Abstracts

INDUSTRIAL MICROBIOLOGY

Screening of filamentous fungi for production of enzymes of biotechnological interest

Seleção de fungos filamentosos para a produção de enzimas de interesse biotecnológico

Luis Henrique S. Guimarães^I; Simone C. Peixoto-Nogueira^II; Michele Michelin^I; Ana Carolina S. Rizzatti^I; Valéria C. Sandrim^III; Fabiana F. Zanoelo^I; Ana Carla M.M. Aquino^I; Altino B. Junior^II; Maria de Lourdes T. M. PolizeliI,^* * 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

^IDepartamento de Biologia - Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP

^IIDepartamento de Bioquímica e Imunologia, Pós Graduação em Bioquímica - FMRP/USP

^IIIInstituto de Química, Pós Graduação em Biotecnologia, Araraquara - UNESP

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 C-8 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

RESUMO

Muitas enzimas produzidas por fungos têm relevantes aplicações em diferentes áreas industriais. O objetivo desse trabalho foi coletar e isolar fungos filamentosos do solo e humus, plantas e bagaço de cana de açúcar de diferentes regiões do Estado de São Paulo. Quarenta isolados foram examinados quanto à sua capacidade de produzir xilanase, glicose-oxidase, fosfatase alcalina, fosfatase ácida, fitase, pectinase e amilase. Entre estes, vinte e três isolados exibiram potencial enzimático. Xilanases produzidas por dois destes isolados (Aspergillus caespitosus e A. phoenicis) mostraram bons resultados no biobranqueamento da polpa de celulose. Entre dezessete isolados, pelo menos três produziram altos níveis de glicose oxidase, sendo Rhizopus stolonifer e Aspergillus. versicolor os melhores produtores. Aspergillus caespitosus, Mucor rouxii e nove outros ainda não identificados foram os melhores produtores de fosfatases em fermentação submersa. Pectinase foi produzida preferencialmente por IF II e C-8 seguida por Rhizopus stolonifer. Níveis significantes de amilases foram produzidos por Paecilomyces variotii e Aspergillus phoenicis. Um notável produtor de diversas enzimas foi Rhizopus microsporus var. rhizopodiformis, que produziu altos níveis de amilase, fosfatase alcalina e ácida e pectinase. Algumas estruturas morfológicas deste fungo estão sendo ilustradas por microscopia de luz e microscopia eletrônica de varredura. Esse estudo contribui para catalogar fungos isolados do estado de São Paulo e fornece informações adicionais para pesquisas futuras sobre o potencial industrial destes microrganismos produtores de enzima e eventualmente também metabólitos secundários com atividade antimicrobiana e antiparasitária.

Palavras-chave: fosfatase, xilanase, glicose oxidase, pectinase, amilase, Rhizopus microsporus var. rhizopodiformis, fungos

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) a-amylase, that cuts the large a-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).

Phosphatases hydrolyze esters and anhydrides of phosphoric acid (7). These enzymes are involved in various biological processes, e.g. in cell cycle, differentiation and others. These enzymes are classified as: i) alkaline phosphatases; ii) high molecular mass acid phosphatases; iii) low molecular mass acid phosphatase; iv) purple acid phosphatases and v) protein phosphatases (8). Alkaline phosphatases are used in enzyme-linked immunoabsorbent assays (ELISA), nonisotopic probing, blotting and sequencing systems (9). Industrial application of acid phosphatase is limited, but phytase, a type of acid phosphatase (EC 3.1.3.8) is used in animal feed (10). These enzymes catalyze the release of phosphate from phytic acid, the major phosphorus storage form in cereal grains, legumes and oilseeds. Pigs and poultry lack phytase and excrete large amounts of phosphorus in the form of phytate into the environment, resulting in pollution by this element.

Xylanases have potential application in food, feed, paper, pulp and textile industries (11). These enzymes degrade plant fibers made of xylan hemicellulose producing xylose monomers. One of the most important xylanase applications is the pretreatment of pulps, prior to bleaching, in pulp and paper industries (12). These enzymes release lignin fragments by hydrolyzing residual xylan and the pretreatment with xylanase reduces the usage of chlorine as the bleaching agent. Not less important is the use of xylanases for bread-making and beer production (13,14). On the other hand, in the industry of animal feed, the xylanases can be added to the ration increasing the intestinal absorption of nutrients (15).

Glucose oxidase (GOD), catalyzes the oxidation of D-glucose to D-gluconic acid and hydrogen peroxide in the presence of oxygen and water. 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). In this study we have explored the biopotential of several filamentous fungi, isolated from soil samples, for the production of some of the above referred enzymes. This study also contributes to cataloguing soil fungi isolated in the state of São Paulo, in order to support future research about industrial application enzymes and eventual secondary metabolites with potential anti-microbial and anti-parasitic activities.

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), as established by the SinBiota - FAPESP program. The best enzyme producer fungi were identified by the André Tosello Foundation, Campinas, SP. 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.

Culture conditions

The enzymes were obtained from cultures in liquid medium of composition adequate to the type of enzymatic activity of interest. To obtain phosphatases the fungi were grown in Adams media (19) with 2% glucose for 48 h. For amylases was used the CP (Carvalho Peixoto) medium (20) with 1% starch, and the fungi were grown for 120 h. For GOD (Glucose oxidase) production the fungi were incubated in medium supplemented with 8% glucose for 72 h (21). Xylanase was obtained in SR (Segato Rizzatti) medium (22) supplemented with 1% birchwood xylan for 72 h. All flasks were inoculated with a spore suspension to give a final concentration of 4 x 10⁵ spores/ml of liquid medium, pH 6.0, at 40ºC, under orbital agitation (100 rpm). Pectinase activity was determined using solid Vogel medium supplemented with 1% pectin (pH adjusted to 8.0). After 5 days of incubation at 40ºC the activities were determined as described.

Production of enzymes by culturing in liquid medium

Mycelia obtained in liquid media were harvested by filtration, rinsed with distilled water, blotted on filter paper and stored at -15ºC until use. The mycelial mass was homogenized by grinding in a mortar with acid-washed sand (sea sand washed with sulfochromic solution and neutralized with water), at 4ºC, resuspended in distilled water and utilized for intracellular activity measurement. The filtrate was dialyzed against the buffer appropriate for each enzyme, in order to measure the extracellular activity.

Enzymatic assays and protein determination

Acid and alkaline phosphatase activities were assayed with p-nitrophenylphosphate as substrate as previously described (23,24). One unit was defined as the amount of enzyme that releases 1 nmol of p-nitrophenolate per min under the assay conditions. Phytase activity was determined using phytic acid as substrate and the inorganic phosphate (Pi) liberated was quantified as Heinonen and Lahti method (25). One unit was defined as the amount of enzyme that releases 1 nmol of inorganic phosphate per min under the assay conditions. Xylanase and amylase activities were assayed by measuring the reducing groups released Miller (26). For amylase, the reaction mixture consisted of 250 µl of 1% (m/v) starch in sodium acetate buffer 100 mM, pH 5.0 and 250 µl of enzyme. The reaction mixture was incubated at 60ºC for 15 min and the reducing sugars formed were quantified by spectrophotometer at 540 nm. One unit was defined as the amount of enzyme that releases 1 µmol of glucose per min under the assay conditions. Xylanase activity was determined according to Rizzatti et al. (22) using 1% birchwood xylan as substrate. The reaction mixture was incubated at 50ºC for 15 min. One unit was defined as the amount of enzyme that releases 1 µmol of xylose per min under the assay conditions. Pectinase activity was determined according to Paterson and Bridge (27), utilizing agar medium supplemented with 0.1% citric pectin as substrate in Petri plates, as described previously. After microorganism growth, a solution of malic acid 0.1M was added and incubated for 1 hour, at room temperature. The solution was removed and 0.01% ruthenium red was added and incubated by 2 days, at 4ºC. The plates were washed with distilled water for 1 hour and the halos revealing activity were measured. For GOD activity assay the reaction mixture was composed of 0.01 M D-glucose, 0.02% o-dianisidine in phosphate buffer pH 7.0, 25 µg of peroxidase and 20 µl of enzyme solution (GOD). It was incubated for 30 min at 40ºC. The reaction was stopped by adding 0.5 ml of HCl 4N, and quantified at 490 nm (28). One unit was defined as the amount of enzyme that liberates one mmol of glucose per minute. Protein was determined according to Lowry (29), using bovine serum albumin as standard, and measured at 620 nm.

Reproducibility of results: All results are the means of at least three (n=3) independent experiments.

RESULTS AND DISCUSSION

Approximately forty filamentous fungi were isolated from various regions of São Paulo State, such as Ilha Solteira, Pereira Barreto, Marília, Ribeirão Preto, Luiz Antônio and Pirassununga, from soil, humus, mushroom compost and several plants from the cerrado, in order to carry out a screen of potential biotechnological enzyme producers. Some of these fungi have been identified, but others are reported here just by the isolation number.

Among sixteen fungi tested the IF-2, Paecilomyces variotii, Rhizopus microsporus var. rhizopodiformis, Aspergillus phoenicis and C-9A were the best amylase producers (Table 1). The production of glucoamylase and a-amylase from Rhizopus microsporus has been recently reported (20). Seventeen isolates were tested for glucose oxidase (GOD) production (Table 1). Among these strains, A. versicolor, C7-B and Rhizopus stolonifer produced de highest levels of intracellular glucose oxidase. In the literature there is no information about species from the Rhizopus genus as GOD producers, but the genus Aspergillus is considered one of the best producers of GOD (30), and the gene sequence and expression has been studied by Malherbe (31).

Alkaline phosphatases from thermophilic and thermotolerant fungi, such as Scytalidium thermophilum (24), Aspergillus caespitosus (23,32) and Humicola grisea var. thermoidea (33) have been characterized in our laboratory. These phosphatases are in general more termostable than those from mesophilic species. Table 2 shows the production of intra- and extracellular alkaline phosphatases, intracellular acid phosphatase and phytase, by various filamentous fungi. Some strains produced high levels of intracellular alkaline phosphatase, as for instance A. caespitosus, C-9A, JI-2, JII-1, JII-2, R. microsporus var. rhizopodiformis and T-I. But only A-2, Mucor rouxii and P-1 secreted high levels of alkaline phosphatase into the medium. Acid phosphatase was present only in intracellular materials and some fungi were more effective, such as R. microsporus var. rhizopodiformis, JII-2, JI-P, or C-9D. Phytase activity was significantly detected in cultures of A. caespitosus, A. niger, C-7A, JI-P, JII-2, P-1 and R. microsporus var. rhizopodiformis. Acid phosphatase with phytase activity from A. caespitosus has been previously characterized in our laboratory (34), moreover, an alkaline phosphatase from the same fungus has been successfully used in cloning protocols (32).

The screening for xylanase activity was performed in eighteen isolates. All fungi tested produced intra- and extracellular xylanases. However, the extracellular levels were highest than intracellular levels. ISA-1 and IF I, among others, were good producers of extracellular xylanases (Table 3). Two thermotolerant Aspergillus strains, identified as A. caespitosus and A. phoenicis, grown at 40ºC, produced the highest xylanase activity thermostable at 50-55ºC. The optimum pH was 6.5 and 3.5 for A. caespitosus and A. phoenicis, respectively. The xylanases produced by A. caespitosus showed good performance for paper pulp bleaching (35).

The screening for pectinase production was carried out with ten isolates. Among them, C-8, IF II, R. microsporus var. rhizopodiformis and R. stolonifer were classified as the best producers, producing clear halos in qualitative cup-plate assay corresponding at 19, 18, 17 and 16 mm, respectively. Strains with halos between and 13-9 mm, as T. reesei, N. crassa, A. niger, and Colletotrichum were classified as good producers. Humicola grisea var. thermoidea and Mucor rouxii produced halos smaller than 10 mm, indicating weak pectinase production (not shown).

Among the isolates, the morphological structures of Rhizopus microsporus var. rhizopodiformis were observed using light microscopy (LM), and scanning electron microscopy (SEM) (Fig. 1). There is no information or illustrations about this fungus in the literature, only some description is reported by Schipper and Stalpers (36). This species supports growth up to 45ºC, and it shows dark, gray brownish colonies. The spores (Fig. 1A) are (sub-)globose, with maximum size 5.7 µm of diameter/length, homogeneous, with slight spinulose ornamentation with ridges. Simple rhizoids (Fig. 1A), slightly or not pigmented, are a characteristic of this species. The ornamentation of sporangiophores (Fig. 1A, B, ^D) is a constant, with sporangia (Fig. 1B, ^D) (sub-)globoses and apparently homogeneous. The sporangiophore shows stolons (Fig. 1A) up to 500 µm in length, brownish, some times grouped. Zigospore (^{Fig. 1C}) is a structure that denotes sexual reproduction, main characteristic of the zygomycetae.

In conclusion, forty isolates of filamentous fungi were obtained from soil and humus, plants and sugar cane bagasse of different regions of São Paulo state. Among these fungi, twenty three exhibited enzymatic potential for industrial uses. Remarkable production was verified for R. microsporus var. rhizopodiformis, but A. caespitosus, A. phoenicis and R. stolonifer also were excellent producers of the enzymes studied. Some studies have already been carried out with fungi from our collection, but we consider that these new isolates may have more potential for industrial uses, for instance IF II and C-9A. All these isolates are available to those interested in secondary metabolites studies to screen for anti- microbial and anti-parasitic activities.

ACKNOWLEDGEMENTS

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho de Desenvolvimento Científico e Tecnológico (CNPq). M.L.T.M.P. is Research Fellows of CNPq. L.H.S.G. is postdoctoral of CNPq. A.C.M.M.A., A.B.J., F.F.Z. and V.C.S. were recipient FAPESP Fellowship. S.C.P., M.M., A.C.S.R. were recipient CNPq. We thank Ricardo F. Alarcon and Mauricio de Oliveira for technical assistance.

Submitted: September 08, 2005; Returned to authors for corrections: January 30, 2006; Approved: July 18, 2006

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