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Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase1 D. F. DAYA N D W. YAP HE^ Department of Microbiology and Itntnrrnology, McGill Utliversity, Montreal, Qrre. H3A 2B4 Accepted June 16, 1975 DAY,D. F., and W. YAPHE.1975. Enzymatic hydrolysisof agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase. Can. J. Microbiol. 21: 1512-1518. The mixture of polysaccharides in the gelling component of agar (agarose) is hydrolyzed to D-galactose and 3,6-anhydro-L-galactose by a series of hydrolytic enzymes obtained from Ps~.rr~lotnot~as ntlnntictr. The final degradative step in the pathway of agarose decomposition is the hydrolysis of the a-linkage in the disaccharide neoagarobiose yielding D-galactose and 3,6-anhydro-L-galactose. Pserrdotnotlns crtlnnticcr when grown on agar produces two specific enzymes, p-nitrophenyl a-galactose hydrolase and neoagarobiose hydrolase. The purification and partial characterization of both enzymes are presented. DAY,D. F., et W. YAPHE. 1975. Enzymatic hydrolysis ofagar: purificationand characterizationof neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase. Can. J. Microbial. 21: 1512-1518. Le melange de polysaccharides dans le composant gelifiant de la gelose (agarose) est hydrolysC en D-galactose et 3,h-anhydro-L-galactosepar une serie d'enzymes hydrolytiques obtenus de Psc.rrdornotzas crtlrrnticrr. La derniere etape de degradation dans la voie de decomposition de I'agarose est I'hydrolyse du lien a du disaccharide neoagarobiose donnant du D-galactose et du 3,h-anhydro-L-galactose. Lorsque Pserrdomot~crsntlnnticn est cultive sur de la gelose, il produit deux enzymes specifiques, la p-nitrophenyl a-galactose hydrolase et la neoagarobiose hydrolase. On presente des donnees sur la purification et la caracterisation partielle des deux enzymes. [Traduit par le journal]
Introduction which hydrolyzes the synthetic substrate for The marine bacterium Pselrdomonas atlantica a-galactosidases, p-nitrophenyl a-D-galactoside, is an agar-degrading microorganism (14). The but does not cleave the a-linkage in neoagaropathway of agar metabolism in this organism biose. involves the initial cleavage of the agarose Materials and Methods moiety of agar by an exoenzyme, P-agarase, Organism and Growth Corzditions yielding neoagarotetraose (0-3,6-anhydro-a-LPseudomonas atlanfica (National Culture Type Collecgalactopyranosyl (1 + 3)-0-P-D-galactopyranosyl tion (NCTC) 18685) stock cultures were kept at room (1 + 4)-0-3,6-anhydro-a-L-galactopyranosyl temperature on slants of agar-salt medium containing (1 + 3)-D-galactose) as one of the major end per litre of distilled water NaCI, 25 g; K2HP04, 0.1 g; . 7 H 2 0 , 5.0 g; CaC12, 0.2 g ; KCI, 1.0 g ; FeS04 . products (15). Neoagarotetraose is cleaved at its 7MgS04 H 2 0 , 0.2 g ; tris(hydroxyrnethyl)aminomethane (Tris), central P-linkage by a neoagarotetraose hydrol- 0.4 g; casamino acids, 2.5 g; and Bacto agar (Difco), 15 g. ase yielding neoagarobiose (0-3,6-anhydro-a-L- The pH of the medium after autoclaving was 7.2. T h e galactopyranosyl (1 + 3)-D-galactose). The a- organism was grown on the surface of 35 x 20 cm agar linkage in neoagarobiose is cleaved by a neo- plates (solid culture) or liquid medium with the substituof 0.1% galactose, 0.1% glucose, o r 0.1% Bacto agar agarobiose hydrolase to the monomeric sugars tion (Difco) for the 1.5% agar as carbon source. Solid culture D-galactose, and 3,6-anhydro-L-galactose (1 5). plates were spread with 5 ml and liquid medium was In this study, the purification and some of the inoculated with 0.5% v/v of a late log-phase culture grown properties of the neoagarobiose hydrolase are in 0.1% agar-salts medium. Liquid cultures were incubated on a rotary shaker (100rpm). All media were presented. Also presented is the purification and incubated a t 25 "C. characterization of an a-galactoside hydrolase 'Received April 25, 1975. 2Person t o whom inquiries regarding the paper should be addressed.
Clzemicals Neoagarobiose and neoagarotetraose were prepared by the method of Duckworth a n d Yaphe (6). Galactostat reagent, hemoglobin, and horseradish peroxidase were
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DAY AND YAPHE: ENZYMATIC HYDROLYSIS O F AGAR
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obtained from the Worthington Biochemical Corp. (Freehold, New Jersey); galactose dehydrogenase and the p-nitrophenyl a-galactoside (and other nitrophenyl compounds) were obtained from the Sigma Chemical Co. (St. Louis, Missouri). Unless specifically listed all other chemicals used were of the best commercially available reagent grade.
Column Cl~rotnatography The G-200 Sephadex column (2.5 x 40 cm) was prepared from dextran which had been thoroughly washed in distilled water and then equilibrated with 0.1 M K 2 H P 0 4buffer, pH 8.1, containing 10-4M dithiothreitol.
Assay for a-Neoagarobiose Hydrolase A quantitative assay for high concentrations of neoagarobiose hydrolase involved coupling the production of galactose produced on cleavage of neoagarobiose with the commercial Galactostat assay and recording the change in absorbance at 425 nm. The assay mixture consisted of neoagarobiose, 1 x M ; NaCI, 2 x l o - * M ; Galactostat reagent, 0.3 ml; and enzyme, to a total assay volume of 0.5 ml. A unit of enzyme activity is defined as that amount of enzyme which will produce a change in absorbance at 425 nm of 0.001/min. Because of the difficulty in obtaining large quantities of neoagarobiose a reducing sugar assay for measuringenzyme activity was not a practical alternative.
PuriJication Cells were harvested from solid culture after 10 h (Fig. 1) by washing the cells off the agar surface with distilled water: the cell suspension was brought to an absorbance of 1.5 at 660 nm with distilled water and then centrifuged at 23 000 x g for 20 min at 4 "C. The cell pellet was resuspended in an equal volume of distilled water. Since P. atlantica is a marine bacterium, this second resuspension in water resulted in lysis of the cells. The cell debris was removed by centrifugation at 23 000 x g for 20 min at 4 "C and the supernatant fraction was adjusted to pH 8.1 and 0.1 M K2HP0, by adding the appropriate amount of 1.0 M K2HP0,; sufficient dithiothreitol was added to give a final concentration of M. This supernatant was used as a source of the neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase. All purification steps were carried out at 4 "C.
Assay for p-Nitrophenyl a-Galactoside Hydrolase Thep-nitrophenyl a-galactoside hydrolase was assayed a t room temperature by measuring the rate of increase in absorbance at 405 nm. The assay mixture consisted of a final concentration of 5 x M p-nitrophenyl a-ogalactoside in 0.1 M K 2 H P 0 4 buffer, pH 8.1, and sufficient enzyme to give a minimum change in absorbance at 405 nm of 0.1 lmin in a total assay volume of 0.5 ml. Qrialitatiue Assays Hydrolysis of agar, neoagarotetraose, neoagarobiose, and melibiose was detected by analysis of hydrolysis products by thin-layer chromatography on microcrystalline cellulose. The assay mixture consisted of enzyme, 0.1 M K,HP04 buffer, pH 7.5, and 0.05 ml each of 1 x M substrate and 1 x M NaCl in a total assay volume of 0.5 ml. Aliquots of the reaction mixture were withdrawn with time and applied to microcrystalline cellulose thin-layer plates, developed, and located as previously described (5).
Results
Purijication of Neoagarobiose Hydrolase Solid ammonium sulfate was added to 70% saturation to the supernatant fluid and the precipitate was removed by centrifugation at 29 000 x g for 20 min. Neoagarobiose hydrolase remained in the supernatant fluid of the 70% ammonium sulfate fraction which was then concentrated from 300 ml to 120 ml by membrane filtration using an Amicon Diaflo PM-I0 Protein Determit~ations Protein was estimated by both the ultraviolet absorp- membrane. Upon a three-fold concentration a tion method of Kalckar (7) and the colorimetric tech- precipitate formed which contained the enzyme. nique of Lowry et ol. (9). The precipitate was collected by centrifugation Disc Gel Electrophoresis at 17 300 x g and resuspended in 10 ml of 0.1 M The gel system consisted of equal parts of stock solu- K2HP0,, 0.1 M NaCI, pH 7.5 buffer. Freezetions A and C, where A was 0.5 M N,N-bis(2-hydroxythawing of this fraction precipitated more proethyl) glycine and 0.006% N,N,N',N'-tetramethylenediamine and C was 30 g of acrylamide, 0.8 g of N,N- tein, leaving the enzyme in the supernatant methylene bis-acrylamide monomer, and 2 mg of K,Fefraction. The supernatant fraction gave a single (CN)6 made up to 100 ml with distilled water. T o this band upon disc acrylamide electrophoresis. The m~xtureof A and C, an equal volume of 0.14% ammonium purified enzyme (0.36 mg proteinlml) was found persulfate was added. The buffer system was composed of 0.1 M N,N-bis (2-hydroxyethyl) glycine - 0.005 M glycine to be unstable. It was inactivated by freezing, adjusted to pH 9.0 with NaOH. Electrophoresis was con- incubation for 48 h at 4 "C, and treatment with ducted at 3 mA per gel tube with a Buchler model 3-1073 M of the sulfhydryl reagent P-mercaptoapparatus. Between 50 and 150 pg of protein was electro- ethanol. The reducing reagent dithiothreitol phoresed per gel. Acrylamide gel electrophoresis in M) had no effect on either stability or sodium dodecyl sulfate was performed according to the activity. At present it has not been possible to method of Leonard (8) in 7% acrylamide gels and 1% sodium dodecyl sulfate (SDS). stabilize the enzyme in its pure form; however, it
CAN. J . MICROBIOL. VOL. 21, 1975 HOURS (liquid culture) 5
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FIG. 1. Growth curve and enzyme production by P. atlantica on solid (lower time scale) or liquid (upper time scale) medium. Growth was determined by the absorbance at 660 nm (a).Enzyme activity for neoagarobiose hydrolase (H)and p-nitrophenyl a-galactoside hydrolase (0) were determined on an aliquot of cells removed from the culture. TABLE 1 Purification of P. atlantica hydrolases
a-Neoagarobiose hydrolase Step
Volume ml
Total protein, mg
Total units"
Sp. act., unitslmg protein
p-Nitrophenyl a-galactoside hydrolase Total units: IU
Sp. act., IU/mg protein
Crude Lysate 70% Am sulfate supernate PM-I0 concn. Precipitate Freeze-thaw supernate 35-70% Am sulfate precipitate Freeze-thaw XM-50 Pooled G-200 concentrated 'One unit is that amount o f enzyme which produces a change in absorbance at 425 nm o f 0.001 per minute. blU = international unit (prnol product/rnin).
Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase1 D. F. DAYA N D W. YAP HE^ Department of Microbiology and Itntnrrnology, McGill Utliversity, Montreal, Qrre. H3A 2B4 Accepted June 16, 1975 DAY,D. F., and W. YAPHE.1975. Enzymatic hydrolysisof agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase. Can. J. Microbiol. 21: 1512-1518. The mixture of polysaccharides in the gelling component of agar (agarose) is hydrolyzed to D-galactose and 3,6-anhydro-L-galactose by a series of hydrolytic enzymes obtained from Ps~.rr~lotnot~as ntlnntictr. The final degradative step in the pathway of agarose decomposition is the hydrolysis of the a-linkage in the disaccharide neoagarobiose yielding D-galactose and 3,6-anhydro-L-galactose. Pserrdotnotlns crtlnnticcr when grown on agar produces two specific enzymes, p-nitrophenyl a-galactose hydrolase and neoagarobiose hydrolase. The purification and partial characterization of both enzymes are presented. DAY,D. F., et W. YAPHE. 1975. Enzymatic hydrolysis ofagar: purificationand characterizationof neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase. Can. J. Microbial. 21: 1512-1518. Le melange de polysaccharides dans le composant gelifiant de la gelose (agarose) est hydrolysC en D-galactose et 3,h-anhydro-L-galactosepar une serie d'enzymes hydrolytiques obtenus de Psc.rrdornotzas crtlrrnticrr. La derniere etape de degradation dans la voie de decomposition de I'agarose est I'hydrolyse du lien a du disaccharide neoagarobiose donnant du D-galactose et du 3,h-anhydro-L-galactose. Lorsque Pserrdomot~crsntlnnticn est cultive sur de la gelose, il produit deux enzymes specifiques, la p-nitrophenyl a-galactose hydrolase et la neoagarobiose hydrolase. On presente des donnees sur la purification et la caracterisation partielle des deux enzymes. [Traduit par le journal]
Introduction which hydrolyzes the synthetic substrate for The marine bacterium Pselrdomonas atlantica a-galactosidases, p-nitrophenyl a-D-galactoside, is an agar-degrading microorganism (14). The but does not cleave the a-linkage in neoagaropathway of agar metabolism in this organism biose. involves the initial cleavage of the agarose Materials and Methods moiety of agar by an exoenzyme, P-agarase, Organism and Growth Corzditions yielding neoagarotetraose (0-3,6-anhydro-a-LPseudomonas atlanfica (National Culture Type Collecgalactopyranosyl (1 + 3)-0-P-D-galactopyranosyl tion (NCTC) 18685) stock cultures were kept at room (1 + 4)-0-3,6-anhydro-a-L-galactopyranosyl temperature on slants of agar-salt medium containing (1 + 3)-D-galactose) as one of the major end per litre of distilled water NaCI, 25 g; K2HP04, 0.1 g; . 7 H 2 0 , 5.0 g; CaC12, 0.2 g ; KCI, 1.0 g ; FeS04 . products (15). Neoagarotetraose is cleaved at its 7MgS04 H 2 0 , 0.2 g ; tris(hydroxyrnethyl)aminomethane (Tris), central P-linkage by a neoagarotetraose hydrol- 0.4 g; casamino acids, 2.5 g; and Bacto agar (Difco), 15 g. ase yielding neoagarobiose (0-3,6-anhydro-a-L- The pH of the medium after autoclaving was 7.2. T h e galactopyranosyl (1 + 3)-D-galactose). The a- organism was grown on the surface of 35 x 20 cm agar linkage in neoagarobiose is cleaved by a neo- plates (solid culture) or liquid medium with the substituof 0.1% galactose, 0.1% glucose, o r 0.1% Bacto agar agarobiose hydrolase to the monomeric sugars tion (Difco) for the 1.5% agar as carbon source. Solid culture D-galactose, and 3,6-anhydro-L-galactose (1 5). plates were spread with 5 ml and liquid medium was In this study, the purification and some of the inoculated with 0.5% v/v of a late log-phase culture grown properties of the neoagarobiose hydrolase are in 0.1% agar-salts medium. Liquid cultures were incubated on a rotary shaker (100rpm). All media were presented. Also presented is the purification and incubated a t 25 "C. characterization of an a-galactoside hydrolase 'Received April 25, 1975. 2Person t o whom inquiries regarding the paper should be addressed.
Clzemicals Neoagarobiose and neoagarotetraose were prepared by the method of Duckworth a n d Yaphe (6). Galactostat reagent, hemoglobin, and horseradish peroxidase were
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/24/14 For personal use only.
DAY AND YAPHE: ENZYMATIC HYDROLYSIS O F AGAR
1513
obtained from the Worthington Biochemical Corp. (Freehold, New Jersey); galactose dehydrogenase and the p-nitrophenyl a-galactoside (and other nitrophenyl compounds) were obtained from the Sigma Chemical Co. (St. Louis, Missouri). Unless specifically listed all other chemicals used were of the best commercially available reagent grade.
Column Cl~rotnatography The G-200 Sephadex column (2.5 x 40 cm) was prepared from dextran which had been thoroughly washed in distilled water and then equilibrated with 0.1 M K 2 H P 0 4buffer, pH 8.1, containing 10-4M dithiothreitol.
Assay for a-Neoagarobiose Hydrolase A quantitative assay for high concentrations of neoagarobiose hydrolase involved coupling the production of galactose produced on cleavage of neoagarobiose with the commercial Galactostat assay and recording the change in absorbance at 425 nm. The assay mixture consisted of neoagarobiose, 1 x M ; NaCI, 2 x l o - * M ; Galactostat reagent, 0.3 ml; and enzyme, to a total assay volume of 0.5 ml. A unit of enzyme activity is defined as that amount of enzyme which will produce a change in absorbance at 425 nm of 0.001/min. Because of the difficulty in obtaining large quantities of neoagarobiose a reducing sugar assay for measuringenzyme activity was not a practical alternative.
PuriJication Cells were harvested from solid culture after 10 h (Fig. 1) by washing the cells off the agar surface with distilled water: the cell suspension was brought to an absorbance of 1.5 at 660 nm with distilled water and then centrifuged at 23 000 x g for 20 min at 4 "C. The cell pellet was resuspended in an equal volume of distilled water. Since P. atlantica is a marine bacterium, this second resuspension in water resulted in lysis of the cells. The cell debris was removed by centrifugation at 23 000 x g for 20 min at 4 "C and the supernatant fraction was adjusted to pH 8.1 and 0.1 M K2HP0, by adding the appropriate amount of 1.0 M K2HP0,; sufficient dithiothreitol was added to give a final concentration of M. This supernatant was used as a source of the neoagarobiose hydrolase and p-nitrophenyl a-galactoside hydrolase. All purification steps were carried out at 4 "C.
Assay for p-Nitrophenyl a-Galactoside Hydrolase Thep-nitrophenyl a-galactoside hydrolase was assayed a t room temperature by measuring the rate of increase in absorbance at 405 nm. The assay mixture consisted of a final concentration of 5 x M p-nitrophenyl a-ogalactoside in 0.1 M K 2 H P 0 4 buffer, pH 8.1, and sufficient enzyme to give a minimum change in absorbance at 405 nm of 0.1 lmin in a total assay volume of 0.5 ml. Qrialitatiue Assays Hydrolysis of agar, neoagarotetraose, neoagarobiose, and melibiose was detected by analysis of hydrolysis products by thin-layer chromatography on microcrystalline cellulose. The assay mixture consisted of enzyme, 0.1 M K,HP04 buffer, pH 7.5, and 0.05 ml each of 1 x M substrate and 1 x M NaCl in a total assay volume of 0.5 ml. Aliquots of the reaction mixture were withdrawn with time and applied to microcrystalline cellulose thin-layer plates, developed, and located as previously described (5).
Results
Purijication of Neoagarobiose Hydrolase Solid ammonium sulfate was added to 70% saturation to the supernatant fluid and the precipitate was removed by centrifugation at 29 000 x g for 20 min. Neoagarobiose hydrolase remained in the supernatant fluid of the 70% ammonium sulfate fraction which was then concentrated from 300 ml to 120 ml by membrane filtration using an Amicon Diaflo PM-I0 Protein Determit~ations Protein was estimated by both the ultraviolet absorp- membrane. Upon a three-fold concentration a tion method of Kalckar (7) and the colorimetric tech- precipitate formed which contained the enzyme. nique of Lowry et ol. (9). The precipitate was collected by centrifugation Disc Gel Electrophoresis at 17 300 x g and resuspended in 10 ml of 0.1 M The gel system consisted of equal parts of stock solu- K2HP0,, 0.1 M NaCI, pH 7.5 buffer. Freezetions A and C, where A was 0.5 M N,N-bis(2-hydroxythawing of this fraction precipitated more proethyl) glycine and 0.006% N,N,N',N'-tetramethylenediamine and C was 30 g of acrylamide, 0.8 g of N,N- tein, leaving the enzyme in the supernatant methylene bis-acrylamide monomer, and 2 mg of K,Fefraction. The supernatant fraction gave a single (CN)6 made up to 100 ml with distilled water. T o this band upon disc acrylamide electrophoresis. The m~xtureof A and C, an equal volume of 0.14% ammonium purified enzyme (0.36 mg proteinlml) was found persulfate was added. The buffer system was composed of 0.1 M N,N-bis (2-hydroxyethyl) glycine - 0.005 M glycine to be unstable. It was inactivated by freezing, adjusted to pH 9.0 with NaOH. Electrophoresis was con- incubation for 48 h at 4 "C, and treatment with ducted at 3 mA per gel tube with a Buchler model 3-1073 M of the sulfhydryl reagent P-mercaptoapparatus. Between 50 and 150 pg of protein was electro- ethanol. The reducing reagent dithiothreitol phoresed per gel. Acrylamide gel electrophoresis in M) had no effect on either stability or sodium dodecyl sulfate was performed according to the activity. At present it has not been possible to method of Leonard (8) in 7% acrylamide gels and 1% sodium dodecyl sulfate (SDS). stabilize the enzyme in its pure form; however, it
CAN. J . MICROBIOL. VOL. 21, 1975 HOURS (liquid culture) 5
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FIG. 1. Growth curve and enzyme production by P. atlantica on solid (lower time scale) or liquid (upper time scale) medium. Growth was determined by the absorbance at 660 nm (a).Enzyme activity for neoagarobiose hydrolase (H)and p-nitrophenyl a-galactoside hydrolase (0) were determined on an aliquot of cells removed from the culture. TABLE 1 Purification of P. atlantica hydrolases
a-Neoagarobiose hydrolase Step
Volume ml
Total protein, mg
Total units"
Sp. act., unitslmg protein
p-Nitrophenyl a-galactoside hydrolase Total units: IU
Sp. act., IU/mg protein
Crude Lysate 70% Am sulfate supernate PM-I0 concn. Precipitate Freeze-thaw supernate 35-70% Am sulfate precipitate Freeze-thaw XM-50 Pooled G-200 concentrated 'One unit is that amount o f enzyme which produces a change in absorbance at 425 nm o f 0.001 per minute. blU = international unit (prnol product/rnin).
