Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 122-132

ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 122-132 http://www.ijcmas.com

Original Research Article Extracellular Enzymatic Profile of Fungal Deteriogens of Historical Palace of

Dubey Shivani1* and Jain Sudhir Kumar2

1Mata Gujri College of Professional Studies, A.B. Road, Indore, M.P., 2School of Studies in Microbiology, Vikram University, Ujjain, M.P., India

*Corresponding author

A B S T R A C T

Present study is aimed to evaluate extracellular enzymatic profiles of seven stone deteriorating fungi, isolated from historically significant Kaliadeh Palace, Ujjain so K e y w o r d s as to provide information about their mode of nutrition and industrial exploitation. The enzymatic activity of fungi wwas performed using specific substrates for the Extracellular analysis of different enzymes such as amylases, cellulases, lipases, caseinase, enzyme, stone catalase and urease. Among the seven test fungi, A. niger, A. flavus, Eurotium deterioration, amsteldomi and Alternaria sp. were found to produce extracellular amylase amylase, enzyme. Eurotium amsteldomi and A. flavus were found positive for cellulase cellulose production and A. niger and A. flavus were positive for lipase production. Catalase activity was shown by Eurotium amsteldomi, Curvularia sp and Talaromyces purpurogenus. While the Urease activity is shown by only A. flavus.

Introduction Significant percentage of world s cultural have demonstrated the involvement of m.o. heritage is made from stones say it be specially fungi in biodeterioration of sandstone, limestone, marble, granite etc. museum collections (Valentin, 2010); The transformation of rocks or stone into library materials (Kowalik, 1980); lower sand and soil is a natural recycling process denomination currency notes (Sharma and which is essential to sustain life on earth. Sumbali, 2014) and in biodeterioration of However, their action on historically even stones (Gadd, 2007). Fungi has the important artifacts in turn may lead to ability to decompose organic matter which permanent loss of nation s heritage leads to destruction of food, textiles, (Scheerer et al., 2009). The stone surfaces plastics, leathers and some other unlikely present a complex ecosystem for a variety of substances (Barron, 1977). Stone by itself biological forms like m.o. such as bacteria, does not favour the growth of fungi unless it fungi, algae, lichens as well as insects, mites has some organic residues of plant leaves, and even higher plants once the bird droppings guano or waste product of colonization take place. Various studies algae or bacteria on it (Sterflinger, 2000). 122

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Evidence of fungi on material of varied complex group of hydrolytic enzymes, composition explores their potential to spoil which add water across amide bonds and the materials aesthetically, physically & thus hydrolyze proteins into small peptides biochemically. The biochemical and amino acid. Previous studies report for deteriorating effect of fungi depicts the synthesis of protease by Aspergillus & involvement of extracellular enzymes Penicillium strains (Chrzanowska et al., secreted by them and causing degradation or 1993). Other m.o. like bacteria also produce deterioration of substrate. protease but fungal proteases are active over a wide pH range and is not so substrate Fungi cause deterioration of stone by means specific (Hankin, 1971). A single organism of both physical and chemical attack which can produce more than one type of protease. more often result synergistic in Fungi are known to produce acid, neutral, deterioration. In fact, several alkaline, and metallo proteases (Teather and cryptoendolithic fungi may actively bore Wood, 1982). In a study on Physiological into the stone and hence physically disrupt diversity of the first filamentous fungi its integrity (Gadd, 2007). It can form isolated from the hypersaline dead sea cracks, fissures, and crevices, extend intensity on the agar media, Molitoris et al. existing ones and lead to the detachment of (2000) described that caseinase showed crystals (Sterflinger, 2000). Biochemical generally the highest activities under all action of fungi may lead to pitting and conditions tested with little effect of salinity etching, mineral dislocation and dissolution and temperature. Due to their ability to (Gadd, 2007). Various metabolic substances survive in this unique ecological niche, these excreted by fungi are colored, leading to fungi may possess novel enzymes with staining of the substrate (Tiano, 2002). improved stability and a specific activity Hyphal sheath polysaccharides are directly that could be exploited for industrial involved in different aspects of stone applications (Kobakhidze et al., 2012). In biodeterioration such as adhesion of the present study seven isolates obtained aerosols; solubilization of stone minerals; from under maintained and deteriorated deposition of carbonates and bicarbonates; historically significant Kaliadeh Palace, formation of complexes with melanin, and Ujjain were evaluated for their extracellular other organic and mineral compounds enzymatic profiles so as to provide (Gutierrez et al., 1995). The production of information regarding their industrial melanins by dematiaceous (dark pigmented exploitation (Table 1). mitosporic) fungi darkens the stone surface, leading to significant aesthetic alterations, Materials and Methods and physical stress. Fungal isolates and Inoculum preparation Number of extracellular hydrolytic enzymes excreted by fungi lead to the formation of Cultures of all the 7 test fungi, A. niger, A. acidic products that cause chemical changes flavus, Eurotium amsteldomi, Curvularia sp, of the material under attack (Koestler, Fusarium sp, Talaromyces purpurogenus 2000). The most prevalent enzymes and Alternaria sp. were maintained on PDA involved in deterioration are Cellulases slants at 28°C. The method employed for which catalyze hydrolysis of cellulose, break inoculum preparation was according to internal bonds to disrupt its crystalline Petrikkou et al. (2001). The isolates were structure (Bhat and Bhat, 1997). Proteases a subcultured on potato dextrose agar and all

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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 122-132 isolates except members of the minimal media plate supplemented with genus Fusarium were incubated at 35°C; carboxymethyl cellulose (CMC) as substrate Fusarium sp was incubated at 30°C. for cellulase. The petriplates were incubated Inoculum suspensions were prepared from in darkness for 7 days at 28°C. The petri fresh, mature (7-days old) cultures grown on plates were then flooded with 2% w/v potato dextrose agar slants. The colonies aqueous Congo red and left for 15 minutes. were covered with 5 ml of sterile distilled After the stain was poured off, agar surface water. 5% of Tween 20 was added to was rinsed with distilled water and again facilitate the preparation of inoculums of flooded with 1M NaCl to destain for about Aspergillus sp. 15 minutes. Finally, NaCl solution was also poured off. The carboxymethyl cellulose The inoculums of Aspergillus sp were degradation around the colony appeared as prepared by gently rubbing the colonies with yellow opaque area against red colour for a sterile loop; the isolates were then shaken undegraded carboxymethyl cellulose test is vigorously for 15 sec with a Vortex mixer considered as positive test. and then transferred to a sterile tube. For slowly sporulating fungi, such as Fusarium, Amylase test the suspensions were obtained by exhaustive scraping of the surface with a sterile loop. Method adapted by Sanivada and Challa Afterwards, the inoculum was filtered using (2014) with some modifications has been a sterile syringe with glass wool so as to used to detect amylase activity. A 1% remove the majority of the hyphae, soluble starch in minimal media (0.003% producing an inoculum mainly composed of NaCl, 0.03% MgSO4 and 0.015% K2HPO4) spore. Then the suspension was collected in in conjunction with 2% agar was used. The a sterile tube. The inoculum size was pH of the medium was adjusted to 7.0 just adjusted to between 1.0 × 106 and 5.0 × 106 before autoclaving. The plates were spores/ml by microscopic enumeration with inoculated with 5 mm inoculum disc with Neubaurs chamber. Sterile 5 mm discs of mycelia and incubated at 28 °C for 7 days. Whatman No. 1 filter paper were soaked in At the end of the incubation period, the the spore suspensions and left to dry and plates were flooded with Lugol s iodine afterwards they were used as culture solution and a yellow colored halo around inoculums. the colony could be seen in an otherwise blue medium indicating amylolytic activity. Enzyme assay Caseinase test For each strain, enzyme hydrolysis was tested in duplicates. As per method of Janda- Method adapted by Sanivada and Challa Ulfig et al. (2009), colony and hydrolysis (2014) with some modifications has been zone diameters on different substrates were used to detect caseinase activity. A 1% milk recorded in mm using a ruler. powder in minimal media (0.003% NaCl, 0.03% MgSO4 and 0.015% K2HPO4) in Cellulase test conjunction with 2% agar was used. The pH of the medium was adjusted to 7.0 just Cellulolytic activity of the test fungi isolates before autoclaving and incubated at 28 °C was determined by following the method of for 7 days. At the end of the incubation Pointing (1999) with some modification. In period, the plates were observed for zone of this method, inoculum discs were kept over 124

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 122-132 clearance after flooding with Coomassie chemicals like Lugol s iodine for detection brilliant blue solution. of amylase, Congo red for detection of cellulose and Coomassie brilliant blue for Urease test caseinase. Other tests performed were lipase test, catalase test and urease test. A. niger As described by Raghuramulu et al, 2011; has shown highest colony diameter on starch Urease test medium (Christensen s urea agar agar & milk agar and zone of hydrolysis on medium) is used for urease activity. During them is also found to be more as compared the preparation of medium phenol red to the other isolates. A. niger, A. flavus, indicator is used and medium was inoculated Eurotium amsteldomi and Alternaria sp. with test fungi. were found to produce extracellular amylase enzyme (Table 1 & 2). Talaromyces The scale for urease was the following: purpurogenus and A. flavus were found (-) no activity; positive for cellulase production and A. (+) weak activity (hydrolysis zone width niger and A. flavus were also positive for <2 mm); producing lipase enzyme. Catalase activity (++) moderate activity (hydrolysis zone was shown by Eurotium amsteldomi, width 2-5 mm); Curvularia sp and Talaromyces (+++) strong activity (hydrolysis zone purpurogenus. While the Urease activity is width >5 mm). shown by A. niger and A. flavus (Table 3).

Catalase test The fungi have shown growth on cellulose agar but hydrolysis zones were not formed Catalase mediates the breakdown of by all test isolates. Slight hydrolytic activity hydrogen peroxide H2O2 into oxygen and of cellulose was observed only in A. flavus water. To determine the catalase producing and Talaromyces purpurogenus. Whereas, potential of test fungi, small inoculums is other studies have demonstrated clear mixed into hydrogen peroxide solution hydrolysis zones on cellulose agar by A. (3%). flavus, A. fumigatus, A. nidulans, A. niger and other species (Gopinath et al., 2005; Catalase activity was evaluated using the Krikstaponis et al., 2001; Ulfig et al., 2009). scale: Alternaria sp is found to show amylase and (-) no activity; caseinase activity. Talaromyces (+) weak reaction (single bubbles); purpurogenus and A. flavus were showed (++) moderate reaction; some diffusion on CMC media, but no (+++) strong reaction (abundant bubbles) significant results obtained. Studies have shown that certain genera such as Results and Discussion Aspergillus, Penicillium and Fusarium have been reported to produce neutral proteases. In present study, the main deteriorating Some acidic and alkaline proteases fungal species isolated from deteriorating producing fungi having commercial values site, A. niger, A. flavus, Eurotium are Aspergillus sp., Penicillium sp. and amsteldomi, Talaromyces purpurogenus, Mucor sp. (Sandhya et al., 2005). All fungal Curvularia sp, Fusarium sp and Alternaria isolates showed growth on media containing sp were evaluated for presence of tributyrin as substrate. A. flavus was extracellular enzyme using specific substrate observed to produce good zone of hydrolysis and the activity is detected by use of certain

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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 122-132 on media with tributyrin as substrate (Chaouche et al., 2013). Studies have whereas A. niger produced comparatively reported that A. fumigatus produces three shorter zone. active catalases; one is produced by conidia, and two by mycelia (Paris et al., 2003). A. Urea hydrolysis was only visualized in case niger has also been shown to produce of A. flavus and A. niger. Urease activity in catalase enzyme with high stability by Isobe A. niger was first reported in 1903, yet the et al. (2006). characteristics of this enzyme have not been determined (Pomar et al., 1970). Smith et al. E. amstelodami showed the lowest colony (1993) worked on first isolation and diameter on milk agar and no zone of characterization of urease from A. niger. hydrolysis were observed in E. amstelodami Deterioration of stored seeds by A. flavus on milk agar (Table 1& 2). Whereas in case and assessment of urease activity was of A. niger, both the colony diameters and reported by Dayal et al. (2001). A. flavus zone of hydrolysis on milk agar was highest. and A. niger and Alternaria sp. found to On tributyrin agar hydrolysis zones around exhibit moderate Urease activity in the colonies were observed in E. comparison to Fusarium sp. amstelodami. No zones were observed on cellulose. Our finding of high amylase and Catalase activity was found to be active in lipase activities in Aspergillus partially case of E. amsteldomi and T. purpurogenus. agrees with Gopinath et al. (2005). They Catalase is used in sterilization and reported the same for A. versicolor from oil- bleaching practices (Akertek and Tarhan, rich environments. The E. amstelodami 1995). A cheap and readily available catalase reaction ranged from weak (single commercial source of catalase can be found bubbles) to strong (abundant bubbles) in the extracellular production by several (Table 2). The urease activities were found microorganisms, especially fungi strains to be weak in E. amstelodami respectively.

Table.1 Colony diameters of various fungi isolated from deteriorating site after 7 days incubation at 25°C

Test fungi Colony diameter* of test fungi on media containing Starch Milk Cellulose Tributyrin A. niger 35.6 ± 3.2 34.4± 3 28.3± 2.6 14.1 ±1.5 A. flavus 29.8 ±0.5 32.8± 1.2 34.3 ± 3 19±0.8 Eurotium amsteldomi 30.2± 4.0 21.2 ±2.3 14.1±2.2 30.2± 3.1 Talaromyces purpurogenus 31.0±0.8 22±2.0 14±0.4 28.5±0.9 Curvularia sp 26.1±0.2 26±1.8 12±0.2 27.1±0.3 Fusarium sp 24.4±3.0 30±1.5 18±2.1 18.5±2.6 Alternaria sp 22.0±2.9 22±1.2 23±2.4 19.8±2.0 *Mean and standard deviation

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Plate.1 Detection of Amylase on minimal agar+Starch (With / without glucose); (a)-(d) Growth of different isolates on media (e)-(i) Addition of iodine to the plate

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i)

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Plate.2 Detection of Casein hydrolyzing enzyme on minimal agar +casein (with/without glucose); (a)-(d) Growth of different isolates on media; (e)-(f) Addition of Coomassie brilliant blue to the plate

(a) (b)

(c) (d)

(e) (f)

Table.2 Hydrolytic activity of stone deteriorating fungi after 7 days incubation at 25°C on agar containing different substrates

Test fungi Hydrolysis zone diameter on media containing Starch Milk Cellulose Tributyrin A. niger 0.9 ±0.1 0.9 ±0.0 0.0 0.7±0.2 A. flavus 1± 0.1 0.8± 0.1 0.8±0.1 1.1 ±0 Eurotium amsteldomi 0.6±0.2 0.0 0.0 0.0 Talaromyces purpurogenus 0.0 0.9±0.2 0.7±2.1 0.0 Curvularia sp 0.0 0.0 0.0 0.0 Fusarium sp 0.0 0.0 0.0 0.0 Alternaria sp 0.5±0.3 0.4±0.3 0.0 0.0 *Mean and standard deviation 128

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Plate.3 Detection of Cellulase enzyme on minimal agar +CMC (with/without glucose) (a)-(d) Growth of different isolates on media; (e)-(f) Addition of Coomassie brilliant blue to the plate

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Table.3 Catalase and Urease tests

Test fungi Catalase Urease A. niger + ++ A. flavus ++ +++ Eurotium amsteldomi + - Talaromyces purpurogenus + - Curvularia sp - - Fusarium sp - + Alternaria sp - ++ Assessed as - = none, + = weak, ++ = moderate, and +++= strong activity

The conclusion is that among seven stone communities for providing varying substrate deteriorating fungi, Aspergillus sp and E. for heterotrophic fungi. Knowledge about amstelodami are the species with the highest the hydrolysis of various substrates will biodeterioration potential to stone artifacts. provide an insight for commercial Extracellular enzymes play an important exploitation of stone deteriorating fungi. Our role in degradation and solubiization of study is aimed to exploit the enzymatic substrate and to make it available for m.o. potential of fungal isolates from deteriorated for nutrition and growth. On the other hand historical palace of Ujjain. However further extracellular enzymatic profile may provide study is required to evaluate the effect of information about the other microbial different pH, temperature, carbon source etc.

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