novel type of chitinase from Vibrio alginolyticus TK-22. Biosci. Biotech. Biochem., 56, 368-369 (1992). 7. Gatz, C., Altschmied, J., and Hillen, W.: Cloning and expression of the Acinetobacter calcoaceticus Mutarotase gene in Escherichia coli. J. Bacterial., 168, 31-38 (1986).