pyranose and 4-0-linked 3,6-anhydro-a-L-galactopyranose. (9). Hydrolytic ..... TABLE 2. Purification of agarase 0107 from Vibrio sp. strain JT0107. Purification ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993, P. 1549-1554
Vol. 59, No. 5
0099-2240/93/051549-06$02.00/0 Copyright © 1993, American Society for Microbiology
Purification and Characterization of a New Agarase from Marine Bacterium, Vibrio sp. Strain JT0107
a
YASUSHI SUGANO,1* ICHIRO TERADA,1 MASATOSHI ARITA,2 MASANA NOMA,' AND TAKASHI MATSUMOTO' Life Science Research Laboratory, Japan Tobacco Inc., 6-2, Umegaoka, Midori-ku, Yokohama, Kanagawa 227,1 and Seawater Science Research Laboratory, Japan Tobacco Inc., 4-13-20, Sakawa, Odawara, Kanagawa 256,2 Japan Received 16 November 1992/Accepted 7 March 1993
A marine bacterial strain that decomposes the cell walls of some seaweeds, including a Laminaria sp. and Undaria pinnatiJida, has been isolated from seawater. This strain has been classified to the genus Vibrio. One of the enzymes which the bacteria secreted into the culture medium was isolated and purified 45-fold from the culture fluid by a combination of ammonium sulfate precipitation and successive rounds of anion-exchange column chromatography. Purified protein migrated as a single band (Mr, 107,000) on sodium dodecyl sulfate-polyacrylamide gels. By amino acid sequence analysis, it was determined that this protein had a single N-terminal sequence that did not exhibit identity with the sequences of other agarases from marine bacteria. This novel enzyme was found to be an endo-type I8-agarase (EC 3.2.1.81) which hydrolyzes the 0-1,4 linkage of agarose to yield neoagarotetraose [0-3,6-anhydro-Ct-L-galactopyranosyl(l-->3)-041-D-galactopyranosyl(l->4)-0-3,6-anhydro-oI-L-galactopyranosyl(l-->3)-D-galactose] and neoagarobiose [O-3,6-anhydro-a-L-galactopyranosyl(l- 3)-D-galactoseI at a pH of around 8. The optimum temperature was 30°C. This enzyme did not decompose sodium alginate or A-, L-, or K-carrageenan. This enzyme may be of practical application in gene technology in the isolation of DNA fragments from agarose gels after electrophoresis.
It is well-known that seaweeds are very valuable marine and contain useful substances, such as unsaturated fatty acids, vitamins, carotenoids, betaine, and so on. One of the main obstacles to making use of these valuable resources is that degradation of the polysaccharides in the cell walls of seaweeds is required during purification of these substances. However, currently available degradation methods, such as acid hydrolysis, are so severe that the labile valuable substances are not extracted intact. Enzymatic degradation of polysaccharides may be a promising alternative, because labile substances may be quite tolerant of the mild conditions produced during enzymatic degradation. So far, few enzymes suitable for degradation of polysaccharides have been isolated. Agar is the most well-known polysaccharide, is found in the cell walls of some red algae, and is composed of agarose and agaropectin. Agaropectin is believed to be composed of a complex range of polysaccharide chains, but the details of the structure are not very well-known. On the other hand, agarose has been shown to have a linear chain structure composed of alternating residues of 3-0-linked P-D-galactopyranose and 4-0-linked 3,6-anhydro-a-L-galactopyranose (9). Hydrolytic enzymes which degrade agarose are classified into two groups in terms of the mode of action on agarose; ot-agarases cleave the a-1,3 linkage of agarose (24), and P-agarases cleave the P-1,4 linkage of agarose (5, 6). It has been predicted that a-agarase exists in a gram-negative bacterium, but a-agarase has not yet been purified or characterized (24). On the other hand, several ,B-agarases have been purified and characterized in the past decade from Streptomyces coelicolor (32 kDa) (4), Pseudomonas atlantica (32 kDa; optimum pH, 7.0) (17), and Vbrio sp. strain
AP-2 (20 kDa; optimum pH, 5.5) (1). The agarases which have been examined so far have two common features; the optimum pH region is neutral or slightly acidic, and the optimum temperature is over 40°C. In contrast, the agarase described in this report produced by the bacterium isolated from seawater works best at alkaline pHs and at a temperature 10°C lower than those of the agarases that have been studied thus far. Here, we report the purification and characterization of this new agarase that has been named agarase 0107 and its application to gene technology.
resources
*
MATERIALS AND METHODS Materials. Low-melting-point agarose, neoagarobiose, neoagarotetraose, neoagarohexaose [0-3,6-anhydro-a-L-galac-
topyranosyl(l- 3)-O-i-D-galactopyranosyl(l-A4)-0-3,6-anhydro-a-L-galactopyranosyl(1--> 3)-O-,-D-galactopyranosyl (1--4)-0-3,6-anhydro-Ct-L-galactopyranosyl(l---3)-D-galactose], and carrageenans were purchased from Sigma Chemical Co. (Tokyo, Japan). Sodium alginate was from Kanto Chemical Co. (Tokyo, Japan). Standard agarose L03, EcoRI, and reaction buffer were from TaKaRa Co. (Tokyo, Japan). Bacto Agar, yeast extract, and marine broth 2216 were from Difco Laboratories (Detroit, Mich.), and all other reagents were of analytical grade and commercially available. Quaternary aminoethyl (QAE)-Toyopearl was obtained from Tosoh (Tokyo, Japan). Mono-Q was from Pharmacia (Tokyo, Japan). Silica gel 60 on glass plates was purchased from E. Merck AG (Darmstadt, Germany). Isolation of strain JT0107. A small amount of seawater collected from Sagami Bay in Kanagawa Prefecture, Japan, was spread on 1.5% agar plates which contained marine broth 2216 (37.4 g/liter of distilled water). After incubation for a few days at 20°C, the organism (strain JT0107) that
Corresponding author. 1549
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SUGANO ET AL.
degraded agar was isolated and stored in the same medium containing 50% glycerol at -80°C. The JT0107 strain was identified on the basis of criteria described in Bergey's Manual of Systematic Bacteriology (2, 19). The motility and morphology of the bacterium were determined with a light microscope and a transmission electron microscope (JEM-1200EX). Enzyme assay. The reducing power of agarase 0107 was determined by the Somogyi-Nelson method (21). Ten microliters of this enzyme was dissolved in 20 mM Tris-HCl buffer (pH 8.0) and then 90 ,ul of this buffer plus 0.2% (wt/vol) neoagarohexaose, neoagarotetraose, or melted low-meltingpoint agarose was added as a substrate. After incubation (30°C, 5 min), 100 RI of copper reagent (100 ml of this reagent contains 0.37 g of CUS04, 2.4 g of anhydrous Na2CO3, 2.4 g of Rochelle salt, 1.92 g of NaHCO3, and 19.2 g of anhydrous Na2SO4) was added to the reaction mixture, and the reaction was stopped by boiling the mixture for 10 min. Nelson reagent was then added to the cooled reaction mixture, and the A660 was measured. One unit of the enzyme was defined as the amount that liberated 1 ,umol of reducing sugar per
min under the conditions described above. Purification of agarase 0107. The culture medium was composed of 0.3% agar, 0.02% K2HPO4, and 0.1% yeast extract in natural seawater and was adjusted to pH 8.0. Strain JT0107 was grown for 6 days on this medium (1 liter) on a rotary shaker (150 rpm) at 20°C. The culture was centrifuged (4,500 x g for 30 min), and 900 ml of clear supernatant fluid was obtained. All the following procedures were carried out in the temperature range of 0 to 4°C unless specified otherwise. The 900-ml culture fluid was adjusted to 90% saturation with solid ammonium sulfate and left to stand overnight. The precipitate collected by centrifugation (5,500 x g, 30 min) was dissolved in 20 ml of 20 mM Tris-HCl buffer (pH 8.0) and dialyzed against 3 liters of the same buffer. In order to remove insoluble particles and proteins not adsorbed to the anion-exchange resin, QAE-Toyopearl chromatography was carried out. A column (1 by 3 cm) of QAE-Toyopearl was equilibrated with 20 mM Tris-HCI buffer (pH 8.0). Thirty milliliters of the dialyzed enzyme solution was applied to the column and washed with 4 ml of the equilibration buffer. The column was eluted with a continuous linear gradient of 0 to 0.5 M NaCl in 20 mM Tris-HCl buffer (pH 8.0; total volume, 4 ml) and then with 4 ml of 0.5 M NaCl in the same buffer. The volume of one fraction was 2 ml. Active fractions detected by the SomogyiNelson method were collected and pooled for subsequent
Mono-Q chromatography.
A column (0.5 by 5 cm) of Mono-Q that had been equilibrated with 20 mM Tris-HCl buffer (pH 8.0) was used. The active fractions (8 ml) were diluted to 32 ml with the equilibration buffer in order to reduce the salt concentration. The fraction was then applied to the column and washed with 3 ml of the same buffer. Elution was performed with a continuous linear gradient of 0 to 0.5 M NaCl in 20 mM Tris-HCl buffer (pH 8.0; total volume, 15 ml) and then with 3 ml of 0.5 M NaCl in 20 mM Tris-HCl buffer (pH 8.0). The volume of one fraction was 1 ml. The active fractions (2 ml) were further purified by rechromatography on the same column and under elution conditions. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the fractions were stored at 4°C and used for characterization of the enzyme. Amino acid composition and sequence analysis. Agarase 0107 was desalted by dialysis, lyophilized, and hydrolyzed with 6 N HCl in a sealed tube at 110°C for 24 h. The amino
APPL. ENVIRON. MICROBIOL.
acids were analyzed with a Hitachi L-8500 amino acid analyzer. The N-terminal amino acid sequence of the enzyme was determined with Applied Biosystems protein sequencers (models 477A and 120A). Analysis of thermostability. Ten microliters of agarase 0107 (0.2 p,g/4l) was placed in a water bath at a temperature of 30, 40, 50, or 60°C for periods of 0, 30, 60, 90, or 120 min. Enzyme activity after treatment was measured at 30°C for 5 min. Time course of enzyme reaction. Ten microliters of the agarase 0107 solution (0.08 ,ug/,ul) and 90 ,ul of 0.2% lowmelting-point agarose in 20 mM Tris-HCl buffer (pH 8.0) were mixed and incubated at 25, 30, 35, or 40°C. The reaction products were measured as the yield of reducing sugar (galactose) at 2, 5, 10, 20, 30, 40, 50, and 60 min. Analysis of pH profile. The pH profile for the activity of agarase 0107 was obtained with the following buffers: 100 mM sodium acetate buffer (pH 3.6 to 5.6); 67 mM phosphate buffer (pH 5.6 to 7.6); 20 mM Tris-HCl buffer (pH 7.5 to 9.0); 50 mM glycine-NaOH buffer (pH 9.0 to 10.0). To each buffer, low-melting-point agarose was added to 0.2% (wt/ vol). Five microliters of agarase 0107 solution (0.4 p,g/,ul) was added to 95 [lI of each substrate solution, and agarase activity was measured for 5 min at 30°C. Other analytical methods. Protein concentrations were determined by UV spectrophotometry by the method of Warburg (11). SDS-PAGE was performed by the method described by Laemmli (12). Isoelectrophoresis was done on a Phast-Gel IEF4-6.5 from PhastSystem (Pharmacia), and the isoelectric point was determined with silver staining. Thin-layer chromatography for the hydrolysate of agarose, neoagarohexaose, or neoagarotetraose by agarase 0107 (20 mU) was performed on a silica gel 60 glass plate and developed with n-butanol-ethanol-water (3:1:1 [by volume] or n-butanol-acetic acid-water (2:1:1 [by volume]). The oligosaccharides that developed were detected with a naphthoresorcinol reagent (7) or a phosphate-molybdenum reagent (18). The Rf values of products were compared with those of authentic neoagarohexaose, neoagarotetraose, neoagarobiose, and galactose. Agarose gel electrophoresis for DNA fragments was performed by the standard method (14). Application experiment using agarase 0107. Electrophoresis of 0.5 ,ug of plasmid pUC19 containing an EcoRI site was carried out on a standard 0.8% agarose gel. A 40-mg section of the gel containing the circular DNA fragment was cut out. Forty microliters of water was added to the gel and heated at 65°C for 5 min. Samples (0.3 U) (50 pl) of agarase 0107, agarase from P. atlantica from Sigma and from Calbiochem, or water (control) were added to the gel and incubated for 180 min at 37°C. Each of these reaction samples was centrifuged (11,000 x g, 10 min), and the supernatant was obtained. Phenol (100 ,ul) was added to the supernatant, and the aqueous layer was recovered and washed with 100 ,ul of chloroform three times. The DNA fragment was precipitated from this aqueous layer by the addition of ethanol by the standard method (20). The DNA fragment was then desiccated and dissolved in 100 ,ul of water. The recovery of DNA was determined byA260. Electrophoresis was performed on a standard 0.8% agarose gel after the isolated DNA fragment was cut with EcoRI.
RESULTS Identification of strain JT0107. The isolated strain had a curved rod form with a sheathed polar flagellum and some small pili (Fig. 1) and had the biochemical and morphological
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A NEW AGARASE FROM V7BRIO SP. STRAIN JT0107
A
0.6
0.5
0
0.4 2 a E
a
0.60co
C
c
0.3 o
o
c I._
FIG. 1. Electron micrograph of Vibno solid medium.
sp.
strain JT0107
grown
G1)
-0.4n
C.)
a ._
6 4 2
Neoagarohexaose Neoagarotetraose Neoagarobiose Galactose Product 1' Product 2c Product 3d Product 4d
60' 40'
co
E
a) 20 cc
0
30
60
90
1 20
solvent':
Degree of polymerization
Oligosaccharide
A
B
0.32 0.44 0.60
0.21 0.34 0.54 0.46
0.43 0.60
0.34 0.53
a Thin-layer chromatography was performed on glass plates containing silica gel 60. b Solvent A is n-butanol-acetic acid-water (2:1:1 [by volume]), and solvent B is n-butanol-ethanol-water (3:1:1 [by volume]). c Obtained from agarose. d Obtained from neoagarohexaose.
Heat in water bath (min.) FIG. 4. Thermostability of agarase 0107. The enzyme activity remaining after heating at 30°C (O), 40'C (D), 50°C (0), or 60'C (-) is shown.
Therefore, this strain requires the salts contained in seawater to grow. This observation means that this strain is a marine bacterium. Strain JT0107 can digest not only agar but also some polysaccharides such as alginic acid that are the components of Laminaria sp. and Undaria pinnatifida (8, 11, 16), from which one can predict that this strain secretes some enzymes that degrade these polysaccharides besides agarase 0107 that cannot hydrolyze alginic acid (data not shown). Agarase 0107 is an enzyme secreted by bacteria and is estimated to have a molecular mass of 107 kDa, which is the largest among the reported agarases such as those of P. atlantica (32 kDa) (17), Cytophaga flevensis (26.5 kDa) (22), Vibrio sp. strain AP-2 (20 kDa) (1), and Pseudomonas sp.
strain PT-5 (31 kDa) (23). Aspartic acid (including asparagine) (15.8%), alanine (12%), and serine (7.8%) are the predominant amino acids in agarase 0107. Alanine accounts for 8.5% of the amino acids in agarase from Pseudomonas sp. strain PT-5. In agarase from a Pseudomonas-like bacterium, aspartic acid (including asparagine) was 11.1% and serine was as much as 13.5% of the amino acids. The first 21-aminoacid sequence has no remarkable feature in comparison with those of other marine bacterial agarases (3, 23). Therefore, agarase 0107 seems to have no homology with other agarases.
Agarase 0107 hydrolyzes agarose and neoagarohexaose. The predominant reaction products are neoagarobiose and neoagarotetraose produced by the cleavage of the 13-1,4 linkage. Therefore, agarase 0107 is a 3-agarase. Agarase 0107 could not hydrolyze the sulfate polysaccharides such as K-, A-, L-carrageenans that are composed of D-galactose and 3,6-anhydro-D-galactose with ,B-1,4 and
50
150
E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1 25
N
E z C.)
100
45.
Cu 0
~~~~~0~~~
40-
35. 30
Cn 0
25
0 C) cn _
60 40 50 20 30 Reaction time (min) FIG. 5. Time course of enzyme reaction. The quantity of reducing sugar (galactose) liberated by the enzyme reaction at 25°C (0), 0
10
30°C (0), 35°C (O), or 40°C (-) is shown time.
as a function of reaction
20-
G
30
60
90
9
120 150 1 80 210 240
Reaction Time(min.) FIG. 6. Changes in viscosity of agarase reaction mixture. The decrease in viscosity of the reaction mixture was measured at regular intervals with a B-type rotational viscometer (Tokimec, Tokyo, Japan) at 30°C. The viscosity of 0.3% agar hydrolyzed by agarase 0107 (0) and 0.3% agar without enzyme (x) is shown.
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SUGANO ET AL.
3.
1 4.
5. FIG. 7. Agarose gel electrophoresis of DNA recovered by agar0107. Lanes: 1, molecular size markers of phage DNA cut by restriction enzyme StyI (shown in kilobase pairs); 2, linear plasmid pUC19 fragment recovered from agarose gel with agarase 0107; 3, linear plasmid pUC19 fragment recovered from agarose gel with commercial agarase; 4, control sample treated with water.
6.
a-1,3 linkages (data not shown). This result suggests that this recognizes the different configurations of 3,6-anhydrogalactose in agar (L) and in carrageenans (D), although the possibility that the sulfate ester groups in carrageenan inhibit the enzyme cannot be ruled out (15). Note that the most interesting characteristics of agarase 0107 are the optimum pH and temperature. The optimum temperature and optimum pH for enzymatic reaction are 30°C and pH 8, respectively, whereas in contrast, those of any other known agarases are over 40°C and below pH 7, respectively. Considering that agar originates from red algae in seawater, which is in a slightly alkaline environment and at a rather low temperature, agarase 0107 seems to have the most suitable characteristics to express maximum activity in a marine environment. On the basis of the characteristics of agarase 0107 mentioned above, the application of the enzyme to the isolation of DNA from an agarose gel, which is very important in gene technology, is promising. Standard agarose gels and lowmelting-point agarose gels have been used mainly for analysis and isolation of DNA, respectively. This may be attributed to the availability of enzymes digesting agarose. Enzymes (commercially available) for which the optimum pH is acidic can hydrolyze completely soluble agar but incompletely hydrolyze insoluble agar, which reduces the recovery of DNA, because these enzymes are forced to work under inappropriate alkaline conditions, such as agarose gels prepared with Tris-acetate buffer (pH 8.0). Agarase 0107, with an optimum pH of 8, allowed us to recover the DNA fragment from standard agarose gels at eightfoldhigher yield than those of commercial agarases. This enzyme may be of practical application in gene technology for the isolation of DNA fragments from agarose gels after electrophoresis.
9.
ase
7. 8.
enzyme
ACKNOWLEDGMENT We thank Shigeru Kuwata for the electron microscopy analysis. REFERENCES 1. Aoki, T., T. Araki, and M. Kitamikado. 1990. Purification and characterization of a novel P-agarase from Vibrio sp. AP-2. Eur. J. Biochem. 187:461-466. 2. Baumann, P., A. L. Furniss, and J. V. Lee. 1984. Genus I. Vibrio Pacini 1854, 411AL, p. 518-538. In N. R. Krieg and J. G. Holt
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(ed.), Bergey's manual of systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore. Belas, R. 1989. Sequence analysis of the agrA gene encoding ,-agarase from Pseudomonas atlantica. J. Bacteriol. 171:602605. Buttner, M. J., I. M. Fernley, and M. J. Bibb. 1987. The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis. Mol. Gen. Genet. 209:101109. Duckworth, M., and J. R. Turvey. 1969. The action of a bacterial agarase on agarose, porphyran and alkali treated porphyran. Biochem. J. 113:687-692. Duckworth, M., and J. R. Turvey. 1969. The specificity of an agarase from a Cytophaga species. Biochem. J. 113:693-697. Duckworth, M., and W. Yaphe. 1970. Thin-layer chromatographic analysis of enzymic hydrolysates of agar. J. Chromatogr. 49:482-487. Fujikawa, T., and M. Wada. 1975. Schleim aus Braunalge Nemacystus decipiens, Mozuku 1. Mitt. Bestandteile des Schleims und ihre Eigenschaften. Agric. Biol. Chem. 39:1109-1114. Hamer, G. K., S. S. Bhattacharjee, and W. Yaphe. 1977. Analysis of the enzymic hydrolysis products of agarose by 13C-n.m.r. spectroscopy. Carbohydr. Res. 54:C7-C10. Harris, E. L. V., and S. Angal. 1989. Protein purification methods, p. 11. IRL Press, Oxford. Haung, A. 1965. Alginic acid. Isolation and fractionation with potassium chloride and manganous ions. Methods Carbohydr. Chem. 5:69-73. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
227:680-685. 13. Macmillan, J. D., H. J. Phaff, and R. H. Vaughn. 1964. The pattern of action of an exopolygalacturonic acid-trans-eliminase from Clostridium multifennentas. Biochemistry 3:572-578. 14. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecular cloning: a laboratory manual, p. 149-172. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. McLean, M. W., and F. B. Williamson. 1979. K-Carrageenase from Pseudomonas carrageenovora. Eur. J. Biochem. 93:553558. 16. Mizuno, H., T. Saito, N. Iso, N. Onda, K. Noda, and K. Takeda. 1983. Mannuronic to guluronic acid ratios of alginic acids prepared from various brown seaweeds. Nippon Suisan Gakkaishi 49:1591-1593. 17. Morrice, L. M., M. W. McLean, F. B. Williamson, and W. F. Long. 1983. 3-Agarase I and II from Pseudomonas atlantica purifications and some properties. Eur. J. Biochem. 135:553558. 18. Perry, E. S., and A. Weissberger. 1967. Thin-layer chromatography, p. 168-169. Interscience Publishers, New York. 19. Reichenbach, H. 1989. Genus I. Cytophaga Winogradsky 1929, 527,AL emend. In J. T. Staley, M. P. Bryant, N. Pfennig, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 3. Williams & Wilkins, Baltimore. 20. Sambrook, J., T. Maniatis, and E. F. Fritsch. 1989. Molecular cloning: a laboratory manual, p. E10-Ell. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19-23. 22. van der Meulen, H. J., and D. C. Harder. 1975. Production and characterization of the agarase of Cytophaga flevensis. Antonie van Leeuwenhoek J. Microbiol. 41:431-447. 23. Yamaura, I., T. Matsumoto, M. Funatsu, H. Shigeiri, and T. Shibata. 1991. Purification and some properties of agarase from Pseudomonas sp. PT-S. Agric. Biol. Chem. 55:2531-2536. 24. Young, K. S., S. S. Bhattacharjee, and W. Yaphe. 1978. Enzymic cleavage of the at-linkages in agarose, to yield agarooligosaccharides. Carbohydr. Res. 66:207-211.
