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[20] Cellulase preparation from Helix pomatia (snails)
[9.0]
MUCOPOLYSACCHARIDASES
173
T h e p H of the above solution is adjusted to 3.72 to 3.78 with 1:1 conc. HC1.
Procedure. Unknown enzyme preparations are dissolved in or diluted with phosphate buffer described above for standard enzyme solution. With each group of assays performed, a standard curve is included containing at least five points; 0, 2, 4, 6, and 8 units of enzyme per tube27 All determinations are performed in duplicate, and assays of unknown enzyme solutions are accepted only if two dilutions fall within the assay range. For best accuracy these should be between 2 and 8 units. T o perform assay, 1.0 ml. of enzyme solution is mixed with 1.0 ml. of HA solution (solutions are previously brought to 38 °) and incubated at 38 ° for 45 minutes. At the end of this time 10 ml. of acid albumin solution (at room temperature) is added rapidly, and exactly 5 minutes later (by stopwatch) the optical density is determined in a photoelectric colorimeter. When a series of determinations is performed, reagents are added to different tubes at 30-second intervals. A Coleman Junior spectrophotometer set at 600 mg with 15-mm. cuvettes has usually been employed, but the method is readily adapted to other comparable instruments. Calculation of Results. A standard curve is constructed b y plotting optical density against enzyme concentration per tube. Unknown samples are then determined from standard curve. The standard curve has been found to be constant from day to day, b u t it is redetermined daily to check on reagents. Accuracy. Accuracy varies at different points on standard curve; a t the 8-unit level the coefficient of variation has been found to be 5 %. 3~The activity is expressed in units per tube containing a reaction mixture of 2.0 ml. Thus the activity per milliliter as used in the previous publication2~is one-half of that given above.
[20] Cellulase Preparation from Helix pomatia (Snails) By GEORGE DE STEVENS1
Assay Method Principle. The development of a method for recording the activity of cellulase preparations in absolute or reproducible terms has not been accomplished, owing to the heterogeneity of the reaction and also to the effect of the physical state of the cellulose substrate. Pringsheim 2 identi1The author wishes to express his appreciation to the authoritiesof Hall Laboratory of Chemistry, Wesleyan University, for the facilities placed at his disposal. z H. Pringsheim, Z. physiol. Chem. 78, 266 (1912).
174
ENZYMES OF CARBOHYDRATE METABOLISM
[20]
fled the degradation product, glucose, as the osazone. Von Euler s and Karrer et al. ~ determined the extent of celluloysis through the Cu numbers. Karrer and Schubert 5 also followed the change by recording the viscosity of the solution at definite intervals. The procedure described herein has been used by Trager 6 and Ziese. 7 It is essentially the Willst~tter-Schudel s method as modified by Kline and Acree. 9 Reagents 100 rag. of cellulose. 2 ml. of phosphate buffer solution, pH 5.28. 3 drops of toluene. 0.4 ml. of enzyme solution. Add 5 ml. of water and store in the incubator at 36 °. Procedure. After the desired interval, titrate the reaction solution with NaOH or HCI solution until it is exactly neutral to phenolphthalein. Add 5 ml. of 0.1 N iodine from a buret, and then introduce dropwise 7.5 ml. of 0.1 N NaOH. Repeat this process until 22 ml. of Is solution and 35 ml. of NaOH have been added during 5 to 6 minutes. Let the reaction mixture stand for 5 minutes, make it acid with 0.1 N HC1, and titrate the liberated Is with Na2S~O3. The amount of glucose present in solution is calculated from the amount of Is liberated. Definition of Unit and Specific Activity. Karrer et al. 4 proposed that the cellulase unit be the amount of cellulase which in 50 ml. at a pH of 5.28 and a temperature of 36 ° will bring about 20% hydrolysis of 1 g. of fibrous cupraammonium rayon (Zellvag) in 96 hours. Purification Procedure Step 1 of the following procedure is based on the method described by Karrer, 1° and step 2 has been outlined by Grassmann et al. ~1 Step 1. The cellulose-splitting enzyme of Helix pomatia is found in the liver. The snails are suffocated by immersion in water in a closed vessel. The darkest-colored skin casing and the underlying light pale viscera H. yon Euler, Z. angew. Chem. 25, 250 (1912). P. Karrer, P. Schubert, and W. Wehrli, Helv. Chim. Aeta 8, 797 (1925). s p. Karrer and P. Schubert, Helv. Chim. Acta 9, 893 (1926). e W. Trager, Biochem. J. 26, 1762 (1932). 7 W. Ziese, Z. physiol. Chem. 205, 87 (1931). 8 R. Willst~tter and G. Schudel, Bet. 51, 780 (1918). 9 G. M. ls'llne and S. F. Acree, Ind. Eng. Chem. Anal. Ed. 2, 413 (1930). 10p. Karrer, Kolloid-Z. 52, 304 (1930). ~l W. Grassmann, L. Zechmeister, G. T6th, and R. Stadler, Ann. 508, 167 (1933).
[20]
CELLULASE PREPARATION FROM SNAILS
175
skin are then cut open. T h e gastrointestinal t r a c t swells out of itself and can be easily detected. I t is drawn out carefully, grasped on the b a c k end with forceps, and separated in the region of the mouth. T h e posterior opening is then closed with pinch-clamps while the anterior opening closes of itself in the area of incision. The correct execution of this operation leads to a m i n i m u m loss of contents. T h e collected intestinal pieces are placed in a m o r t a r with toluene, ground v e r y fine with sand, covered with a small a m o u n t of water, and filtered through asbestos. The resulting brown solution consists of a mixture of various enzymes plus intestinal m a t t e r and sugar. TABLE I SPECIFICITY OF FRACTIONATED ENZYME PREPARATIONSa
Cellulase Cellobiase Amount of hydrolysis in ml. of 0.02 N I~ Substrate Hydratcellulose CeUodextrin Cellotriose Cellobiose
Mg.
8 hours
24 hours
8 hours
24 hours
13.6 13.6 21.0 13.6
0.40 2.17 0.00 0.00
0.55 2.57 0.05 0.00
0.05 0.05 1.85 1.90
0.10 0.20 2.70 2.90
Note: Each solution contains 1.2 ml. of enzyme solution eluted to 4 ml. with 0.25 M phosphate buffer, pH 5.2. Temperature, 30 °. W. Grassmann, L. Zechmeister, G. T6th, and R. Stadler, Ann. 503, 167 (1933). T h e extraneous substituents are removed b y dialyzing the solution for several days. Cellulase has the r e m a r k a b l e p r o p e r t y of remaining quite stable in aqueous solution. Step 2. 33 ml. of the dialyzed enzyme solution is combined with 20 ml. of 0.2 N acetate mixture at p H 3.5. This is then treated with a suspension of a l u m i n u m m e t a h y d r o x i d e (corresponding to 370 mg. of A12Os), and water is added to m a k e a volume of 100 ml. After centrifugation, the residual solution is t r e a t e d with 10 ml. of an alumina suspension (corresponding to 250 mg. of Al~08) and centrifuged again. In this w a y a residual solution of high cellulase content with cellobiase being adsorbed on the A1~08 is obtained. I n Table I are recorded the d a t a demonstrating the degree of separation of enzymes attained.
Properties of Enzyme Specificity. Since, to date, no m e t h o d has been devised to isolate pure crystalline cellulase enzyme, v e r y little can be said concerning its absolute
176
ENZYMES OF CARBOHYDRATE METABOLISM
[9.0]
specificity. However, the evidence does demonstrate that cellulase catalyzes the breakdown of polysaccharides (cellulose) to glucose. That this enzyme is also instrumental in the hydrolysis of cellobiose to glucose has not, as yet, been proved unequivocally, although Grassmann's 11 work does indicate that such is not the case. Activators and Inhibitors. It has been shown 7 that 1% CuSO4 and 3% t t C N separately have practically no effect on the activity of snail ceUulase, but the mixture is strongly inhibitory. Glutathione and cysteine, but not H~S or Na2S203, are inhibitory when phosphate buffer is used, but not when citrate buffer of the same pH is used. In contrast to the snail enzyme, malt cellulase is not inhibited by CuSO4 plus HCN. Glutathione and cysteine, in phosphate or citrate buffer, also have no inhibitory effect on this enzyme preparations. Snail cellulase is also resistant to papain-HCN. Walseth 12 demonstrated that Aspergillus niger cellulase preparation is resistant to pentachlorophenol but is inactivated by other antiseptics. Effect of Temperature. The maximum temperature at which cellulase retains its activity has been found to differ with the source of the enzyme as well as with the method of preparation. Pringsheim ~ reports that bacterial cellulase has a temperature optimum at 46 °, but its activity extends through a temperature range of 20 to 70 °. The Helix pomatia cellulase 4 was found to exert its optimum activity at 36 °, with complete deactivation at 60 °. However, the snail cellulase prepared by Ziese 7 was found to be stable up to 100°. Walseth ~2maintained his A. niger cellulase preparation at a temperature of 47 ° for maximum enzyme activity. Kristiansson ~3 found that barley malt cellulase loses two-thirds of its activity on heating at 60° and pH 5 for 5 minutes. The cellulase from the roach, Cryptoeercus punctulatus, 6 was reported to give favorable results at a temperature of 26 °, but inactivation occurred readily at 100° or heating for 1 hour at 60°. Effect of pH. The data in Table II illustrate the optimal pH of cellulases from different sources. The apparent discrepancies outJined in this table are probably due to the fact that no pure enzyme preparation has been obtained. This becomes notable especially with regard to differences within the same species. Kinetics of Enzyme Reaction. Although crude enzyme preparations have been used, several workers, 6,7n°.~2 in the field have demonstrated that the reaction follows the unimolecular law. Karrer 1° followed the course of the reaction by determining the change in viscosity of the reaction mixture. 12 C. Walseth, Thesis, Institute of Paper Chemistry, Appleton, Wisconsin, 1948. '~ I. Kristiansson, Svensk Kern. Tidskr. 62, 133 (1950).
[20]
CELLULASE PREPARATION FROM SNAILS
177
TABLE II OPTIMAL PH OF VARIOUS CELLULASE PREPARATIONS Source Asperffillus oryzae Aspergillus oryzae Aspergillus niger Helix pomatia Helix pomatia Barley malt eellulase Helix pomatia Malt cellulase Merulius-lacrymans Cryptocerous punctulatus Earthworms
Optimum pH
Reference
4.5 4.7 4.5 5.2-5.5 5.28 5.0 4.51 3.68 4.7 5.3 5.5
Grassmann el al. a Freudenberg and Ploetzb Walsethc Freudenberg and Ploetzb Karrer et al. ~ Kristiansson° Ziese! ZieseY Ploetzg Tragerh Tracey i
W. Grassmann, L. Zeehmeister, G. TTth, and R. Stadler, Ann. 503, 167 (1933). b K. Freudenberg and T. Ploetz, Z. physiol. Chem. 259, 19 (1939). c C. Walseth, Thesis, Institute of Paper Chemistry, Appleton, Wisconsin, 1948. d p. Karrer, P. Shubert, and W. Wehrli, Helv. Chim. Acta 8, 797 (1925). I. Kristiansson, Svensk Kern. Tidskr. 62, 133 (1950). f W. Ziese, Z. physiol. Chem. 203, 87 (1931). g T. Ploetz, Z. physiol. Chem. 281, 183 (1939). W. Trager, Biochem. J. 26, 1762 (1932). M. V. Tracey, Biochem. J. 47, 431 (1950); Nature 167, 776 (1951).
Sources of Enzyme. The cellulase enzyme has also been reported to be present in potato sprouts, 14 in Neurospora sitophila,'S and in numerous wood-destroying fungi of the brown rot type--e.g., Polyporus sulfureous, Lenzites sepiaria, Daedalea quercina, and Lentinus lepideus. 16.i~ Cellobiase
The literature presents evidence for and against the separation of cellobiase from other enzymes. Pringsheim and his associates 17 found 14B. M. Singh, P. B. Mathar, and M. L. Mehta, Current Sci. (India) 7, 281 (1938). 15E. Tamura and Y. Takai, Japan. J. Nutrilion 8, 129 (1950). ,6 G. de Stevens and F. F. Nord, Fortschritte Chem. Forsch. 3, 70 (1954). 1~ While this manuscript was in press, the work of D. R. Whitaker [Arch. Biochem. and Biophys. 43, 253 (1953) ; 49, 259 (1954)] on the purification of Myrothecium verrucaria cellulase was brought to the author's attention. Whitaker reports that his enzyme preparation is homogeneous electrophoretically and in the ultracentrifuge. His cellulase preparation is purified by a sequence of concentration, precipitation by ammonium sulfate, fractionation with ethanol, and precipitation with polymethacrylic acid. " H . Pringsheim and J. Leibowitz, Z. physiol Chem. 131, 267 (1923) I H, Pringsheim and W. Kusenack, ibid. 137, 265 (1924).
178
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
that aged solutions of malt retained considerable power to hydrolyze lichenin, whereas the cellobiase activity was lost. The work of Karrer and Lier 18 indicated that lichenase and cellobiase could be separated by fractional absorption on aluminum hydroxide. However, the products of hydrolysis were very complex and a simple product was not identified. Freudenberg and Ploetzl* present similar results. Grassmann et al.~l and later Zechmeister et al. ~° applied the adsorption technique for the solution of a cellobiase solution. The Grassmann procedure is a continuation of step 2 for the purification of cellulase.
Purification Procedure The combined absorbed material (Al~03 plus adsorbed enzyme fraction) was stirred with 20 ml. of 0.20 M sodium bicarbonate solution and 80 ml. of water and centrifuged. This neutralized supernatant solution was employed for the activity determination. The results of this determination are outlined in Table I. Grassmann 11 further indicated that the cellobiase preparation from A s p e r g i l l u s oryzae exhibited some lichenase activity ( ~ cellobiase potency to 1/~ lichenase). The pH optimum of cellobiase is between 4 and 5. Enders and Saji 21 found that the enzyme from malt and barley exhibited optimum activity at pH 4 to 4.5 and a temperature of 37 °, whereas Grassmann et al. 11 and Zechmeister et al. 2° suggest working in the pH range of 4.5 to 5 and a temperature of 30 °. Smith, ~s working in Nord's laboratory, found that the mold M e r u l i u s l a c h r y m a n s hydrolyzed cellobiase initially to glucose at pH 4.28 and a temperature of 22°. Pringsheim 2 reports that cellobiase is deactivated at temperatures above 67 ° . 18 p. Karrer and H. Lier, HelD. Chim. Acta 8, 248 (1925). 19 K. Freudenberg and T. Ploetz, Z. physiol. Chem. 259, 19 (1939). s o L. Zechmeister, G. Tdth, P. Fiirth, and J. B£rsony, Enzymologia 9, 155 (1941). 21 C. Enders and T. Saji, Biochem. Z. 306, 430 (1940). 2~ V. M. Smith, Arch. Bioehem. 28, 446 (1949).
[21] Polysaccharide Synthesis from Disaccharides B y E D W A R D J. H E H R E
Introductory Note This section deals exclusively with enzymes known to catalyze, at least under certain conditions, the synthesis of high molecular weight polysaccharides from disaccharides. No attempt has been made to treat related biological systems that effect syntheses of series of oligosaccharides
MUCOPOLYSACCHARIDASES
173
T h e p H of the above solution is adjusted to 3.72 to 3.78 with 1:1 conc. HC1.
Procedure. Unknown enzyme preparations are dissolved in or diluted with phosphate buffer described above for standard enzyme solution. With each group of assays performed, a standard curve is included containing at least five points; 0, 2, 4, 6, and 8 units of enzyme per tube27 All determinations are performed in duplicate, and assays of unknown enzyme solutions are accepted only if two dilutions fall within the assay range. For best accuracy these should be between 2 and 8 units. T o perform assay, 1.0 ml. of enzyme solution is mixed with 1.0 ml. of HA solution (solutions are previously brought to 38 °) and incubated at 38 ° for 45 minutes. At the end of this time 10 ml. of acid albumin solution (at room temperature) is added rapidly, and exactly 5 minutes later (by stopwatch) the optical density is determined in a photoelectric colorimeter. When a series of determinations is performed, reagents are added to different tubes at 30-second intervals. A Coleman Junior spectrophotometer set at 600 mg with 15-mm. cuvettes has usually been employed, but the method is readily adapted to other comparable instruments. Calculation of Results. A standard curve is constructed b y plotting optical density against enzyme concentration per tube. Unknown samples are then determined from standard curve. The standard curve has been found to be constant from day to day, b u t it is redetermined daily to check on reagents. Accuracy. Accuracy varies at different points on standard curve; a t the 8-unit level the coefficient of variation has been found to be 5 %. 3~The activity is expressed in units per tube containing a reaction mixture of 2.0 ml. Thus the activity per milliliter as used in the previous publication2~is one-half of that given above.
[20] Cellulase Preparation from Helix pomatia (Snails) By GEORGE DE STEVENS1
Assay Method Principle. The development of a method for recording the activity of cellulase preparations in absolute or reproducible terms has not been accomplished, owing to the heterogeneity of the reaction and also to the effect of the physical state of the cellulose substrate. Pringsheim 2 identi1The author wishes to express his appreciation to the authoritiesof Hall Laboratory of Chemistry, Wesleyan University, for the facilities placed at his disposal. z H. Pringsheim, Z. physiol. Chem. 78, 266 (1912).
174
ENZYMES OF CARBOHYDRATE METABOLISM
[20]
fled the degradation product, glucose, as the osazone. Von Euler s and Karrer et al. ~ determined the extent of celluloysis through the Cu numbers. Karrer and Schubert 5 also followed the change by recording the viscosity of the solution at definite intervals. The procedure described herein has been used by Trager 6 and Ziese. 7 It is essentially the Willst~tter-Schudel s method as modified by Kline and Acree. 9 Reagents 100 rag. of cellulose. 2 ml. of phosphate buffer solution, pH 5.28. 3 drops of toluene. 0.4 ml. of enzyme solution. Add 5 ml. of water and store in the incubator at 36 °. Procedure. After the desired interval, titrate the reaction solution with NaOH or HCI solution until it is exactly neutral to phenolphthalein. Add 5 ml. of 0.1 N iodine from a buret, and then introduce dropwise 7.5 ml. of 0.1 N NaOH. Repeat this process until 22 ml. of Is solution and 35 ml. of NaOH have been added during 5 to 6 minutes. Let the reaction mixture stand for 5 minutes, make it acid with 0.1 N HC1, and titrate the liberated Is with Na2S~O3. The amount of glucose present in solution is calculated from the amount of Is liberated. Definition of Unit and Specific Activity. Karrer et al. 4 proposed that the cellulase unit be the amount of cellulase which in 50 ml. at a pH of 5.28 and a temperature of 36 ° will bring about 20% hydrolysis of 1 g. of fibrous cupraammonium rayon (Zellvag) in 96 hours. Purification Procedure Step 1 of the following procedure is based on the method described by Karrer, 1° and step 2 has been outlined by Grassmann et al. ~1 Step 1. The cellulose-splitting enzyme of Helix pomatia is found in the liver. The snails are suffocated by immersion in water in a closed vessel. The darkest-colored skin casing and the underlying light pale viscera H. yon Euler, Z. angew. Chem. 25, 250 (1912). P. Karrer, P. Schubert, and W. Wehrli, Helv. Chim. Aeta 8, 797 (1925). s p. Karrer and P. Schubert, Helv. Chim. Acta 9, 893 (1926). e W. Trager, Biochem. J. 26, 1762 (1932). 7 W. Ziese, Z. physiol. Chem. 205, 87 (1931). 8 R. Willst~tter and G. Schudel, Bet. 51, 780 (1918). 9 G. M. ls'llne and S. F. Acree, Ind. Eng. Chem. Anal. Ed. 2, 413 (1930). 10p. Karrer, Kolloid-Z. 52, 304 (1930). ~l W. Grassmann, L. Zechmeister, G. T6th, and R. Stadler, Ann. 508, 167 (1933).
[20]
CELLULASE PREPARATION FROM SNAILS
175
skin are then cut open. T h e gastrointestinal t r a c t swells out of itself and can be easily detected. I t is drawn out carefully, grasped on the b a c k end with forceps, and separated in the region of the mouth. T h e posterior opening is then closed with pinch-clamps while the anterior opening closes of itself in the area of incision. The correct execution of this operation leads to a m i n i m u m loss of contents. T h e collected intestinal pieces are placed in a m o r t a r with toluene, ground v e r y fine with sand, covered with a small a m o u n t of water, and filtered through asbestos. The resulting brown solution consists of a mixture of various enzymes plus intestinal m a t t e r and sugar. TABLE I SPECIFICITY OF FRACTIONATED ENZYME PREPARATIONSa
Cellulase Cellobiase Amount of hydrolysis in ml. of 0.02 N I~ Substrate Hydratcellulose CeUodextrin Cellotriose Cellobiose
Mg.
8 hours
24 hours
8 hours
24 hours
13.6 13.6 21.0 13.6
0.40 2.17 0.00 0.00
0.55 2.57 0.05 0.00
0.05 0.05 1.85 1.90
0.10 0.20 2.70 2.90
Note: Each solution contains 1.2 ml. of enzyme solution eluted to 4 ml. with 0.25 M phosphate buffer, pH 5.2. Temperature, 30 °. W. Grassmann, L. Zechmeister, G. T6th, and R. Stadler, Ann. 503, 167 (1933). T h e extraneous substituents are removed b y dialyzing the solution for several days. Cellulase has the r e m a r k a b l e p r o p e r t y of remaining quite stable in aqueous solution. Step 2. 33 ml. of the dialyzed enzyme solution is combined with 20 ml. of 0.2 N acetate mixture at p H 3.5. This is then treated with a suspension of a l u m i n u m m e t a h y d r o x i d e (corresponding to 370 mg. of A12Os), and water is added to m a k e a volume of 100 ml. After centrifugation, the residual solution is t r e a t e d with 10 ml. of an alumina suspension (corresponding to 250 mg. of Al~08) and centrifuged again. In this w a y a residual solution of high cellulase content with cellobiase being adsorbed on the A1~08 is obtained. I n Table I are recorded the d a t a demonstrating the degree of separation of enzymes attained.
Properties of Enzyme Specificity. Since, to date, no m e t h o d has been devised to isolate pure crystalline cellulase enzyme, v e r y little can be said concerning its absolute
176
ENZYMES OF CARBOHYDRATE METABOLISM
[9.0]
specificity. However, the evidence does demonstrate that cellulase catalyzes the breakdown of polysaccharides (cellulose) to glucose. That this enzyme is also instrumental in the hydrolysis of cellobiose to glucose has not, as yet, been proved unequivocally, although Grassmann's 11 work does indicate that such is not the case. Activators and Inhibitors. It has been shown 7 that 1% CuSO4 and 3% t t C N separately have practically no effect on the activity of snail ceUulase, but the mixture is strongly inhibitory. Glutathione and cysteine, but not H~S or Na2S203, are inhibitory when phosphate buffer is used, but not when citrate buffer of the same pH is used. In contrast to the snail enzyme, malt cellulase is not inhibited by CuSO4 plus HCN. Glutathione and cysteine, in phosphate or citrate buffer, also have no inhibitory effect on this enzyme preparations. Snail cellulase is also resistant to papain-HCN. Walseth 12 demonstrated that Aspergillus niger cellulase preparation is resistant to pentachlorophenol but is inactivated by other antiseptics. Effect of Temperature. The maximum temperature at which cellulase retains its activity has been found to differ with the source of the enzyme as well as with the method of preparation. Pringsheim ~ reports that bacterial cellulase has a temperature optimum at 46 °, but its activity extends through a temperature range of 20 to 70 °. The Helix pomatia cellulase 4 was found to exert its optimum activity at 36 °, with complete deactivation at 60 °. However, the snail cellulase prepared by Ziese 7 was found to be stable up to 100°. Walseth ~2maintained his A. niger cellulase preparation at a temperature of 47 ° for maximum enzyme activity. Kristiansson ~3 found that barley malt cellulase loses two-thirds of its activity on heating at 60° and pH 5 for 5 minutes. The cellulase from the roach, Cryptoeercus punctulatus, 6 was reported to give favorable results at a temperature of 26 °, but inactivation occurred readily at 100° or heating for 1 hour at 60°. Effect of pH. The data in Table II illustrate the optimal pH of cellulases from different sources. The apparent discrepancies outJined in this table are probably due to the fact that no pure enzyme preparation has been obtained. This becomes notable especially with regard to differences within the same species. Kinetics of Enzyme Reaction. Although crude enzyme preparations have been used, several workers, 6,7n°.~2 in the field have demonstrated that the reaction follows the unimolecular law. Karrer 1° followed the course of the reaction by determining the change in viscosity of the reaction mixture. 12 C. Walseth, Thesis, Institute of Paper Chemistry, Appleton, Wisconsin, 1948. '~ I. Kristiansson, Svensk Kern. Tidskr. 62, 133 (1950).
[20]
CELLULASE PREPARATION FROM SNAILS
177
TABLE II OPTIMAL PH OF VARIOUS CELLULASE PREPARATIONS Source Asperffillus oryzae Aspergillus oryzae Aspergillus niger Helix pomatia Helix pomatia Barley malt eellulase Helix pomatia Malt cellulase Merulius-lacrymans Cryptocerous punctulatus Earthworms
Optimum pH
Reference
4.5 4.7 4.5 5.2-5.5 5.28 5.0 4.51 3.68 4.7 5.3 5.5
Grassmann el al. a Freudenberg and Ploetzb Walsethc Freudenberg and Ploetzb Karrer et al. ~ Kristiansson° Ziese! ZieseY Ploetzg Tragerh Tracey i
W. Grassmann, L. Zeehmeister, G. TTth, and R. Stadler, Ann. 503, 167 (1933). b K. Freudenberg and T. Ploetz, Z. physiol. Chem. 259, 19 (1939). c C. Walseth, Thesis, Institute of Paper Chemistry, Appleton, Wisconsin, 1948. d p. Karrer, P. Shubert, and W. Wehrli, Helv. Chim. Acta 8, 797 (1925). I. Kristiansson, Svensk Kern. Tidskr. 62, 133 (1950). f W. Ziese, Z. physiol. Chem. 203, 87 (1931). g T. Ploetz, Z. physiol. Chem. 281, 183 (1939). W. Trager, Biochem. J. 26, 1762 (1932). M. V. Tracey, Biochem. J. 47, 431 (1950); Nature 167, 776 (1951).
Sources of Enzyme. The cellulase enzyme has also been reported to be present in potato sprouts, 14 in Neurospora sitophila,'S and in numerous wood-destroying fungi of the brown rot type--e.g., Polyporus sulfureous, Lenzites sepiaria, Daedalea quercina, and Lentinus lepideus. 16.i~ Cellobiase
The literature presents evidence for and against the separation of cellobiase from other enzymes. Pringsheim and his associates 17 found 14B. M. Singh, P. B. Mathar, and M. L. Mehta, Current Sci. (India) 7, 281 (1938). 15E. Tamura and Y. Takai, Japan. J. Nutrilion 8, 129 (1950). ,6 G. de Stevens and F. F. Nord, Fortschritte Chem. Forsch. 3, 70 (1954). 1~ While this manuscript was in press, the work of D. R. Whitaker [Arch. Biochem. and Biophys. 43, 253 (1953) ; 49, 259 (1954)] on the purification of Myrothecium verrucaria cellulase was brought to the author's attention. Whitaker reports that his enzyme preparation is homogeneous electrophoretically and in the ultracentrifuge. His cellulase preparation is purified by a sequence of concentration, precipitation by ammonium sulfate, fractionation with ethanol, and precipitation with polymethacrylic acid. " H . Pringsheim and J. Leibowitz, Z. physiol Chem. 131, 267 (1923) I H, Pringsheim and W. Kusenack, ibid. 137, 265 (1924).
178
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
that aged solutions of malt retained considerable power to hydrolyze lichenin, whereas the cellobiase activity was lost. The work of Karrer and Lier 18 indicated that lichenase and cellobiase could be separated by fractional absorption on aluminum hydroxide. However, the products of hydrolysis were very complex and a simple product was not identified. Freudenberg and Ploetzl* present similar results. Grassmann et al.~l and later Zechmeister et al. ~° applied the adsorption technique for the solution of a cellobiase solution. The Grassmann procedure is a continuation of step 2 for the purification of cellulase.
Purification Procedure The combined absorbed material (Al~03 plus adsorbed enzyme fraction) was stirred with 20 ml. of 0.20 M sodium bicarbonate solution and 80 ml. of water and centrifuged. This neutralized supernatant solution was employed for the activity determination. The results of this determination are outlined in Table I. Grassmann 11 further indicated that the cellobiase preparation from A s p e r g i l l u s oryzae exhibited some lichenase activity ( ~ cellobiase potency to 1/~ lichenase). The pH optimum of cellobiase is between 4 and 5. Enders and Saji 21 found that the enzyme from malt and barley exhibited optimum activity at pH 4 to 4.5 and a temperature of 37 °, whereas Grassmann et al. 11 and Zechmeister et al. 2° suggest working in the pH range of 4.5 to 5 and a temperature of 30 °. Smith, ~s working in Nord's laboratory, found that the mold M e r u l i u s l a c h r y m a n s hydrolyzed cellobiase initially to glucose at pH 4.28 and a temperature of 22°. Pringsheim 2 reports that cellobiase is deactivated at temperatures above 67 ° . 18 p. Karrer and H. Lier, HelD. Chim. Acta 8, 248 (1925). 19 K. Freudenberg and T. Ploetz, Z. physiol. Chem. 259, 19 (1939). s o L. Zechmeister, G. Tdth, P. Fiirth, and J. B£rsony, Enzymologia 9, 155 (1941). 21 C. Enders and T. Saji, Biochem. Z. 306, 430 (1940). 2~ V. M. Smith, Arch. Bioehem. 28, 446 (1949).
[21] Polysaccharide Synthesis from Disaccharides B y E D W A R D J. H E H R E
Introductory Note This section deals exclusively with enzymes known to catalyze, at least under certain conditions, the synthesis of high molecular weight polysaccharides from disaccharides. No attempt has been made to treat related biological systems that effect syntheses of series of oligosaccharides