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Polygalacturonase from Corticium rolfsii
POLYGALACTURONASE FROM Corticium rolfsii
[39]
361
described procedure shows a very slight pectin lyase activity (increase in absorbance at 235 nm of 0.018 within 1 hr). Assay for Glycoproteins. PE I and PE II give positive reactions in a modified silver staining procedure for detecting glycoproteins on SDS-electrophoresis gels. 11 Furthermore, PE I is adsorbed to Con A-Sepharose (for PE II this was not tested). This indicates that pectinesterases from P. infestans are glycoproteins. Further Purification of PE I. The chemical properties of both proteins of PE I seem to be very similar. The double protein band cannot be separated by chromatography on hydroxylapatite, different ion exchangers (DEAE-Sepharose, CM-Sepharose), Con A-Sepharose and cross-linked polypectate, ~2 hydrophobic interaction chromatography on octyl sepharose, or adsorption on bentonite and chromatofocusing with Pharmacia Polybuffer exchanger PBE 118. H G. Dubray and G. Bezard, Anal Biochem. 119, 325 (1982). 12 L. Marcus and A. Schejter, Physiol. Plant PathoL 22, 1 (1983).
[39] Polygalacturonase from Corticium rolfsii
By KIYOSHI T A G A W A and AKIRA KAJI Polygalacturonase [poly(l,4-ot-o-galacturonide) glycanohydrolase, EC 3.2.1.15) catalyzes the random hydrolytic splitting of internal glycosidic a-1,4 linkages in t)-galacturonan chains of pectic substances. In Corticium rolfsii, this enzyme is produced inducibly in a medium containing D-galacturonic acid or pectin. The enzyme is unusually stable in acidic conditions and active at pH 2.5.1,2
Assay Method
Principle Two assay methods have generally been employed for the estimation of polygalacturonase activity: release of reducing groups from pectic acid and viscosity reduction of pectic acid. The viscometric assay is more sensitive than the reducing group assay, so it is convenient for estimation of the 1A. Kaji and K. Tagawa, J. Agric. Chem. Soc. Jpn. 40, 325 (1966). 2 A. Kaji and T. Okada, Arch. Biochem. Biophys. 131, 203 (1969).
METHODS IN ENZYMOLOGY, VOL. 161
Copyrisht© 1988by AcademicPress,Inc. All rightsof r~productionin any formre.fred.
362
PECTIN
[39]
minor activity. It cannot, however, be used for estimation of the activity in acidic conditions such as at pH below 3.0, where pectic acid forms a gel. In these conditions, it is better to use the reducing group assay using acid-soluble pectic acid 3 as a substrate.
Reducing Group Assay Method Reagents 1.0-0.8% acid-soluble pectic acid solution, pH 2.5 0.1 M citrate-phosphate buffer, pH 2.5 Suitably diluted enzyme solution 1 M Na2CO 3 Somogyi's copper reagent and arsenomolybdate reagent 4,5
Procedure. The reaction mixture contains 2 ml of the pectic acid solution, 0.5 ml of buffer, and 0.5 ml of enzyme solution. The reaction proceeds at 30 ° for 30 min. At the end of the time, 0.1 ml of Na2CO3 is added to stop further reaction. Reducing groups released are measured colorimetrically with the Somogyi-Nelson reagent 4,5 using D-galacturonic acid as a standard. Definition of Unit. One unit of polygalacturonase activity is defined as the amount of enzyme which will produce 1 #mol of reducing groups per minute at 30 ° . Viscometric Assay Method The optimum pH for activity of the polygalacturonase from C. rolfsii lies at 2.5. 2 Therefore, it is not possible to estimate accurately the enzyme activity by the viscometric assay. It is, however, useful for characterizing the enzyme in order to evaluate the viscosity reducing activity on pectic acid at pH 4.0.1,2 Reagents
1.0-0.8% sodium pectate solution, pH 4.0 0.1 M citrate-phosphate buffer, pH 4.0 Suitably diluted enzyme solution
Procedure. Sodium pectate solution (3.5 ml) and 1 ml of buffer are pipetted into an Ostwald viscometer (capillary length, 9 cm) kept at 30 °, 3Acid-solublepectic acid is prepared from pectin NF by the method ofR. M. McCreadyand C. G. SeegmiUer[Arch. Biochem. Biophys. 50, 440 (1954)]. Its average degree of polymerization is about 10. 4M. Somogyi,J. Biol. Chem. 195, 19 (1952). 5N. Nelson, J. Biol. Chem. 153, 375 (1944).
[39]
POLYGALACTURONASE FROM Corticium rolfsii
363
followed by addition of 0.5 ml of enzyme solution. Initial flow time is obtained by substituting water for enzyme solution. The flow time of the reaction mixture is taken at suitable intervals and the time for 50% viscosity reduction is determined graphically. A linear relationship is obtained between the enzyme concentration and the reciprocal of the time required to reach 50% of the original viscosity. Definition of Unit. One viscosimetric unit is arbitarily defined as the amount of enzyme that will reduce the viscosity of pectic acid by 50% in l min. Preparation and Purification Procedure
Cultivation of Organism Corticium rolfsii IFO 6146 (supplied by the Institute for Fermentation, Osaka, Japan) is cultured in a medium composed of 12.5 g of pectin, 10 g of peptone, 0.5 g of K2HPO4, 0.5 g of NH4NO3, 0.2 g of MgSO4" 7 H20, and 0.3 ml of 2% FeC1, in 1 liter of water. The initial pH is adjusted to 5.4 by addition of 1 N HC1. Cultivation is carried out at 28 ° for 72 hr under aerobic conditions in a jar fermenter. The mycelia are filtered off through cloth and the solution is centrifuged at 5 °. The crude enzyme solution thus obtained is purified as follows. Purification Procedure Unless otherwise specified, purification of the enzyme is carried out at 5 °"
Step 1: Salting Out with Ammonium Sulfate. To 10 liters of the crude enzyme solution, 6100 g of (NH4)2SO4 (0.85 saturation) is added with constant stirring. After standing for several hours, the resulting precipitate is collected by centrifugation, dissolved in 100 ml of water, and the solution dialyzed for 24 hr against water and then for 24 hr against 0.01 M citrate-phosphate buffer, pH 6.0. Step 2: DEAE-Cellulose Column Chromatography. The dialyzed solution (130 ml) is chromatographed on a column (4 × 50 cm) of DEAE-cellulose equilibrated with 0.01 M citrate-phosphate buffer, pH 6.0. After washing with the equilibrating buffer (600 ml), elution is performed with the same buffer containing 0.2 M NaC1. The active fraction (200 ml) is collected, dialyzed against water for 48 hr, and concentrated with polyethylene glycol (Mr 60,000). The concentrate is dialyzed against 0.1 M acetate buffer, pH 4.0, for 48 hr. Step 3: Affinity Chromatography on Crosslinked Pectic Acid. The concentrated enzyme solution (30 ml) is purified by affinity chromatography
364
PECTIN
[39]
TABLE I PURIFICATIONOF POLYGALACTURONASEFROMC. rolfsii
Specific Step Culture filtrate Salting out with ammonium sulfate DEAE-cellulos¢ chromatography Affinity chromatography Sephadex G-100 gel filtration
Volume (ml)
Activity (U/ml)
Protein (mg/ml)
10,000 130
1.8 72
3.4 17.1
activity (U/mg protein) 0.53 4.2
Yield (%) 100 52
30
208
5.2
40.0
34.7
10 12
332 265
3.0 2.1
110.7 126.2
18.4 17.7
on a column (2 × 20 cm) packed with pectic acid crosslinked by epichlorhydrin. 6 The column is equilibrated with 0.1 M acetate buffer, pH 4.0, and then the enzyme solution is added. After washing the column with the same buffer (100 ml), the enzyme is eluted with 0.2 M acetate buffer, pH 6.0. The active fraction (40 ml) is collected, dialyzed against water for 24 hr, and then against 0.01 M acetate buffer, pH 6.0, for 24 hr. On lyophilization, the enzyme gives a white powder and it is dissolved in l0 ml of water. Step 4: Gel Filtration on Sephadex G-IO0. The enzyme solution (10 ml) is applied to a Sephadex G-100 column (2 × 40 cm) that was previously equilibrated with 0.05 M acetate buffer, pH 6.0, and eluted with the same buffer. The enzyme is eluted as a single peak coincident with the elution of protein. The active fraction (12 ml) is collected and stored at - 2 0 o. The purified enzyme thus obtained appears homogeneous when examined by ultracentrifugation analysis 2 and has a specific activity of 126 U / m g of protein, representing an overall 240-fold purification. A summary of the above purification procedure is presented in Table I. Properties
Optimum pH. Maximum enzyme activity is obtained at pH 2.5 with acid-soluble pectic acid. It is also confirmed from measurement of the release of pectin ~ that the enzyme is most active at this pH region when it acts on plant tissues.l,2,a 6 L. Rexov~-Benkov[t and V. Tibensk~, Biochim. Biophys. Acta 268, 187 (1972). K. Tagawa and A. Kaji, Tech. Bull. Fac. Agric., Kagawa Univ. 17, 104 (1966). 8 A. Kaji, N. Mikuni, and K. T agawa, Tech. Bull. Fac. Agric., Kagawe Univ. 16, 137 (1965).
[39]
POLYGALACTURONASE FROM Corticium rolfsii
365
Stability. The purified enzyme is stable in solutions having a wide range of pH values from 2.0 to 8.0, 2 and can be stored below 5 ° for a month without any appreciable loss of the activity. 2 Substrate Specificity. The real substrate for the purified enzyme is pectic acid (nonmethylated polygalacturonan), because, at pH 4.0 where pectic acid is soluble, it hydrolyzes pectic acid more rapidly than acid-soluble pectic acid or pectin. A limited hydrolysis of pectin is attainable, 2 depending on its degree of esterification: pectin NF (68% esterified) 12%, methylated pectin (91% esterified) 1.5%. With prolonged incubation pectic acid is completely hydrolyzed to mono- and digalacturonic acids and the extent of its hydrolysis reaches 70%. 2 The enzyme attacks t~-l,4-1inked D-galacturonide chains of pectic substances in plant tissues. For this reason fungal maceration is observed in fruits, vegetables, and barks of young trees. ~,2,8 Comment. The fungal polygalacturonases have been used for production of pulps, juices, 9 and concentrates from fruits and vegetables, and baby and geriatric foods) ° Recently, this hydrolytic ability has been applied to biomass utilization and biotechnology for extraction of leaf proteins, m~liquiefaction of agricultural biomass,12 and isolation of plant protoplasts. ~3 For these purposes, the enzyme of C. rolfsii is particularly useful owing to the activity in low pH regions where the enzyme reaction proceeds nearly aseptically. Although pectolytic enzymes other than polygalacturonases from various origins show some activity in plant tissue maceration, singly or in combination, an enzyme preparation from Clostridiumfelsineum ~4reveals a strong macerating activity in spite of a lack of activity against purified pectin or pectic acid. This enzyme presumably acts on native pectin where galacturonan combines with other polysaccharides and should be useful for vegetable biomass utilization.
9 M. Manabe, A. Kaji, and T. Tarutani, J. Foodlnd. Soc. Jpn. 13, 269 (1966). ~oH. K. Sreenath, M. D. Frey, H. Scherz, and B. J. Radola, Biotechnol. Bioeng. 26, 788 0984). 1~ M. Oshima and K. Tagawa, unpublished observations. ~2G. Beldman, M. J. F. Leeuwen, A. G. J. Searle-Van Voragen, F. M. Rombouts, and W. Pilnir, Comm. Eur. Commun. EUR9940, 41 0985). ~aA. Ruesink, this series, Vol. 69, p. 69. 14A. Kaji, Bull. Agric. Chem. Soc. Jpn. 20, 8 (1956).
[39]
361
described procedure shows a very slight pectin lyase activity (increase in absorbance at 235 nm of 0.018 within 1 hr). Assay for Glycoproteins. PE I and PE II give positive reactions in a modified silver staining procedure for detecting glycoproteins on SDS-electrophoresis gels. 11 Furthermore, PE I is adsorbed to Con A-Sepharose (for PE II this was not tested). This indicates that pectinesterases from P. infestans are glycoproteins. Further Purification of PE I. The chemical properties of both proteins of PE I seem to be very similar. The double protein band cannot be separated by chromatography on hydroxylapatite, different ion exchangers (DEAE-Sepharose, CM-Sepharose), Con A-Sepharose and cross-linked polypectate, ~2 hydrophobic interaction chromatography on octyl sepharose, or adsorption on bentonite and chromatofocusing with Pharmacia Polybuffer exchanger PBE 118. H G. Dubray and G. Bezard, Anal Biochem. 119, 325 (1982). 12 L. Marcus and A. Schejter, Physiol. Plant PathoL 22, 1 (1983).
[39] Polygalacturonase from Corticium rolfsii
By KIYOSHI T A G A W A and AKIRA KAJI Polygalacturonase [poly(l,4-ot-o-galacturonide) glycanohydrolase, EC 3.2.1.15) catalyzes the random hydrolytic splitting of internal glycosidic a-1,4 linkages in t)-galacturonan chains of pectic substances. In Corticium rolfsii, this enzyme is produced inducibly in a medium containing D-galacturonic acid or pectin. The enzyme is unusually stable in acidic conditions and active at pH 2.5.1,2
Assay Method
Principle Two assay methods have generally been employed for the estimation of polygalacturonase activity: release of reducing groups from pectic acid and viscosity reduction of pectic acid. The viscometric assay is more sensitive than the reducing group assay, so it is convenient for estimation of the 1A. Kaji and K. Tagawa, J. Agric. Chem. Soc. Jpn. 40, 325 (1966). 2 A. Kaji and T. Okada, Arch. Biochem. Biophys. 131, 203 (1969).
METHODS IN ENZYMOLOGY, VOL. 161
Copyrisht© 1988by AcademicPress,Inc. All rightsof r~productionin any formre.fred.
362
PECTIN
[39]
minor activity. It cannot, however, be used for estimation of the activity in acidic conditions such as at pH below 3.0, where pectic acid forms a gel. In these conditions, it is better to use the reducing group assay using acid-soluble pectic acid 3 as a substrate.
Reducing Group Assay Method Reagents 1.0-0.8% acid-soluble pectic acid solution, pH 2.5 0.1 M citrate-phosphate buffer, pH 2.5 Suitably diluted enzyme solution 1 M Na2CO 3 Somogyi's copper reagent and arsenomolybdate reagent 4,5
Procedure. The reaction mixture contains 2 ml of the pectic acid solution, 0.5 ml of buffer, and 0.5 ml of enzyme solution. The reaction proceeds at 30 ° for 30 min. At the end of the time, 0.1 ml of Na2CO3 is added to stop further reaction. Reducing groups released are measured colorimetrically with the Somogyi-Nelson reagent 4,5 using D-galacturonic acid as a standard. Definition of Unit. One unit of polygalacturonase activity is defined as the amount of enzyme which will produce 1 #mol of reducing groups per minute at 30 ° . Viscometric Assay Method The optimum pH for activity of the polygalacturonase from C. rolfsii lies at 2.5. 2 Therefore, it is not possible to estimate accurately the enzyme activity by the viscometric assay. It is, however, useful for characterizing the enzyme in order to evaluate the viscosity reducing activity on pectic acid at pH 4.0.1,2 Reagents
1.0-0.8% sodium pectate solution, pH 4.0 0.1 M citrate-phosphate buffer, pH 4.0 Suitably diluted enzyme solution
Procedure. Sodium pectate solution (3.5 ml) and 1 ml of buffer are pipetted into an Ostwald viscometer (capillary length, 9 cm) kept at 30 °, 3Acid-solublepectic acid is prepared from pectin NF by the method ofR. M. McCreadyand C. G. SeegmiUer[Arch. Biochem. Biophys. 50, 440 (1954)]. Its average degree of polymerization is about 10. 4M. Somogyi,J. Biol. Chem. 195, 19 (1952). 5N. Nelson, J. Biol. Chem. 153, 375 (1944).
[39]
POLYGALACTURONASE FROM Corticium rolfsii
363
followed by addition of 0.5 ml of enzyme solution. Initial flow time is obtained by substituting water for enzyme solution. The flow time of the reaction mixture is taken at suitable intervals and the time for 50% viscosity reduction is determined graphically. A linear relationship is obtained between the enzyme concentration and the reciprocal of the time required to reach 50% of the original viscosity. Definition of Unit. One viscosimetric unit is arbitarily defined as the amount of enzyme that will reduce the viscosity of pectic acid by 50% in l min. Preparation and Purification Procedure
Cultivation of Organism Corticium rolfsii IFO 6146 (supplied by the Institute for Fermentation, Osaka, Japan) is cultured in a medium composed of 12.5 g of pectin, 10 g of peptone, 0.5 g of K2HPO4, 0.5 g of NH4NO3, 0.2 g of MgSO4" 7 H20, and 0.3 ml of 2% FeC1, in 1 liter of water. The initial pH is adjusted to 5.4 by addition of 1 N HC1. Cultivation is carried out at 28 ° for 72 hr under aerobic conditions in a jar fermenter. The mycelia are filtered off through cloth and the solution is centrifuged at 5 °. The crude enzyme solution thus obtained is purified as follows. Purification Procedure Unless otherwise specified, purification of the enzyme is carried out at 5 °"
Step 1: Salting Out with Ammonium Sulfate. To 10 liters of the crude enzyme solution, 6100 g of (NH4)2SO4 (0.85 saturation) is added with constant stirring. After standing for several hours, the resulting precipitate is collected by centrifugation, dissolved in 100 ml of water, and the solution dialyzed for 24 hr against water and then for 24 hr against 0.01 M citrate-phosphate buffer, pH 6.0. Step 2: DEAE-Cellulose Column Chromatography. The dialyzed solution (130 ml) is chromatographed on a column (4 × 50 cm) of DEAE-cellulose equilibrated with 0.01 M citrate-phosphate buffer, pH 6.0. After washing with the equilibrating buffer (600 ml), elution is performed with the same buffer containing 0.2 M NaC1. The active fraction (200 ml) is collected, dialyzed against water for 48 hr, and concentrated with polyethylene glycol (Mr 60,000). The concentrate is dialyzed against 0.1 M acetate buffer, pH 4.0, for 48 hr. Step 3: Affinity Chromatography on Crosslinked Pectic Acid. The concentrated enzyme solution (30 ml) is purified by affinity chromatography
364
PECTIN
[39]
TABLE I PURIFICATIONOF POLYGALACTURONASEFROMC. rolfsii
Specific Step Culture filtrate Salting out with ammonium sulfate DEAE-cellulos¢ chromatography Affinity chromatography Sephadex G-100 gel filtration
Volume (ml)
Activity (U/ml)
Protein (mg/ml)
10,000 130
1.8 72
3.4 17.1
activity (U/mg protein) 0.53 4.2
Yield (%) 100 52
30
208
5.2
40.0
34.7
10 12
332 265
3.0 2.1
110.7 126.2
18.4 17.7
on a column (2 × 20 cm) packed with pectic acid crosslinked by epichlorhydrin. 6 The column is equilibrated with 0.1 M acetate buffer, pH 4.0, and then the enzyme solution is added. After washing the column with the same buffer (100 ml), the enzyme is eluted with 0.2 M acetate buffer, pH 6.0. The active fraction (40 ml) is collected, dialyzed against water for 24 hr, and then against 0.01 M acetate buffer, pH 6.0, for 24 hr. On lyophilization, the enzyme gives a white powder and it is dissolved in l0 ml of water. Step 4: Gel Filtration on Sephadex G-IO0. The enzyme solution (10 ml) is applied to a Sephadex G-100 column (2 × 40 cm) that was previously equilibrated with 0.05 M acetate buffer, pH 6.0, and eluted with the same buffer. The enzyme is eluted as a single peak coincident with the elution of protein. The active fraction (12 ml) is collected and stored at - 2 0 o. The purified enzyme thus obtained appears homogeneous when examined by ultracentrifugation analysis 2 and has a specific activity of 126 U / m g of protein, representing an overall 240-fold purification. A summary of the above purification procedure is presented in Table I. Properties
Optimum pH. Maximum enzyme activity is obtained at pH 2.5 with acid-soluble pectic acid. It is also confirmed from measurement of the release of pectin ~ that the enzyme is most active at this pH region when it acts on plant tissues.l,2,a 6 L. Rexov~-Benkov[t and V. Tibensk~, Biochim. Biophys. Acta 268, 187 (1972). K. Tagawa and A. Kaji, Tech. Bull. Fac. Agric., Kagawa Univ. 17, 104 (1966). 8 A. Kaji, N. Mikuni, and K. T agawa, Tech. Bull. Fac. Agric., Kagawe Univ. 16, 137 (1965).
[39]
POLYGALACTURONASE FROM Corticium rolfsii
365
Stability. The purified enzyme is stable in solutions having a wide range of pH values from 2.0 to 8.0, 2 and can be stored below 5 ° for a month without any appreciable loss of the activity. 2 Substrate Specificity. The real substrate for the purified enzyme is pectic acid (nonmethylated polygalacturonan), because, at pH 4.0 where pectic acid is soluble, it hydrolyzes pectic acid more rapidly than acid-soluble pectic acid or pectin. A limited hydrolysis of pectin is attainable, 2 depending on its degree of esterification: pectin NF (68% esterified) 12%, methylated pectin (91% esterified) 1.5%. With prolonged incubation pectic acid is completely hydrolyzed to mono- and digalacturonic acids and the extent of its hydrolysis reaches 70%. 2 The enzyme attacks t~-l,4-1inked D-galacturonide chains of pectic substances in plant tissues. For this reason fungal maceration is observed in fruits, vegetables, and barks of young trees. ~,2,8 Comment. The fungal polygalacturonases have been used for production of pulps, juices, 9 and concentrates from fruits and vegetables, and baby and geriatric foods) ° Recently, this hydrolytic ability has been applied to biomass utilization and biotechnology for extraction of leaf proteins, m~liquiefaction of agricultural biomass,12 and isolation of plant protoplasts. ~3 For these purposes, the enzyme of C. rolfsii is particularly useful owing to the activity in low pH regions where the enzyme reaction proceeds nearly aseptically. Although pectolytic enzymes other than polygalacturonases from various origins show some activity in plant tissue maceration, singly or in combination, an enzyme preparation from Clostridiumfelsineum ~4reveals a strong macerating activity in spite of a lack of activity against purified pectin or pectic acid. This enzyme presumably acts on native pectin where galacturonan combines with other polysaccharides and should be useful for vegetable biomass utilization.
9 M. Manabe, A. Kaji, and T. Tarutani, J. Foodlnd. Soc. Jpn. 13, 269 (1966). ~oH. K. Sreenath, M. D. Frey, H. Scherz, and B. J. Radola, Biotechnol. Bioeng. 26, 788 0984). 1~ M. Oshima and K. Tagawa, unpublished observations. ~2G. Beldman, M. J. F. Leeuwen, A. G. J. Searle-Van Voragen, F. M. Rombouts, and W. Pilnir, Comm. Eur. Commun. EUR9940, 41 0985). ~aA. Ruesink, this series, Vol. 69, p. 69. 14A. Kaji, Bull. Agric. Chem. Soc. Jpn. 20, 8 (1956).