Isolation, identification and optimization of

Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2011, 1 (4):137-147

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ISSN : 2231 –3168 CODEN (USA) : JMBRB4

Isolation, identification and optimization of xylanase enzyme produced by aspergillus niger under submerged fermentation Padmavathi Tallapragada* and Kavya Venkatesh Department of Microbiology, Centre for PG Studies, Jain University, Bangalore, Karnataka India

______________________________________________________________________________ ABSTRACT The objectives of the present study were isolation, identification and characterization of xylanase producing fungi, optimization of carbon and nitrogen sources and cultural conditions for xylanase enzyme production. A variety of microorganisms were reported to produce endoxylanases, which can degrade ß -1, 4-xylan in a random fashion, yielding a series of linear and branched oligosaccharide fragments. The fungal strains were isolated from garden soil by serial dilution technique and Aspergillus niger was identified and isolated in pure form. In conformation screening by congo red test, based on the reddish zone of enzyme activity formation in oat spelt xylan agar plates, Aspergillus niger was selected and optimized for xylanase enzyme production in czapek dox medium using different carbon and nitrogen sources. Maximum enzyme activity was observed in xylan as carbon source and yeast extract as nitrogen source. Optimum pH and temperature for xylanase activity were found to be 8 and 28°C, Thus the present study proved that the fungal strain A .niger used was potential and useful for xylanase production. Key words: Xylanases, submerged fermentation, Aspergillus, optimization. ______________________________________________________________________________ INTRODUCTION There are several applications of xylanases in industry [1]. Currently, the major applications of xylanases are in pulp and paper, feed, and baking industries. Xylanases are used in the prebleaching of kraft pulp to reduce the use of harsh chemicals in the subsequent chemical bleaching stages. The enzymatic treatments improve the chemical liberation of lignin by hydrolyzing residual xylan. This reduces the need for chlorine-based bleaching chemicals, which is beneficial for the environment [2]. In feed formulations, cooperation of xylanases, glucanases, proteinases and amylases reduces viscosity of the feed and increases the adsorption of nutrients. Enzymes liberate nutrients either by hydrolysis of non-degradable fibers or by liberating nutrients blocked by these fibers. In the food industry, xylanases are used to improve the dough 137

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Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________ properties and baking quality of bread and other baked goods by breaking down the polysaccharides in the dough. The enzyme treatment has favourable effects on dough handing, bread volume, texture and stability [3]. In combination with pectinases and other enzymes, xylanases have also been used in other processes such as clarification of juices, extraction of coffee, and extraction of plant oils and starch. Other potential applications include the conversion of agricultural waste and the production of fuel ethanol [4]. Filamentous fungi are particularly useful producers of xylanases from the industrial point of view, due to the high production level and extra cellular secretion of enzymes, as well as relative ease of cultivation. In general, xylanase activity levels from fungal cultures are typically much higher than those from yeasts or bacteria [5]. Xylanolytic enzymes are receiving increasing attention because of their potential applications in improving digestibility of animal feed [6], pulp bleaching [7] and bioconversion of lignocelluloses into feed-stocks and fuels [7]. Principal xylanolytic enzymes are endo- betaxylanases, which attack the main chain of xylans, and beta-xylosidases, which hydrolyze xylooligosaccharides into D-xylose. Several studies have shown that the xylanases are coinduced in response to xylan or natural substrates containing hemicellulose or even by pure cellulose [8]. Xylanases have been produced by a variety of microbes on different sources that is of great importance and application. In the view of this, there is a need to search new sources of xylanases and their characterization. The present paper reports on the isolation and optimization of xylanase from a fungal source Aspergillus niger in Czapek’s dox medium. MATERIALS AND METHODS Collection of samples The garden soil samples were collected using pre- sterilized sample bottles and sterile spatula from Bangalore south of India. Precautionary measures were taken to minimize the contamination. The soil was collected from 5 to 6 places in and around Bangalore and the collected soil samples were pooled. The mixed soil sample was taken or isolating xylanase producing fungi. Isolation and Identification of fungi Preliminary screening was done by following serial dilution technique [9]. A total of 7 fungal strains were isolated from the pooled soil sample from Bangalore south of India, they are Aspergillus niger, Trichoderma, Cladosporium, Rhizopus, Fusarium, Aspergillus sp, Mucor. Aspergillus niger being the most predominant was used for xylanase production. Fungi isolated were identified using standard reference manuals [10] by wet mount preparation. Aspergillus niger isolates were preserved on Czapek’s dox agar plates as pure culture [11] for further studies. Secondary screening Aspergillus niger isolated from the preliminary screening were cultured in liquid Media (Czapek’s dox broth) in Erlenmeyer flasks. After incubation on a rotary shaker (28°C, 180 rpm) for 6 days, the culture broth was filtered using whatmann filter paper 1and the supernatant was collected for enzyme assay for xylanase production. Its ability to produce xylanase enzyme was further confirmed by Congo red test by growing Aspergillus niger on oat spelt xylan agar plates. 138


Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________ After incubation plate was treated with Congo red and washed with 1 M NaCl [12]. This confirmed Aspergillus niger was xylanase producing strain and were preserved on Czapek’s dox agar plates as pure culture for further studies. Xylanase assay Xylanase activity was assayed by using oat spelt xylan (Fluka) as enzyme substrate. The reaction mixture contained 0.5 ml of substrate solution of 0.25 g of xylan wetted with 2ml 95% ethanol and 22.5ml of distilled water in a beaker and covered with alluminium foil and boiled to dissolve on hot plate at 100°C for 10 min and made up the volume to 25ml with distilled water. To this solution, 0.1 ml of the culture filtered supernatant crude enzyme and 1ml sodium acetate buffer was added. The mixture was incubated at 40°C in water bath with shaking for 15 min. Released reducing sugar was measured using 3, 5-dinitrosalicylic acid [13] and glucose as standard. Colour was developed by boiling in water bath for 5 min and read, using spectrophotometer at 540 nm. One unit of activity was defined as amount of enzyme required to liberate 1µmol of xylose per minute under the assay conditions. Characterization of xylanase production The characterization of xylanase production was carried out based on stepwise modification of the governing parameters for xylanase production. The effect of various carbon sources: lactose, sucrose, maltose, dextrose, xylan, cellulose, and mannitol were examined using czapek’s dox broth, cultivation was carried out at ambient temperature (28°C ± 3°C) for 6 days [14]. Similarly nitrogen sources: peptone, yeast extract, sodium nitrate, ammonium sulphate, ammonium nitrate, meat extract and beef extract were used. [15]. The optimization of salt concentration was carried out using NaCl and KCl, among which NaCl was used at different concentrations, varying from 1 to 6% [14]. The effect of agitation was important as it is directly related to oxygen supply, thus the effect was examined by using regular incubator for non agitating flasks and shaker incubator for agitating flasks [16] .The effects of incubation period were evaluated by 24 h interval by checking the enzyme activity [17]. The effect of pH was observed changing the pH 5 to 10 in the production medium [18]. The effect of cultivation temperature on the enzyme production was examined at different temperatures starting from 25 to 60oC for enzyme assay and 28oC, 32oC, 37oC, 40oC for growth [19]. The effects of incubation period were evaluated by 24 h interval by checking the enzyme activity [17]. Optimization of inoculum size for xylanase production was carried out by adding approximately 100 to 150 spores of A.niger to 9 ml of sterile water blank. This suspension was serially diluted -1 -6 to 10 to 10 . 1ml of the diluted sample from each dilution was transferred to sterile media and incubated at 28°C ± 2°C for 5-7 days. Optimization of growth conditions for xylanase production by Aspergillus niger in Czapek’s dox broth before and after optimization for the above parameters were also carried out [20]. Statistical Analysis: All the above readings obtained at 540 nm absorbance were subjected to student t-test.

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Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________ RESULTS AND DISCUSSION Identification of fungi was done based on colony characters and microscopic examination .The most predominant fungi being Aspergillus niger. The confirmative congo red test was positive by forming reddish orange halo-zone of hydrolysis this confirmed Aspergillus niger was xylanase producing strain and was preserved on Czapek’s dox agar plates as pure culture by point inoculation for further studies. Standard glucose was estimated by DNSA method at 540 nm for the further enzyme activity. When different carbon sources were used in the production medium, maintaining other physical parameters and nitrogen source constant, the highest enzyme activity was obtained in oat spelt xylan (13.88 Unit/ml), and least in mannitol (2 Unit/ml) while maltose, cellulose, sucrose, lactose and dextrose did not show any significant production of xylanase (Figure 1).

Soluble substrates have some advantages compared to the cellulosic materials. A series of different carbohydrates have been studied for Aspergillus niger growth [21]. Hydrolysates of many different bulk materials have been used for xylanase production for decades. Hemicelluloses were suitable substrates for the xylanase production with concomitant low production level [22]. Notably, Aspergillus niger produces higher xylanase activities when cultured on pure xylan [23]. Since xylan is unable to enter the microbial cell, it has been suggested that low molecular weight degradation products of xylan hydrolysis penetrate into the cells and induce the production of hydrolytic enzymes [24]. xylanases are induced by monosaccharides and disaccharides. There are only a few reports regarding the use of different soluble carbon sources for enzyme induction in Aspergillus niger. Xylan seems to be efficient inducers of xylanase activity. Several substances have been indicated in the literature as suitable carbon sources for xylanase producing microorganisms, oat wheat [25], birchwood xylan [26], oat spelt xylan [27], bagasse xylan .wheat bran arabino-xylan [28], wheat bran [29], and rice bran [30] are few recommended sources. Typical nitrogen sources for Aspergillus niger cultivation was found to be yeast extract (14.37 Unit/ml) as it shows high yield. Whereas peptone, ammonium, beef extract and nitrates can 140


Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________ stimulate enzyme production [31] but not as high as yeast extract (Figure 2). In the shake flask cultivations, the initial 30 g/l xylan and 2 g/l yeast extract concentration was considered the best [32]. The Maillard reaction may have a bad influence on the cultivation, while using few carbon sources, since autoclaving involves high temperatures, the colour-forming Maillard reaction plays a significant role in this process. The Maillard reaction consumes nutrients such as amino acids and saccharides, and the colour products can be harmful for microbes and cell growth [33]. High temperature, high substrate concentration and alkaline conditions greatly increase the intensity of the colour reaction [34]. Our results suggest that yeast extract is a better nitrogen source and nutrient material than other common organic or inorganic nitrogen sources [15].

Among the 2 salts used (Na Cl and K Cl), Na Cl gave high value (8.33 Unit/ml) when compared to KCl (7.33 Unit/ml) .Varying percentage of Na Cl salt concentration was used to determine the optimum enzyme production, was confirmed to be highest at 1% of salt concentration (3.39 Unit/ml) (Figure 3). To date there is no literature available on the xylanase production by A.niger, but similar work was carried out using Penicillium oxalicum for xylanase production [14]. The enzyme production by Aspergillus niger was seriously affected by agitation [35]. Low xylanase activities were obtained in non agitated flasks, most probably due to oxygen or mass transfer limitations, while on agitation high xylanase was produced, probably due to good oxygen supply. The effect of oxygen saturation has greater impact on xylanase production. For proper growth and enzyme production, agitation is important as it ensures aeration. Aeration has essential functions: oxygen supply for aerobic metabolism, and removal of CO2, heat, water vapor, and volatile components produced during the metabolism. Aeration also has very important effects on hydration properties [36]. For good ventilation, agitated and non-agitated culture conditions on xylanase production were studied. The result revealed that agitated flasks 141


Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________ showed better enzyme activity of 12.65 Unit/ml than non-agitated flasks (6.08 Unit/ml). This revealed good ventilation contributed to higher enzyme activity (Figure 4).

The time course of xylanase production was investigated and maximum production was observed after 6 days (6.11 Unit/ml) while minimum was noted at 24 h (0.66 Unit/ml) (Figure 5). Further incubation after this did not show any increment in the level of enzyme production, probably due to increase in toxic unwanted wastes and depletion of nutrients in the media, which leads to decreased growth and enzyme.

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Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________

pH is an important parameter in the production of enzymes by Aspergillus niger optimum pH with high xylanase production was found to be pH 8 (5.51 Unit/ml) and minimum was observed at pH 5 (3.33 Unit/ml) (Figure 6). Earlier reports indicated that a rather high pH (7.0) is essential for good production of xylanases production, although growth (broth viscosity) was evidently better at pH 8.0 than at pH 7.0.

A high pH (8.0) was essential for high xylanase production in cellulose medium. During the course of the fermentation, the nitrogen source can significantly influence the pH of the medium [37]. The growth and enzyme production dropped significantly above pH 8. Optimum pH not

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Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________ only provides suitable condition for growth and enzyme production, but will also determine the enzymatic action on the substrates and enhance the enzyme stability.

The cultivation temperature does not only affect the growth rate of an organism, but it also has a marked effect on the level of xylanase production. Aspergillus niger grew well at 28oC (8.98U/ml) (Figure 7) when cultivated in Czapex dox media, but xylanase production was significantly decreased at higher temperature [24]. The net temperature is influenced not only by the environmental temperature, but also by the increase in temperature generated from the metabolic activities of the fungi growing on the solid substrates. It might be due to that at high or low temperature than optimum, the growth of the fungus was inhibited and hence the xylanase production was decreased [38].

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Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________ Incubation temperature for enzyme substrate reaction plays a critical role in enzyme productivity [39]. So, the enzyme activity in present studies was investigated at different temperatures ranging from 25-60°C (Figure 8). The Aspergillus niger gave promising results, with maximum activity of xylanase, when incubated at 45°C (16.00 Unit/ml) and minimum was observed at 25°C (7.33 Unit/ml). When the temperature was increased or decreased from 45oC, the activity of xylanase was gradually reduced. Probably due to enzyme denaturation, conformation change, as enzymes are proteins. -1 The effect of inoculum size was examined using the spore suspension of concentration from 10 to 10 -6 dilution for a fermentation period of up to 6 days. The results of the enzyme productivity -1 profiles at different inoculum sizes are shown in Figure 9, higher inoculum size of 10 dilution resulted in a higher xylanase productivity compared to other inoculum sizes with the maximum productivity of 5.37 Unit/ml. At lower inoculum sizes, it was observed that the time taken to achieve maximum growth or enzyme productivity was much longer. This is clearly shown that with the inoculum size of 10-6 dilution, the enzyme has not achieved maximum productivity even after 6 days of fermentation. Higher enzyme production at higher inoculum is related to the rapid growth of the fungus, which resulted higher degradation of the substrates and increase availability of the nutrients.

Results show the profiles of the xylanase production by A. niger in Czapek dox medium before and after optimization of the cultural conditions and medium composition. As shown in the Figure 10, xylanase production is high (5.79 unit/ml) in optimized conditions when compared to unoptimized (4.80 unit/ml) conditions. 145


Padmavathi Tallapragada et al J. Microbiol. Biotech. Res., 2011, 1 (4):137-147 ______________________________________________________________________________

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