Philippine Journal of Science 135 (2): 113-119, December 2006 ISSN 0031 - 7683

A Local Isolate of Non-Cellulolytic, Xylanolytic, and Pectinolytic Thermophilic

Thermomyces lanuginosus Cla

Natividad S. Mendoza1*, Maria Trisette E. Caliwara2, and Noel M. Unciano1

1Industrial Technology Development Institute 2Philippine Textile Research Institute Department of Science and Technology Taguig City, Metro Manila, Philippine

Twenty strains of thermophilic fungi were screened for their production of xylanolytic and cellulolytic enzymes in liquid medium containing birchwood as substrate. All strains were

found to be xylanolytic but only 3 strains were proven to be non-cellulolytic. One of the strain C1a was finally selected based on the highest production and absence of cellulolytic activity in culture filtrates. The selected thermophilic fungus, identified as a strain of Thermomyces lanuginosus has optimum temperature and pH for growth at 55°C and pH 6.0, respectively. It grew well on both Potato Dextrose Agar and Mandels & Sternberg medium in which cellulose was substituted with xylan. Among various cellulosic, sugars and xylan-based substrates as carbon sources, xylan and corn cob induced simultaneous production of high levels of xylanase and pectinase. Maximum enzyme activities of 5,846 and 840 IU/mL xylanase and pectinase, respectively, were obtained in liquid Mandels and Reese medium with 3% corn cob at pH 6.0 after 7 d incubation at 50°C and 150 rpm.

Key Words: thermophilic, Thermomyces lanuginosus, -free, xylanolytic, pectinolytic

INTRODUCTION are also believed to be essential in improving the nutritional quality of animal feed and in the recovery The , of which a major part is composed of textile fibers (Kalogeris et al. 1998; Subramaniyan of the , constitute 20-35% by weight of wood and & Prema 2002 ). In addition, increasing concern over agricultural residues that could serve as an abundant and preserving the environment from industrial wastes has inexpensive source of fermentable carbohydrates. The raised interest in applying microbial xylanases in both the most important enzymes for the degradation of xylans pulp and paper, and fiber processing industries (Paice et al. are the endoxylanases, which randomly attack the xylan 1988; Viikari et al. 1991). Naturally-occurring microbial chain (Dekker & Richards 1976). ß-1,4-Xylanases are strains capable of secreting xylanases free of cellulase used in bread making, clarification of beer and juices, activity would be attractive for such applications as they and the conversion of xylan-containing lignocellulosic can remove xylan selectively from lignocellulose without materials to D-, that can be converted to a variety affecting cellulose fiber length. of bioproducts with high aggregate value (Biely 1985). Xylanase production has been reported in bacteria, yeasts, and fungi. Xylanase-producing microorganisms *Corresponding author: [email protected] also include thermophilic fungi, of which strains of

113 Philippine Journal of Science Mendoza et al.: Non-Cellulolytic, Xylanolytic, and Pectinolytic Vol. 135 No. 2, December 2006 Thermophilic Fungus Thermomyces lanuginosus

Thermomyces lanuginosus were reported to be among medium (Mandels & Reese 1957) in 500 mL Erlenmeyer the best cellulase-free xylanase producers in nature flasks at pH 6.0 using birchwood xylan (1% w/v) as (Purkarthofer et al. 1993; Singh et al. 2000; Damaso et al. substrate. A piece of PDA (1 cm2) with an actively 2002). Little is known about other types of - growing 4-5-d old colony of fungal strain was inoculated hydrolyzing enzymes from Thermomyces lanuginosus on the cultivation medium. The flasks were incubated at There are only two reports on the production in T. 50°C with shaking at 150 rpm. Samplings of triplicate lanuginosus of pectinolytic enzymes (Hoq et al. 1994; flasks per 24 h for 7 d incubation period were done. Puchart et al. 1999; Gomes et al. 1992). The present paper Cultures were then filtered out through fluted Whatman reports on the isolation of a local strain of Thermomyces No. 1 filter paper and the filtrates were assayed for the lanuginosus simultaneously producing high levels of activities of xylanase and cellulase. cellulase-free xylanase and pectinase. The effect of carbon sources on the co-production of pectinase and xylanase enzymes by the selected strain was studied using different carbon sources at 1% (w/v): MATERIALS AND METHODS xylan, pectin, sugarcane bagasse, corn cob, cellulose, carboxymethyl cellulose, and . The lignocellulosic substrates, sugarcane bagassse, and corn cob were milled Isolation, screening, and selection of xylanase (mesh size = 2.0 mm), washed thoroughly with distilled producing fungi water and oven-dried before using. For maximizing the Soil samples collected under woodchip pile, a self-heated production of xylanase and pectinase enzymes by the environment inside Paper Industries Corporation of the selected strain, 3% corn cob was used as the carbon Philippines (PICOP) paper mill factory in Agusan Del source. In all cases, similar cultivation conditions Sur was the fungal source. One (1) g of soil mixture (medium, pH, temperature, shaking speed, and sampling was inoculated into test tubes containing 10 mL of the procedures) and crude enzyme preparations were carried enrichment medium and incubated at 50°C for 1 wk out as described earlier. Xylanase and pectinase activities, or until fungal mats were formed on the surface of the pH, reducing sugar, total dry matter, and protein levels in broth. The composition of the enrichment (Mandels the culture filtrates were determined as a function of time & Sternberg) medium was as described (Mandels after 7 d incubation. & Sternberg 1976): 1% proteose peptone, 1.4 g/L

(NH4)2SO4, 2.0 g/L KH2PO4, 0.3 g/L , 0.3 g/L CaCl2, 0.3 g/L MgSO .7H 0, 5.0 mg/L FeSO .7H O, 1.6 mg/L Morphological and physiological studies 4 2 4 2 Potato Dextrose Agar was used for morphological MnSO4.H2O, 1.4 mg/L ZnSO4.7H2O, 2.0 mg/L CoCl2, pH 6.0, in which cellulose was substituted with (1%) studies. Malt extract agar, Czapek-Dox agar, and birchwood xylan (Sigma). One (1) mL of the culture Mandels and Sternberg media were used for selecting the broth was then transferred aseptically into new fresh best culture and stock culture media. Cover slip cultures medium every week for a period of one month and (Banaek & Rogers 1970) were prepared for microscopic incubated at the same temperature. A small portion of observation of the organism. the fungal mat was taken in distilled water, shaken well, diluted, inoculated in Potato Dextrose Agar (PDA) (pH Enzyme assays 6.0) and incubated at 50°C for 5 -7 d. The colonies were Xylanase activity was determined using 1% birchwood subsequently transferred at an early stage of growth onto xylan in 50 mM citrate-phosphate, pH 6.5 as substrate. agar plates of above enrichment medium and incubated The assay mixture, containing 1.8 mL of the substrate at 50°C in moisture-saturated plastic bags to minimize and 0.2 mL of suitably diluted enzyme solution in buffer desiccation due to the high incubation temperature. was incubated at 50°C for 5 min (Bailey et al. 1992). Fungal colonies with high growth rates and clearing Pectinase activity was determined by incubating 0.9 zone formation around the colonies were then selected. mL of culture filtrate with 10 mg of pectin in 0.05 M They were screened further for the best fungal strain citrate-phosphate buffer (pH 6.5) at 50°C for 20 min. secreting extracellular xylanase without cellulase in Each enzyme reaction was stopped by adding 3 mL 3,5- liquid shake cultures. dinitrosalicylic acid reagent. Tubes were held at 100°C for 5 min, cooled immediately and the absorbance of Shake-flask cultures, media, and cultivation conditions the reaction mixture measured at 547 nm. Endo-β- Studies on the production of cellulase and xylanase by 1,4-glucanase or cellulase as filter paper (FPase) and the selected xylanolytic strains in the culture filtrates carboxymethyl cellulase (CMCase) activities were were carried out in 100 mL of liquid Mandels & Reese assayed under the conditions previously established

114 Philippine Journal of Science Mendoza et al.: Non-Cellulolytic, Xylanolytic, and Pectinolytic Vol. 135 No. 2, December 2006 Thermophilic Fungus Thermomyces lanuginosus

(Mandels et al. 1976). One unit of pectinase, xylanase, RESULTS AND DISCUSSION FPase and CMCase activity was defined as the amount of enzyme which liberated 1µmol of reducing group Initial screening of xylanolytic thermophilic fungus (galacturonic, xylose or glucose equivalent) released Initially, 20 thermophilic fungal strains out of 100 per min and expressed as IU/mL. colonies that grew in solid Mandels & Sternberg medium containing xylan were selected based on clearing zone Other analyses formation around the colonies indicating the presence Reducing sugars were quantified as xylose equivalent of endoxylanase activities. To ensure that the clearing in mg/ml culture filtrate (Miller 1959). Soluble protein zone formation is a true reflection of the abilities of the in culture filtrate was determined using Bovine Serum organisms to produce extracellular xylanase and also Albumin (BSA) as standard (Lowry et al. 1951). Biomass to check the cellulase activities, the 20 thermophilic or total dry matter was estimated by filtering a known fungi were grown in shake cultures in Mandels & volume of culture broth through a pre-weighed Whatman Reese medium containing 1% xylan (Table 1). General No. 1 filter paper, washing several times with distilled correlation between clear zone test for estimating water, and drying the filter cake at 85°C for 24 h. xylanolytic potential of the fungal strains and the actual xylanase production in liquid cultures was obtained.

Table 1. Production of xylanolytic and cellulolytic enzymes by thermophilic fungi in liquid culturea Enzyme Activity (U/mL)b Cellulase Fungal Strain Xylanase CM Case Filter Paper

NM2 362 0.02 0.02

119P2R 198 0.12 0.47

NM1a 378 0.03 0.04

C1a 487 0.02 0.02

C1b 183 0.14 0.67

NM1b 147 0.02 0.03 11a 72 0.10 0.44

C3a 96 0.04 0.02 J 72 0.12 0.74

C3 96 0.22 0.16 13 37 0.20 0.67

NM2a 41 0.27 0.17

118b2 65 0.84 0.16

118b12 56 2.20 0.29

118b1c 39 2.79 0.18

118A1L 49 4.25 0.16

118A2W 43 5.56 0.17

118b2W 35 0.14 0.29

118b3W 71 3.40 0.16

119P2 284 2.75 0.19 a Cultures were grown on birchwood xylan (1% w/v) in liquid Mandel & Reese medium, pH 6.0, and incubated at 50oC with shaking at 150 rpm for 7 days b Enzyme activities listed were the maximum levels obtained

115 Philippine Journal of Science Mendoza et al.: Non-Cellulolytic, Xylanolytic, and Pectinolytic Vol. 135 No. 2, December 2006 Thermophilic Fungus Thermomyces lanuginosus

Seven strains, NM2, 119P2R, 119P2, NM1a, C1a, C1b (Cooney & Emerson 1964; Hudson 1992) which and NM1b exhibited high xylanase activities (>100 IU/ reproduces asexually by forming aleurioconidia. mL). However, only three hihger xylanase-producing strains NM , NM and C exhibited almost negligible 2 1a 1a Induction of xylanase, pectinase, and cellulase cellulase activities in their culture filtrates. On the basis activities with various carbon sources of the highest level of xylanase activity (487 IU/mL) and To determine if the selected cellulase-free xylanolytic absence of cellulase, the strain C was finally selected. 1a fungus C could grow and produce xylanase, It is apparent that both the agar plate clearing assay and 1a pectinase and cellulase using different carbon sources, the liquid culture studies are useful in screening work Thermomyces lanuginosus C was grown in shake-flask except that the choice of each approach might depend 1a cultures of Mandels & Reese medium containing (1% on the number of cultures to be studied. The authors find w/v) birchwood xylan, corn cob, pectin, sugarcane it convenient to screen the strains first for xylanolytic bagasse, cellulose, carboxymethyl cellulose, cellobiose, activity using agar clearing assay method then followed xylose, and glucose at pH 6.0, 50°C and 150 rpm for by checking for their cellulase and xylanase activities in 7 d. As shown in Figure 1, no measurable amounts of liquid cultures to determine which among the xylanolytic xylanase and pectinase were observed after growth strains are non-cellulolytic. on cellulose, CMCellulose, cellobiose and glucose. Xylose induced xylanase but without pectinase. Pectin,

Characterization of the fungus C1a on solid media on the other hand induced only pectinase and did not Media studies showed that the fungus has the highest support production of xylanase. Similar observation was growth rate in both PDA and Mandels and Sternberg noted in the case of thermophilic T. lanuginosus RT9 medium with xylan. No growth was observed with which produced only pectinase but without xylanase cellulose as carbon source. Table 2 shows the effects of when grown in two different media each containing pH and temperature on growth of strain C1a when grown 1% pectin (Gomes et al. 1992). A better production in PDA. The optimum temperature for growth was 55°C of xylanase on xylan and xylan-based lignocelluloses and very poor growth occurred at 30°C. The strain grew such as corn cob and sugarcane bagasse rather than on in the pH range from pH 5.0 to 9.0. Maximum growth pectin indicates that the T. lanuginosus strain prefers was obtained at pH 6.0 and growth was absent at pH natural plant materials although the oligosaccharides below 5.0 and above 9.0. In PDA, the strain grew rapidly from these carbohydrates may be considered an efficient reaching 2.5 to over 5 cm in diameter at 50°C within 2 d. inducer also. Xylanase and pectinase activities were Initially, colonies appear white then turn grey to greenish observed to be produced simultaneously when the strain grey, starting from the center of the colony. Colonies was grown on commercial xylan (from birchwood) or turn dark brown to black as they mature after 5-7 d on lignocellulosic residues (corn cob and sugarcane staining the whole agar dark red due to some substances bagasse). Co-production of xylanase and pectinase by secreted by the colony. the fungus RT9 in medium containing 1% jute cuttings powder, another lignocellulosic material from raw jute Microscopically, the fungus has immature conidia fiber was also noted (Gomes et al. 1992). which are dark brown, round, globose and single. Aleuriospores are generally unbranched, septate and Although the strain exhibited growth in the liquid hyaline. The mycelium is partly found on the surface. media containing all the above carbon sources, no Aleuriospores are straight, colorless, brown, and cellulase activity was detected that suggests that this smooth. Based on the above, the strain is a member of activity is not related to utilization of the carbon sources. the Deuteromycete (imperfect fungus). It is classified Corn cob was found to be the most effective substrate as a strain of thermophilic Thermomyces lanuginosus both for xylanase and pectinase production. High

Table 2. Effects of pH and temperature on growth of thermophilic C1a strain* pH Temperature (°C)

5 6 7 8 9 30 35 40 45 50 55

5.8 37.5 24.8 14 1.2 2.0 3.0 5.8 8.5 15.4 40.6

*Cultures were grown on potato dextrose agar plates at various pHs and incubated at various temperatures for up to 2 wk. Maximum hyphal growth in mm (in bold) was measured

116 Philippine Journal of Science Mendoza et al.: Non-Cellulolytic, Xylanolytic, and Pectinolytic Vol. 135 No. 2, December 2006 Thermophilic Fungus Thermomyces lanuginosus

400 350 300 250 200 150 100 50

Pectinase Activity (IU/mL) Activity Pectinase 0 3000 2500 2000 1500 1000 500

Xylanase Activity (IU/mL) Activity Xylanase 0 xylan corncob sugarcane xylose pectin glucose cellobiose cellulose carboxy bagasse methyl cellulose Figure 1. Co-production of xylanase and pectinase by Thermomyces lanuginosus in Mandels & Reese’s medium containing various carbon sources (1% w/v) such as birch wood xylan, pectin, sugar cane bagasse, glucose, cellobiose,cellulose and carboxymethyl cellulose xylanase production on corn cobs has been attributed to Maximum xylanase and pectinase activities of 5,846 and their greater particle size (between 2-7 mm) that leads 840 units/mL, respectively were obtained after 7 days of to slower solubilization of reducing sugars creating a culture. Both enzyme syntheses seem to be non-growth support system for fungal growth and enzyme release associated with the pH increasing up to 5th day then by several authors (Gomes et al. 1993; Purkarthofer & decreasing till the end of cultivation. The final cell and Steiner 1995; Purkarthofer et al. 1993) and this has been soluble protein concentrations were 11.82 and 1.78 mg/ confirmed in this study. mL, respectively after 7 d. Throughout the cultivation period, no cellulase activity was detected. When the selected strain was grown on corn cob at increased concentration of 3%, higher co-production of The T. lanuginosus strain studied here proved to xylanase and pectinase was obtained as seen in Figure 2. be non-cellulolytic as the other T. lanuginosus strains

900 2 14 7000 14 7 800 12 6000 12 700 1.5 6 10 5000 10 600 4000 500 5 8 8 1 400 6 3000 6 RS (mg/mL) TDM (mg/mL) 300 (mg/mL) Protein 2000 Xylanse (IU/mL) Pectinase (IU/mL) 4 0.5 4 200 2 1000 100 2 0 0 0 0 0 1 2 3 4 5 6 7 Day protein RS TDM xylanase pectinase pH

Figure 2. Shake-culture of the fungus Thermomyces lanuginosus C1a with 3% (w/v) corncob in Mandels & Reese medium. RS - reducing sugar; TDM – total dry matter

117 Philippine Journal of Science Mendoza et al.: Non-Cellulolytic, Xylanolytic, and Pectinolytic Vol. 135 No. 2, December 2006 Thermophilic Fungus Thermomyces lanuginosus studied earlier. It showed to be a promising fungus for 1992. Isolation and characterization of cellulase-free xylanase production even when compared with other pectinolytic and hemicellulolytic thermophilic fungi. strains cultivated under similar conditions as strains: World J Microbiol Biotechnol 8:559-562. RT9 (Hoq et al. 1994), DSM 5826 (Purkarthofer et al. GOMES J, GOMES I, KREINER W, ESTERBAUER 1993), ATCC 22083, ATCC 58160 and ATCC 34626 H, SINNER M and Steiner W. 1993. Production of (Singh et al. 2000) that produced 425, 1,600, 455, 377, high level xylanase by a wild strain of Thermomyces and, 1,170 U/mL, respectively. It is not known whether lanuginosus using beechwood xylan. J Biotechnol the above strains also co-produce high levels of pectinase 30:283-297. when grown in corn cob medium. Pectinase activities of T. lanuginosus previously reported (Gomes et al. HOQ MM, HEMPEL C, DECKWER W. 1994. Cellulase- 1992; Puchart et al. 1999) have considerably lower free xylanase by Thermomyces lanuginosus RT9; pectinase activities (> 100 units/mL) compared with that effect of agitation, aeration and medium components of the strain studied (> 100 units/mL) here eventhough on production. J Biotechnol 37:49-58. different conditions for enzyme production and activity Hudson HJ. 1992. Fungal Biology, Cambridge, USA: assay were used. Cambridge University Press. 106-170p. Since this thermophilic fungus produces high levels KALOGERIS E, CHRISTAKOPOULOS P, KEKOS D of cellulase-free xylanase and pectinase using cheap and MACRIS BJ. 1998. Studies on the solid-state xylan-rich agricultural waste material as corn cob, production of thermostable endoxylanases from the technical and economic potential of the fungus Thermoascus aurantiacus: characterization of two maybe great in biotechnological processes requiring isozymes. J Biotechnol 60:155-163. large scale application of the enzymes in both pulp bleaching and fiber processing in the pulp and paper LOWRY OH, ROSEBROUGH NS, FARR AL and and textile industries. Further studies on optimization Randall, R.J. 1951. Protein measurement with the Folin of condition for enzyme production, and purification and phenol reagent. J Biol Chem 193:265-275. characterization of the enzymes are in progress. MANDELS M, ANDREOTTI R and Roche C. 1976. Measurement of saccharifying cellulase. Biotechnol Bioeng Symposium 6:21-33. REFERENCES MANDELS M and REESE ET. 1957. Induction of cellulase in Trichoderma viride as influenced by carbon BAILEY MJ, BIELY P and POUTANEN K. 1992. sources and metals. J Bacteriol 73:269-278. Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23:257-270. MANDELS M and STERNBERG D. 1976. Recent advances in cellulose technology. J Ferment Technol BANAEK ES and ROGERS AL. 1970 Medical 54:267-286. Mycology Manual. 3rd ed. Minneapolis: Burgess Publishing. 457 p. MILLER GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugars. Annal Chem BIELY P. 1985. Microbial xylanolytic systems. Trends in 31:426-428. Biotechnol 3:286-290. PAICE MG, BERNIER R and JURASEK I. 1988. COONEY DG and Emerson R. 1964. Thermophilic Viscosity enhancing bleaching of hardwood kraft pulp fungi: an account of their biology, activities and with xylanase from a cloned . Biotechnol Bioeng classification, San Francisco, CA. Freeman, 80-88 p. 32:235-239. DAMASO MCT, ANDRADE CMC and PEREIRA JR. PUCHART V, KATAPODIS P, BIELY P, KREMNICKY N. 2002. Production and properties of cellulase-free L, CHRISTAKOPOULOS P, VRSANSKA M, xylanase from Thermomyces lanuginosus 10C-4145. KEKOS D, MACRIS B and BHAT M. 1999. Brazil J Microbiol 33:4-13. Production of xylanases, mannanases, and pectinases DEKKER RFH and RICHARDS GN. 1976. by the thermophilic fungus Thermomyces lanuginosus. Hemicellulases: their occurrence, purification, Enz Microb Technol 24:355-361. properties and mode of action. Adv Carbo Chem PURKARTHOFER H, SINNER M and Steiner W. Biochem 32:277-352. 1993. Cellulase-free xylanase from Thermomyces GOMEZ I, RAAJIT KS, MOHIUDDIN G and HOQ MM. lanuginosus: optimization of production in submerged and solid state culture. Enz Microbiol Technol 15:677-

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680. PURKARTHOFER H and STEINER W. 1995. Induction of endo-β-xylanase in the fungus, Thermomyces lanuginosus. Enz Microbiol Technol 17:114-118. SINGH S, REDDY P, HAARHOFF J, BIELY P, JANSE B, PILLAY B, PILLAY D and PRIOR BA. 2000. Relatedness of Thermomyces lanuginosus strains producing a thermostable xylanase. J Biotechnol 8:119-128. SUBRAMANIYAN S and PREMA P. 2002. Bio/ Technology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22:33-64. VIIKARI L., SUNDQVIST J and KETTUNEN J. 1991. Xylanase enzyme promotes bleaching. Paper puu (Paper Timber) 73:384-389.

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