JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 86, No. 4, 403-405. 1998

Purification and Characterization of ,&Xylosidase from Thermoascus sp. MASARU MATSUO,‘* AK10 ENDOU,’ TAKAHIRO OKADA,’ AND YUUICHI YAMAOKA*

Institute of Applied Biochemistry’ and Institute of Agriculture and Forestry,= Tsukuba University, 1-1-I Tennodai, Tsukuba, Zbaraki 305-8572, Japan

Received 25 May 1998/Accepted 18 July 1998

A fixylosidase was purified from the culture filtrate of the thermophilic fungus Thermoascus sp. by ultrafiltration, ethanol precipitation, and chromatography with DEAEToyopearl650M, Mono Q HRW5, and Phenyl Superose HR5/5. The purified ,&xylosidase was found to be homogeneous on sodium dodecyl sul- phate-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was estimated to be 107 kDa by gel filtration chromatography (Superdex 200 HR) and 100 kDa by SDS-PAGE. The optimum activity of the enzyme was observed at pH 4.5 and 55°C. The enzyme was stable up to 60°C at pH 4.5 for 1 h. The enzyme exhibited hydrolytic activity on phenyl ,&o-xyfoside and xylan (birch wood). Fifteen of the amino acid residues in the amino terminai region of the Thermoascus sp. ,%xylosidase were homologous with residues in the equivalent region of the maturation protein bglucosidase from Aspergillus aculeatus.

[Key words: /3-xylosidase, Thermoascus sp., xylan]

Many microorganisms are known to elaborate p-xylosi- dase, and several reviews of xylanolytic enzymes of microbial origin have been published (l-3). In studies of enzymatic hydrolysis of ,&l,Cxylans (which consist of p-1 ,Clinked D-xylosyl residues), the xylan-degrading enzyme from microorganisms has been found to involve two kinds of xylanase, an endo-type xylanase and a ,B- xylosidase. The complete degradation of xylan requires the synergistic action of the two enzymes (endo-type xylanase and fl-xylosidase). To date, no enzyme reaction has been utilized in the hydrolysis of xylan to xylose. In earlier attempts (4-6, 9) to produce xylose from xylan by enzymic hydrolysis, it was difficult to achieve the enzyme reaction using any of the available fungal ,%xylosidases except that from Malbranchea. In order to obtain a ,% xylosidase other than the Malbranchea enzyme suitable for use in producing xylose from xylan, we screened a number of P-xylosidase-producing fungi from soils col- lected from various places in Okinawa prefecture, Japan. We finally selected a thermophilic fungus (desig- nated as strain no. 16-2-A), which was identified as Ther- moascus sp., and purified the /3-xylosidase from this strain in order to clarify its properties. Here, we describe the purification procedure and some of the properties of the P-xylosidase.

P-Xylosidase activity was determined from the amount of phenol liberated with a phenol reagent (Nakalai Tesque, Kyoto) at 660 nm using 20mM phenyl B-D- xyloside (Nakalai Tesque) as a substrate. One unit of enzyme activity was defined as the amount of enzyme liberating 1 pmol of phenol per min from the substrate. Protein concentrations were measured by the method of Lowry et al. with bovine serum albumin (Wako Pure Chemicals, Osaka) as the standard (7), and the reducing sugar concentrations were measured by the Somogyi method with xylose as the standard (8). Insoluble and soluble xylan from birch wood were previously prepared in our laboratory (9, 10). The other chemicals were of reagent grade. A p-xylosidase was produced from an aerobic flask culture of Thermoascus sp. strain 16-2-A for 7 d at 45°C under the conditions described below. The

* Corresponding author.

medium contained per liter of distilled water: insoluble xylan 10 g, Polypepton 3.3 g, yeast extract 1 g, KH2P04 5 g. The pH was adjusted to 5.3 before sterilization.

With the exception of HPLC (Tosoh, Tokyo) and FPLC (Pharmacia, Uppsala, Sweden), all the purification proce- dures were performed at 4°C. The culture filtrate solu- tion was concentrated with a UK-50 membrane (Advan- tee, Tokyo). Cold ethyl alcohol (-SO’C) was added to the concentrated solution to 70% (v/v). The solution was left for 24 h and then centrifuged at 10,000~ g for 30 min. The precipitate dissolved in 10 mM acetate buffer (PH 5.5) was dialyzed against a 10 mM buffer for 24 h and then applied on -to

212 kDa +

170lcDa -----)

116 kDa +

76lcDa -

a DEAF-Toyopearl 650M

53lcDa --+

1 2 Lane

FIG. 1. SDS-PAGE of purified Thermoascus ,@.-xylosidase. Lane 1, Molecular weight markers (myosin, 212 kDa; a-macroglobulin, 170 kDa; P-galactosidase, 116 kDa; transferrin, 76 kDa; glutamic dehydrogenase, 53 kDa); lane 2, purified ,%xylosidase. The proteins were treated with SDS-sample buffer and analyzed by 7.5% SDS- PAGE. The gel was stained by the silver staining method.

403

404 MATSUO ET AL. J. FERMENT. BIOENG.,

~_I 100 S ,i

g 5 80

:: $ 60 .* s 5 40 Z s li: _$ 20 z 13

0 Xl x2 x3 x4 x5

15 min 30 min lh 3h

Time

FIG. 2. Xylobiose hydrolyzates by Thermoascus ,%xylosidase. The reaction mixture contained 25 mg xylobiose and 0.4 U purified $xylosi- dase in 10 ml of 10 mM acetate buffer @H 4.5). The reaction was carried out at 45°C. An aliquot of the reaction mixture was taken out at each of the times indicated in the figure and boiled to inactivate the enzyme. Samples were chromatographed by HPLC on a 4.6 x 250 mm Amide 80 (Tosoh) column at 40°C with a GL Sciences 556 instrument. The column was eluted by acetonitrille : water (70 : 30, v/v) at 0.8 ml/min with detection by a RI detector (GL Sciences; 504). Abbreviations: Xl, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose.

column (2 x 8 cm; Tosoh), equilibrated with 10mM acetate buffer (pH 5.5). The DEAE-Toyopearl 650M chromatography was carried out twice under the same conditions. The enzyme was eluted in a linear gradient manner with 0 to 200 mM NaCl in the same buffer. The P-xylosidase fraction was eluted at 50mM NaCl and the eluate was concentrated with a UK-10 membrane (Advan- tee). The concentrated solution was applied on to an FPLC instrument (Pharmacia) equipped with a Mono Q HR 5/5 column equilibrated with 1OmM acetate buffer (pH 5.5). The collected and concentrated ,&xylosidase solution was applied on to a Phenyl Superose HR 5/5 column (Pharmacia) equilibrated with 2 M (NH&SO4 in 20mM acetate buffer (pH 5.5) and eluted with a linear gradient of 0.5-OM (NH&SO4 in the same buffer. The /3-xylosidase from Thermoascus sp. 16-2-A was purified 36.7-fold from the crude enzyme solution (culture filtrate), as shown in Table 1. The purified enzyme was detected as a single protein band on sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE), as shown in Fig. 1.

The molecular weight of purified enzyme was estimat- ed to be 107 kDa by Superdex 200HR lo/30 column (Pharmacia) gel-filtration chromatography and 100 kDa by SDS-PAGE, suggesting that the purified enzyme might be a monomer. Enzyme protein from the SDS- PAGE gel was transferred to a Clearblot P-membrane

TABLE 1, Summary of a-xylosidase purification steps

Purification step Total Total

protein ,9-xylosjdase Specific activity Yield

(mg) aci$y (U/mg protein) (%)

Culture filtrate 7106.4 1410 0.2 Ultrafiltration 2631.1 1108 0.4 78.6 Ethanol precipitation 628.2 832 1.3 59.0 DEAE-Toyopearl 224.3 404 1.8 28.7

650M (1) DEAE-Toyopearl 122.8 344 2.8 24.4

650M (2) Mono Q HR 5/5 29.2 169 5.8 12.0 Phenyl Superose HR 5/5 6.2 81 7.0 5.7

(ATTO, Tokyo) and applied to a protein sequencer (477A; Applied Biosystems). The amino terminal amino acid sequence of the Thermoascus /3-xylosidase was determined to be KDDLAYSPPFYPSPWMNGNG (12). A homol- ogy search in the BLASTN database (13) revealed 15 homologous amino acid residues in the amino terminal region of the maturation protein P-glucosidase from Aspergillus aculeatus ( 14).

The optimum pH and temperature were pH 4.5 and 55”C, respectively. The thermal stability of the enzyme was examined at pH 4.5 in 10 mM acetate buffer; it was found to be stable up to 60°C for 1 h. The enzyme activ- ity was strongly inhibited by HgZ+, N-bromosuccinimide, and p-CMB at a concentration of 2 mM. The substrate specificity of the Thermoascus j?-xylosidase was investi- gated with various synthetic glycosides. The purified enzyme acted preferentially on b-1,4-xylooligosaccharides and was practically free of P-glucosidase activity (using cello-oligosaccharides as substrates). The B-xylosidase showed no action on the following substrates: phenyl n-D-glucoside, phenyl /?-D-glucoside, phenyl u-o-galacto- side, phenyl ,6-D-galactoside, phenyl a-D-xyloside, p- nitrophenyl a+arabinoside, p-nitrophenyl P-L-arabino- side, p-nitrophenyl a-D-mannoside, and p-nitrophenyl ,9- D-mannoside.

In an analysis of the transfer products hydrolyzed by the Thermoascus P-xylosidase using xylobiose as a sub- strate, transxylosidation products were observed (Fig. 2). In the hydrolysis of xylan by Thermoascus j-xylosidase, soluble xylan was hydrolyzed to the extent of 38% under the same reaction conditions as those used for xylobiose hydrolysis. The degree of hydrolysis (%) was calculated by the method of Kusakabe et al. (15). When xylan was used as a substrate, the ,&xylosidase of Malbranchea pulchella var. sulfrea could act on xylan (16), but other /3-xylosidases from Trichoderma viride (17), Emericella nidulans (18), and Penicillium wortmanii (6) hardly had an effect.

We are grateful to Dr. Tomohiko Kuwabara of Tsukuba Univer- sity for his helpful advice in analysis of the amino terminal amino acid sequence. We also thank Dr. Norihiko Oku of Ryukyu Univer-

VOL. 86, 1998 NOTES 405

sity for supplying soil samples.

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