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Brazilian Journal of Microbiology (2006) 37:474-480 ISSN 1517-8382

SCREENING OF FILAMENTOUS FUNGI FOR PRODUCTION OF ENZYMES OF BIOTECHNOLOGICAL INTEREST
Luis Henrique S. Guimarães1; Simone C. Peixoto-Nogueira2; Michele Michelin1; Ana Carolina S. Rizzatti1; Valéria C. Sandrim3; Fabiana F. Zanoelo1; Ana Carla M.M. Aquino1; Altino B. Junior2; Maria de Lourdes T.M. Polizeli1* Departamento de Biologia - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP; 2Departamento de Bioquímica e Imunologia, Pós Graduação em Bioquímica - FMRP/USP; 3Instituto de Química, Pós Graduação em Biotecnologia, Araraquara - UNESP
Submitted: September 08, 2005; Returned to authors for corrections: January 30, 2006; Approved: July 18, 2006
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ABSTRACT Many enzymes produced by fungi have relevant biotechnological applications in several industrial areas. The purpose of this study was to collect and isolate filamentous fungi from soil and humus, plants and sugar cane bagasse of different regions of the São Paulo state. Forty isolates were examined for their ability to produce xylanase, glucose-oxidase, alkaline phosphatase, acid phosphatase, phytase, pectinase and amylase. Among these, twenty three isolates exhibited enzymatic potential. The xylanases produced by two of these isolates (Aspergillus caespitosus and A. phoenicis) showed good potential for pulp bleaching. Among seventeen isolates, at least three produced high levels of glucose-oxidase, being Rhizopus stolonifer and A. versicolor the best producer strains. A. caespitosus, Mucor rouxii, and nine others still not identified were the best producers of phosphatases in submerged fermentation. Pectinase was best produced by IF II and C8 belong R. stolonifer. Significant levels of amylase were produced by Paecilomyces variotii and A. phoenicis. A remarkable enzyme producer was Rhizopus microsporus var. rhizopodiformis that produced high levels of amylase, alkaline and acid phosphatases, and pectinase. Some morphological structures of this fungus were illustrated using light microscopy (LM) and scanning electron microscopy (SEM). This study contributes to catalogue soil fungi isolated in the state of São Paulo, and provides additional information to support future research about the industrial potential of these microorganisms that may produce enzymes and, eventually, also secondary metabolites with anti-microbial or anti-parasitic activities.
Key words: phosphatase, xylanase, glucose oxidase, pectinase, amylase, Rhizopus microsporus var. rhizopodiformis, fungi

INTRODUCTION Among a large number of non-pathogenic microorganisms capable of producing useful enzymes, filamentous fungi are particularly interesting due to their easy cultivation, and high production of extracellular enzymes of large industrial potential. These enzymes are applied in the industrialization of detergents, starch, drinks, food, textile, animal feed, baking, pulp and paper, leather, chemical and biomedical products. The use of starch degrading enzymes was the first large-scale application of microbial enzymes in the food industry (1,2). Two enzymes carry out the conversion of starch to glucose, namely: (i) α-amylase,

that cuts the large α-1,4-linked glucose polymers into shorter oligomers and (ii) glucoamylase that hydrolyses the oligomers to glucose. Amylases have applications in food, detergents, drinks, animal feed and baking (3,4). Pectinases are used in the preparation of wine and fruit juices (5,6). Fruit cell wall needs to be broken down to improve juice liberation. Pectins are polymeric substances in the fruit lamella and cell walls. Pectinases were classified according to its specificity to attack pectin, pectic acid and oligo-D-galacturonate. Pectin pectinesterases, depolymerizing enzymes and protopectinase are the three major types of pectinases. Presently, pectinases have been used in textile industries (5,6).

*Corresponding Author. Mailing address: Departamento de Biologia - FFCLRP - USP - Av. Bandeirantes, 3900 Monte Alegre cep: 14040-901 Ribeirão Preto, SP - Brasil. Tel.: (16) 3602-4680 ou (16) 602-3666. E-mail: polizeli@ffclrp.usp.br

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0 and 250 µl of enzyme. prior to bleaching. The organisms were maintained on slants of solid 4% oatmeal baby food (Quaker) medium or in slants of Vogel complete solid medium (18) with 2% glucose as the carbon source. Xylanases have potential application in food. at 40ºC. These enzymes release lignin fragments by hydrolyzing residual xylan and the pretreatment with xylanase reduces the usage of chlorine as the bleaching agent.24). One unit was defined as the amount of enzyme that releases 1 µmol of xylose per min under the assay conditions. Industrial application of acid phosphatase is limited. Glucose oxidase (GOD). On the other hand. blotted on filter paper and stored at -15ºC until use. Pectinase activity was determined according to Paterson and Bridge (27). The best enzyme producer fungi were identified by the André Tosello Foundation. iii) low molecular mass acid phosphatase. iv) purple acid phosphatases and v) protein phosphatases (8). isolated from soil samples.Production of enzymes by filamentous fungi Phosphatases hydrolyze esters and anhydrides of phosphoric acid (7). in order to support future research about industrial application enzymes and eventual secondary metabolites with potential anti-microbial and anti-parasitic activities. pulp and textile industries (11). To obtain phosphatases the fungi were grown in Adams media (19) with 2% glucose for 48 h. All flasks were inoculated with a spore suspension to give a final concentration of 4 x 105 spores/ml of liquid medium. The reaction mixture was incubated at 50ºC for 15 min. These enzymes are classified as: i) alkaline phosphatases. and the fungi were grown for 120 h. pH 5.14). pH 6. the xylanases can be added to the ration increasing the intestinal absorption of nutrients (15). For GOD (Glucose oxidase) production the fungi were incubated in medium supplemented with 8% glucose for 72 h (21). utilizing agar medium supplemented with 0. Pigs and poultry lack phytase and excrete large amounts of phosphorus in the form of phytate into the environment. One of the most important xylanase applications is the pretreatment of pulps. catalyzes the oxidation of D-glucose to D-gluconic acid and hydrogen peroxide in the presence of oxygen and water. resuspended in distilled water and utilized for intracellular activity measurement. ii) high molecular mass acid phosphatases. Campinas. MATERIALS AND METHODS Microorganisms and maintenance The fungi used in this study were collected from samples of soil or decomposing organic material from several regions of the State of São Paulo (Brazil). (22) using 1% birchwood xylan as substrate. The mycelial mass was homogenized by grinding in a mortar with acid-washed sand (sea sand washed with sulfochromic solution and neutralized with water). in the industry of animal feed. as established by the SinBiota – FAPESP program. Xylanase was obtained in SR (Segato Rizzatti) medium (22) supplemented with 1% birchwood xylan for 72 h. Culture conditions The enzymes were obtained from cultures in liquid medium of composition adequate to the type of enzymatic activity of interest. Xylanase and amylase activities were assayed by measuring the reducing groups released Miller (26). Enzymatic assays and protein determination Acid and alkaline phosphatase activities were assayed with p-nitrophenylphosphate as substrate as previously described (23.g. but phytase. blotting and sequencing systems (9). These enzymes catalyze the release of phosphate from phytic acid. at 4ºC. The reaction mixture was incubated at 60ºC for 15 min and the reducing sugars formed were quantified by spectrophotometer at 540 nm. These enzymes are involved in various biological processes. One unit was defined as the amount of enzyme that releases 1 nmol of p-nitrophenolate per min under the assay conditions. the major phosphorus storage form in cereal grains.0. Its major use is in the quantification of free glucose (16) but it has also been used for labeling antibodies used in the detection of tumor marker and viral antigens (17). the reaction mixture consisted of 250 µl of 1% (m/v) starch in sodium acetate buffer 100 mM. One unit was defined as the amount of enzyme that releases 1 nmol of inorganic phosphate per min under the assay conditions. in pulp and paper industries (12). These enzymes degrade plant fibers made of xylan hemicellulose producing xylose monomers. After 5 days of incubation at 40ºC the activities were determined as described. in order to measure the extracellular activity.1% citric pectin as 475 . Production of enzymes by culturing in liquid medium Mycelia obtained in liquid media were harvested by filtration. Not less important is the use of xylanases for bread-making and beer production (13. for the production of some of the above referred enzymes. in cell cycle. Phytase activity was determined using phytic acid as substrate and the inorganic phosphate (Pi) liberated was quantified as Heinonen and Lahti method (25). Xylanase activity was determined according to Rizzatti et al. differentiation and others. SP. paper. For amylases was used the CP (Carvalho Peixoto) medium (20) with 1% starch. nonisotopic probing. a type of acid phosphatase (EC 3.1. feed. Alkaline phosphatases are used in enzymelinked immunoabsorbent assays (ELISA). The filtrate was dialyzed against the buffer appropriate for each enzyme. In this study we have explored the biopotential of several filamentous fungi.0). This study also contributes to cataloguing soil fungi isolated in the state of São Paulo. resulting in pollution by this element. One unit was defined as the amount of enzyme that releases 1 µmol of glucose per min under the assay conditions. e. For amylase. under orbital agitation (100 rpm). Pectinase activity was determined using solid Vogel medium supplemented with 1% pectin (pH adjusted to 8.3.8) is used in animal feed (10). legumes and oilseeds. rinsed with distilled water.

d. flavus (mushroom compost) and A. The solution was removed and 0.7 2. C-7B (soil).d. flavus A. R.2 2. niveus (Mangifera hispida L. et al.4 1. mushroom compost and several plants from the cerrado. variotii R. rhizopodiformis was collected from Kielmeyera sp in Pirassununga .1 10. 2. niveus M. A. humus. R. 1. 1 0.8 n.5 3 n. n. 4. grisea var.6 1. microsporus var.T.5 57. 1. at 4ºC.8 n.1 n. Rhizopus microsporus var. M.3 n.d.2 5. variotii (Psidium guajava L. Some of these fungi have been identified. It was incubated for 30 min at 40ºC.8 n. stolonifer (soil and humus).1 5.d. microsporus var.d.1 4.6 Extra Activity (total U) 32. Marília.5 2. A.4 n.0 110. phoenicis (Sacharum officinarum bagasse). but others are reported here just by the isolation number. After microorganism growth.3 3. Ribeirão Preto. 102. The reaction was stopped by adding 0. Luiz Antônio and Pirassununga. A. phoenicis A.7 n. such as Ilha Solteira.M. 11. SP: P.L. Ten fungi were collected from Ribeirão Preto.). Two fungi were isolate from Luis Antônio.3 8964 274 n.7 5 n. Amylase Fungus A-1 A.2 n. The plates were washed with distilled water for 1 hour and the halos revealing activity were measured. 7.2 n. Table 1.8 6.d. The type of colleted sample was described between parentheses. Protein was determined according to Lowry (29).d.1M was added and incubated for 1 hour.d.7 19. 118.d. Five fungi were collected from Marilia: C. C-9A (soil) and C-9D (soil).9 4. rouxii (collection) and N.2 95.d.02% o-dianisidine in phosphate buffer pH 7. 25 µg of peroxidase and 20 µl of enzyme solution (GOD). n.0.01% ruthenium red was added and incubated by 2 days. lindemuthianum (Phaseolus vulgaris L. lindemuthianum C-7B C-8 C-9A C-9D H. and quantified at 490 nm (28).8 n.1 8.d.d.3 97 n. RESULTS AND DISCUSSION Approximately forty filamentous fungi were isolated from various regions of São Paulo State. not determined.2 310 714. 95. Reproducibility of results: All results are the means of at least three (n=3) independent experiments. rhizopodiformis. versicolor Chaetomium thermophilum C. caespitosus (Solanium tuberosum). SP: A.8 5. H.5 8. at room temperature. 607 274 95.8 Glucose oxidase Protein (total mg) 19. Chaetomium thermophilum (collection). and measured at 620 nm. in order to carry out a screen of potential biotechnological enzyme producers.6 n.d.d. C-8 (soil). The production of glucoamylase and α-amylase from Rhizopus microsporus has been recently reported (20). n. 476 .4 7. rhizopodiformis R.Polizeli.d. M.2 54. versicolor (collection). grisea var. Aspergillus phoenicis and C-9A were the best amylase producers (Table 1). Amylase and glucose oxidase production by filamentous fungi. from soil.d. stolonifer S-1 Protein (total mg) 2. niger (Eucaliptus).8 n.d.d. crassa P. niger A.) and S-1 (Glycine indica L. 143 2297 0 n. Three fungi were collected from Ilha Solteira. 2.d.d.2 n. SP: A-1 (Arachis sp). n. Two fungi were collected from Pereira Barreto. Paecilomyces variotii. a solution of malic acid 0.4 6. substrate in Petri plates. 1. One unit was defined as the amount of enzyme that liberates one mmol of glucose per minute.d.3 86. thermoidea (collection). using bovine serum albumin as standard.43 9. 0 0 7523. 8. as described previously.). 130. Pereira Barreto.5 7.d. n.).3 4.d. crassa (collection). Seventeen isolates were tested for glucose oxidase (GOD) production (Table 1).01 M Dglucose.8 n. IF I and IF II (Inga heterophyla). rouxii N.8 160.d. 88. thermoidea IF I IF II JII-1 A. SP: A. 91 n.d. 9.d. Among sixteen fungi tested the IF-2. caespitosus A.2 Intra Activity (total U) 536 0 n.5 ml of HCl 4N. 0. JII-1 (soil and humus).5 97. For GOD activity assay the reaction mixture was composed of 0.

d.05 13.Production of enzymes by filamentous fungi Among these strains. The type of colleted sample was described between parentheses. caespitosus A.d. microsporus var. alkaline and acid phosphatases by filamentous fungi. 2626 676 0 806 n. SP. intracellular acid phosphatase and phytase. Alkaline phosphatases from thermophilic and thermotolerant fungi. A. Some strains produced high levels of intracellular alkaline phosphatase. versicolor. JII-2 (soil and humus). thermoidea (33) have been characterized in our laboratory. 200 580 1130 320 360 1500 240 1260 3760 n. JII-2.57 5. niger C-7A C-7B C-8 C-9A C-9C C-9D C. microsporus var. n. R.84 10. n. Seven fungi were collected in Ribeirão Preto. microsporus var. lindemuthianum F2P JI-2 JI-3 JI-P JII-1 JII-2 IF I IF II ISA I LH-4 Mucor rouxii P-1 R. SP. such as Scytalidium thermophilum (24).40 33. A. In the literature there is no information about species from the Rhizopus genus as GOD producers. JII-2.80 7. JII-2. SP: A-2 (soil). C-9A. caespitosus. by various filamentous fungi. or C-9D.30 11. JI-2 (soil and humus).d.d. C7-B and Rhizopus stolonifer produced de highest levels of intracellular glucose oxidase.35 29. Aspergillus caespitosus (23. 4654 949 0 Fungus Protein (total mg) 8. SP. JI-3 (soil and humus). 2314 n. These phosphatases are in general more termostable than those from mesophilic species. rhizopodiformis S-1 T-I n.40 8.00 32.d. SP.30 7.80 8.13 11. T-I was collected from Triparis sp of Pereira Barreto. as for instance A. niger.80 14. 1612 2340 2028 0 1170 455 0 0 n.40 16. C-9C was collected from soil of Marilia. JI-P.d.d.and extracellular alkaline phosphatases.50 10. P-1 and R. Production of phytases. ISA I was collected from soil and humus of Ilha Solteira.17 13. Mucor rouxii and P-1 secreted high levels of alkaline phosphatase into the medium. = not determined. P-1 was collected from soil Pirassununga.40 1. Acid phosphatase was present only in intracellular materials and some fungi were more effective. rhizopodiformis.29 13. but the genus Aspergillus is considered one of the best producers of GOD (30).60 A-1 A-2 A. stolonifer R. rhizopodiformis and T-I. LH-4 (soil and humus). such as R. microsporus var. 477 . Table 2 shows the production of intra. 0 325 325 2054 n.d. and the gene sequence and expression has been studied by Malherbe (31).01 14. Phytase activity was significantly detected in cultures of A.66 13.30 9.50 11. caespitosus. C7A. Alkaline Phosphatase Intra (total U) 1540 2110 6270 660 780 3150 460 4390 1270 230 860 90 4290 1720 2000 3920 3920 1510 2015 180 1830 3000 2110 920 7410 1740 5680 Extra (total U) 90 3900 470 450 500 370 220 230 40 250 120 60 20 0 0 20 0 0 200 340 160 1350 1930 720 330 600 100 Acid phosphatase Intra (total U) n. F2P (Ficus carica). But only A-2.d. JI-P. JI-2.45 10. Table 2.d. 2720 810 590 3830 2740 3510 2640 0 2510 1380 880 80 240 4390 0 500 Phytase Intra (total U) n.15 31.32) and Humicola grisea var. JII-1. rhizopodiformis. JI-P (soil and humus).40 17.07 9.

1A). Xylanase production by filamentous fungi.05 2.5 15. C-8. In conclusion.01 2. niger P.Polizeli. but A. The optimum pH was 6. Four fungi were collected from Ribeirão Preto: T. = not determined. identified as A.L. rhizopodiformis. indicating weak pectinase production (not shown). microsporus var.51 3. slightly or not pigmented.and extracellular xylanases.06 348.6 81. 1C) is a structure that denotes sexual reproduction.32 2. V-2 (soil and humus).4 83.35 3. Simple rhizoids (Fig. The screening for pectinase production was carried out with ten isolates.33 247.05 128. IF II.d.18 381. some times grouped. 1). for instance IF II and C-9A. There is no information or illustrations about this fungus in the literature. caespitosus and A. homogeneous. Among these fungi. are a characteristic of this species. ISA-1 and IF I. and it shows dark.55 3. caespitosus A. caespitosus. rhizopodiformis were observed using light microscopy (LM). microsporus var. 1B. Among the isolates. and Colletotrichum were classified as good producers. niveus A.5 and 3.4 753.47 490. SP. 1A) up to 500 µm in length.15 38. phoenicis and R.71 743.5 for A.55 39. Humicola grisea var. n. crassa with halos between and 13-9 mm.8 100 109.65 3.7 µm of diameter/length.69 2. Acid phosphatase with phytase activity from A. Two thermotolerant Aspergillus strains. producing clear halos in qualitative cup-plate assay corresponding at 19. versicolor A. Fungus Protein (total mg) 0. C-10 gray (soil). rhizopodiformis and R. The sporangiophore shows stolons (Fig. D) is a constant. respectively. with maximum size 5. reesei (soil and humus).3 473. This species supports growth up to 45ºC. et al. phoenicis. M. 18. the morphological structures of Rhizopus microsporus var. niger. Among them. main characteristic of the zygomycetae. Some studies have already been carried out with fungi from our collection.98 3. B. A. were good producers of extracellular xylanases (Table 3).55 24. All fungi tested produced intra.06 3. only some description is reported by Schipper and Stalpers (36).36 3. variotii T.48 4.95 63. Zigospore (Fig. stolonifer also were excellent producers of the enzymes studied.8 31 49. R. A. grown at 40ºC.5 7. The type of colleted sample was described between parentheses.4 416.II was collected from Triparis in Pereira Barreto. as T. phoenicis. plants and sugar cane bagasse of different regions of São Paulo state. caespitosus showed good performance for paper pulp bleaching (35).7 143. forty isolates of filamentous fungi were obtained from soil and humus.1 56. caespitosus has been previously characterized in our laboratory (34). Strains Table 3. The xylanases produced by A.12 3. 478 . among others. T.M. moreover.45 5. V-1 (soil and humus). 1A) are (sub-)globose. crassa.6 101. brownish.4 1255. Figure 1. However. stolonifer were classified as the best producers. The screening for xylanase activity was performed in eighteen isolates. caespitosus and A. 1A. D) (sub-)globoses and apparently homogeneous.8 A.4 16.4 1407.15 4.86 5. Remarkable production was verified for R.II A-1 ISA I C-10 gray JI-2 JII-1 IF I IF II V-1 V-2 N.24 Xylanase Intra (total U) 5. reesei T. 420. The spores (Fig. respectively. 17 and 16 mm. but we consider that these new isolates may have more potential for industrial uses. The ornamentation of sporangiophores (Fig.35 208. with slight spinulose ornamentation with ridges. gray brownish colonies.3 Extra (total U) n. Guimarães et al.33 2. twenty three exhibited enzymatic potential for industrial uses.2 56. the extracellular levels were highest than intracellular levels. thermoidea and Mucor rouxii produced halos smaller than 10 mm. with sporangia (Fig. and scanning electron microscopy (SEM) (Fig.9 108. phoenicis A. an alkaline phosphatase from the same fungus has been successfully used in cloning protocols (32).T. reesei.09 2. N. (2006). produced the highest xylanase activity thermostable at 50-55ºC.77 907.d.8 129.

Jorge. 2002. Safety evaluation of a xylanase expressed in Bacillus subtilis.C.M. Lee. D. 2. 66.. 19-24.F. 269-273.C. Dervilly. S.. W. Review. Peixoto.R. were recipient FAPESP Fellowship. pectinase.T. Food Chem. 21. Dong. Palavras-chave: fosfatase..C. Handbook of Fungal Biotechnology. Appl. Marks. 1964. In: Torriani-Gorini A.G.S. 10. C. Purification and characterization of alkaline phosphatase from Thermotoga neapolitana. 1997. 77. C. vinte e três isolados exibiram potencial enzimático. Tewari. Advances in microbial amylases . Microbiol. Toxicol.. A. L. 1-52. Polizeli. F. industrial applications and molecular biology . 18. Roue. Humicola and Papulospora. G. James. 3. Jorge. O objetivo desse trabalho foi coletar e isolar fungos filamentosos do solo e humus.A..F. J.a review.T. 2003..N. 1990. 72. p.T. Vogel. Biotechnol. 251. 67. glicose-oxidase.M.. Mcconnell.. 2002.. J. Panda. Sci.V. J. 98. 15. 2003.K.. 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