Production, partial purification and characterization of xylanase from Trichosporon cutaneum SL409

Process Biochemistry Vol. 33, No. 3, pp. 331-336, 1998 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain (1032-9592/98 $19.(10+ 0.00 ELSEVIER

PII:

S0032-9592(97)00071-X

Production, partial purification and characterization of xylanase from Trichosporon cutaneum SL409 Wen Liu*, Wenmiao Zhu, Yanling Lu, Jian Kong and Guirong Ma The Institute of Microbiology, Shandong University, Jinan, Shandong 250100, People's Republic of China (Received 10 June 1997; accepted 3 August 1997)

Abstract

The effects of different parameters on extracellular xylanase biosynthesis by Trichosporon cutaneum SL409 were studied. Addition of wheat bran and Tween 80 to the medium stimulated enzyme biosynthesis significantly. The highest xylanase activity obtained in liquid culture was 74 IU/ml. The xylanase appeared to be homogeneous after ethanol precipitation and chromatography on DEAE-cellulose and Sephadex G-75 but it exhibited some microheterogeneity on polyacrylamide gel electrophoresis. Enzyme activity was optimal at pH 6-5 and 50°C, and completely inhibited by Hg2÷. Cu 2÷, Fe 2÷, Zn 2÷ and Mn 2÷ also showed significant inhibitory effects. No inhibitation was observed with Mg2+, Ca2÷ and EDTA at 1 mM. © 1998 Elsevier Science Ltd. All rights reserved Keywords: xylanase, Trichosporon

cutaneum,

enzyme purification, enzyme characterisation,

enzyme

biosynthesis.

hydrolysis of plant heteroxylans involves the action of several enzymes, of which endo-l,4-/~-xylanase (1,4-//-xylan xylohydrolase, EC 3.2.1.8) is the crucial enzyme for general xylan depolymerization. Endo1,4-/~-xylanase (xylanase) hydrolyses xylosidic linkages. Since xylan constitutes 15-30% of all plant biomass, its efficient utilization by enzymic procedures could enhance the economic competitiveness of bioconversion processes intended to compete with the petrochemical industry. Xylanases from numerous fungi and bacteria have been characterized and some of their genes cloned. Many of these xylanases show sequence homology and structural similarity 13]. Several /%l,4-glycanases showing both xylanase and cellulose activities have been found in bacteria [4-6] and fungi [7]. Enzymes that degrade only xylan have also been characterized and purified [8,9]. This type of cellulasefree xylanase may be of particular interest for the treatment of pulp. In this paper, we describe the production, partial purification and characterization of cellulase-free xylanase from a yeast, Tricho.sporon cutaneum SL409.

Introduction

Xylan, the most plentiful of the hemicelluloses, is present in the cell walls of all land plants and is particularly abundant in tissues that have undergone secondary thickening [1]. It is composed of 150-200 D-xylonopyranose units joined by /%l,4-1inkages and, depending on the source and method of extraction, it is substituted with acetyl, arabinosyl and methy [1] glucuronosyl residues and may be linear or branched [2]. Xylanolytic enzymes of microorganisms have received a great deal of attention in the last ten years mainly due to their potential application in the food, feed, and pulp and paper industries. They can be used in processes where xylan is to be depolymerized, including hydrolysis of xylan-lignin complexes in pulp to facilitate chemical pulp bleaching or the partial hydrolysis of xylans to xylo-oligosaccharides, which can be used as moisture-preserving food additives. Complete enzymic *Mr Wen Liu, The Institute of Microbiology, Shangdong University, Jinan, Shandong, 250100, People's Republic of China. 331

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Wen Liu et al.

Materials and methods

Chemicals All chemicals used were reagent grade unless otherwise stated. The polysaccharide substrates used, hemicellulose A and wheat bran, were local isolates. Commercial xylan was obtained from Sigma. DEAE-cellulose and Sephadex G-75 were purchased from Pharmacia. Microorganism, media and cultivation conditions T. cutaneum SL409, a yeast capable of degrading hemicellulose, was isolated from rotting beet residue by the enrichment-culture technique. Stock cultures were maintained on malt extract agar at 4°C and transferred every three months. A vegetative culture was cultivated for 12 h at 35°C in a seed medium containing glucose (10 g/litre), yeast extract (5 g/litre), (NH4)2504 (4 g/litre), KH2PO 4 (1 g/litre), CaC12 (0-02 g/litre) and MgSO4. 7H20 (0.89 g/litre). The initial pH was adjusted to 5-4. Cells were harvested and used for inoculation of solid-state and liquid cultures. Solidstate cultures were carried out in 500 ml wide-mouthed Erlenmeyer flasks plugged with standardized cotton and inoculated in static conditions at 32°C. Each flask contained 10g wheat bran, 0-1g sucrose and 1 ml Mandels mineral solution described previously [10]. The initial pH was adjusted to 6"5. Moisture was added at 2 ml/g of solids. For liquid cultures, media consisting of 5.0 g/litre (NH4)2504, 0.2 g/litre yeast extract and 1.0 g/litre KH2PO4 were prepared. Certain media were supplemented with one of the following carbohydrates: glucose, glycerol, sucrose, maltose, xylose, cellobiose, xylan, mannitol, wheat bran and hemicellulose A. The total culture volume was 75 ml in 300 ml Erlenmeyer flasks. The initial pH was adjusted to 6.5. Flasks were incubated at 32°C and 160 rpm on an incubator shaker. Preparation of crude extracts For solid-state cultures, flasks were removed after the stipulated period and the enzyme extracted by a wash of 50 ml distilled water per flask. The flask contents were stirred for 30 min on a magnetic stirrer and the supernatant, obtained after centrifugation at 4000 × g for 20 min, was used as the crude enzyme preparation. For liquid cultures, the culture medium was centrifuged (3000 x g, 15 min) and the clear supernatant used for the determination of enzyme activities.

Mandels [12] using filter paper (Whatman No. 1) and CMC-Na salt (1%), respectively, fl-Glucosidase activity was assayed according to Ohmiya et al. [13] using p-nitrophenyl fl-D-glucopyranoside as substrate. 0t-Amylase activity was estimated using a 1% solution of soluble starch in 100 mM sodium acetate buffer (pH 4-8) with a reaction time of 30 min. All enzyme activities were expressed as the amount of enzyme releasing 1 pmol of reducing sugar as xylose (for xylanase assay) and glucose (for FPase, CMCase and a-amylase assays) in 1 min. Protein concentration The protein content was determined by the method of Lowry et al. [14] with crystalline bovine serum albumin as standard. The protein content in the chromatographic fractions was estimated by measuring the absorbance at 280 nm. Purification Column chromatography was carried out on DEAEcellulose and Sephadex G-75. Details of the purification are given in the results and discussion section. Polyacrylamide gel electrophoresis (PAGE) PAGE by the method of Maurer et al. [15] was used to monitor the purity of the purified xylanase. The gels were stained with Coomassie Brilliant Blue R250. p H and temperature relationships The optimal pH for xylanase activity was estimated using the xylanase assay in 50 mM sodium citrate-phosphate buffers, pH 2.2-8.0. For determination of pH stability, the purified enzyme preparation was diluted in 50mM sodium citrate-phosphate buffers (pH 2-2-8.0) and in 50mM glynine-sodium hydroxide buffers (pH 8"4-11-6), and incubated for 24 h at 30°C. The enzyme activity was also assayed at 30 to 70°C, while the pH was maintained at pH 6-0. Thermal stability was determined by incubating the enzyme preparation in 50 mM sodium phosphate, pH 6.0, at 50 and 70°C. In the stability studies, samples were analyzed for residual xylanase activity immediately after incubation. Results and discussion

Influence of the carbon source Enzyme assays Xylanase was assayed by the DNS method described previously [11] using 1% (w/v) oat spelts xylan in 50 mM sodium phosphate buffer, pH 6-0, as substrate. Filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase) activities were estimated according to

Xylanase activity was measured in the supernatant of T. cutaneum SL409 grown at 32°C for 60 h in liquid medium supplemented with various carbohydrates (Table 1). Of the carbohydrates tested, hemicellulose and wheat bran supported the best xylanase production. Cultures grown in medium with xylose showed

Production, partial purification and characterization of xylanase Table 1. Effect of different carbon sources on production of xylanase by Trichosporon cutaneum SL409 a'b

Carbon source

Final pH

Xylanase activity (IU/mL culture)

4.0 5.0 4.5 4-5 4.5 6.5 6.5 6.5 6-5

0 0 0 0 6.0 1"1 14.3 19.5 39-7

Glucose Glycerol Sucrose Maltose Xylose Mannitol Xylan Hemicellulose A Hemicellulose A 1% (wheat bran 1%)

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three of the nitrogen sources tested yielded more than 70% enzymic activity. Two of these were organic nitrogen sources (peptone, yeast extract) and one was a mineral nitrogen source (NH4NO3). For further experiments, (NH4)2SO4 was chosen as the nitrogen source.

Initial p H in the culture as a function of xylanase production The effect of initial pH in liquid culture on the production of xylanase was investigated. The pH in the initial culture was adjusted in the range 3-5 to 9.5 by using hydrochloric acid or sodium hydroxide solution of suitable concentration. The results of a 60 h culture are shown against the pH recorded after sterilization in Fig. 2. The maximum enzyme production was at pH

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The influence of eight nitrogen sources on xylanase biosynthesis was studied. The following were all tested at the same nitrogen concentration of 0.5% (w/v): (NH4)2SO4, NaNO3, NH4NO3, (NH4)2HPO4, urea, peptone, yeast extract and beef extract. Figure 1 shows the enzyme biosynthesis measured after 60 h cultivation with the different nitrogen sources. On a basis of 100% for the highest enzymic activity produced, only

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lower xylanase activity. Xylanase activity was not found when the cells were grown in media containing glucose, glycerol, sucrose, maltose or cellobiose.

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6.5. Xylanase production declined sharply after p H 7.5. Further studies were carried out at pH 6"5.

Influence of Tween 80 The experiment was carried out with different Tween 80 concentrations from 0 to 1.0% (v/v) in liquid cultures. Tween 80 can stimulate xylanase production significantly. After 60 h of cultivation a maximum enzyme production for Tween 80 of 0.6% was observed (Fig.

3).

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Influence of water content The effect of initial moisture on the biosynthesis of xylanase in solid-state cultures was studied by carrying out experiments at six different initial water contents (50, 55, 60, 65, 70 and 75%). The highest xylanase activities measured after 96h of cultivation were obtained for cultures with an initial water content of 60%. Under this culture condition enzyme production was about two times higher than for a water content of 75% (data not shown). In addition to xylanase activities, the crude enzyme

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Production,partialpurification and characterizationof xylanase Table 2. Effect of various compoundsa on the activity of xylanaseb Compound Control (none) MgCI2 CuCI2 CaCI2 BaCI2 ZnCI2 FeCI2 HgC12 MnC12 EDTA SDS

Activity (%) 100 98 25 92 74 56 41 0 54 94 80

"As a concentration of 1 mM. bIncubation for 30 min.

335

after 3 h of incubation. Xylanase was assayed in the normal manner in the presence of 1 mM EDTA, SDS and a number of metal chlorides (Table 2). The enzyme activity was completely inhibited by Hb 2+, and Cu 2÷, Fe 2÷, Mn 2÷ and Zn 2÷ also had significant inhibitory effects. Ca 2÷, Mg 2÷ and EDTA were not inhibitory at all.

Acknowledgements The technical assistance of the State Key Laboratory of Microbiology of Shandong University is gratefully acknowledged. This work was supported by a grant from the Natural National Foundation of China.

References preparation exhibited some pectinase activity, but no /~-glucosidase, CMCase, FPase or a-amylase activity at all (data not shown).

Partial purification and characterization of xylanase 4-day-old solid-state cultures were collected and the enzyme was extracted as described in the materials and methods section. Unless otherwise stated, all subsequent steps involved in enzyme purification were conducted at 5-10°C. The enzyme extract (21itres) was precipitated by slow addition of 2.5 litres of cold ethanol ( - 1 6 ° C ) with gentle stirring. The precipitate was collected by centrifugation at 12000 × g for 10 min, dissolved in 10 ml of 50 mM sodium acetate buffer, pH 5.8, and dialysed against this buffer overnight. The enzyme preparation was then applied to a column (2.6 x 20 cm) of DEAE-cellulose pre-equilibrated with 5 mM sodium acetate buffer, pH 5-8, and eluted with a linear NaCI concentration gradient (0-0.8 M in acetate buffer) at 25 ml/h. The eluate showing xylanase activity was applied to a column (2.5 × 100 cm) of Sephadex G-75 pre-equilibrated with 50mM sodium acetate buffer, pH 5"8. The column was eluted with the same buffer at the rate of 20 ml/h. The appropriate fractions were pooled and dialysed against the same buffer. The elution profiles of protein and enzyme activity are shown in Fig. 4. The enzyme obtained from the Sephadex G-75 step was subjected to PAGE at pH 8.0. It showed a single band but also exhibited a little microheterogeneity. The enzyme had a pH optimum of 6.5 and a temperature optimum of 50°C (Fig. 5). The pH stability was studied between 2.2 and 11.6. The enzyme was stable between 4-5 and 8.5 (Fig. 5), even after 24 h of incubation. Temperature stability was studied at 50 and 70°C. At 50°C, 78% of the initial activity was measured after 3 h of incubation, whereas at 70°C only 8% of the initial activity was measured

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tion and properties of fl-glucosidase from Ruminococcus albus. Journal of Bacteriology, 1985, 161, 432-437. 14. Lowry, O. H., Rosenbrough, N. H. and Farr, A. L., Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 1951, 193, 265-275. 15. Maurer, H. R., Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis. Springer, Berlin, 1971.