Isoenzyme characterization of proteases and amylases and partial purification of proteases from filamentous fungi causing biodeterioration of industrial paper

International Biodeterioration & Biodegradation 63 (2009) 169–175

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Isoenzyme characterization of proteases and amylases and partial purification of proteases from filamentous fungi causing biodeterioration of industrial paper Jorge Alejandro Rojas a, *, Carlos Cruz b, Jose´ Fernando Mika´n b, Luz Stella Villalba c, Marı´a Caridad Cepero de Garcı´a a, Silvia Restrepo a a b c

´ D.C., Colombia Mycology and Plant Pathology Laboratory, Los Andes University, Bogota ´ D.C., Colombia Research Laboratory, Medicine Faculty, Nueva Granada Military University, Bogota ´ Archive, Bogota ´ D.C., Colombia Chemistry and Biology Laboratory, Bogota

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 January 2008 Received in revised form 7 July 2008 Accepted 19 July 2008 Available online 18 October 2008

Biodeterioration is an undesirable process that can affect cultural heritage and economically important materials. Although several biotic and abiotic conditions can accelerate this process, microorganisms are perhaps its main promoters. Fungi are the most important microbial agents of biodeterioration of industrial paper stored in archives. The high genetic plasticity of these organisms allows them to adapt to different environments, using almost any class of materials as substrate. Fungi produce a wide array of enzymes, including cellulases, amylases, and proteases, which are responsible for their gross biodeterioration activity. Thirty-two morphotypes of filamentous fungi were isolated on different media from industrial paper at an advanced stage of biodeterioration. The isolates showed different degrees of cellulolytic, proteolytic, and amylolytic activities on plate assays. The highest proteolytic and amylolytic activities were selected for isoform characterization, which provided an indication of the biochemical diversity that allowed them to colonize these materials. Eladia sacculum was the morphotype selected for partial purification of basic proteases since it has three basic isoforms, simplifying the purification process. We obtained a protein of 35 kDa with a pI of 8.9. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.

Keywords: Partial purification Filamentous fungi Hydrolytic activity Biodeterioration Industrial paper

1. Introduction Fungi show remarkable plasticity in adapting to different environments. They can alter their metabolism to make use of different substrates and can colonize materials with historical or economic value for humans (Cappitelli and Sorlini, 2005), such as written or printed documents, causing various levels of biodeterioration. The widely distributed filamentous fungi, mostly saprophytes, readily degrade heritable important industrial paper, which consists mainly of organic matter, especially cellulose (Teygeler, 2001; Eggins and Oxley, 2001; Bueno et al., 2003). During the biodeterioration process, a number of indicators of biodeteriorated paper appear such as fungal stains and somatic and reproductive structures. These indicators may be a sign of the wide diversity of fungal activity, most of the biological agents in which are not well

* Corresponding author. Carrera 1, No. 18A-10, Biological Sciences Department, Faculty of Science, Los Andes University, Bogota´ D.C., Colombia. Tel.: þ57 1 3394949x2768; fax: þ57 1 3394949x2817. E-mail address: [email protected] (J.A. Rojas).

recognized. For this reason, interest focuses on determining the causal agents as well as the deterioration mechanism. Knowledge about biodeterioration of industrial document material and the agents involved is still poor. Most previous studies have focused on the diversity of organisms found on different kinds of paper. Fungxi of the genera Chaetomium (Szczepanowska and Cavaliere, 2000), Penicillium, Aspergillus, Stachybotrys, Paecilomyces, Cladosporium, and Alternaria (Gallo, 1992) are commonly found. The authors of another report detected and characterized volatile metabolic compounds produced by fungal activity on paper (Canhoto et al., 2004). In addition, the antifungal properties of different compounds designed to control the presence of filamentous fungi on industrial paper or archives have been evaluated (Gilbert and Brown, 1995; Rakotonirainy et al., 1999; Clausen, 2000). However, few studies have assessed the mechanisms by which filamentous fungi damage the support, ultimately degrading it. Studies conducted to characterize the biodeterioration of documents in archives in Colombia have been very limited. Interest is growing and work on the problem has acquired momentum. Previous studies focused on establishing the diversity of fungi that colonize stored documents and on investigating antifungal

0964-8305/$ – see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2008.07.009

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products (Mateus et al., 2004; Villalba et al., 2004). Nonetheless, knowledge about the causal biological factors has been limited to studies evaluating the enzymes involved (mainly cellulases), taking into account the fact that cellulose is the main component of paper (Villalba et al., 2004). The objective of this study was to determine the fungal hydrolytic activities (proteolytic and amylolytic) that may act on historically important industrial papers. The diversity of fungi isolated from paper was evaluated on different media. In order to assess how these enzymes participate in fungal exploitation of a carbon source, their hydrolytic activities were screened on plates and variation was assessed. Among the particular protease isoforms, alkaline proteases were partially purified to explore possible applications in restoration work or other biotechnological uses. 2. Materials and methods 2.1. Sample collection and recovery of filamentous fungi Three folios at an advanced stage of biodeterioration (according to criteria already established in the Bogota´ Archive) were selected and samples were collected from them. These documents are made of industrial paper. The samples were taken with a cotton swab or scraped with a scalpel, followed by pre-incubation in Letheen broth plus chloramphenicol (500 mg/l) for 24 h at 25  C. 2.2. Evaluation of cultivable diversity on different media In order to optimize growth conditions, the pre-incubated samples were cultured on the surfaces of four different media: (1) malt extract agar (MEA) plus V8 juice; (2) MEA plus yeast nitrogen base; (3) MEA; and (4) potato dextrose agar (PDA), which was considered as a control medium commonly used for isolating fungi. The media were adjusted to pH 5.5 with citrate buffer and the cultures were incubated for 15 days at 25  C. Periodic observations were made at 72, 96, and 168 h. Triplicate cultures were used for statistical analysis. Morphotypes recovered and colonyforming units (CFU) were recorded for each medium. Data normality was evaluated with a Shapiro–Wilk test. For normal data, significant differences were established by one-way analysis of variance; otherwise, Kruskal–Wallis one-way analysis of variance and all-pairwise comparisons of mean ranks were used to determine differences. The analysis software used was Statistix v8.0. 2.3. Selection and maintenance of isolates Based on the diversity recovered on the different media, 32 representative morphotypes were selected from the cultured populations. The selected isolates were subcultured on PDA, and monosporic cultures were obtained to ensure their purity (Ho and Ko, 1997; Mika´n, 2001). Briefly, a propagule suspension was cultured on water agar (final agar concentration was 1.5% without any supplement) and incubated for 24 h. Using the stereomicroscope, the germination process was monitored until a germ tube formed. The germinated propagule was then extracted with a fine needle and cultured on PDA, and the isolates were preserved from this culture. Thirty-two morphotypes were identified to genera on the basis of macroscopic and microscopic features (Carmichael et al., 1980; Domsch et al., 1980; Kiffer and Morelet, 2000; Watanabe, 2002).

Data were evaluated graphically by scatter plots; and on the basis of a normality test, significant differences were analyzed to select the morphotypes with the greatest hydrolytic activity. 2.5. Preparation of crude extracts for assessing proteolytic and amylolytic activities In order to analyze proteolytic and amylolytic activities, a protocol similar to that described by Cooper was followed (Cooper and Wood, 1980). From the selected morphotypes, biomass was produced by inoculating 105 propagules/ml in 25-ml Erlenmeyer flasks containing Saboraud broth and culturing at 25  C for 3 days with shaking at 150 rpm. The whole content of the flask (8 ml) was then filtered and the biomass recovered was inoculated into a new 25-ml flask containing 8 ml minimal medium [0.1% KH2PO4, 0.2% NaNO3, 0.05% MgSO4, 1% (v/v) trace element solution (0.1% K2HPO4, 0.002% NaMoO4, 0.002% MnCl2, 0.001% FeSO4, 0.0003% ZnSO4, 0.002% CuSO4)] for 1 day. Finally, the biomass was recovered again and inoculated into 8 ml minimal medium supplemented with 0.4% skim milk (for proteases) or 0.1% soluble potato starch (for amylases) for 7 days, after which the culture was centrifuged at 6500  g for 15 min and the supernatant was recovered and stored at 20  C for later evaluation of hydrolytic activities. The protein content of the crude extracts was determined by a modified Bradford protocol (Bradford, 1976; Stoscheck, 1990). On a 96-well plate, 10 ml of crude extract was mixed with 20 ml 1 M NaOH and 200 ml Bradford reagent. After the colour developed, the plate was read at 595 nm. To determine the protein concentration, a standard curve of protein concentrations over the range 0.06–2.5 mg/ml was used. 2.6. Enzymatic activities 2.6.1. Proteolytic activity Initially, proteolytic activity was determined using 2% denatured haemoglobin, pH 8.5, as substrate (Mika´n, 2001). The substrate was pre-incubated for 10 min at 37  C; then the enzyme was added and incubated for 30 min. The reaction was stopped by adding 305 mM trichloroacetic acid (TCA) followed by centrifugation at 8000  g for 15 min. The supernatant was recovered, neutralised with 500 mM NaOH, and developed with Folin–Ciocalteu reagent followed by incubation for 15 min and reading at 750 nm (Folin and Ciocalteu, 1927). Proteolytic activity was established using a tyrosine standard curve over the range 0.028–0.700 mM. Enzyme activity was defined in units per microliter of extract based on micromoles of tyrosine liberated. Proteolytic activity was confirmed using azocasein, which was hydrolyzed by the enzyme to yield a coloured product that can be read at 540 nm (Sapan et al., 1999). The reaction mixture [20 ml enzyme extract, 190 ml substrate (1.5% azocasein in 0.1 M bicarbonate buffer pH 8.3)] was incubated at 37  C for 60 min, then centrifuged at 8000  g for 15 min. The supernatant was recovered and plated on a microplate to be read at 405 nm. Enzyme activity was defined as relative units of proteinase K under the same conditions. 2.6.2. Amylolytic activity A 1-ml solution of Remazol brilliant blue (RBB) coupled with starch was used as substrate at 0.1% in acetate buffer, pH 6.0, to which 0.05 ml crude extract was added. The absorbance of the blue reaction product was read at 595 nm after 30 min. Units of absorbance were used as relative units of amylase (Akpan et al., 1999). Amylolytic activity was also determined by measuring the release of reducing sugars from soluble starch using the dinitrosalicylic acid (DNS) method (Miller, 1959; Ghose, 1987). A standard curve was produced using known concentrations of glucose over the range 0.2–0.5 mg/ml. 2.7. Determination of protease and amylase isoforms

2.4. Screening of hydrolytic activity on plates Propagules of the selected isolates were harvested from 8-day-old PDA cultures and the suspension was adjusted to a final concentration of 105 propagules/ml. The suspensions were used to inoculate specific substrate plates in order to evaluate hydrolytic activities, following the method proposed by Hankin and Anagnostakis (1975). A minimal medium was prepared by adding 0.1% KH2PO4, 0.2% NaNO3, 0.05% MgSO4, 1% (v/v) trace element solution (0.1% K2HPO4, 0.002% NaMoO4, 0.002% MnCl2, 0.001% FeSO4, 0.0003% ZnSO4, 0.002% CuSO4) to 1.5% bacteriological agar. To induce each specific activity, we used the minimal medium as a basis and supplemented it with one of the following substrates: (i) carboxymethylcellulose (CMC) at 1% (w/v) for cellulases (Mika´n Venegas and Castellanos Suarez (2004); Villalba and Mika´n, 2005; Villalba et al., 2004), to evaluate degradation halos using 1% (w/v) Congo red with contrast by 2% acetic acid; (ii) skim milk (2%) for proteases (St Leger et al., 1997; Ten et al., 2005) to evaluate halos directly on the media, or 0.8% casein (Cowan and Daniel, 1982; Montville, 1983), verifying the proteolytic activity by Ponceau S staining (Green, 1991); or (iii) starch–RBB (0.1%) (Remazol brilliant blue starch complex) for amylolytic activity (Akpan et al., 1999; Omemu et al., 2005), visualizing the clear zone around the colonies against the blue background of substrate. Triplicate cultures were incubated for 96 h at 25  C, after which the diameters of the colony and the clear zone were measured with a Vernier caliper. Activity was determined by the formula: activity ¼ clear zone diameter  colony diameter.

To analyze protease isoforms, isoelectric focusing was performed on polyacrylamide gels, running crude extracts that showed high activities. The crude extracts were desalted, concentrated, and resuspended in deionised water (1/10 of the original volume of extract). Aliquots around 0.5–1 mg protein/ml were applied to a 125  105  0.4-mm polyacrylmide gel and run on the MultiphorÔ II electrophoresis system (LKB Bromma 2117 Multiphor, Pharmacia) connected to a MultiTempÔ III Thermostatic Circulator at 10  C. Running conditions were as described by Mika´n Venegas and Castellanos Suarez (2004), and pI markers (broad pI kit 3.5–9.3, Pharmacia-Biotech) were run together with the samples. After focusing, amylolytic activity was measured using a method proposed by Biely et al. (1985) with some modifications. The gel was immersed in 1% soluble starch in 20 mM phosphate buffer, pH 6.9, and incubated for 40 min at 37  C. Proteolytic isoforms were detected by immersing the gel in 1.5% azocasein in 0.1 M bicarbonate buffer, pH 8.3, and incubating for 1 h at 37  C (Va´zquez Peyronel and Cantera, 1995). 2.8. Purification of alkaline proteases After plate screening for protease activities and protease isoforms among the morphotypes, those showing the highest proteolytic activities and high pI were selected for purification. A crude extract was prepared as described above and was fractionated by ammonium sulphate saturation at 0  C (Scopes, 1982). A bulk precipitate was obtained at 85% saturation and centrifuged at 8000  g for 30 min;

J.A. Rojas et al. / International Biodeterioration & Biodegradation 63 (2009) 169–175 then the pellet was resuspended in a 65% saturated solution and centrifuged again. The supernatant was retained and the pellet was resuspended in a 35% saturated solution. After centrifugation, the supernatant was retained and the pellet was resuspended in 2 mM phosphate buffer, pH 7.4. All fractions were dialyzed against this buffer to remove excess salts. The crude extracts were taken as the purification reference and analyzed together with the fractions to determine enzyme activity and quantity of protein. The 35% saturated fraction, which contained high proteolytic activity, was chromatographed on a 5-ml DEAE Sepharose fast flow column preequilibrated with 25 mM potassium phosphate buffer, pH 8.9. The fraction was concentrated by precipitation with cold acetone and resuspended in the same buffer. The sample (0.7 ml; 0.1120 mg/ml protein) was applied to a column containing 3 ml of the same matrix and eluted with 15–30 ml buffer at a flow rate of 0.5 ml/min to remove unbound protein. The bound enzyme was then eluted with a linear NaCl gradient (0–0.5 M) in 25 mM potassium phosphate buffer (pH 8.9) at the same flow rate. The collected fractions were monitored at 220 and 280 nm with a UV–vis spectrophotometer (Pharmacia) and those with specific activity were pooled, dialyzed extensively against 2 mM potassium phosphate buffer, pH 7.4, concentrated by lyophilization and stored at 70  C. Protein concentration was determined as previously described, and enzyme activity was measured using the azocasein assay (Charney and Tomarelli, 1947; Iversen and Jørgensen, 1995). 2.9. SDS-PAGE in a discontinuous buffer system The bound and unbound fractions were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous buffer system (Laemmli, 1970). The resolving gel was composed of 0.375 M Tris–HCl buffer, pH 8.8, acrylamide-bis T12C2.6, 10% (w/v) SDS and glycerol, and the stacking gel was composed of 0.125 M Tris– HCl, pH 6.8, acrylamide-bis T3.7-C2.45 and 10% (w/v) SDS. The reservoir buffer (pH 8.3) contained 25 mM Tris and 192 mM glycine. Enzyme samples (5-ml aliquots) were loaded onto the wells along with sample buffer [0.08 M Tris–HCl pH 6.8, 10% (w/v) SDS, 12.5% (v/v) glycerol, 0.002% (v/v) bromophenol blue] and b-mercaptoethanol in the ratio 50:1000 to sample buffer. Gels were run at a constant current of 4 mA in resolving gel until the dye reached the end of the cassette (around 3 h at room temperature); then they were silver-stained as described by Rabilloud et al. (1988).

3. Results 3.1. Sample collection and recovery of filamentous fungi Three different folios dating from 1920 to 1930 were evaluated. These units were in advanced stages of deterioration, and different areas of biodeteriorated paper were selected to recover the filamentous fungi. Selection was based on indicators not produced by abiotic factors but related to filamentous fungi. Some of the biodeterioration indicators selected are shown in Fig. 1, corresponding to fungi propagules on the surface of the paper, the material stained and destroyed by fungal action. 3.2. Evaluation of culturable diversity on different media The selected areas of biodeteriorated paper were sampled and the samples were pre-incubated in Letheen broth using the

171

methods described. Each sample obtained from the different folios was cultured to estimate culturable diversity. The diversities obtained with each medium were compared. The criteria for selection were based on supplements added to the cultures (taking MEA as the initial medium) that promoted the growth of different fungi. PDA was used as a control because it is a common and standard medium for isolating and maintaining fungi. No more than 12 different morphotypes were obtained per medium. Statistical analysis showed no significant differences between the media (data not shown) for CFU or for the different morphotypes. However, the morphotypes isolated varied between the media; e.g., malt agar recovered morphotypes that were different from those recovered with V8 supplement. Also, the morphology and growth characteristics varied with the supplement. Morphotype data were obtained from differential counting; the media evaluated showed no significant differences, although recovery in CFU was lower in PDA than in the other three media. Taking into account that the media evaluated were intended to recover greater diversity, the different morphotypes isolated reflect some of the fungal diversity associated with biodeterioration. 3.3. Selection and maintenance of isolates In this phase, we selected representative morphotypes recovered on the different media to evaluate their role in the biodegradation of the stored documents. Thirty-two morphotypes were cultivated on PDA in order to maintain, purify (monosporic cultures) and identify them. Among these 32 morphotypes, we identified several genera of filamentous fungi. For example, the fungus Eladia sacculum, a micromycete previously identified in Czechoslovakia (Kuba´tova´, 1989), was isolated. Other genera found were Chaetomium, Penicillium, Aspergillus, Cladosporium, Alternaria, and the teleomorphic state of Penicillium. 3.4. Screening of hydrolytic activity on plates Three hydrolytic activities were evaluated on plates: proteolytic, amylolytic, and cellulolytic. The screening data enabled us to obtain measurements indicating the activities of each morphotype. In Fig. 2, the three hydrolytic activities of each morphotype are represented by clear zones on the plates. A well-established group for each activity was determined on the basis of statistically significant differences, using a Kruskal–Wallis test and all-pairwise comparison (a ¼ 0.05), and those with higher activities were selected. Among the 32 morphotypes there are isolates with all three hydrolytic activities, but it is notable that isolates with cellulases

Fig. 1. Areas of biodeteriorated paper caused by filamentous fungi; the main indicator is the presence of propagules on the surface of the paper. Other areas of biodeteriorated paper are the staining produced by fungi and the destruction of the paper.

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Fig. 2. Screening on plates of the three hydrolytic activities (cellulases, amylases, and proteases) for the different morphotypes.

also have amylases. Isolates with proteases have amylases as well (Fig. 2). The initial substrate used for protease screening was skim milk. However, we observed that this substrate masked the real activity, so a new substrate, casein, was used on plates to unmask the proteolytic activity as evidenced by Ponceau S staining. As a result, seven morphotypes with higher proteolytic and amylolytic activities were selected for further quantitative analysis (Table 1). 3.5. Enzyme activity and isoelectric focusing (IEF) profile Crude extracts of the selected morphotypes were obtained and hydrolytic activities were determined spectrophotometrically. The haemoglobin and azocasein techniques differed in sensitivity; e.g., E. sacculum activity was 10 times higher when denatured azocasein was used as substrate instead of haemoglobin. Aspergillus sp. and E. sacculum showed the highest proteolytic activities, while Aspergillus and Penicillium showed lower levels (Table 2). Amylases were evaluated by two different methods to corroborate the starchhydrolysis activity accurately. Among the morphotypes studied, Penicillium and Aspergillus appeared to degrade starch efficiently

Table 1 Morphotypes with higher hydrolytic activity selected on the basis of Tukey allpairwise comparison Hydrolytic activity Amylases

Proteases

Morphotype

Organism

Morphotype

Organism

M M M M M M M

Penicillium sp. Chaetomium sp. Penicillium sp. Penicillium sp. Aspergillus sp. Aspergillus sp. Penicillium sp.

M M M M M M M M

Aspergillus sp. Aspergillus sp. Penicillium sp. Aspergillus sp. Penicillium sp. Eladia sacculum Aspergillus sp. Aspergillus sp.

13 15 16 20 25 26 28

1 9 16 19 20 21 25 30

while Chaetomium showed the lowest amylolytic activity. Only Aspergillus sp. (morphotype 25) expressed both amylase and protease activities; the amylase activity was more pronounced (Table 3). Crude extracts with higher activities were selected to determine the isoform profiles of each morphotype. Amylase isoform patterns in the selected morphotypes are summarized in Table 3. Amylases are usually acidic proteins, so most of the amylase patterns observed had several isoforms with low pI (6–3). Also, each morphotype presented multiple acidic isoformsdfor instance, Penicillium sp. (morphotype M 13 has pIs of 3.60, 3.97, 4.19 and 4.33). On the other hand, Chaetomium sp. has almost neutral isoforms (pIs of 6.36 and 6.89). Other morphotypes evaluated (M 25 and M 26), identified as Aspergillus sp., have a single isoform with an acidic pI. The protease isoform patterns of each morphotype are summarized in Table 2. Zymograms for proteases showed basic and acidic isoforms as indicated by the pI values. Aspergillus sp. (M 1) presented two basic isoforms (8.53 and 7.67) and one acidic isoform, while E. sacculum presented the most isoforms with light and dense bands, two with acidic pIs and three with basic ones (8.53– 7.60). Aspergillus (M 25) expressed one band with an acidic pI (5.84). 3.6. Purification of an alkaline protease Different substrates were evaluated as possible carbon sources for protease production, among them were collagen (from bovine leg), skim milk, bovine serum albumin, and casein. Skim milk showed quantitatively the highest total protein production but the specific protease activity was low, probably because of the presence of lactose in this substrate. Casein was chosen as substrate for the induction of protease activity. The morphotype selected for purification was E. sacculum because its isoenzyme profile facilitated the partitioning of an alkaline protease, in contrast to Aspergillus, which has similar isoforms but with lower purification (Table 4). Purification of a single band (silver-stained SDS-PAGE) was

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Table 2 Proteolytic activities obtained from crude extracts of the selected morphotypes, using denatured haemoglobin and azocasein as substrates Proteolytic activity

Isoforms

Morphotype

Organism

Denatured haemoglobin

Azocasein

Specific activitya

Specific activityb

pI

M1

Aspergillus sp.

0.001864

0.0430

8.53 7.67 3.58

M9

Aspergillus sp.

0.000013

0.0002

ND

M 16

Penicillium sp.

0.001344

0.0055

ND

M 20

Penicillium sp.

0.000228

0.0033

ND

M 21

Eladia sacculum

0.000157

0.0612

8.53 8.12 7.60 3.95 3.51

M 25

Aspergillus sp.

0.000331

0.0002

5.84

M 30

Aspergillus sp.

0.001860

0.0467

ND

ND: not determined. a Specific activity: nKats/mg of protein. b Specific activity: absorbance/mg of protein.

achieved by cation exchange chromatography of the 35% ammonium sulphate pellet at high pH, and corresponded to the unbound fraction eluted from the column. This protein has an MW of 35 kDa and a pI of 8.9. 4. Discussion One of the main objectives of this study was to devise a medium adequate for recovering the mycobiota associated with the

biodeterioration process. However, there were no significant differences among the media evaluated. Many of the fungi isolated in this research have been reported in previous studies (Zyska, 1997; Florian and Manning, 2000; Jellison and Jasalavich, 2000; Szczepanowska and Cavaliere, 2000; Gorbushina et al., 2004). Also, the diversity of fungi affecting the documents investigated was maintained because of the storage conditions in the archive. The fact that we isolated a rare micromycete, E. sacculum, suggests that fungal diversity in the archives must be analyzed more thoroughly,

Table 3 Amylolytic activities obtained from crude extracts of the selected morphotypes, based on RBB–starch and reducing sugar using DNS methods, and isoforms of amylolytic activity detected in seven morphotypes Morphotype

Organism

DNS reducing sugars a

RBB–starch

Isoforms b

Specific activity

Specific activity

pI

10.128

1.068

3.60 3.97 4.19 4.33

Chaetomium sp.

0.739

0.278

6.36 6.89

M 16

Penicillium sp.

2.674

0.813

3.55 3.78 4.00 4.19

M 20

Penicillium sp.

8.675

0.551

3.52 3.78 3.97 4.17 6.50

M 25

Aspergillus sp.

6.380

0.523

3.35

M 26

Aspergillus sp.

18.690

1.577

3.89

M 28

Penicillium sp.

22.048

3.973

3.50 3.72 3.94 4.08 4.29

M 13

Penicillium sp.

M 15

a b

Specific activity: nKats/mg of protein. Specific activity: absorbance/mg of protein.

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Table 4 Protease isoelectric points determined from crude extracts of Aspergillus sp. (M 1) and Eladia sacculum (M 21) liquid cultures Sample

No. of bands

Distance from cathode (cm)

pI

M 1 Aspergillus sp.

3

1 6.2 7.5

8.92 5.58 4.75

M 21 E. sacculum

4

0.9 6.1 6.7 7.4

8.98 5.64 5.26 4.81

M 1 Aspergillus sp.

5

0.9 4.9 6.1 6.7 7.4

8.98 6.41 5.64 5.26 4.81

Trypsin (Gibco)

2

7 7.5

5.07 4.75

Proteinase K (Invitrogen)

1

0.6

9.17

M 1 means crude extract from Aspergillus sp. precipitated with cold acetone.

particularly since our study is the first on the Bogota´ Archive. The mycobiota established could include a wide diversity of non-culturable and culturable morphotypes, the latter having different requirements for successful isolation. This could explain the recovery of distinct morphotypes in the supplemented media. The genera identified in this study were characterized by the production of different enzymes involved in plant pathogenesis, which may be extrapolated to the paper biodeterioration process. Paper consists mainly of cellulose, plus minor components that could be the targets of degradation by accessory enzyme activities, e.g., proteases and amylases acting on glues or additives. For example, enzyme production by genera such as Alternaria, Chaetomium, and Cladosporium has been reported (Das et al., 1997; Baraznenok et al., 1999). Also, we obtained environmental genera that have been implicated in the biodeterioration process, such as the classically named Penicillium and Aspergillus (Zyska, 1997). Plate screening of hydrolytic activities allows isolates to be evaluated rapidly and those with higher activities to be identified. In this research, we evaluated celullolytic, proteolytic, and amylolytic activities, first to establish the morphotypes with higher proteolytic and amylolytic activities, and secondly to elucidate the distribution of hydrolytic activities in the population. The isolates selected showed high activity, where the clear zone exceeded the area occupied by the colony. This indicates enzyme production or substrate degradation with high efficiency, attributable to high rates of enzyme secretion. Consequently, the isolates selected on the basis of plate screening seem to be optimal for enzyme production in liquid culture for analysis. We also developed or modified three protocols for evaluating three kinds of enzymes (cellulases, amylases, and proteases) on plates, facilitating selection for production in liquid culture. Our methods could be useful in similar studies or in diversity studies relating to different environments. Similarly, the establishment of a method for quantifying proteolytic and amylolytic activities provides a basis for future studies and enables us to identify fungal species with the capacity to produce extracellular enzymes that contribute to biodeterioration. These determinations also corroborate the qualitative results obtained on plates. The use of these techniques employing liquid culture shows the real activity, particularly specific protein activity. Surprisingly, in addition to genera such as Penicillium and Aspergillus, isolates such as Eladia produce proteases that could act on paper and additives.

Using qualitative and quantitative determinations of enzyme activities, we proceeded to evaluate the isoelectric focusing (IEF) profiles using the morphotypes with higher activities. The IEF profile indicates whether degradation is a result of processes catalyzed by multiple enzymes or just one enzyme complex. Thus, it indicates the biochemical diversity involved in biodeterioration. For that reason, fungi with wide enzymatic profiles, probably extracellular, have an enzyme repertoire designed to adapt to any substrate; in contrast, a limited IEF profile could indicate specialized fungi that have developed specificity for certain paper components or additives. Finally, it reveals aspects of the ecology and diversity of the biodeteriorant population for the Bogota´ Archive; we observed similar phenomena for the isolates evaluated. For example, E. sacculum has a reduced profile of proteases with basic and acidic behaviours that probably act on different paper components, suggesting that this micromycete has become specialized for a specific substrate mainly composed of protein. In contrast, the amylase IEF profiles show that most of the isolates have different isoforms, suggesting that each has become specialized for degrading specific substrates. These phenomena could result from the quantitative distribution of different components or additives in the paper. On the other hand, the protease purification process was aimed at characterizing a protease profile, exploring this type of activity in the biodeterioration process. For this reason, the experiments were designed to establish the presence of alkaline proteases, and we duly established their presence. At the same time, we found the most useful ways of purifying the enzymes involved in this process. 5. Conclusions Biodeterioration is an important process, and thorough study is required to discover the microorganism mechanisms and pathways that act on industrial paper. Enzymes have a key role in this process, facilitating fungal attack on the documents and their components. Therefore, a study of fungal proteases and amylases revealed how fungi could use the paper as a nutrient source for survival and maintenance. Also, we were able to relate proteolytic activity to other enzyme activities such as cellulases. References Akpan, I., Bankole, M.O., Adesemowo, A.M., 1999. A rapid plate culture method for screening of amylase producing micro-organisms. Biotechnology Techniques 13, 411–413. Baraznenok, V.A., Becker, E.G., Ankudimova, N.V., Okunev, N.N., 1999. Characterization of neutral xylanases from Chaetomium cellulolyticum and their biobleaching effect on eucalyptus pulp. Enzyme and Microbial Technology 25, 651. Biely, P., Markovic, O., Mislovicova, D., 1985. Sensitive detection of endo-1,4-betaglucanases and endo-1,4-beta-xylanases in gels. Analytical Biochemistry 144, 147–151. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Bueno, D.J., Silva, J.O., Oliver, G., 2003. Hongos ambientales en una biblioteca: un ˜ o de estudio. Anales de Documentacio´n 6, 27–34. an Canhoto, O., Pinzari, F., Fanelli, C., Magan, N., 2004. Application of electronic nose technology for the detection of fungal contamination in library paper. International Biodeterioration & Biodegradation 54, 303. Cappitelli, F., Sorlini, C., 2005. From papyrus to compact disc: the microbial deterioration of documentary heritage. Critical Reviews in Microbiology 31, 1–10. Carmichael, J.W., Kendrick, W.B., Conners, I.L., Sigler, L., 1980. Genera of Hyphomycetes. University of Alberta Press. Clausen, C.A., 2000. Recognize, remove, and remediate mold and mildew. In: Proceedings of the Second Annual Conference on Durability and Disaster Mitigation in Wood-Frame Housing, pp. 231–234. Cooper, R.M., Wood, R.K.S., 1980. Cell wall degrading enzymes of vascular wilt fungi. III. Possible involvement of endo-pectin lyase in Verticillium wilt of tomato. Physiological Plant Pathology 16, 285–300. Cowan, D.A., Daniel, R.M., 1982. A modification for increasing the sensitivity of the casein–agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases. Journal of Biochemical and Biophysical Methods 6, 31–37.

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