274s
Biochemical SocietyTransacttons ( 1 992) 20
Hydrolysis of xylo-oligosaccharides b y a B-xylosidese f r o m t h e rumen anaerobic f u n g u s Neocallimaatix frontalis VICENTA GARCIA-CAMPAYO and THOMAS M. WOOD Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland, U.K. Xylan polysaccharides are major hemicellulose components of wood and agricultural residues. The xylans consist of a backbone of 1,4-D-linked Dxylopyranose residues substituted by L-arabinose and 4-0-methyl-D-glucuronic acids, which a r e linked glycosidically to t h e backbone of t h e polysaccharide, and ferulic, p c o u m a r i c and acetic acids which are attached by e s t e r links [ l ] . Because of t h e complexity of t h e xylan molecule, complete hydrolysis requires t h e action of several different enzymes acting in sequence and in concert. The enzymes include endo-1,4-D-Dxylanases, which cleave t h e glycosidic linkages in t h e xylan backbone to yield oligosaccharides of various length; L-arabinofuranosidases, 4-O-methyl-a-Dglucuronidases, acetyl esterases, ferulic and p c o u m a r i c acid e s t e r a s e s which are capable of cleaving t h e s u b s t i t u e n t s from t h e xylan chain; and B-xylosidases, which hydrolyse t h e soluble xylo-oligomers generated by t h e o t h e r enzymes. The importance of D-xylosidases in t h e saccharification of xylan by fungi and bacteria has been clearly demonstrated [2, 3, 41. Many of these D-xylosidases have been purified and characterized b u t t h e r e a r e few r e p o r t s on t h e kinetics and quantitative analysis of products of hydrolysis of xylooligosaccharides of different degrees of polymerization. This paper focusses on t h e mechanism of action on several xylo-oligosaccharide s u b s t r a t e s of a O-xylosidase purified from t h e anaerobic rumen f u n g u s Neocallimastix frontalis. This f u n g u s has recently been shown to produce highly active extracellular cellulase and xylanase systems [5, 61. Kinetic experiments were carried o u t in 1 mM phosphate buffer pH 6.4 at 37OC with homogeneous unbranched xylo-oligosaccharides u p to degree of polymerization (d.p.) 4. Samples were incubated f o r 30 min and t h e reaction was terminated by immersing t h e t u b e s in a boiling-bath f o r 5 min. Time course experiments were performed i n a similar way except t h a t xylo-oligosaccharides u p to d.p. 7 were used as substrates. Samples were taken at different time intervals. Acidic s u g a r s and residual ions were removed from t h e hydrolysates by different ion-exchange resins prior to chromatography. The products of hydrolysis w e r e analysed by HPLC using a HICHROM Spherisorb detector. S50DS2 column and a refractive-index Acetonitri1e:water:amine modifier 1 at 65:35:0.01 (v/v) w a s used as eluent when analysing xylose and xyloligosaccharides u p to d.p. 4 and at 58:42:0.01 ( v / v ) when analysing longer xylo-oligosaccharides. The D-xylosidase purified from N. frontalis was able to hydrolyse xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and xyloheptaose, b u t not t h e xylan polymer. The specific activity of t h e enzyme was highest on xylobiose and decreased as t h e chain length of t h e s u b s t r a t e increased. The time course of hydrolysis of t h e xylo-oligosaccharides u p to d.p. 4 (Fig. 1) showed t h a t t h e enzyme hydrolysed xylobiose at a slightly higher r a t e t h a n xylotriose during t h e f i r s t 15 min incubation. After that, t h e rate, which remained roughly constant with t h e xylobiose s u b s t r a t e , increased in t h e case of xylotriose. Xylotetraose was degraded more rapidly and to a relatively g r e a t e r extent than xylobiose and xylotriose during t h e period of time tested. The extent of hydrolysis of xylo-oligosaccharides longer than xylotetraose a f t e r incubation for 30 min did not follow a n y distinct pattern: t h u s xylopentaose was degraded to t h e extent of 33% while xylohexaose and hydrolysed by 57% and 29% xyloheptaose w e r e
Fig. 1. Time course of hudrolvsis of xulo-oliaosaccharides by D-xulosidase from N. frontalis. Initial concentration of s u b s t r a t e , 1 mg/ml. Abbreviations: X2, xylobiose; X3, xylotriose; X4, xylotetraose. respectively. In all cases mentioned, xylose and a xylooligomer with a degree of polymerization one unit smaller than t h e s u b s t r a t e were virtually t h e only products detected and no transfer-products were observed. These r e s u l t s indicate t h a t t h e enzyme a p p e a r s to act endwise on t h e xylo-oligosaccharides, i.e. cleaving a single unit of xylose from t h e chain and, contrary to o t h e r D-xylosidases reported [?I, lacks transferase activity. The Km values (pH 6.4 a n d 37OC) for xylooligosaccharides u p t o d.p. 4, calculated from t h e initial rates of release of xylose, indicated a lower affinity of t h e enzyme f o r t h e s u b s t r a t e as t h e chain length increased. Specifically, t h e values were 2.42 mM f o r xylobiose, 1.24 mM for xylotriose and 1.18 mM f o r xylotetraose. Vmax was also affected by d.p. of t h e substrate. Thus, maximum velocities were as much as 1.5 and 3 t i m e s lower for xylotriose and xylotetraose, respectively, t h a n f o r xylobiose. Similar r e s u l t s have been reported f o r t h e B-xylosidases from Trichoderma viride and Emericella nidulans IS]. This work h a s been supported by E.E.C. g r a n t contract SC 1000205. 1. Biely, P. (1985) Trends in Biotechnology 3, 286-290 2. Mishra, Ch., Seeta, R. & Rao, M. (1985) Enzyme Microb. Technol. 7, 295-299 3. Dekker, R.F.H. (1983) Biotechnol. Bioeng. 26, 1127-1146 4. Kitpreechavanich, V., Hayashi, M. & Nagai, S. (1986) Agr. Biol. Chem. 50, 1703-1711 5. Wood, T.M., Wilson, C.A., McCrae, S.I. & Joblin, K.N. (1986) FEMS Microbiol. Lett. 34, 37-40 6. Mountfort, D.O. EL Asher, R.A. (1989) Appl. Environ. Microbiol. 55, 1016-1022 7. Oguntimein, G.B. & Reilly, P.J. (1980) Biotechnol. Bioeng. 22, 1143-1154 8. Matsuo, M. & Yasui, T. (1988) in Methods in Enzymology (Wood, W.A. & Kellog, S.T., e d s ) , vol. 160, Academic Press, London and New York.
Biochemical SocietyTransacttons ( 1 992) 20
Hydrolysis of xylo-oligosaccharides b y a B-xylosidese f r o m t h e rumen anaerobic f u n g u s Neocallimaatix frontalis VICENTA GARCIA-CAMPAYO and THOMAS M. WOOD Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland, U.K. Xylan polysaccharides are major hemicellulose components of wood and agricultural residues. The xylans consist of a backbone of 1,4-D-linked Dxylopyranose residues substituted by L-arabinose and 4-0-methyl-D-glucuronic acids, which a r e linked glycosidically to t h e backbone of t h e polysaccharide, and ferulic, p c o u m a r i c and acetic acids which are attached by e s t e r links [ l ] . Because of t h e complexity of t h e xylan molecule, complete hydrolysis requires t h e action of several different enzymes acting in sequence and in concert. The enzymes include endo-1,4-D-Dxylanases, which cleave t h e glycosidic linkages in t h e xylan backbone to yield oligosaccharides of various length; L-arabinofuranosidases, 4-O-methyl-a-Dglucuronidases, acetyl esterases, ferulic and p c o u m a r i c acid e s t e r a s e s which are capable of cleaving t h e s u b s t i t u e n t s from t h e xylan chain; and B-xylosidases, which hydrolyse t h e soluble xylo-oligomers generated by t h e o t h e r enzymes. The importance of D-xylosidases in t h e saccharification of xylan by fungi and bacteria has been clearly demonstrated [2, 3, 41. Many of these D-xylosidases have been purified and characterized b u t t h e r e a r e few r e p o r t s on t h e kinetics and quantitative analysis of products of hydrolysis of xylooligosaccharides of different degrees of polymerization. This paper focusses on t h e mechanism of action on several xylo-oligosaccharide s u b s t r a t e s of a O-xylosidase purified from t h e anaerobic rumen f u n g u s Neocallimastix frontalis. This f u n g u s has recently been shown to produce highly active extracellular cellulase and xylanase systems [5, 61. Kinetic experiments were carried o u t in 1 mM phosphate buffer pH 6.4 at 37OC with homogeneous unbranched xylo-oligosaccharides u p to degree of polymerization (d.p.) 4. Samples were incubated f o r 30 min and t h e reaction was terminated by immersing t h e t u b e s in a boiling-bath f o r 5 min. Time course experiments were performed i n a similar way except t h a t xylo-oligosaccharides u p to d.p. 7 were used as substrates. Samples were taken at different time intervals. Acidic s u g a r s and residual ions were removed from t h e hydrolysates by different ion-exchange resins prior to chromatography. The products of hydrolysis w e r e analysed by HPLC using a HICHROM Spherisorb detector. S50DS2 column and a refractive-index Acetonitri1e:water:amine modifier 1 at 65:35:0.01 (v/v) w a s used as eluent when analysing xylose and xyloligosaccharides u p to d.p. 4 and at 58:42:0.01 ( v / v ) when analysing longer xylo-oligosaccharides. The D-xylosidase purified from N. frontalis was able to hydrolyse xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and xyloheptaose, b u t not t h e xylan polymer. The specific activity of t h e enzyme was highest on xylobiose and decreased as t h e chain length of t h e s u b s t r a t e increased. The time course of hydrolysis of t h e xylo-oligosaccharides u p to d.p. 4 (Fig. 1) showed t h a t t h e enzyme hydrolysed xylobiose at a slightly higher r a t e t h a n xylotriose during t h e f i r s t 15 min incubation. After that, t h e rate, which remained roughly constant with t h e xylobiose s u b s t r a t e , increased in t h e case of xylotriose. Xylotetraose was degraded more rapidly and to a relatively g r e a t e r extent than xylobiose and xylotriose during t h e period of time tested. The extent of hydrolysis of xylo-oligosaccharides longer than xylotetraose a f t e r incubation for 30 min did not follow a n y distinct pattern: t h u s xylopentaose was degraded to t h e extent of 33% while xylohexaose and hydrolysed by 57% and 29% xyloheptaose w e r e
Fig. 1. Time course of hudrolvsis of xulo-oliaosaccharides by D-xulosidase from N. frontalis. Initial concentration of s u b s t r a t e , 1 mg/ml. Abbreviations: X2, xylobiose; X3, xylotriose; X4, xylotetraose. respectively. In all cases mentioned, xylose and a xylooligomer with a degree of polymerization one unit smaller than t h e s u b s t r a t e were virtually t h e only products detected and no transfer-products were observed. These r e s u l t s indicate t h a t t h e enzyme a p p e a r s to act endwise on t h e xylo-oligosaccharides, i.e. cleaving a single unit of xylose from t h e chain and, contrary to o t h e r D-xylosidases reported [?I, lacks transferase activity. The Km values (pH 6.4 a n d 37OC) for xylooligosaccharides u p t o d.p. 4, calculated from t h e initial rates of release of xylose, indicated a lower affinity of t h e enzyme f o r t h e s u b s t r a t e as t h e chain length increased. Specifically, t h e values were 2.42 mM f o r xylobiose, 1.24 mM for xylotriose and 1.18 mM f o r xylotetraose. Vmax was also affected by d.p. of t h e substrate. Thus, maximum velocities were as much as 1.5 and 3 t i m e s lower for xylotriose and xylotetraose, respectively, t h a n f o r xylobiose. Similar r e s u l t s have been reported f o r t h e B-xylosidases from Trichoderma viride and Emericella nidulans IS]. This work h a s been supported by E.E.C. g r a n t contract SC 1000205. 1. Biely, P. (1985) Trends in Biotechnology 3, 286-290 2. Mishra, Ch., Seeta, R. & Rao, M. (1985) Enzyme Microb. Technol. 7, 295-299 3. Dekker, R.F.H. (1983) Biotechnol. Bioeng. 26, 1127-1146 4. Kitpreechavanich, V., Hayashi, M. & Nagai, S. (1986) Agr. Biol. Chem. 50, 1703-1711 5. Wood, T.M., Wilson, C.A., McCrae, S.I. & Joblin, K.N. (1986) FEMS Microbiol. Lett. 34, 37-40 6. Mountfort, D.O. EL Asher, R.A. (1989) Appl. Environ. Microbiol. 55, 1016-1022 7. Oguntimein, G.B. & Reilly, P.J. (1980) Biotechnol. Bioeng. 22, 1143-1154 8. Matsuo, M. & Yasui, T. (1988) in Methods in Enzymology (Wood, W.A. & Kellog, S.T., e d s ) , vol. 160, Academic Press, London and New York.
