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Synthesis of alkyl β-mannosides from mannobiose by Aspergillus niger β-mannosidase
JOURNALOF FERMENTATIONAND Vol. 80, No. 5, 510-512. 1995
BIOENGINEERWC
Synthesis of Alkyl ,&Mannosides from Mannobiose Aspergillus niger ,&Mannosidase HIROTAKA Institute of Applied Biochemistry,
by
ITOH AND YOSHI KAMIYAMA* University of Tsukuba, Tsukuba-shi,
Ibaraki 305, Japan
Received 8 March 1995/Accepted 4 September 1995
The reaction conditions for synthesis of various alkyl ,B-mannosides by transmannosylation with Aspergillus were studied. Maximum yield of methyl /I-mannoside expressed in mol% based on initial mannobiose was 81% in the presence of 50% (v/v) methanol. The longer carbon chains were, the less the yield of corresponding mannoside became. Octyl ,&mannoside, a new glycoside, was successfully synthesized in a 2 mol% yield in the presence of 90% (v/v) octanol.
niger ,&mannosidase
[Key words:
alkyl $-mannosides,
transmannosylation
reaction,
Aspergillus niger $mannosidase,
crystalline
mannobiose]
alkyl ,3-mannosides have not been investigated. In the present paper, we described the suitable reaction conditions in water-miscible alcohols (methanolpropanol) and water-immiscible alcohols (butanoloctanol) using Cellulosin HC I?-mannosidase. Crystalline mannobiose (j-l,4 mannodisaccharide) as substrate was prepared by the hydrolysis of copra meal (gift from San. Migel. Corp., Philippines) by ,3-mannanase from Penicillium purprogenum no. 617 (12, 13). Cellulosin HC was dissolved in 0.1 M acetate buffer (pH 4.0) and used as crude ,3-mannosidase. One unit of ;3-mannosidase activity was defined as the amount of enzyme releasing one ;tmol of p-nitrophenol per min (11). The reaction mixture (2 ml) of water-miscible alcohol containing ;3-mannosidase (4 units) and mannobiose (3.42-342 mg) in 0.1 M acetate buffer (pH 4.0) and alcohol (O-l.5 ml) was incubated at 30°C in screw-capped tube. The reaction mixture (l-10 ml) of water-immiscible alcohol containing 1.0 ml of 0.1 M acatete buffer solution (pH 4.0) having $mannosidase (2 units), mannobiose (17.1-171 mg) and alcohol (O-9ml) was incubated at 30°C in hermetically sealed IOml-glass flask with magnetic stirrer. Samples were collected at appropriate intervals, heated at 100°C for 5 min for inactivation of the enzyme, and, to which, internal standard (inositol) for quantitative analysis was added, then filtered by membrane (Ultrafree C3GC, Millipore Ltd., Tokyo), dried at reduced pressure, and analyzed by GLC (14). The amounts of transfer products were determined according to a calibration curve prepared by a ratio of peak area of methyl a-mannoside to inositol. At the initial stage of the reaction, a transfer product was formed and was followed by gradual hydrolysis. The times at which the transfer product formation reached a maximum were 3-6 h in water-miscible alcohol and 3 h in water-immiscible alcohol, regardless of those alcohol concentrations. Figure 1 shows the effect of methanol concentration on the maximum yield of methyl j-mannoside expressed in mol,%’ based on initial mannobiose. The highest yield of methyl ,+mannoside, 81%, was obtained in the presence of 50% (v/v) methanol. In this condition, 74% of the residual activity was retained after 24 h. The yield increased with increasing concentration of the acceptor (alcohol), however, further the increase of the acceptor
From the standpoint of the effective utilization of waste materials, the enzymatic hydrolyses of copra meal has been studied in our laboratory. Copra meal is the residual cake of coconut oil extraction and contains about 50% mannan. To utilize the hydrolysis products, mannooligosaccharides, synthesis of alkyl $mannoside was investigated. Higher alkyl glycosides are of interest in connection with surfactants (1). Generally, the chemical approach for synthesizing these compounds gives a low yield with the mixture of (Y-and i3-anomers (1). Especially, mannoside is difficult to synthesize because of its 1,2-cis-arrangement and the stereoelectronically disfavored anomeric equatorial C-O linkage (2, 3). Recently, their preparation by chemical methods was well developed with stereoselectivity but still requires multi-step procedures (2). On the other hand, the enzymatic method for the synthesis of glycoside (4) is now considered very promising since it is carried out under mild conditions by a simple one-step reaction process with regio- and stereoselectivities. In general, two types of enzymes are used for this purpose: the glycosyltransferases and the glycosylhydrolases. The former is expensive and its use is confined to a limited number of substrates. On the other hand, the latter is extensivelly used and many of them are commercially available from different sources (5). Various glycosidases have been shown to transglycosylate effectively using alcohols as acceptors (6-10). In our laboratory for example, ,3-xylosidase has been used for the preparation of various alkyl ,9-xylosides using xylobiose as a substrate (10). In previous studies, we reported screening for ,3-mannosidase (EC 3.2.1.25) with transmannosylation capacity and f-mannosidase of crude commercial enzyme, Cellulosin HC (Hankyu Bioindustry Co. Ltd., Osaka) derived from Aspergillus niger, was shown to have the best capacity for transmannosylation in various alcohols (11). Recently, synthesis of several alkyl (methyl, ethyl, ipropyl and butyl) and hydroxyalkyl $-mannosides was also reported from p-nitrophenyl $-mannoside and corresponding alcohols using $mannosidase from snails (3). However, reaction conditions for synthesis of these * Corresponding author. 510
NOTES
0
20 Cont.
40 of methanol
I
60
5
( %.viv )
FIG. 1. Effect of methanol concentration on the maximum yield of methyl F-mannoside. The reaction mixture (2.0ml), containing 34.2 mg of mannobiose, 0.5 ml of enzyme solution (4 units), 1.5 ml of methanol (O-75%, v/v) and 0.1 M acetate buffer (pH 4.0), was incubated at 30°C.
concentration then inactivated the enzyme which resulted in decreasing the yield. The same results were also obtained when ethanol and propanol were used as acceptors for the reaction. Figure 2 shows the effect of hexanol (as water-immiscible alcohol) concentration on the maximum yield of hexyl /Gmannoside. The highest yield of hexyl $-mannoside, 18%, was obtained in the presence of 90% (v/v) hexanol. In this condition, the enzyme was stable. The yield increased with increasing the acceptor concentration up to 90% (v/v). Synthesized hexyl ,3-mannoside had high affinity for hexanol than water and moved into organic (hexanol) phase, but on the other hand, the substrate and the enzyme had high affinity for water than organic phase to proceed in the direction of hydrolysis of substrate. This fact resulted in decreasing the yield of transfer product compared to that from water-miscible alcohol. Taking into account the solubilities of mannobiose and the enzyme in reaction mixture, the optimum concentration of hexanol was assumed to be 90% (v/v). In the case of water-miscible alcohol, a maximum yield (%) of transfer product was independent of initial mannobiose concentration (5-500 mM). On the other
20 Cont.
40 of hexanol
60
10
20
15
Reaction Iline
80
511
25
( ti )
FIG. 3. Time course of the formation of alkyl ,3-mannoside in various alcohols. The reaction carried out at optimum condition. Symbols: 0, methyl B-mannoside; 0, ethyl p-mannoside; q , propyl +mannoside; W, butyl B-mannoside; Q, pentyl ;3-mannoside; A, hexyl $-mannoside; A, heptyl $mannoside; 3, octyl ,3-mannoside.
hand, in water-immiscible alcohol, the yield slightly decreased when the concentration of mannobiose in the aqueous phase is more than 100mM. Possibly, this result is due to the excess of substrate against the acceptor in water phase. When the transfer product formation reached a maximum, about 10% of mannobiose still remained unreacted. As a result of this investigation, the maximum yields (%) of alkyl p-mannosides relating to acceptors of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol and octanol were 81, 65, 59, 42, 34, 18, 5 and 2%, respectively. The longer carbon chains were, the less the yield of corresponding mannoside became. Figure 3 shows the formations of the transfer products from various alcohols. Octyl +mannoside so far has not been reported to be synthesized (11). According to this optimum condition, the synthesis of octyl jmannoside, a new product, was successful. In a two phase system using an acceptor of a waterimmiscible alcohol, solubility of alcohol to water phase is considered to be the most important factor for yield of a transfer product. As mentioned above, the yield of octyl $-mannoside was 2%. In case of octyl p-galactoside (l), its yield was reported in 50.4% when o-nitrophenyl ,V-galactoside and octanol were incubated in 55 ~01% acetone in phosphate buffer using j-galactosidase from Escherichia coli. The effect of organic cosolvent on the yield of glycosides was reviewed (15). In the case of octyl ,3-mannoside, acetone, acetonitrile, N,N-dimethylformamide, and dimethylsulfoxide were also investigated but no effect was observed. To increase the yield of a transfer product, especially from higher alcohol, some further investigations have to be carried out to find suitable condition for the reaction.
80
( %, v/v )
FIG. 2. Effect of hexanol concentration on the maximum yield hexyl ,3-mannoside. The reaction mixture, containing 17.1 mg mannobiose, 0.25 ml of enzyme solution (2 units), 0.75 ml of 0.1 acetate buffer (pH 4.0) and hexanol (S-90%, v/v), was incubated 30°C.
REFERENCES of of M at
1. Matsumura, S., Kubokawa, H., and Yosbikawa, S.: Enzymatic synthesis of w-hydroxyalkyl and n-alkyl +-D-galactopyranosides by the transglycosylation reaction of $-galactosidase. Chem. Lett., 945-948 (1991). 2. Ito, Y. and Ogawa, T.: A novel approach to the stereoselective
512
3.
4.
5.
6.
7.
8.
ITOH
AND KAMIYAMA
synthesis of p-mannosides. Angew. Chem. Int. Ed. Engl., 33, 1765-1767 (1994). Taubken, N. and Thiem, J.: Enzymatic synthesis of alkyl and hydroxyalkyl ,3-D-mannopyranosides. Synthesis, 6, 517-518 (1992). Wong, C.-H., Halcomb, R. L., Ichikawa, Y., and Kajimoto, T.: Enzymes in organic synthesis: application to the problems of carbohydrate recognition. II. Angew. Chem. Int. Ed. Engl., 34, 521-546 (1995). Vie, G. and Crout, D. H. G.: Synthesis of glucosidic derivatives with a spacer arm by reverse hydrolysis using almond [Y-Dalucosidase. Tetrahedron: Asvmmetrv. 5. 2513-2516 (1994). Goi, Y., Hashimoto, T., Mituo, N:,‘and Satoh, T.: Eniymic formation of ,i-alkyl glycosides by ;3-galactosidase from Aspergillus oryzue and its application to the synthesis of chemically unstable cardiac glycosides. Chem. Pharm. Bull., 33, 18081814 (1985). Vulfson, E. N., Pate], R., and Law, B. A.: Alkyl-,j-glucoside synthesis in a water-organic two-phase system. Biotechnol. Lett., 12, 397-402 (1990). Tricone, A., Nicolaus, B., Lima, L., Morzillo, P., de Rosa, M., and Gambacorta, A.: Enzyme-catalyzed synthesis of alkyl ,3-pglycosides with crude homogenate of Sulfolobus solfataricus. Biotechnol. Lett., 13, 235-240 (1991).
J. FERMENT. BIOENG..
9. Selisko, B., Ulbrich, R., and Schellenberger, A.: Invertase-catalyzed reactions in alcoholic solutions. Biotechnol. Bioeng., 35, 1006-1010 (1990). 10. Shinoyama, H., Kamiyama, Y., and Yasui, T.: Enzymatic synthesis of alkyl ,%xylosides from xylobiose by application of the transxylosylation reaction of Aspergillus niger $xylosidase. Agric. Biol. Chem., 52, 2197-2202 (1988). 11. Holazo, A., Shinoyama, H., Kamiyama, Y., and Yasui, T.: Screening for ;3-mannosidase with transmannosylation capacity. Biosci. Biotech. Biochem., 56, 822-824 (1992). 12. Kusakabe, I., Park, G. G., Kumita, N., Yasui, T., and Murakami, K.: Specificity of 3-mannanase from Peniciliium purpurogenum for konjac glucomannan. Agric. Biol. Chem., 52, 519-524 (1988). 13. Kusakabe, I., Zama, M., Park, G. G., Tubaki, K., and Murakami, K.: Preparation of ,3-1,4-mannobiose from copra meal by a mannanase from Penicillium purpurogenum. Agric. Biol. Chem., 51, 2825-2826 (1987). 14. Iizuka, Y., Kamiyama, Y., and Yasui, T.: Kinetic comparison of fungal ,3-xylosidases in the condensation reaction of xylose. Seibutsu-koaaku. 71. 325-331 (1993). 15. Yasui, T. aid Iiiuka, Y.: Transglycosylation and glucosyl condensation by enzyme in organic cosolvent systems. Nippon Nogeikagaku Kaishi, 65, 1107-l 110 (1991). (in Japanese)
BIOENGINEERWC
Synthesis of Alkyl ,&Mannosides from Mannobiose Aspergillus niger ,&Mannosidase HIROTAKA Institute of Applied Biochemistry,
by
ITOH AND YOSHI KAMIYAMA* University of Tsukuba, Tsukuba-shi,
Ibaraki 305, Japan
Received 8 March 1995/Accepted 4 September 1995
The reaction conditions for synthesis of various alkyl ,B-mannosides by transmannosylation with Aspergillus were studied. Maximum yield of methyl /I-mannoside expressed in mol% based on initial mannobiose was 81% in the presence of 50% (v/v) methanol. The longer carbon chains were, the less the yield of corresponding mannoside became. Octyl ,&mannoside, a new glycoside, was successfully synthesized in a 2 mol% yield in the presence of 90% (v/v) octanol.
niger ,&mannosidase
[Key words:
alkyl $-mannosides,
transmannosylation
reaction,
Aspergillus niger $mannosidase,
crystalline
mannobiose]
alkyl ,3-mannosides have not been investigated. In the present paper, we described the suitable reaction conditions in water-miscible alcohols (methanolpropanol) and water-immiscible alcohols (butanoloctanol) using Cellulosin HC I?-mannosidase. Crystalline mannobiose (j-l,4 mannodisaccharide) as substrate was prepared by the hydrolysis of copra meal (gift from San. Migel. Corp., Philippines) by ,3-mannanase from Penicillium purprogenum no. 617 (12, 13). Cellulosin HC was dissolved in 0.1 M acetate buffer (pH 4.0) and used as crude ,3-mannosidase. One unit of ;3-mannosidase activity was defined as the amount of enzyme releasing one ;tmol of p-nitrophenol per min (11). The reaction mixture (2 ml) of water-miscible alcohol containing ;3-mannosidase (4 units) and mannobiose (3.42-342 mg) in 0.1 M acetate buffer (pH 4.0) and alcohol (O-l.5 ml) was incubated at 30°C in screw-capped tube. The reaction mixture (l-10 ml) of water-immiscible alcohol containing 1.0 ml of 0.1 M acatete buffer solution (pH 4.0) having $mannosidase (2 units), mannobiose (17.1-171 mg) and alcohol (O-9ml) was incubated at 30°C in hermetically sealed IOml-glass flask with magnetic stirrer. Samples were collected at appropriate intervals, heated at 100°C for 5 min for inactivation of the enzyme, and, to which, internal standard (inositol) for quantitative analysis was added, then filtered by membrane (Ultrafree C3GC, Millipore Ltd., Tokyo), dried at reduced pressure, and analyzed by GLC (14). The amounts of transfer products were determined according to a calibration curve prepared by a ratio of peak area of methyl a-mannoside to inositol. At the initial stage of the reaction, a transfer product was formed and was followed by gradual hydrolysis. The times at which the transfer product formation reached a maximum were 3-6 h in water-miscible alcohol and 3 h in water-immiscible alcohol, regardless of those alcohol concentrations. Figure 1 shows the effect of methanol concentration on the maximum yield of methyl j-mannoside expressed in mol,%’ based on initial mannobiose. The highest yield of methyl ,+mannoside, 81%, was obtained in the presence of 50% (v/v) methanol. In this condition, 74% of the residual activity was retained after 24 h. The yield increased with increasing concentration of the acceptor (alcohol), however, further the increase of the acceptor
From the standpoint of the effective utilization of waste materials, the enzymatic hydrolyses of copra meal has been studied in our laboratory. Copra meal is the residual cake of coconut oil extraction and contains about 50% mannan. To utilize the hydrolysis products, mannooligosaccharides, synthesis of alkyl $mannoside was investigated. Higher alkyl glycosides are of interest in connection with surfactants (1). Generally, the chemical approach for synthesizing these compounds gives a low yield with the mixture of (Y-and i3-anomers (1). Especially, mannoside is difficult to synthesize because of its 1,2-cis-arrangement and the stereoelectronically disfavored anomeric equatorial C-O linkage (2, 3). Recently, their preparation by chemical methods was well developed with stereoselectivity but still requires multi-step procedures (2). On the other hand, the enzymatic method for the synthesis of glycoside (4) is now considered very promising since it is carried out under mild conditions by a simple one-step reaction process with regio- and stereoselectivities. In general, two types of enzymes are used for this purpose: the glycosyltransferases and the glycosylhydrolases. The former is expensive and its use is confined to a limited number of substrates. On the other hand, the latter is extensivelly used and many of them are commercially available from different sources (5). Various glycosidases have been shown to transglycosylate effectively using alcohols as acceptors (6-10). In our laboratory for example, ,3-xylosidase has been used for the preparation of various alkyl ,9-xylosides using xylobiose as a substrate (10). In previous studies, we reported screening for ,3-mannosidase (EC 3.2.1.25) with transmannosylation capacity and f-mannosidase of crude commercial enzyme, Cellulosin HC (Hankyu Bioindustry Co. Ltd., Osaka) derived from Aspergillus niger, was shown to have the best capacity for transmannosylation in various alcohols (11). Recently, synthesis of several alkyl (methyl, ethyl, ipropyl and butyl) and hydroxyalkyl $-mannosides was also reported from p-nitrophenyl $-mannoside and corresponding alcohols using $mannosidase from snails (3). However, reaction conditions for synthesis of these * Corresponding author. 510
NOTES
0
20 Cont.
40 of methanol
I
60
5
( %.viv )
FIG. 1. Effect of methanol concentration on the maximum yield of methyl F-mannoside. The reaction mixture (2.0ml), containing 34.2 mg of mannobiose, 0.5 ml of enzyme solution (4 units), 1.5 ml of methanol (O-75%, v/v) and 0.1 M acetate buffer (pH 4.0), was incubated at 30°C.
concentration then inactivated the enzyme which resulted in decreasing the yield. The same results were also obtained when ethanol and propanol were used as acceptors for the reaction. Figure 2 shows the effect of hexanol (as water-immiscible alcohol) concentration on the maximum yield of hexyl /Gmannoside. The highest yield of hexyl $-mannoside, 18%, was obtained in the presence of 90% (v/v) hexanol. In this condition, the enzyme was stable. The yield increased with increasing the acceptor concentration up to 90% (v/v). Synthesized hexyl ,3-mannoside had high affinity for hexanol than water and moved into organic (hexanol) phase, but on the other hand, the substrate and the enzyme had high affinity for water than organic phase to proceed in the direction of hydrolysis of substrate. This fact resulted in decreasing the yield of transfer product compared to that from water-miscible alcohol. Taking into account the solubilities of mannobiose and the enzyme in reaction mixture, the optimum concentration of hexanol was assumed to be 90% (v/v). In the case of water-miscible alcohol, a maximum yield (%) of transfer product was independent of initial mannobiose concentration (5-500 mM). On the other
20 Cont.
40 of hexanol
60
10
20
15
Reaction Iline
80
511
25
( ti )
FIG. 3. Time course of the formation of alkyl ,3-mannoside in various alcohols. The reaction carried out at optimum condition. Symbols: 0, methyl B-mannoside; 0, ethyl p-mannoside; q , propyl +mannoside; W, butyl B-mannoside; Q, pentyl ;3-mannoside; A, hexyl $-mannoside; A, heptyl $mannoside; 3, octyl ,3-mannoside.
hand, in water-immiscible alcohol, the yield slightly decreased when the concentration of mannobiose in the aqueous phase is more than 100mM. Possibly, this result is due to the excess of substrate against the acceptor in water phase. When the transfer product formation reached a maximum, about 10% of mannobiose still remained unreacted. As a result of this investigation, the maximum yields (%) of alkyl p-mannosides relating to acceptors of methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol and octanol were 81, 65, 59, 42, 34, 18, 5 and 2%, respectively. The longer carbon chains were, the less the yield of corresponding mannoside became. Figure 3 shows the formations of the transfer products from various alcohols. Octyl +mannoside so far has not been reported to be synthesized (11). According to this optimum condition, the synthesis of octyl jmannoside, a new product, was successful. In a two phase system using an acceptor of a waterimmiscible alcohol, solubility of alcohol to water phase is considered to be the most important factor for yield of a transfer product. As mentioned above, the yield of octyl $-mannoside was 2%. In case of octyl p-galactoside (l), its yield was reported in 50.4% when o-nitrophenyl ,V-galactoside and octanol were incubated in 55 ~01% acetone in phosphate buffer using j-galactosidase from Escherichia coli. The effect of organic cosolvent on the yield of glycosides was reviewed (15). In the case of octyl ,3-mannoside, acetone, acetonitrile, N,N-dimethylformamide, and dimethylsulfoxide were also investigated but no effect was observed. To increase the yield of a transfer product, especially from higher alcohol, some further investigations have to be carried out to find suitable condition for the reaction.
80
( %, v/v )
FIG. 2. Effect of hexanol concentration on the maximum yield hexyl ,3-mannoside. The reaction mixture, containing 17.1 mg mannobiose, 0.25 ml of enzyme solution (2 units), 0.75 ml of 0.1 acetate buffer (pH 4.0) and hexanol (S-90%, v/v), was incubated 30°C.
REFERENCES of of M at
1. Matsumura, S., Kubokawa, H., and Yosbikawa, S.: Enzymatic synthesis of w-hydroxyalkyl and n-alkyl +-D-galactopyranosides by the transglycosylation reaction of $-galactosidase. Chem. Lett., 945-948 (1991). 2. Ito, Y. and Ogawa, T.: A novel approach to the stereoselective
512
3.
4.
5.
6.
7.
8.
ITOH
AND KAMIYAMA
synthesis of p-mannosides. Angew. Chem. Int. Ed. Engl., 33, 1765-1767 (1994). Taubken, N. and Thiem, J.: Enzymatic synthesis of alkyl and hydroxyalkyl ,3-D-mannopyranosides. Synthesis, 6, 517-518 (1992). Wong, C.-H., Halcomb, R. L., Ichikawa, Y., and Kajimoto, T.: Enzymes in organic synthesis: application to the problems of carbohydrate recognition. II. Angew. Chem. Int. Ed. Engl., 34, 521-546 (1995). Vie, G. and Crout, D. H. G.: Synthesis of glucosidic derivatives with a spacer arm by reverse hydrolysis using almond [Y-Dalucosidase. Tetrahedron: Asvmmetrv. 5. 2513-2516 (1994). Goi, Y., Hashimoto, T., Mituo, N:,‘and Satoh, T.: Eniymic formation of ,i-alkyl glycosides by ;3-galactosidase from Aspergillus oryzue and its application to the synthesis of chemically unstable cardiac glycosides. Chem. Pharm. Bull., 33, 18081814 (1985). Vulfson, E. N., Pate], R., and Law, B. A.: Alkyl-,j-glucoside synthesis in a water-organic two-phase system. Biotechnol. Lett., 12, 397-402 (1990). Tricone, A., Nicolaus, B., Lima, L., Morzillo, P., de Rosa, M., and Gambacorta, A.: Enzyme-catalyzed synthesis of alkyl ,3-pglycosides with crude homogenate of Sulfolobus solfataricus. Biotechnol. Lett., 13, 235-240 (1991).
J. FERMENT. BIOENG..
9. Selisko, B., Ulbrich, R., and Schellenberger, A.: Invertase-catalyzed reactions in alcoholic solutions. Biotechnol. Bioeng., 35, 1006-1010 (1990). 10. Shinoyama, H., Kamiyama, Y., and Yasui, T.: Enzymatic synthesis of alkyl ,%xylosides from xylobiose by application of the transxylosylation reaction of Aspergillus niger $xylosidase. Agric. Biol. Chem., 52, 2197-2202 (1988). 11. Holazo, A., Shinoyama, H., Kamiyama, Y., and Yasui, T.: Screening for ;3-mannosidase with transmannosylation capacity. Biosci. Biotech. Biochem., 56, 822-824 (1992). 12. Kusakabe, I., Park, G. G., Kumita, N., Yasui, T., and Murakami, K.: Specificity of 3-mannanase from Peniciliium purpurogenum for konjac glucomannan. Agric. Biol. Chem., 52, 519-524 (1988). 13. Kusakabe, I., Zama, M., Park, G. G., Tubaki, K., and Murakami, K.: Preparation of ,3-1,4-mannobiose from copra meal by a mannanase from Penicillium purpurogenum. Agric. Biol. Chem., 51, 2825-2826 (1987). 14. Iizuka, Y., Kamiyama, Y., and Yasui, T.: Kinetic comparison of fungal ,3-xylosidases in the condensation reaction of xylose. Seibutsu-koaaku. 71. 325-331 (1993). 15. Yasui, T. aid Iiiuka, Y.: Transglycosylation and glucosyl condensation by enzyme in organic cosolvent systems. Nippon Nogeikagaku Kaishi, 65, 1107-l 110 (1991). (in Japanese)