JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 79, No. I, 17-22. 1995

Purification and Some Properties of Aspergillus pulverulentus P-Xylosidase with Transxylosylation Capacity

JOKO SULISTYO,’ YOSHI KAMIYAMA,‘* AND TSUNEO YASUF

Institute of Applied Biochemistry, University of Tsukuba, Tsukuba-shi, Ibaraki 305l and Seitoku Junior College of Nutrition, Shinkoiwa, Katsushika-ku, Tokyo 124,2 Japan

Received 8 July 1994/Accepted 26 September 1994

Two &xylosidases [EC 3.2.1.371, ,&Xyl I (molecular mass 180 kDa, pI 4.7) and BXyl II (molecular mass 190 kDa, p1 3.5), derived from Aspergillus pulverulentus were separated and purified by successive chroma- tographies and their characterization and traasxylosylation were studied. ,%$yl I and BXyl II were stable at temperatures up to 50°C and from pH 1.5 to 6.5 and 2.5 to 7.0, respectively, while their highest activities were in the pH ranges 2.5-3.5 and 4.0-5.0 at 60%. Although both enzymes were strongly inhibited by N-bromosuc- cinimide, the inhibitory effect of HgCl2 was not significant on either. The two enzymes exhibited different resist- ances against AgN03, glucoao 1,5-la&one and nojirlmycin. They were shown to have broad acceptor spe- cificity in transferring the xylosyl residue of xyloollgosaccharldes to various alcohol and phenolic compound ac- ceptors. In the presence of 25% or more 2-propanol, the synthesis of the transfer product, 2-propyl hxyloside, was closely consistent with the theoretical yield.

[Key words: ,kxylosidase, AspergiNus pulverulentus, transxylosylation]

Glycosides such as alkyl ,&xylosides (l), ascorbic acid glucoside (2) and a-arbutin (3), which have attracted spe- cial interest because of their physiological and physical usefulness, can be prepared and synthesized by applica- tion of the transfer reaction of P-xylosidase, a-glucosi- dase and sucrose phosphorylase.

Many studies on transxylosylation have shown that p- xylosidase from Aspergillus niger is effective for the prep- aration of transfer products from xylooligosaccharides to alcohols because of certain advantageous properties such as its high stability in organic solvents (4), strong transfer activity and the simplicity of the operation (1, 5).

Recently, we reported the synthesis of hydroquinone P-xyloside (HQX), by the transxylosylation of crude Aspergillus pulverulentus /3-xylosidase [EC 3.2.1.371 from xylooligosaccharides (6). We found that this A. pul- verulentus P-xylosidase could synthesize HQX more effec- tively than A. niger P-xylosidase. This crude p-xylosi- dase, however, showed high activities on phenyl p-gluco- side, PNP cY-xyloside and soluble xylan.

To investigate the transfer reaction in the presence of alcohols and phenolic compounds and to determine the enzyme which is actually involved in the reaction, the purification and characterization, especially with respect to its transfer reaction, of A. pufverulentus P-xylosidase are important. However, no such studies on this p-xylosi- dase have been reported to date. Moreover, in a prelimi- nary experiment we observed that the crude enzyme was highly stable in the present of alcohols, especially 2- propanol, which raised the possibility that it might syn- thesize the transfer product at a high concentration.

Here, the purification and characterization of A. pul- verulentus ,E-xylosidase with respect to its transxylosyla- tion ability on the formation of 2-propyl ,%xyloside are reported.

* Corresponding author.

17

MATERIALS AND METHODS

Preparation of enzymes The main source of p- xylosidase was Pectinase G (Amano Pharmaceutical Co. Ltd., Nagoya, Lot PNH 105226), a creamy powder de- rived from A. pulverulentus. Commercial products from Sigma Chemical Co. Ltd. (St. Louis, MO, USA), Ueda Chemical Industry Co. Ltd. (Osaka), Kyowa Hakko Kogyo Co. Ltd. (Tokyo), Kumiai Chemical Industry Co. Ltd. (Tokyo), Sankyo Co. Ltd. (Tokyo), and Meiji Seika Kaisha Ltd. (Tokyo), were also tested, as shown in Table 1.

Enzyme assays P-Xylosidase activity was measured by determining the amount of phenol liberated from phenyl P-xyloside. The reaction mixture contained 0.5 ml of 0.02 M substrate dissolved in 0.1 M sodium acetate buffer (pH 4.0) and 0.5 ml of enzyme preparation. After incubation at 40°C for 15 min, the reaction was stopped by the addition of 5 ml of 0.55 M Na2C0,, followed by the addition of 1.0 ml phenol reagent in 0.5 N HCl, and the absorbance at 660 nm was measured (1). One unit (U) of enzyme activity was defined as the amount of enzyme which produced 1 pmol of phenol per minute under the above conditions. Protein was measured by the Lowry method using bovine serum albumin (BSA, Sigma) as standard (7).

Purification of pxylosidase Twenty grams of crude enzyme was dissolved in 100 ml of 0.05 M sodium ace- tate buffer and stirred for 1 h at 4°C. After centrifugation at 800 x g for 15 min, the supernatant was fractioned by step-wise addition of solid ammonium sulfate to 50 and 80% saturation. The 50-80x precipitate was dissolved in a small amount of 0.05 M sodium acetate buffer at pH 4.0, applied onto a column of Biogel P-6 DC (2.6 x 38 cm), and eluted with 30 ml/h of the same buffer.

Enzyme fraction tubes 17-25 (containing 37 ml) were pooled and concentrated into 10 ml with an Advantec UK-10 ultrafiltration membrane. The concentrated solu- tion was applied onto a column of DEAE-Toyopearl 650 M anion exchanger (2.5 x 20cm), and eluted at

18 SULISTYO ET AL.

24 ml/h with the same buffer. Tubes 22 - 28 (28 ml) with high concentrations of p-Xyl I were pooled and concen- trated as described above. After elution with a linear gradient increasing from 0 to 0.4M NaCl, tubes 152- 156 (30 ml) containing ,&Xyl II was also pooled and concentrated, as well as being electrofocused to deter- mine the isoelectric point (PI) of the two P-xylosidases.

Fractions containing fi-Xyl I and II were separately applied onto a column of hydroxyapatite (2.0 x 41 .O cm) and eluted at 60 ml/h with 750 ml of 0.007 M Na-K-phos- phate buffer at pH 6.6. After a linear gradient elution with an increasing concentration (0.007 M to 0.4M) of the same buffer, p-Xyl I, fractions 77-95 (37 ml) and ,& Xyl II, fractions 97 - 117 (64 ml), were separately pooled and concentrated as described above.

p-Xyl I was purified to homogeneity using fast protein liquid chromatography (FPLC) through a Superose 12 column (Pharmacia, Uppsala, Sweden) equilibrated with 0.05 M Na-K-phosphate buffer at pH 6.6 (buffer A). /3- Xyl II was also loaded onto a Phenyl Sepharose HP (Pharmacia) column which was pre-equilibrated with 0.4 M ammonium sulfate in buffer A (buffer B). The column was then eluted step-wisely with 8 : 2, 6 : 4, 4 : 6, 2:8 and 0: 10 of buffer B to A.

Chromatofocusing The fractions containing is-Xyl I and ,&Xyl II were separately electrofocused in a 1 lo-ml column (LKB-Produkter) with a voltage of 850V for 48 h at 4°C. The carrier Bio-Lyte (Bio-Rad, California, USA) (pH 3.0-10.0) was used in a glycerine density gradient.

Polyacrylamide gel electrophoresis SDS (sodium dodecyl sulfate)-PAGE was done by the method of Weber and Osborn (8). The slab was run at a constant current of 30 mA per slab for approximately 2 h at room tem- perature. The slab was stained with 0.25% Coomassie brilliant blue R-250, and then destained with a 25% methanol and 7% acetic acid solution.

Molecular mass determination The molecular mass- es of the /3-xylosidases were estimated by FPLC using a column of Superose 12 with the following protein stan- dards: ferritin (450 kDa), catalase (240 kDa), I-amylase (200 kDa), aldolase (158 kDa), alcohol dehydrogenase (150 kDa) and bovine serum albumin (67 kDa). The sub-

J. FEWNT. BIOENG.,

unit molecular masses were measured by SDS-PAGE with the following standard proteins: myosin (200 kDa), P-galactosidase (116 kDa), phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa).

Determination of carbohydrate content The amount of carbohydrate in the purified enzyme was estimated by the phenol-sulfuric acid method (9) and was expressed as glucose.

pH and temperature relationships The pH optima of the P-xylosidase activities were determined in a mix- ture of 20 mM phenyl I-xyloside and 0.35 U/ml p-xylosi- dases over a pH range from 1.0 to 10.0. The buffer sys- tems used included 100mM citrate buffer @H l.O-3.0), McIlvaine buffer (pH 3.0-7.0), sodium acetate buffer (pH 4.0-5.5), Na-K-phosphate buffer (pH 6.0-7.5) and TRIS-HCl buffer (pH 8.0-10.0). Temperature optima were measured over the range 30 to 70°C using the stan- dard reaction conditions.

Effects of various compounds on /%xylosidase activ- ities The effects of metal ions and various reagents on the activities of p-Xyl I and II were determined by incubating the enzyme solutions (0.35 U/ml) in 0.1 M acetate buffer, pH 4.0, containing various compounds (1 mM) at 40°C for 30 min. The relative activities were expressed as a percentage against the control.

Reaction mixtures for synthesis of 2-propyl ,&xyloside (2PX) For the synthesis of 2PX, reaction mixtures containing the substrates, 150mM xylobiose or 70 mM xylotriose, were incubated with 25% (v/v) 2-propanol and an appropriate amount (0.7 U/ml) of p-Xyl I or p- Xyl II at 40°C for 24 h. Aliquots of 5 ~1 were subjected to TLC and lOO-,ul aliquots were chromatographed with HPLC after inactivating the enzymes by heating at 100°C for 5 min and filtration through a Milipore filter with a 0.45 pm pore size.

Thin layer chromatogaphy (TLC) TLC was per- formed on a Kiesel Gel 60 plate (Merck, Darmstadt, Germany), developed with solvent A: I-propanol-water (85 : 15, v/v) for reaction mixtures containing alcohol, or solvent B: ethyl acetate-acetic acid-water (3 : 1 : 1, v/v) for the mixture containing phenol compounds, respectively. The sugars on plates were visualized by heat-

TABLE 1. ,%Xylosidase activity in commercial enzyme preparations

Trade name Source Supplier ,b-Xylof$i;aictivity

1. a-Amylase 2. Biodiastase 3. Cellulosin PC 4. Cellulase TAP-4 5. Deamizyme 6. Drisselase 7. Glucozyme AF-6 8. Kitalase 9. Kokukase

10. Lactase F 1 I. Lipase M-AP 10 12. Meicelase P-l 13. Lipase AP-6 14. Newrase 15. Pectinase G 16. Protease A 17. Prozyme 6 18. Saccharase 19. Takadiastase 20. YL-5 PAT 808989

Bacillus subtilis Aspergillus oryzae Aspergillus niger Aspergillus niger Aspergillus meleus Irpex lacteus Rhizopus niveus Rhizopus solani Aspergillus oryzae Aspergillus oryzae Mucor javanicus Trichoderma viride Aspergillus niger Rhizopus niveus Aspergillus pulverulentus Aspergillus sp. Aspergillus sp. Conythyrium sp. Aspergillus sp. Achromobacter sp.

Sigma Amano Ueda Amano Amano Kyowa Amano Kumiai Sankyo Amano Amano Meiji Seika Amano Amano Amano Amano Amano Sankyo Sankyo

0.00 2.34 2.54 1.66 2.05 1.97 0.00 0.00 1.68 0.52 0.00 0.34 5.55 0.00 5.96 0.70 2.48 1.31 3.11 0.00

VOL. 79, 1995 TRANSXYLOSYLATION BY ,&XYLOSIDASE 19

Purification step

TABLE 2. Summary of purification of A. pulverulentus /3-xylosidases

Total protein Total activity Specific activity Recovery Purification P-Glucosidase (mg) (D) Wmg) (%) (-fold) activity

Crude enzyme 1680 SO-SO% (NH&SO, precipitation 944 DEAE-Toyopearl 650 M ion-exchanger

p-Xyl I 95 p-Xyl II 117

Electrofocusing p-Xyl I 16 p-Xyl II 24

Hydroxyapatite p-Xyl I 6 B-Xyl II 13

Superose 12 (FPLC) p-Xyl I 2

Phenyl Sepharose HP /9-Xyl II 4

1110 0.7 100 796 0.8 72

549 5.8 49 195 1.7 18

163 74

10.2 3.1

13.3 4.0

15 7

80 52

7 19 5 6 +

64 32.0 6 46 _

48 12.0 4 17 _

1 ttt 1 ttt

8 + 2 tt

14 +- 4 +

ing at I40-150°C for 5min, after spraying with 20 or 50% (v/v) H2S04 in methanol for plates developed in sol- vent A or B, respectively.

Measurement of reaction products The reaction products were measured by HPLC with a Waters Associ- ates M-45 solvent delivery system and Waters Differen- tial Refractometer R401 detector, under the following conditions: column, Shodex Sugar SPO810 (Pb*+) (8 mm x 30 cm); solvent, double distilled water; flow rate, 0.8 ml/min; temperature, 80°C. The amount of 2PX was estimated according to the calibration curve ob- tained by employing the ratio of the peak area of authen- tic 2PX to that of arbutin (internal standard).

RESULTS AND DISCUSSION

,9-Xylosidase activity in various commercial enzymes Table 1 shows the results of the screening test conducted on various commercial products. Fourteen of the twenty products tested were found to contain a considerable amount of P-xylosidase activity. The highest was that of Pectinase Cl (5.96 U/mg of solid), which is produced from A. pulverulentus. This product was therefore se- lected for further experiments.

Enzyme purification ,&Xylosidase was concentrat-

TABLE 3. Enzymatic properties of purified A. pulverulentus P-xylosidases

p-Xyl I p-Xyl II

Xylanase activity - - a-Xylosidase activity - - p-Glucosidase activity - - M, (Gel filtration) 180000+2000 190000~2000

(SDS-PAGE) 65OOO-t2000 1OOOOOir2000 Subunits 3 2 Sugar content (%) 4.2 3.6 Isoelectric point 4.7 3.5 Optimum temperature (“C) 60 60 Temperature stability (“C) -50 -50 Optimum pH 2.5-3.5 4.0-5.0 pH stability 1.5-6.5 2.5-7.0 Inhibitor (Relative activity, %)

HgCl, 79 77 AgNO, 23 95 Glucono 1,5-lactone 92 12 Nojirimycin 46 11 N-Bromosuccinimide 0 0

ed and purified by ammonium sulfate fractionation, gel filtration on Biogel P-6DG, DEAE-Toyopearl 650M chromatography, isoelectric focusing, and chroma- tographies on hydroxyapatite, Superose 12 and Phenyl Sepharose HP.

Two P-xylosidases were separated by the DEAE ion- exchange column, p-Xyl I and II. The two purified /3-xylo- sidases did not exhibit detectable activities of xylanase, (Y- xylosidase or ,&glucosidase. A summary of the purifica- tion of p-Xyl I and II is given in Table 2. p-Xyl I and II were purified 46-fold with 6% recovery and 17-fold with 4% recovery, respectively. The molecular masses of the purified p-Xyl I and II were respectively estimated to be 180 kDa and 190 kDa by gel filtration chromatography and 65 kDa and 100 kDa by SDS-PAGE (Table 3 and Fig. 1). The results suggest that the two enzymes are a trimer and dimer, respectively. The estimated molecular masses of the A. pulverulentus j-xylosidases are similar to those of P-xylosidases (IO, 11) reported for other fungi. ,&Xylosidases of A. niger (12), Emericella nidu- lans (lo), Penicillium wortmanni (13) and Chaetomium trilaterate (14) have also been reported as dimers; how- ever, a very small number of P-xylosidases possessing a trimeric structure have been found (15).

The purified j-Xyl I and p-Xyl II contained 4.2 and 3.6% carbohydrate residue, respectively. In this regard, /3-Xyl I and p-Xyl II are quite similar to the E. nidulans

94,000 -

67,000-- :

A B 200,wo -

i 116,000- f"~ 94,000 - - 64,600

-67,000

-443,000

-30,000

1 2 1 2 3

FIG. 1. SDS-PAGE of purified p-Xyl I and p-Xyl II. Lanes 1 (A), 1 (B) and 3 (B), protein markers (described in Materials and Methods); lane 2 (A), purified ,&Xyl I; lane 2 (B), purified ,&Xyl II.

20 SULISTYO ET AL. J. FERMENT. BIOENO.,

(4

2PX

Xl

x2

x3

PC

AR

Xl

x2

x3

FIG. 2. Thin layer chromatography of reaction products by ,%Xyl I. Reaction mixtures (1 .O ml) containing 100 mM xylobiose and 25% (v/v) of various kinds of alcohols (A) or 50 mM xylotriose and 5% of certain phenolic compounds (B), were incubated with ,9-xyl I (0.7 U/ml) in 0.1 M sodium acetate buffer (pH 4.0) at 40°C without shaking. The reaction of mixture A was ended after 24 h, and of B after 3 h by boiling, and analysis by TLC was performed directly after emulsification by the addition of acetonitrile. S, Standards QPX, 2-propyl P-xyloside; Xl, xylose; X2, xylobiose; X3, xylotriose; AR, arbutin); PC, phenolic compounds.

and Trichoderma viride enzymes which contain 4 and 4.5% (10, 11).

Characterization of purified @-Xyl I and II Table 3 shows that the maximum activities of /3-Xyl I and II were observed at pH2.5-3.5 and 4.0-5.0, respectively. The enzymes were stable for 30min at temperatures up to 5O”C, and at pH values between 1.5-6.5 and 2.5-7.0, respectively. Under the standard reaction conditions, maximum activity occurred at 60°C and pH 4.0. These two ,&xylosidases exhibit considerably wider pH stability ranges rather than those of other P-xylosidases so far re- ported. Although /3-Xyl I and II were stable up to 5O”C, most of their activities were lost when incubated at 70°C for 30 min. They appear, therefore, to be enzymes of the kind that are not thermostable, but that show activity across a broad pH spectrum.

The pIs of ,&Xyl I and II were estimated by chroma- tofocusing as 4.7 and 3.5, respectively. The A. pul- verulentus p-Xyl I is therefore similar in p1 to T. reesei /3-xylosidase described by Poutanen and Puls (16), while p-Xyl II is similar to Tyromyces palustris /3-xylosidase reported by Ishihara and Shimizu (17).

The effects of metal ions and chemical reagents on the activites of the /3-xylosidases are also shown in Table 3. The two /3-xylosidases were completely inhibited by N- bromosuccinimide, even at a low concentration (1 mM). Their activities were inhibited 54 and 89% by nojirimy- tin, and slightly (21 and 23%) by mercuric ions. B-Xyl II

was strongly inhibited (88%) by glucono 1,5-lactone but not by silver nitrate. In contrast, p-Xyl I was inhibited 77% by silver nitrate but not by glucono 1,5-lactone. It is supposed that tryptophan is related to the activities of these enzymes, but that cysteine is not. The fact that the two ,&xylosidases show significantly different inhibition patterns against particular inhibitors indicates that the structure of /3-Xyl I is likely to differ from that of p-Xyl II. In the presence of xylooligosaccharides and various alcohols, the enzymes resembled one another in the trans- fer reaction.

Transxylosylation catalyzed .by crude and purified en- zymes Optimum activities of crude and purified ,?.- Xyl II at 40°C were observed at pHs4.0 and 4.0-5.0, respectively, whereas the optimum activity of purified p- Xyl I was seen at pH2.5-3.5. Using the optimum condi- tion of the first two enzyme preparations, the reaction was carried out at pH4.0.

Both P-xylosidases showed broad specificities toward acceptors. p-Xyl I rapidly converted 100 mM xylobiose into xylose and transfer products in the presence of vari- ous kinds of alcohol-acceptors after 24 h incubation at 40°C (Fig. 2A). Figure 2B shows the TLC of the trans- xylosylation products from 50mM xylotriose and 5% phenolic compounds after 3-h incubation at 40°C. ,&Xyl II and /3-Xyl I exhibited similar capacities for trans- xylosylation in the presence of alcohol-acceptors, but since p-Xyl II appeared to be slightly more unstable than

0 10 20

Reaction time ( h )

30

FIG. 3. Time courses of transxylosylation reaction in the presence of 2-propanol. Reaction mixtures (1 ml) containing 150mM xylobiose, 25% (v/v) 2-propanol and /3-Xyl I or crude ,f3- xylosidase (0.7 U/ml) in 0.1 M sodium acetate buffer (pH 4.0) were in- cubated at 40°C for 24 h. Unbroken lines and closed symbols indicate reactions by the purified enzyme; broken lines and open symbols indicate those by the crude enzyme. Symbols: A, xylobiose; n , xylose; 0, 2PX.

,&Xyl I in the presence of 25% of these alcohols, only ,!3- Xyl I was used in the subsequent experiment.

Figure 3 shows the time courses of the formation of 2PX from 150mM xylobiose by purified /3-Xyl I and by the crude enzyme. Within 12 h, maximum concentrations of 2PX (135 and 132mM) and xylose (147 and 156mM) with a 2PX to xylose molar ratio of approximately 1 : 1 were obtained by the crude and purified enzymes. These values were in close agreement with the theoretical yields.

Effect of donor and acceptor concentrations on the formation of 2PX The amounts of 2PX which accu- mulated at various concentrations of xylobiose or xylotriose were measured by HPLC. Figure 4A shows the result after 12 h of reaction. The formation of 2PX increased in direct proportion to the xylobiose concentra- tion (O-150 mM). The effects of the 2-propanol concentra- tion (O-40%) on the formation of 2PX were studied (Fig. 4 B). In the presence of 25% 2-propanol, p-Xyl I retained 96% of its activity after 24 h incubation at 4O”C, indicating that it is a highly stable enzyme in the

0

Concn. of xylobiosa (mM)

TRANSXYLOSYLATION BY /3-XYLOSIDASE 21

*O”. 9 g E $ 3 100

6

E

s

0 0 10 20

Reaction time (h)

30

FIG. 5. Time course of formation of 2PX from xylotriose. Reac- tion mixtures (2.0 ml) containing 70 mM xylotriose and 25% (v/v) 2-propanol were incubated with /3-xyl I (0.7 U/ml) at 40°C for 24 h. Symbols: o , xylose; q , xylobiose; n , xylotriose; 0, 2PX.

presence of this alcohol-acceptor. The formation of transfer products increased with increasing concentration of the xylosyl-acceptor, and at 25% or more 2-propanol the maximum yields of 2PX and xylose, which were close to the theoretical yield, were achieved. This indi- cates that the enzyme had good enough activity for the transfer reaction, although the reaction is done in the presence of 40% 2-propanol. At a lower concentration, however, hydrolysis occurred simultaneously with the transfer reaction, which resulted in the low product yield.

Yields of 2PX and xylose of approximately 140 mM and 77mM, respectively, were obtained from 70mM of xylotriose (Fig. 5). This gives a 2PX to xylose molar ratio of 2 : 1, which is also in close agreement with the theoretical yield.

The above results show that the A. pulverulentus p- xylosidases have sufficient activities for the transxylosyla- tion reaction of xylooligosaccharides, and that there is a possibility of obtaining a higher molar ratio of 2PX from xylooligosaccharide mixtures of DP longer than 3.

Important findings were made by employing purified as well as crude A. pulverulentus ,&xylosidases. Both en- zyme preparations had good enough activity for trans-

0 10 20 30 40 50

Concn. of 2propanol (%, v/v)

FIG. 4. Effect of donor and acceptor concentrations on formation of 2PX. Reaction mixtures (2.0 ml) containing 25% (v/v) 2-propanol, p-xyl I (0.7 U/ml) and various concentrations of xylobiose (O-150 mM) (A) or 140 mM xylobiose and various concentrations of 2-propanol (O-40%, v/v) (B) in 0.1 M sodium acetate buffer pH 4.0 were incubated at 40°C for 12 h. Samples were collected and then analyzed by HPLC after filtration through 0.45 pm (Milipore Co.). Symbols: 0, xylose; 0, xylobiose; 0, 2PX.

22 SULISTYO ET AL. J. FERMENT. BIOENG.,

xylosylation to 2-propanol, while xylotriose was found to be a better substrate than xylobiose for the produc- tion of P-xylosides such as 2PX. These findings indicate that the preparation of the 2PX from xylooligosaccha- rides on a large scale may be achievable using the crude enzyme. However, in order to eliminate the presence of any unexpected enzymes which may interfere with the re- actions, further purification of the crude preparation is necessary.

6.

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9.

ACKNOWLEDGMENTS 10.

We wish to thank Dr. Yoshie Iizuka, Department of Food and Nutrition, School of Life Studies, Sugiyama Jogakuen University, Nagoya, for fruitful consultations. 11.

Sulistyo, J., Kamiyama, Y., Ito, H., and Yasui, T.: Enzymatic synthesis of hydroquinone /3-xyloside from xylooligosaccha- rides. Biosci. Biotech. Biochem., 58, 1311-1313 (1994). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). Weber, K. and Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electro- phoresis. J. Biol. Chem., 244, 4406-4412 (1969). Dubois, M., Giles, K., Hamilton, J. K., Rebers, P., and Smith, F.: Calorimetric method for determination of sugars. Anal. Chem., 28, 350-356 (1956). Matsuo, M. and Yasui, T.: Purification and some properties of @-xylosidase from Emericalla nidulans. Agric. Biol. Chem., 48, 1853-1860 (1984). Matsuo, M. and Yasui, T.: Purification and some properties of P-xylosidase from Trichoderma viride. Agric. Biol. Chem., 48, 1845-1852 (1984). John, M., Schmidt, B., and Schmidt, J.: Purification and some properties of five endo-1,4-,5-D-xylanases and a @-D-xylosidase produced by a strain of Aspergillus niger. Can. J. Biochem., 57, 125-134 (1979). Matsuo, M., Fujie, A., Win, M., and Yasui, T.: Four types of ,%xylosidases from Penicillium wortmanni IF0 7237. Agric. Biol. Chem., 51, 2367-2379 (1987). Uziie, M., Matsuo, M., and Yasui, T.: Purification and some properties of /%xylosidase from Chaetomium trilaterale. Agric. Biol. Chem., 49, 1159-1166 (1985). Bachmann, S.L. and MacCarthy, A. J.: Purification and characterization of a thermostable ,+xylosidase from Thermo- monospora fusca. J. Gen. Microbial., 135, 293-299 (1989). Poutanen, K. and Puls, J.: Characteristics of Trkhoderma reesei ,%xylosidase and its use in the hydrolysis of solubilized xylans. Appl. Microbial. Biotechnol., 28, 425-432 (1988). Ishihara, M. and Shimizu, K.: Hemicellulases of the brown rot- ting fungus, Tyromyces palustris. III. Purification and some properties of an extracellular p-xylosidase and a-glucosidase. Mokuzai Gakkaishi, 29, 315-323 (1983).

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