JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 77, No. 1, 109-111. 1994

Application of Thermostable Xylanases from Humicola sp. for Pulp Improvement

ROBERTO DA SILVA, DONG K. YIM, AND YONG K. PARK*

College of Food Engineering, State University of Campinas (UNICAMP), Campinas, SP., Brazil

Received 20 May 1993/Accepted 24 September 1993

Thermophilic Humicola sp. secreted thermostable xylanases when grown on wheat bran medium at 50°C. DEAE-Sephadex A-50 column chromatography of the crude xylanase separated three fractions of xylanase (I, II and lIl), xylanase I being homogeneous in polyacrylamide gel electrophoresis after CM-Sephadex column chromatography. The respective xylanases, including the crude xylanase, increased pulp brightness but xylanases II and HI decreased the viscosity of the pulp due to CMCase activity. The crude xylanase contained lower CMCase activity than xylanases II and III.

In recent years, there has been an increasing interest in applying xylanases to pulp bleaching processes. These enzymes can be used to facilitate the bleaching of Kraft pulp (1-3) and to improve fiber properties (4, 5). The use of thermostable xylanases at high temperatures over pro- longed periods of time might enhance both the technical and economic feasibilities of the hydrolysis process (6).

The objective of this research was to screen thermophilic strains of microorganism for highly thermostable xylanase and to purify the enzyme for characterization. An attempt was also made to improve the properties of Kraft pulp derived from eucalyptus using both crude and purified xylanases with reduced chlorine dioxide during bleaching.

Isolation of thermophilic fungus producing xylanase For isolation of xylanase-producing thermostable microor- ganisms, approximately 1 g of soil or decayed wood was in- oculated into test tubes containing 10 ml of the enrichment medium and incubated at 50°C for one week. The culture was then inoculated onto potato dextrose agar plates and the plates were incubated at the same temperature. The colonies were subsequently transferred onto slant cultures. The composition of the enrichment medium was as de- scribed by Mandels and Sternberg (7): 1% xylan (Birch- wood, Sigma), 0.1% proteose peptone, 0.1% Tween 80, 1.4 g/l (NH4)2SO4, 2.0 g/l KH2PO4, 0.3 g/l urea, 0.3 g/l CaCI2, 0 .3g/ l MgSO4.7H20, 5 .0mg/l FeSO4.7H20, 1.6 mg/l MnSO4. HzO, 1.4 mg/l ZnSOa.7H20, 2.0 mg/l COC12 and 2% agar, pH 5-6 (only 1% of cellulose being substituted by xylan). Isolated thermophilic microorgan- isms from the slant cultures were inoculated into 100 ml of same culture medium without agar in 500-ml Erlenmeyer flasks and incubated at 50°C with shaking at 200rpm. After incubation for 5 d, the culture filtrates were collect- ed for analysis of xylanase activity. It was found that one strain of thermophilic fungus, isolated from decayed wood, produced high xylanase activity with low CMCase activity. Furthermore, this strain did not produce avicelase and maximal xylanase production could be obtained by solid state fermentation for 4 d at 50°C. When the isolated fungus was grown on potato dextrose agar, the mycelium exhibited the following characteristics: (i) colonies reach- ing 2-2.5 cm in diameter in 7 d at 50°C, greyish white, cottony, later becoming dark yellowish-brown; and (ii)

* Corresponding author.

109

brown, solitary aleurioconidia, globose to elongated (10- 17/zm in diameter) and surrounded by a slimy melanizing sheath, formed laterally or sometimes intercalarily; and (iii) growth temperatures between 40-55°C and an opti- mum temperature of 500C. From these observations, in accordance with the Manual of Thermophilic Fungi (8), the fungus strain was identified as Thermophilic Humicola sp.

Fractionation of thermostable xylanases Enzyme production by solid-state fermentation was carried out using the isolated Humicola sp. Solid culture medium was prepared by placing 10g of wheat bran and an equal weight of water in 500-ml Erlenmeyer flasks. After steriliza- tion, the spore of the fungus was inoculated and then incu- bated at 50°C for 4 d. After incubation, 100 ml of water was added to the flasks to extract the enzyme, and the ex- tracts were filtered. The extracted enzymes were precipi- tated by the addition of abolute ethanol to 70% (vol) and the precipitates were collected by centrifugation and freeze dried. This was crude xylanase. The crude enzyme was ap- plied on a DEAE-Sephadex A-50 column which has been equilibrated with 0.05 M acetate buffer pH 5.0 and then the column was eluted by a saline concentration gradient, 0.1-1.0 M. The DEAE-Sephadex A-50 column chromatog- raphy separated the crude enzyme into three fractions, with xylanase activity as shown in Fig. 1. The three frac- tions of xylanase were designated as xylanases I, II and III. The respective fractions of xylanases were further purified using a CM-Sephadex C-50 column. The specific activities of xylanases I, II and III after final purification were 585, 430 and 109 units mg -~, respectively. Xylanase I was homo- geneous by polyacrylamide gel electrophoresis and the molecular weight was 45,000 by SDS-PAGE. The other two xylanases contained substantial CMCase activity. Fur- thermore, paper chromatography analysis indicated that xylanase I hydrolyzed xylans to produce mainly xylobiose and some xylose and xylotriose, resembling endoxylanase activity. Xylanase II hydrolyzed p-nitrophenyl /%D-xylo- pyranoside, indicating xylosidase activity (Table 1) and this result was further confirmed by paper chromatog- raphy. Xylanase II also hydrolyzed oat spelt xylan to yield trace amounts of arabinose and glucose due to activity of arabinosidase and possibly ot-glucosidase, as shown in Table 1. On the other hand, xylanase III hydrolyzed oat spelt xylan to yield a remarkable amount of xylose,

110 SILVA ET AL. J. F ~ N T . BIO~NG.,

1 .4

~ 1 . (

~ 0 . 6

~ 0 . 4

0.2

• s / ! "!5 ~

/ \ ~ . - / , . o z \ \ ,, / i o I" . ' " / n i I i - o - - . 42 • / \ \.

/ * I~ I I I I I I I I I I I ' 20 40 60 80 100 120 140

Fractions

FIG. 1. DEAE-Sephadex A-50 column chromatography of crude xylanases. NaC1. I, xylanase I; II, xylanase II; III, xylanase III.

t . 0

Z

0 - 5 4 t~ Z

Symbols: O, protein; ©, xylanase activity. Line: ---, M

TABLE 1. Activities of crude and purified xylanases against various substrates

Substrate Crude enzyme (units/ml) (units mg-')

Xylanase I Xylanase II Xylanase III Xylan 23 585 430 109 p-Nitrophenyl-~-o-xylopyranoside 0.1 < 0.01 2.7 ( 0.01 p-Nitrophenyl-a-L-arabinofuranoside 0.21 < 0.01 2.0 5.5 CMC 2.5 <0.01 140.0 180.0 Avicel 0.02 <0.01 <0.01 <0.01 p-Nitrophenyl-t~-D-glucopyranoside 0.7 < 0.01 5.9 2.0 p-Nitrophenyl-a-D-glucopyranoside 0.05 < 0.01 1.6 3.0

xylobiose, xylotriose, arabinose, glucose, whereas the purified larchwood xylan was hydrolyzed to mainly xylose, xylobiose and xylotriose, resembling endoxylanase. For- mation of arabinose and glucose from oat spelt xylan is due to activities of arabinosidase and the other enzymes indicated in Table 1. Oat spelt xylan contains approxi- mately 10% arabinose and 15% glucose residues according to information from Sigma Co.

Characterization of purified xylanases The pH optima of xylanases I, II and III were between 5.0 and 5.6, while the pH stabilities of the xylanases were in the ranges of 5-8, 4.5-9 and 5-8, respectively. The optimum temperature for the activity of the three xylanases was 75°C. The activities of endo-l,4-t~-D-xylanase (1,4-t%D- xylan xylohydrolase, EC 3.2.1.8), carboxymethylcellulase (endo-l,4-t~-D-glucan glucanohydrolase, EC 3.2.1.4) and avicelase (exo-l,4-tg-D-cellobiohydrolase, EC 3.2.1.91) were determined by incubating a mixture of 0.9 ml of a 1% substrate solution (larchwood xylan, CMC and avicel, respectively; Sigma Co.) in 0.1 M acetate buffer, pH 5.0, and 0.1 ml of enzyme at 60°C for 10 rain. Reduc- ing substances were then quantified using a dinitrosalicylic acid (DNS) solution. Activities of exo-l,4-t%D-xylosidase (t~-D-xylan xylohydrolase, EC 3.2.1.37), t~-glucosidase (t % D-glucoside glucohydrolase, EC 3.2.1.21) and ct-glucosi- dase were determined by incubating a mixture of 0.9 ml of the respective substrate at 1 mM concentration in 0.1 M acetate buffer, pH 5.0, and 0.1 ml enzyme solution at 60°C for 10m in. The respective substrates were p- nitrophenyl t~-D-xylopyranoside, pNPX, p-nitrophenyl ~-D-glucopyranoside, pNPG, and p-nitrophenyl a-D-glu- copyranoside. The p-nitrophenol released was measured. The activity of a-L-arabinofuranosidase, EC 3.2.1.55, was similarly determined using p-nitrophenyl a-L-arabino- furanoside, pNPA, as a substrate. One unit of the respec-

tive enzyme activity was defined as the amount of enzyme needed to liberate 1 pmol of p-nitrophenol, xylose and glucose per min under the assay conditions. The xylan hy- drolysates were examined by descending paper chroma- tography using a Butanol-Pyridine-Water ( 6 : 4 : 3 ) sol- vent system and silver nitrate staining (9). Standards for xylotriose and xylotetraose were prepared as described by Lee et al. (10). Larchwood xylan was purified as described by Talz and Honigman (11).

Enzymatic treatment of pulp Kraft pulp and bleached Kraft pulp derived from eucalyptus were donated by Champion Paper and Cellulose Ltd., Mogi Guasu, Brazil. Bleached Kraft pulp was obtained by bleaching the Kraft pulp using a five-stage process (C, Ep, H, D and H) which involved treatment with free chlorine (C), NaOH and peroxide (Ep), calcium hypochlorite (H) and chlorine dioxide (D). The bleached Kraft pulp was mixed with en- zyme solution (xylanases I, II, III and crude xylanase, respectively) to give a suspension containing 1.S°/00 pulp (dry weight) and 3 units per ml of xylanase activity at pH 5.0. The respective suspension was incubated at 60°C for 5 h without agitation, and then washed with distilled water by filtration. The viscosity of the enzyme-treated

TABLE 2. Enzyme-treated bleached kraft eucalyptus pulp

Brightness of Viscosity of pulp pulp mPa~ (%) (cp)

Xylanase I 87. l 20.0 Xylanase II 87.3 16.0 Xylanase III 86. l 13.0 Crude enzyme 87.1 19.5 Bleached Kraft pulp as control 85.8 19.0

Bleached Kraft pulp used as a control was not treated with enzyme.

VOL. 77, 1994 NOTES 111

TABLE 3. Effects of chlorine dioxide concentration on bleaching of enzyme-treated Kraft pulp

Chlorine dioxide added Brightness Viscosity (rag/25 g) (ISO%) Kappa number (mPa~)

Kraft pulp Not bleached 35.2 15.1 60.0 Kraft pulp 825 52.4 4.0 32.5 Enzyme-treated Kraft pulp 701 57.6 2.8 30.0 Enzyme-treated Kraft pulp 577 55.2 3.7 31.2

Pulp weight, 25 g.

pulps was measured according to the T A P P I (Technical Associa t ion of Pulp and Pulp Industry) method T-230 OM-82. The brightness of the pulps was measured by direc- t ional reflectance at 457 nm as described in the T A P P I method T-452 OM-87. Enzyme t reatment for Kraft pulp was per formed as described in reference 12. The Kraf t pulp (25 g dry weight) was di luted with water to 10% solid consistency and then adjus ted to p H 5.0 with sulfuric acid in plastic bags. The crude xylanase (500 units) was added to the bags and the bags were then placed in a 60°C water bath for 5 h. Untrea ted pulp used as a control underwent the same procedure, except that no enzyme was added. Af te r incubat ion, the pulps were washed with distilled water and then bleaching of the pulps (10% solid consis- tency) was carried out using chlorine dioxide at 40°C for 20 min; 10~0 N a O H was then added to give a p H of 10- 11. Af ter 35 min N a O H treatment the pulps were washed with water, and the k a p p a number , brightness and visco- sity were measured. Measurement o f the kappa number was per formed as described in the T A P P I method T-236 OM- 85. The enzyme-treated bleached Kraft pulps were gen- erally brighter than the control pulp (Table 2). This was p robab ly caused by a loss of xylan during enzyme treat- ment, because xylan hydrolysates were found f rom the

FIG. 2. Paper chromatography of xylan hydrolysates obtained using purified xylanases I, II and III. (A) Oat spelt xylan; (B) purified larchwood xylan. X, Xylose; A, arabinose; G, glucose; X2, xylobiose; X3, xylotriose.

supernatant o f a mixture o f pulp and enzyme solut ion by paper chromatography . The viscosity o f the pulp was slightly higher after t reatment with xylanase I and crude xylanase, whereas the viscosities o f pulps treated with xyla- nase II and II I decreased considerably, indicat ing degra- da t ion of cellulose chains. As shown in Table 1, xylanases II and III contained significant amounts o f CMCase ac- tivity. The viscosity of crude enzyme-treated pulp was not altered because o f lower CMCase activity than xylanases II and III . These results indicated that crude enzyme could be applied to pulp improvement . Therefore, the Kraft pulp was t reated with only crude enzyme and then bleach- ed in two stages using various concentrat ions of chlorine dioxide and N a O H . The results are shown in Table 3. I t is apparent that enzyme pret reatment o f Kraf t pulp can achieve higher brightness with a reduction o f about 30% in the chlorine dioxide charge during bleaching.

REFERENCES

1. Jurasek, L. and Paice, M.G.: Biological bleaching of pulp, p. 11-13. In Salvo, T. J. (ed.), International pulp bleaching confer- ence, Orlando, Fla. TAPPI proceeding. Technical Association of the Pulp and Paper Industry, Atlanta (1988).

2. Kantelinen, A., Ratto, M., Sundquist, J., Ranua, M., Viikari, L., and Linko, M.: Hemicellulase and their potential role in bleaching, p. 1-4. In Salvo, T. J. (ed.), International pulp bleach- ing conference, Orlando, Fla. TAPPI proceeding. Association of the Pulp and Paper Industry, Atlanta (1988).

3. Viikari, L., Kantelinen, K., Poutanen, K., and Rauna, M.: Characterization of pulps treated with hemicellulolytic enzymes prior to bleaching, p. 145-151. In Kirk, K. and Chang, H.M. (ed.), Biotechnology in pulp and paper manufacture. Butter- worth-Heinemann Stoneham, Mass. (1990).

4. Mora, F., Comtat, J., Barnoud, F., Pla, F., and Noe, P.: Action of xylanases on chemical pulp fibers. I. Investigation on cell-wall modification. J. Wood Chem. Technol., 6, 147-165 (1986).

5. Noe, P., Chevalier, F., Mora, F., and Comtat, J.: Action of xylanases on chemical pulp fibers. II. Enzymatic beating. J. Wood Chem. Technol., 6, 167-187 (1986).

6. Yu, E. K. C., Tan, L. U. L., Chan, M. K. H., Desehatelets, L., and Saddler, J.N.: Production of thermostable xylanase by a thermophilic fungus, Thermoascus aurantiacus. Enzyme Microb. Technol., 9, 16-24 (1987).

7. Mandels, M. and Sternberg, D.: Recent advances in cellulase technology. J. Ferment. Technol., 54, 267-286 (1976).

8. Cooney, D.C. and Emerson, R.: Humicola insolens and Humicola grisea var. thermoidea, p. 72-79. In Freeman, W. H. (ed.), Thermophilic fungi, chap. 8. Publ. Co., San Francisco (1964).

9. Trevelyan, W. E., Procter, D. P., and Harrison, J. G.: Detection of sugars on paper chromatograms. Nature, 166, 444--445 (1950).

10. Lee, S.F., Forsberg, C.W., and Rattray, J.B.: Purification and characterization of two endoxylanases from Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol., 53, 644- 650 (1987).

11. Taiz, L. and Honigman, W.A.: Production of cell wall hydro- lyzing enzymes by barley aleurone layers in response to gibberel- lic acid. Plant Physiol., 58, 380-386 (1976).

12. Tolan, J.S. and Vega Canovas, R.: The use of enzymes to decrease the C12 requirements in pulp bleaching. Pulp Paper Canada, 93, 39-42 (1992).