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Xylanase production by a newly isolated Aspergillus foetidus strain and its characterization
Process Biochemistry 40 (2005) 1763–1771 www.elsevier.com/locate/procbio
Xylanase production by a newly isolated Aspergillus foetidus strain and its characterization Amita R. Shah, Datta Madamwar* Post Graduate Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India Received 24 December 2003; accepted 17 June 2004
Abstract Xylanase production by a newly isolated Aspergillus foetidus MTCC 4898 was studied using submerged fermentation under shaking. The maximum xylanase yield (210 U/ml) was obtained with negligible cellulase activity at 30 8C using 1% birchwood xylan as substrate in 3 days of cultivation. The strain was able to produce extracellular xylanases on birchwood xylan as well as on cellulose rich substrates also with very poor cellulase production (<0.1 FPU/ml). The partially purified enzyme recovered by ammonium sulphate fractionation showed maximum activity at 50 8C and at pH 5.3. The enzyme was highly stable at 40 8C but retained 36% of its activity at 50 8C after 3 h. The presence of KCl, CaCl2, glycerol, xylan and trehalose significantly improved thermostability at 50 and 60 8C. Zymogram analysis indicated three isoenzymes. The enzyme preparation was completely stable at freezing temperature for up to 6 months and reasonably stable at refrigeration temperatures for more than 2 weeks. # 2004 Elsevier Ltd. All rights reserved. Keywords: Xylanase; Cellulase; Aspergillus foetidus; Birchwood xylan; Thermostability
1. Introduction In recent years, increasing concern over preserving resources and environment has initiated a growing interest in producing microbial enzymes. Xylanases from microorganisms have attracted a great deal of attention in the last decade because of their biotechnological potential in various industrial processes such as food, feed and paper-pulp industries. Moreover, xylanases have immense potential for increasing the production of several valuable products like xylitol and ethanol in a most economic way [1]. Hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of plant polysaccharides. Xylan is the major constituent of hemicellulose. Endo-xylanase (b-1,4-Dxylan-xylanohydrolase, EC 3.2.1.8) is the key enzyme for xylan depolymerization. Xylanases are produced by both prokaryotes and eucaryotes. A large number of bacteria and * Corresponding author. Tel.: +91 2692 226863; fax: +91 2692 226865. E-mail address: [email protected] (A.R. Shah), [email protected] (D. Madamwar). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.06.041
fungi are known to produce xylanases [2,3]. Filamentous fungi are interesting producers of this enzyme from an industrial point of view due to extracellular release of xylanases, higher yield compared to yeast and bacteria and production of several auxiliary enzymes that are necessary for debranching of the substituted xylans [4]. However, fungal xylanases are generally associated with concurrent production of cellulases [5]. Characterization of xylanolytic enzymes is important for its biotechnological application. The cost of enzyme is one of the main factors determining the economics of a process. This can be partially achieved by optimising fermentation medium. Several industrial processes are carried out using whole cells as the source of enzymes but the efficiency can be improved by using isolated and purified enzymes. Criteria for selection of particular method for isolation and purification depend on its end use. A high state of purity is generally not required in food processing, detergent as well as paper pulp industry, however it may be necessary to exclude certain contaminating enzymes [6]. Many of the fungal enzymes are extracellular and can easily be separated from the mycelia by filtration. It is usually necessary to concentrate the crude
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enzyme extract by spray drying, lyophilization or precipitation by ammonium sulphate or solvents. In industrial applications, the enzymes should be ideally tolerant to many physico-chemical environmental factors, viz. temperature, pH, metal ions, etc. as well as should have high catalytic efficiency. The stability of enzymes can be improved using several methods/techniques like chemical modification, immobilization or treatment with additives, viz. polyols or osmolytes or by protein engineering. Among various methods available for enhancing the enzyme functional stability, addition of low molecular weight additives to change the microenvironment of the enzyme solution is a relatively simpler, feasible as well as a cheaper and practical means [7]. In a screening programme, we isolated a new strain of Aspergillus from Vallabh Vidyanagar (Gujarat, India) showing xylanase producing ability. In the present investigation we report on production of xylanases using this strain and optimization of various physico-chemical parameters. The biochemical properties of the partially purified enzyme were also examined.
2. Materials and methods 2.1. Materials All chemicals used were of analytical grade. Potato dextrose agar (PDA) was obtained from Hi-media (India). Birchwood xylan was obtained from Fluka, Germany. Wood pulp (60% cellulose, 30% hemicellulose) was procured from Rohit pulp and paper mill, Vapi, India and other lignocellulosic substrates like wheat straw (30% cellulose, 50% hemicellulose and 15% lignin) and corncobs (45% cellulose, 35% hemicellulose, 15% lignin) were from local farms.
Tween 80, 0.1% (v/v); (NH4)2SO4, 1.4 g/l; KH2PO4, 2.0 g/l; urea, 0.3 g/l; CaCl2, 0.3 g/l; MgSO47H2O, 0.3 g/l; FeSO47H2O, 5.0 mg/l; MnSO4H2O, 1.6 mg/l; ZnSO47H2O, 1.4 mg/l; CoCl2, 2.0 mg/l; adjusted to pH 5.0. Birchwood xylan (1% (w/v)) was used as a substrate. Erlenmeyer flasks (500 ml) containing 100 ml of medium were inoculated with 2 ml of 2 106 spores/ml suspension prepared from a week old potato dextrose agar slants of A. foetidus. Spore count was done microscopically using Neubauer’s chamber. Inoculated flasks were incubated on a rotary shaker at 180 rpm at 30 8C. Samples were withdrawn at regular intervals and filtered through Whatman filter No. 1 and filtrate was used for analysis of extracellular xylanase, cellulase, and total soluble protein. 2.5. Enzyme activity assays Xylanase (EC 3.2.1.8) activity was measured using 1% birchwood xylan (4-O-methyl glucuronoxylan) solution (prepared in 0.05 M citrate buffer, pH 5.3) as a substrate [9]. The release of reducing sugars in 10 min at 50 8C at pH 5.3 were measured as xylose equivalent using dinitrosalysylic acid (DNS) method [10]. One unit of xylanase activity (U) is defined as the amount of enzyme liberating 1 mmol of xylose per minute. Filter paper cellulase activity was measured according to IUPAC recommendations, employing filter paper (Whatman No. 1) as substrate [11]. The release of reducing sugars in 60 min at 50 8C at pH 4.8 (0.05 M citrate buffer) was measured as glucose equivalent using DNS method [10]. One unit of filter paper activity is defined as the amount of enzyme liberating one mmol of glucose per minute. 2.6. Protein estimation Soluble protein from filtered fermentation broths was determined by following Lowry’s method [12].
2.2. Microorganism 2.7. Study of physico-chemical factors Aspergillus foetidus MTCC 4898 was isolated from decaying agricultural waste and identified by the Institute of Microbial Technology (IMTECH), Chandigadh, India. It was maintained on PDA slants and stored at 4 8C. 2.3. Pretreatment of lignocellulosic substrates Wheat straw and corncobs were washed, dried, milled and sieved before use to obtain a 50 mesh size. Each substrate was given alkali treatment with 1N NaOH in a 5% (w/v) slurry for 18 h at room temperature. After treatment, the substrates were thoroughly washed with water to neutral pH, dried and reground to 50 mesh size.
For studies on the effect of pH on xylanase production, the isolate Aspergillus foetidus was grown in the MS medium containing 1% birchwood xylan with different initial pH in the range of 3.0–9.0. Xylanase production was also studied at different temperatures, viz. 20, 25, 30, 37 and 42 8C. Influence of size of inoculum was carried out using 104 to 1010 spores/ml in the medium. Influence of various substrates and nitrogen sources was studied by growing the culture in the basal medium with different carbon/nitrogen source. Various combinations of inorganic and organic nitrogen sources were also tried.
2.4. Xylanase production by submerged fermentation
2.8. Partial purification of xylanases by ammonium sulphate fractionation
For submerged fermentation, Mandels and Sternburg’s (MS) basal medium [8] containing proteose-peptone, 1 g/l;
A calculated amount of solid ammonium sulphate was added to the culture supernatant with constant stirring at
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10 8C to achieve 40% saturation. After centrifugation at 8000 rpm at 4 8C for 20 min the precipitate was discarded and the supernatant was subsequently adjusted to 40–70% saturation by addition of calculated amounts of ammonium sulphate. The precipitate was dissolved in small volume of buffer. The enzyme solution was subjected to dialysis for about 18–24 h at 10 8C against 0.05 M citrate buffer (pH 5.3) fortified with 100 ppm sodium azide, with three intermittent changes of the buffer and xylanase activity and protein estimation were carried out. 2.9. Activity detection by zymogram staining Zymogram analysis was done according to Schwarz et al. (1987) [13]. Native poly acrylamide gel electrophoresis (native-PAGE) at 10% gel was performed after incorporating birchwood xylan solution (final concentration 0.1% (w/ v)) in the separating gel before addition of ammonium persulphate and polymerization. After the run, the gel was submerged in 0.1% (w/v) Congo red solution for about 10 min. The excess dye was removed by washing in 0.1 M NaCl and then the gel was transferred to 5% (v/v) acetic acid. 2.10. Determination of optimum temperature and pH, thermostability and pH stability
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refrigeration temperature (5–7 8C) and room temperature (25–30 8C). The enzyme solution was stored in sterile Eppendorf vials. Xylanase activity was assayed at regular intervals.
3. Results and discussion 3.1. Time course of xylanase production Xylanase production was studied at 30 8C for 7 days in liquid MS medium containing 1% birchwood xylan as substrate (Fig. 1). Xylanase production started from the second day and reached a maximum on the fourth day (80.5 U/ml). The volumetric productivity (U/(l h)), which is an important parameter to assess the effectiveness of the process [4], was highest on the third day (1047 U/(l h)). From the sixth day onwards, a significant decrease in production was observed. Xylanases are usually expressed at the end of the exponential phase and harvesting time is correlated to the medium under consideration [2]. The phenomenon of sudden increase and subsequent decrease in enzyme activities during the cultivation period has also been noted in xylanase produced by Aspergillus sydowii MG-49 [14] and Streptomyces sp. CH-M-035 [15]. 3.2. Influence of temperature and pH
The xylanase activity of the partially purified enzyme was assayed at different temperatures ranging from 35 to 65 8C. The energy of activation of the xylanase catalysed reaction was determined by measuring the maximum velocity, V, at different temperatures and plotting log10 V against 1/T. The thermal stability was determined at the temperatures 30, 40, 50, 60 and 70 8C after incubation of suitably diluted enzyme samples in absence of substrate for 0.0, 0.5, 1.0, 2.0 and 3.0 h. The optimum pH was determined using buffers ranging from 3.0 to 8.0. Citrate buffer (0.05 M) was used in the pH range of 3.0–5.6 and phosphate buffer (0.02 M) was used for pH 6.0–8.0. To study the stability of partially purified xylanase at different pH, appropriate dilutions of xylanase solutions were made in above buffers in the range of 4–8 pH and incubated at 50 8C for 30 min. After cooling, residual activity was estimated under standard conditions of xylanase assay. To study the influence of various additives on heat stability, appropriate dilution of xylanase solution was incubated in the presence of various additives, viz. KCl (50 mM), CaCl2 (50 mM), polyethylene glycol-3300 (50 mM), birchwood xylan (3.0% (w/v)), Tween 80 (1.0% (v/v)) and glycerol (10.0% (v/v)) at 50 8C for 30 min and 60 8C for 15 min.
Xylanase production at different temperatures was examined for 4 days keeping the other fermentation conditions constant. Xylanase production increased with increase in temperature from 20 to 30 8C. Maximum production of xylanase (80.5 4.0 U/ml) was obtained at 30 8C (Fig. 2). At 37 8C a significant decline (36%) in xylanase activity was evident. Growth and xylanase production totally ceased at higher temperature (42 8C). Similar observations were shown by Suh et al. for Trichoderma reesei [16]. It was revealed that environmental
2.11. Storage stability The storage stability of purified xylanase was studied by keeping the membrane sterilized enzyme solution in 0.05 M citrate buffer (pH 5.3) at deep freeze ( 10 to 20 8C),
Fig. 1. Time course of xylanase production by Aspergillus foetidus MTCC 4898 on xylan based media under submerged fermentation at 30 8C.
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A.R. Shah, D. Madamwar / Process Biochemistry 40 (2005) 1763–1771 Table 1 Xylanases and cellulases production by Aspergillus foetidus MTCC 4898 on various substrates at 30 8C under submerged fermentation Substrate
Xylanase activity (U/ml)
Protein (mg/ml)
Cellulase activity (U/ml)
0.5% Xylan 1% Xylan 2% Wheat straw Alkali treated 2% wheat straw 2% Corn cobs Alkali treated 2% corn cobs 2% Wood pulp 1% Glucose !% Lactose 1% Xylose
59.0 80.5 20.1 19.2 29.0 25.1 51.2 2.9 3.5 25.6
0.39 0.61 0.74 0.47 0.58 0.43 0.64 0.31 0.29 0.34
0.03 0.05 0.10 0.09 0.09 0.02 0.01 0.02 0.03 0.03
Fig. 2. Influence of temperature on xylanase production by Aspergillus foetidus MTCC 4898 under submerged fermentation.
temperature not only affects growth rates of organisms but also exhibit marked influence on the levels of xylanase production. Another important factor significantly affecting the production of xylanases is the initial pH of medium. Xylanase production by this isolate was observed in the range 2.0–7.0 pH (Fig. 3). Growth and xylanase production completely ceased at 8.0 and 9.0 pH. Maximum xylanase production (80.5 U/ml) was found at pH 5.0. A marginal decrease in xylanase yield was seen at lower pH, i.e. at pH 3.0 and 4.0. Decline in xylanase production was remarkable at pH 6.0 and 7.0. The results clearly indicated acidophilic nature of the fungus. With rare exceptions, fungi reported to produce xylanases at initial pH lower than 7.0. There has been increased usage of xylanase preparations having an optimum pH around 5.0 invariably from fungi [17]. 3.3. Xylanase production on various substrates Among the various substrates tested, 1% birchwood xylan was found to be the most effective for xylanase production (Table 1). Maximum xylanase production (80.5 U/ml) was achieved in 4 days of incubation at
Fig. 3. Influence of pH of the medium on xylanase production by Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation.
30 8C (pH 5.0) with only 0.05 U/ml filter paper activity. By reducing the concentration of the xylan (0.5%) xylanase production was significantly reduced. Wood pulp (2.0%) was also found to be a good substrate, which yielded 51.2 U/ ml xylanase activity. It was striking that, even in presence of such a cellulose rich substrate also, cellulase production was negligible. Wheat straw (2.0%) and corncobs (2.0%), individually showed relatively lesser xylanase production 20.1 and 29.0 U/ml, respectively but again with negligible cellulase activity. In A. terreus, selective induction of xylanases occurred by employing xylan or xylobiose or Dxylose as the substrate whereas in presence of cellulose or cellobiose both are produced [18]. In order to increase the accessibility of the cellulosic and hemicellulosic substrates, mild alkali treatment with1 M NaOH is a widely accepted method for solubilizing lignin from lignocellulose. In this study, xylanase activity did not increased even after mild alkali treatment, rather, it was marginally reduced. Such negative effects of alkali treatment on xylanase production were also reported by studies on Aspergillus ochraceus. A possible explanation for this observation has been suggested that rapid consumption of carbon sources and concurrent release of monomeric sugars would lead to a repression in enzyme synthesis [19]. Use of soluble sugars can reduce certain problems during large-scale fermentation. In this study pure sugars gave good growth but xylanase production was very poor. With 1% glucose, 1% lactose and 1% xylose, xylanase production was 2.9, 3.5 and 25.6 U/ml, respectively. Glucose, xylose and lactose (1.0% (w/v)) proved to be ineffective as inducers for xylanase production in Fusarium oxysporum as reported by Christakopoulos et al. [20]. Smith and Wood [21] also reported very low titres of xylanases on glucose and lactose, while xylose gave slightly better production (25.0% production of the control). As reviewed by Bajpai [22], fungal xylanase appear to be inducible or under derepression control, which includes enzyme production on carbon sources that are used slowly. In some fungi, high xylanase production has been shown to be linked strictly to cellulase production [5]. But
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Aspergillus foetidus MTCC 4898 did not produce high amount cellulases in spite of using cellulose rich substrates like wood pulp, wheat bran and corncobs. This is one of the attractive features of this isolate.
Table 3 Effect of combination of nitrogen sources on xylanase production by Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation
3.4. Influence of nitrogen sources
Ammonium sulphate (1.4) + urea (0.3) + proteose peptone (1.0) Ammonium sulphate (1.4) + urea (0.3) Ammonium sulphate (1.4) + urea (0.3) + proteose peptone (0.5) Urea (0.3) + proteose peptone (1.0) Ammonium sulphate (1.4) + proteose peptone (1.0) Ammonium sulphate (1.4) Ammonium sulphate (2.8) Proteose peptone (1.0) Proteose peptone (2.0) Urea (0.3) Urea (0.6) Calcium nitrate (2.0) + urea (0.3) + proteose peptone (1.0) Sodium nitrate (1.8) +urea (0.3) + proteose peptone (1.0) Ammonium nitrate (1.2) +urea (0.3) + proteose peptone (1.0)
The mechanisms that govern the formation of extracellular enzymes are influenced by the availability of precursors for protein synthesis [2]. Furthermore, the nitrogen source can significantly affect the pH of the medium during the course of fermentation [23]. The effect of different organic nitrogen sources were also studied. Maximum xylanase activity (105.4 U/ml) was evident when 0.05% proteose peptone was added (Table 2). Organism could show significantly good activity even in the absence of any organic nitrogen source. These results are in contrast with many investigators, who have reported that fungi produce more xylanases on addition of complex organic nitrogen sources [24–27]. Although Kelley has shown that higher amounts of easily fermentable carbon in the complex nitrogen sources could enhance growth rather than enzyme production [28]. When different combinations of organic and inorganic nitrogen sources were incorporated in the Mandels and Sternburg’s medium (Table 3), maximum yield of xylanases (105.4 U/ml) was obtained using a combination of ammonium sulphate (1.4 g/l), urea (0.3 g/l) and proteose peptone (0.5 g/l). Excluding either urea or ammonium sulphate from the combination produced a significant decrease in the yield of the enzyme. It was also observed that ammonium sulphate could be replaced by calcium nitrate and sodium nitrate without much loss of the xylanase yield. 3.5. Influence of size of inoculum The inoculum size must be large enough to colonize all the substrate particles [29]. In the present investigation, four
Table 2 Effect of complex organic nitrogen sources on xylanase production by Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation Source of organic nitrogen
Rate of addition (g/l)
Xylanase activity (U/ml)
Proteose-peptone
0.0 0.2 0.5 1.0
102.6 104.6 105.4 81.5
Peptone (bacteriological)
0.5 1.0
84.2 75.6
Yeast extract
0.5 1.0
73.1 78.0
Tryptone
0.5 1.0
84.6 73.8
Combination of nitrogen sources (g/l)
Xylanase activity (U/ml) 81.7 102.6 105.4 65.2 71.8 50.6 64.5 27.8 69.7 65.9 56.9 96.4 95.3 70.1
different levels of inoculum were studied (Fig. 4). Maximum xylanase production (210 U/ml) was found using 1.5 108 spores/ml inoculum. Further increase in the size of inoculum was not beneficial for xylanase production. By increasing the size of inoculum from 1.5 104 to 1.5 108 spores/ml, the enzyme production increased to about two fold. It was also noticed that maximum enzyme production occurred in a shorter time (3 days), when 1.5 108 spores/ml inoculum was used. Studies on Aspergillus awamori by Smith and Wood [21] indicated optimum inoculum size 106 to 107 spores/ml for xylanase production. Lower than 106 spores/ml inoculum showed a significant lag in xylanase production. 3.6. Partial purification of xylanase Ammonium sulphate fractionation (40–70% saturation) of crude xylanase yielded 84.7% of the enzyme with 4.98fold purification (Table 4). Compared to many reports good yield and purification were achieved. Purification of xylanase from A. ochraceus NG-13 was done using 30– 60% ammonium sulphate fractionation, which yielded 56% xylanase with 2.6-fold purification [30]. Xylanases from two Aspergillus sp. were concentrated from 30 to 80% ammonium sulphate saturation with 62 and 67% yield [31]. 3.7. Detection of xylanase activity by zymogram staining Native-PAGE (incorporated with 0.1% xylan) of partially purified xylanases and subsequent activity staining revealed three clear and distinct bands, indicating the presence of multiple forms of xylanases (Fig. 5). Such multiple forms of xylanases have also been reported in different Aspergillus
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Fig. 4. Influence of size of inoculum on xylanase production by the Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation. Table 4 Partial purification of xylanase produced under SmF by the Aspergillus foetidus MTCC 4898
Crude broth Dialyzed ammonium sulphate fraction (40–70%)
Volume (ml)
Total xylanase units
Total protein (mg)
Specific activity
Yield (%)
Purification
75.0 6.7
15068 12768
56.4 9.6
267.2 1330.0
100.0 84.7
1.00 4.98
strains [32,33] as well as in other organisms such as Clostridium, Aeromonas and Streptomyces sp. [34]. Gawande and Kamat [31] reported three and two forms of xylanases in two Aspergillus sp., respectively. Several factors could be responsible for the multiplicity of xylanases, viz. differential m-RNA processing, post secretional modification by proteolytic digestion and posttranslational modification such as glycosylation and autoaggregation [35]. Multiple xylanases can also be the product from different alleles of the same gene [34]. However, some of the multiple xylanases are the result of independent gene [36].
range (4.5–7.5), with optimum pH 6.0 was suitable for xylanase activity [30]. Stability of partially purified xylanase was studied by preincubating the enzyme in suitable buffers ranging from 4.0 to 8.0 at 50 8C for 30 min (Fig. 7). Minimum thermoinactivation was found at pH 5.0. A
3.8. Properties of partially purified xylanases 3.8.1. Effect of pH on enzyme activity and stability Enzyme activity is markedly affected by pH. This is because substrate binding and catalysis are often dependent on charge distribution on both, substrate and, in particular enzyme molecules. The favourable pH range for xylanase activity of A. foetidus MTCC 4898 was 4.6–5.6, with optimum pH 5.3 (Fig. 6). A significant decrease drop in enzyme activity was observed below 4.6 and above 5.6 pH. Sudden drop in the relative enzyme activity was seen at pH 7.0 (19.4%) and negligible at pH 8.0 (0.1%). Unlike neutral and alkaline pH, around 40% activity was retained at pH 3.0. The enzyme behaviour clearly indicates that it is more suitable for any application in the pH range of 3.0–6.0. Costa Ferreira et al. [37] also reported optimum pH around 5.0 for xylanases of A. niger isolate, with favourable pH range between 4.0 and 6.0. In another investigation on A. ochraceus NG-13 mutant strain, a relatively broader pH
Fig. 5. Zymogram staining on native PAGE of partially purified xylanases obtained from SmF on xylan based medium.
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Fig. 8. Influence of temperature on activity of partially purified xylanases of Aspergillus foetidus MTCC 4898. Fig. 6. Influence of pH on the activity of partially purified xylanases of Aspergillus foetidus MTCC 4898.
Marginal decrease (8.9%) in stability was found at pH 4.0 and about 17% decrease in stability was seen at pH 6.0, 7.0 and 8.0, compared to enzyme stability at pH 5.0. Thus, higher pH (5.0–8.0) caused little adverse effect on xylanase stability. 3.8.2. Effect of temperature on xylanase activity and stability Initial reaction rates for partially purified xylanases were determined at temperatures between 35 and 65 8C. For 10 min reactions, the optimum temperature was 50 8C (Fig. 8). Enzyme activity was reduced below 48 8C and above 55 8C. Drastic reduction in enzyme activity was found at 65 8C (18.4% residual activity). Three distinct xylanases from A. aculeatus showed optimum temperature between 50 and 70 8C [32]. Activation energy in the temperature range 35–50 8C was calculated to be 58 kJ/mol for the xylanase For A. ochraceus xylanase activation energy reported was 45 kJ/mol measured in the range of 30–45 8C [30].
Fig. 7. Influence of pH on stability (50 8C/30 min) of partially purified xylanases produced by Aspergillus foetidus MTCC 4898.
Utilization of enzymes in industrial processes often encounters the problem of thermal inactivation of the enzyme. Thermal stability studies were carried out by pre incubating the enzymes up to 3.0 h. in the range of 30– 70 8C. The xylanase retained 100% activity at 30 8C after 3.0 h, while at 40 8C only marginal decrease was found. The enzyme was sensitive to 50 8C, retaining 71% after 30 min exposure and only 36% activity after 3.0 h. At 60 8C the residual xylanase activity was less than 20% at the end of 0.5 h, while at 70 8C the enzyme was completely inactivated within 0.5 h (Fig. 9). The results clearly indicate that the suitable temperature range for industrial application for xylanase of A. foetidus MTCC 4898 is 30–50 8C. In another study, the xylanase from A. niger was found to be very stable at 40 8C with no decrease in activity up to 3.0 h and losing one-third of its activity at 50 8C. This lower activity was attributed to the presence of proteolytic enzymes [37].
Fig. 9. Themostability of partially purified xylanases of Aspergillus foetidus MTCC 4898.
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Substrate molecules have long been known to stabilize enzymes specifically by stabilizing their active site. Substrate provides conformational stability, which prevents unfolding [43]. 3.9. Storage stability For industrial applications, storage of the enzymes at room and/or refrigerated temperature, without appreciable loss of activity is one of the most important and desirable characteristics. The enzyme retained full activity when stored in deep freeze up to 6 months. At refrigeration temperature no loss of activity was found up to 2 weeks but after 4 weeks a marginal decrease (5–10%) was found. At room temperature decrease in activity was not observed for up to 2 days but at the end of a week 10% loss in enzyme activity was found. The present work has established the potential of locally isolated strain A. foetidus for production of xylanases. The properties of partially purified enzyme indicates its possible use in processes operated at moderate temperatures and pH which may include preparation of baked cereal food products and saccharification of agroresidues.
References
Fig. 10. (a) Influence of certain additives on heat stability (50 8C/30 min) of partially purified xylanases of Aspergillus foetidus MTCC 4898. (b) Influence of certain additives on heat stability (60 8C/15 min) of partially purified xylanases of Aspergillus foetidus MTCC 4898.
In the present study additives like KCl, CaCl2, glycerol and xylan showed remarkable improvement in thermostability of partially purified enzyme at 50 8C and at 60 8C (Fig. 10a and b). Polyethylene glycol (50 mM) did not give protection against thermoinactivation and Tween 80 a nonionic detergent showed only marginal improvement. Similar studies on T. lanuginosus indicated thermostabilization by glycerol, b-mercaptoethanol and polyethylene glycol [38]. Ghosh and Nanda [39] reported increase in enzyme stability of A. sydowii MG49 at 60 8C in presence of 10% glycerol. Bandivadekar and Deshpande [40] found increased half life of xylanase of Chainia sp. at 60 8C upon addition of Ca2+ (10 mM), polyethylene glycol (10 mM), cysteine (10 mM), Tween 80 and xylan (3%). Inorganic salts, one of these additives influence protein stability directly by preferential binding to the folded or unfolded protein, and/or indirectly by changing the properties of solvent water, mainly the water activity [41]. The protective effect of polyols like glycerol is the result of their capability to form hydrogen bonds with the solvent [42].
[1] Beg QA, Kapoor M, Mahajan G, Hoondal S. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 2001;56:326–38. [2] Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 1999;23:411–56. [3] Subramaniyan S, Prema P. Biotechnology of microbial xylanases; enzymology, molecular biology, and application. Crit Rev Biotechnol 2002;22:33–64. [4] Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Zupancic S. Production of fungal xylanases. Bioresour Technol 1996;58: 137–161. [5] Steiner W, Laffarty RM, Gomes I, Esterbauer H. Studies on a wild type strain of Schizophyllum commune, cellulase and xylanase production and formation of the extracellular polysaccharide schizophyllan. Biotechnol Bioeng 1987;30:169–78. [6] Price NC, Stevens L. Enzyme technology. In: Fundamentals of enzymology: the cell and molecular biology of catalytic proteins. . third ed. UK: Oxford University Press; 1999. p. 430–49. [7] Park MT, Lee MS, Choi JY, Kim SC, Lee GM. Orthophosphate anion enhances the stability and activity of endoxylanase from Bacillus sp. Biotechnol Bioeng 2001;72:434–40. [8] Mandels M, Sternburg D. Recent advances in cellular technology. J Ferment Technol 1976;54:267–86. [9] Bailey MJ, Biely P, Poutanen K. Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 1992;23:257–70. [10] Miller GL. Use of dinitrosalycylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [11] Ghosh T. Measurement of cellulase activities. Commission on Biotechnology, IUPAC; 1994. p. 1–12. [12] Lowry OH, Rousebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193: 265–75. [13] Schwarz WH, Bronnenmeier K, Grabnitz F, Staudenbauer WL. Activity staining of cellulase in poly acrylamide gels containing mixed linkage b-glucans. Anal Biochem 1987;164:72–7.
A.R. Shah, D. Madamwar / Process Biochemistry 40 (2005) 1763–1771 [14] Ghosh M, Das A, Mishra AK, Nanda G. Aspergillus sydowii MG 49 is a strong producer of thermostable xylanolytic enzymes. Enzyme Microbial Technol 1993;15:703–9. [15] Flores ME, Perea M, Rodriguez O, Malvaez A, Huitron C. Physiological studies on induction and catabolite repression of b-xylosidase and endoxylanases in Streptomyces sp. CH-M-1035. J Biotechnol 1996;49:179–87. [16] Suh DH, Becker TC, Sands JA, Montenecourt BS. Effects of temperature on xylanase secretion by Trichoderma reesei. Biotechnol Bioeng 1988;32:821–5. [17] Subramaniyan S, Prema P. Cellulase free xylanase from Bacillus and other microorganisms. FEMS Microbiol Lett 2000;183:1–7. [18] Hrmova M, Beily P, Vrsanka M. Cellulose and xylan degrading enzymes of Aspergillus terreus. Enzyme Microbiol Technol 1989; 11:610–6. [19] Biswas SR, Jana SC, Mishra AK, Nanda G. Production, purification and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus. Biotechnol Bioeng 1988;35:244–51. [20] Christakopoulos P, Kekos D, Macris BJ, Claeyssens M, Bhat MK. Purification and characterisation of a major xylanase with cellulase and transferase activities from Fusarium oxysporum. Carbo Res 1996;289:91–104. [21] Smith DC, Wood TM. Xylanase production by Aspergillus awamori. Development of a medium and optimisation of extracellular xylanase and b-xylosidases while maintaining low protease production. Biotechnol Bioeng 1991;38:883–90. [22] Bajpai P. Microbial xylanolytic enzyme system: properties and application. Adv Appl Microbiol 1997;43:141–94. [23] Haapala R, Linko S, Parkkinen E, Suominen P. Production of endo1,4-L-glucanase and xylanase by Trichoderma reesei immobilized on polyurethane foam. Biotechnol Tech 1994;8:401–6. [24] Bansod SM, Dutta Choudhury M, Srinivasan MC, Rele MV. Xylanase active at high pH from an alkalotolerant Cephalosporium species. Biotechnol Lett 1993;15:965–70. [25] Gomes DJ, Gomes J, Steiner W. Factors influencing the induction of endoxylanases by Thermoascus aurantiacus. J Biotechnol 1994; 33:87–94. [26] Hoq MM, Hempel C, Deckwer WD. Cellulase free xylanase by Thermomyces lanuginosus RT9: effect of agitation, aeration and medium components on production. J Biotechnol 1994;37:49–58. [27] Gamerith G, Groicher R, Zeilinger S, Herzog P, Kubicek CP. Cellulase-poor xylanases produced by Trichoderma reesei Rut C-30 on hemicellulose substrates. Appl Microbiol Biotechnol 1992;38: 315–22.
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[28] Kelley M. Yeast extract. In: Godfrey T, Reichelt J, editors. Industrial enzymology. New York: Stockpon Press; 1983. p. 457–65. [29] Sikyta B. Development of microbial processes. In: Method in industrial microbiology. Chichester, UK: Ellis Horwood Ltd.; 1983. p. 250–74. [30] Biswas SR, Jana SC, Mishra AK, Nanda G. Production, purification and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus. Biotechnol Bioeng 1990;35:244–51. [31] Gawande P, Kamat MY. Purification of Aspergillus sp. xylanase by precipitation with an anionic polymer Eudrajit S100. Process Biochem 1999;34:577–80. [32] Fugimoto H, Ooi T, Wahg SL, Takizawa T, Hidaka H, Murao S, Arai M. Purification and properties of three xylanases from Aspergillus aculeatus. Biosci Biotechnol Biochem 1995;59:538–40. [33] Chandra RK, Chandra TS. Purification and characterization of xylanase from alkali tolerant Aspergillus fischeri FXN 1. FEMS Microbiol Lett 1996;145:457–61. [34] Wong KKY, Tan LUL, Saddler JN. Multiplicity of b-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev 1988; 52:305–17. [35] Biely P. Microbial xylanolytic systems. Trends Biotechnol 1985; 3:286–90. [36] Hazlewood GP, Gilbert HJ. Molecular biology of hemicellulases. In: Coughlan MP, Hazlewood GP, editors. Hemicelluloses and hemicellulases. London: Portland Press; 1993. p. 103–26. [37] Costa Ferreira M, Dias A, Maximo C, Morgado MJ, Sena-Martins G, Cardosoduarte J. Xylanolytic enzyme production by an Aspergillus niger isolate. Appl Biochem Biotechnol 1994;44:231–42. [38] Cesar T, Mrsa V. Purification and properties of the xylanase produced by Thermomyces lanuginosus. Enzyme Microbial Technol 1996; 4:289–96. [39] Ghosh M, Nanda G. Purification and some properties of a xylanase from Aspergillus sydowii MG49. Appl Environ Microbiol 1994;60:4620–3. [40] Bandivadekar KR, Deshpande VV. Enhanced stability of cellulase-free xylanase from Chainia sp. (NCL 82.5.1). Biotechnol Lett 1994; 16:179–82. [41] Timasheff SN. Water as legend: preferential binding and exclusion of denaturants in protein unfolding. Biochemistry 1992;31:9857–64. [42] Lozano P, Combes D, Iborra JL. Effect of polyols on a-chymotrypsin thermostability: a mechanistic analysis of the enzyme stabilization. J Biotechnol 1994;35:9–18. [43] Kibanov AM. Immobilized enzymes and cells as practical catalysts. Science 1983;219:722–7.
Xylanase production by a newly isolated Aspergillus foetidus strain and its characterization Amita R. Shah, Datta Madamwar* Post Graduate Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India Received 24 December 2003; accepted 17 June 2004
Abstract Xylanase production by a newly isolated Aspergillus foetidus MTCC 4898 was studied using submerged fermentation under shaking. The maximum xylanase yield (210 U/ml) was obtained with negligible cellulase activity at 30 8C using 1% birchwood xylan as substrate in 3 days of cultivation. The strain was able to produce extracellular xylanases on birchwood xylan as well as on cellulose rich substrates also with very poor cellulase production (<0.1 FPU/ml). The partially purified enzyme recovered by ammonium sulphate fractionation showed maximum activity at 50 8C and at pH 5.3. The enzyme was highly stable at 40 8C but retained 36% of its activity at 50 8C after 3 h. The presence of KCl, CaCl2, glycerol, xylan and trehalose significantly improved thermostability at 50 and 60 8C. Zymogram analysis indicated three isoenzymes. The enzyme preparation was completely stable at freezing temperature for up to 6 months and reasonably stable at refrigeration temperatures for more than 2 weeks. # 2004 Elsevier Ltd. All rights reserved. Keywords: Xylanase; Cellulase; Aspergillus foetidus; Birchwood xylan; Thermostability
1. Introduction In recent years, increasing concern over preserving resources and environment has initiated a growing interest in producing microbial enzymes. Xylanases from microorganisms have attracted a great deal of attention in the last decade because of their biotechnological potential in various industrial processes such as food, feed and paper-pulp industries. Moreover, xylanases have immense potential for increasing the production of several valuable products like xylitol and ethanol in a most economic way [1]. Hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of plant polysaccharides. Xylan is the major constituent of hemicellulose. Endo-xylanase (b-1,4-Dxylan-xylanohydrolase, EC 3.2.1.8) is the key enzyme for xylan depolymerization. Xylanases are produced by both prokaryotes and eucaryotes. A large number of bacteria and * Corresponding author. Tel.: +91 2692 226863; fax: +91 2692 226865. E-mail address: [email protected] (A.R. Shah), [email protected] (D. Madamwar). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.06.041
fungi are known to produce xylanases [2,3]. Filamentous fungi are interesting producers of this enzyme from an industrial point of view due to extracellular release of xylanases, higher yield compared to yeast and bacteria and production of several auxiliary enzymes that are necessary for debranching of the substituted xylans [4]. However, fungal xylanases are generally associated with concurrent production of cellulases [5]. Characterization of xylanolytic enzymes is important for its biotechnological application. The cost of enzyme is one of the main factors determining the economics of a process. This can be partially achieved by optimising fermentation medium. Several industrial processes are carried out using whole cells as the source of enzymes but the efficiency can be improved by using isolated and purified enzymes. Criteria for selection of particular method for isolation and purification depend on its end use. A high state of purity is generally not required in food processing, detergent as well as paper pulp industry, however it may be necessary to exclude certain contaminating enzymes [6]. Many of the fungal enzymes are extracellular and can easily be separated from the mycelia by filtration. It is usually necessary to concentrate the crude
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enzyme extract by spray drying, lyophilization or precipitation by ammonium sulphate or solvents. In industrial applications, the enzymes should be ideally tolerant to many physico-chemical environmental factors, viz. temperature, pH, metal ions, etc. as well as should have high catalytic efficiency. The stability of enzymes can be improved using several methods/techniques like chemical modification, immobilization or treatment with additives, viz. polyols or osmolytes or by protein engineering. Among various methods available for enhancing the enzyme functional stability, addition of low molecular weight additives to change the microenvironment of the enzyme solution is a relatively simpler, feasible as well as a cheaper and practical means [7]. In a screening programme, we isolated a new strain of Aspergillus from Vallabh Vidyanagar (Gujarat, India) showing xylanase producing ability. In the present investigation we report on production of xylanases using this strain and optimization of various physico-chemical parameters. The biochemical properties of the partially purified enzyme were also examined.
2. Materials and methods 2.1. Materials All chemicals used were of analytical grade. Potato dextrose agar (PDA) was obtained from Hi-media (India). Birchwood xylan was obtained from Fluka, Germany. Wood pulp (60% cellulose, 30% hemicellulose) was procured from Rohit pulp and paper mill, Vapi, India and other lignocellulosic substrates like wheat straw (30% cellulose, 50% hemicellulose and 15% lignin) and corncobs (45% cellulose, 35% hemicellulose, 15% lignin) were from local farms.
Tween 80, 0.1% (v/v); (NH4)2SO4, 1.4 g/l; KH2PO4, 2.0 g/l; urea, 0.3 g/l; CaCl2, 0.3 g/l; MgSO47H2O, 0.3 g/l; FeSO47H2O, 5.0 mg/l; MnSO4H2O, 1.6 mg/l; ZnSO47H2O, 1.4 mg/l; CoCl2, 2.0 mg/l; adjusted to pH 5.0. Birchwood xylan (1% (w/v)) was used as a substrate. Erlenmeyer flasks (500 ml) containing 100 ml of medium were inoculated with 2 ml of 2 106 spores/ml suspension prepared from a week old potato dextrose agar slants of A. foetidus. Spore count was done microscopically using Neubauer’s chamber. Inoculated flasks were incubated on a rotary shaker at 180 rpm at 30 8C. Samples were withdrawn at regular intervals and filtered through Whatman filter No. 1 and filtrate was used for analysis of extracellular xylanase, cellulase, and total soluble protein. 2.5. Enzyme activity assays Xylanase (EC 3.2.1.8) activity was measured using 1% birchwood xylan (4-O-methyl glucuronoxylan) solution (prepared in 0.05 M citrate buffer, pH 5.3) as a substrate [9]. The release of reducing sugars in 10 min at 50 8C at pH 5.3 were measured as xylose equivalent using dinitrosalysylic acid (DNS) method [10]. One unit of xylanase activity (U) is defined as the amount of enzyme liberating 1 mmol of xylose per minute. Filter paper cellulase activity was measured according to IUPAC recommendations, employing filter paper (Whatman No. 1) as substrate [11]. The release of reducing sugars in 60 min at 50 8C at pH 4.8 (0.05 M citrate buffer) was measured as glucose equivalent using DNS method [10]. One unit of filter paper activity is defined as the amount of enzyme liberating one mmol of glucose per minute. 2.6. Protein estimation Soluble protein from filtered fermentation broths was determined by following Lowry’s method [12].
2.2. Microorganism 2.7. Study of physico-chemical factors Aspergillus foetidus MTCC 4898 was isolated from decaying agricultural waste and identified by the Institute of Microbial Technology (IMTECH), Chandigadh, India. It was maintained on PDA slants and stored at 4 8C. 2.3. Pretreatment of lignocellulosic substrates Wheat straw and corncobs were washed, dried, milled and sieved before use to obtain a 50 mesh size. Each substrate was given alkali treatment with 1N NaOH in a 5% (w/v) slurry for 18 h at room temperature. After treatment, the substrates were thoroughly washed with water to neutral pH, dried and reground to 50 mesh size.
For studies on the effect of pH on xylanase production, the isolate Aspergillus foetidus was grown in the MS medium containing 1% birchwood xylan with different initial pH in the range of 3.0–9.0. Xylanase production was also studied at different temperatures, viz. 20, 25, 30, 37 and 42 8C. Influence of size of inoculum was carried out using 104 to 1010 spores/ml in the medium. Influence of various substrates and nitrogen sources was studied by growing the culture in the basal medium with different carbon/nitrogen source. Various combinations of inorganic and organic nitrogen sources were also tried.
2.4. Xylanase production by submerged fermentation
2.8. Partial purification of xylanases by ammonium sulphate fractionation
For submerged fermentation, Mandels and Sternburg’s (MS) basal medium [8] containing proteose-peptone, 1 g/l;
A calculated amount of solid ammonium sulphate was added to the culture supernatant with constant stirring at
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10 8C to achieve 40% saturation. After centrifugation at 8000 rpm at 4 8C for 20 min the precipitate was discarded and the supernatant was subsequently adjusted to 40–70% saturation by addition of calculated amounts of ammonium sulphate. The precipitate was dissolved in small volume of buffer. The enzyme solution was subjected to dialysis for about 18–24 h at 10 8C against 0.05 M citrate buffer (pH 5.3) fortified with 100 ppm sodium azide, with three intermittent changes of the buffer and xylanase activity and protein estimation were carried out. 2.9. Activity detection by zymogram staining Zymogram analysis was done according to Schwarz et al. (1987) [13]. Native poly acrylamide gel electrophoresis (native-PAGE) at 10% gel was performed after incorporating birchwood xylan solution (final concentration 0.1% (w/ v)) in the separating gel before addition of ammonium persulphate and polymerization. After the run, the gel was submerged in 0.1% (w/v) Congo red solution for about 10 min. The excess dye was removed by washing in 0.1 M NaCl and then the gel was transferred to 5% (v/v) acetic acid. 2.10. Determination of optimum temperature and pH, thermostability and pH stability
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refrigeration temperature (5–7 8C) and room temperature (25–30 8C). The enzyme solution was stored in sterile Eppendorf vials. Xylanase activity was assayed at regular intervals.
3. Results and discussion 3.1. Time course of xylanase production Xylanase production was studied at 30 8C for 7 days in liquid MS medium containing 1% birchwood xylan as substrate (Fig. 1). Xylanase production started from the second day and reached a maximum on the fourth day (80.5 U/ml). The volumetric productivity (U/(l h)), which is an important parameter to assess the effectiveness of the process [4], was highest on the third day (1047 U/(l h)). From the sixth day onwards, a significant decrease in production was observed. Xylanases are usually expressed at the end of the exponential phase and harvesting time is correlated to the medium under consideration [2]. The phenomenon of sudden increase and subsequent decrease in enzyme activities during the cultivation period has also been noted in xylanase produced by Aspergillus sydowii MG-49 [14] and Streptomyces sp. CH-M-035 [15]. 3.2. Influence of temperature and pH
The xylanase activity of the partially purified enzyme was assayed at different temperatures ranging from 35 to 65 8C. The energy of activation of the xylanase catalysed reaction was determined by measuring the maximum velocity, V, at different temperatures and plotting log10 V against 1/T. The thermal stability was determined at the temperatures 30, 40, 50, 60 and 70 8C after incubation of suitably diluted enzyme samples in absence of substrate for 0.0, 0.5, 1.0, 2.0 and 3.0 h. The optimum pH was determined using buffers ranging from 3.0 to 8.0. Citrate buffer (0.05 M) was used in the pH range of 3.0–5.6 and phosphate buffer (0.02 M) was used for pH 6.0–8.0. To study the stability of partially purified xylanase at different pH, appropriate dilutions of xylanase solutions were made in above buffers in the range of 4–8 pH and incubated at 50 8C for 30 min. After cooling, residual activity was estimated under standard conditions of xylanase assay. To study the influence of various additives on heat stability, appropriate dilution of xylanase solution was incubated in the presence of various additives, viz. KCl (50 mM), CaCl2 (50 mM), polyethylene glycol-3300 (50 mM), birchwood xylan (3.0% (w/v)), Tween 80 (1.0% (v/v)) and glycerol (10.0% (v/v)) at 50 8C for 30 min and 60 8C for 15 min.
Xylanase production at different temperatures was examined for 4 days keeping the other fermentation conditions constant. Xylanase production increased with increase in temperature from 20 to 30 8C. Maximum production of xylanase (80.5 4.0 U/ml) was obtained at 30 8C (Fig. 2). At 37 8C a significant decline (36%) in xylanase activity was evident. Growth and xylanase production totally ceased at higher temperature (42 8C). Similar observations were shown by Suh et al. for Trichoderma reesei [16]. It was revealed that environmental
2.11. Storage stability The storage stability of purified xylanase was studied by keeping the membrane sterilized enzyme solution in 0.05 M citrate buffer (pH 5.3) at deep freeze ( 10 to 20 8C),
Fig. 1. Time course of xylanase production by Aspergillus foetidus MTCC 4898 on xylan based media under submerged fermentation at 30 8C.
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A.R. Shah, D. Madamwar / Process Biochemistry 40 (2005) 1763–1771 Table 1 Xylanases and cellulases production by Aspergillus foetidus MTCC 4898 on various substrates at 30 8C under submerged fermentation Substrate
Xylanase activity (U/ml)
Protein (mg/ml)
Cellulase activity (U/ml)
0.5% Xylan 1% Xylan 2% Wheat straw Alkali treated 2% wheat straw 2% Corn cobs Alkali treated 2% corn cobs 2% Wood pulp 1% Glucose !% Lactose 1% Xylose
59.0 80.5 20.1 19.2 29.0 25.1 51.2 2.9 3.5 25.6
0.39 0.61 0.74 0.47 0.58 0.43 0.64 0.31 0.29 0.34
0.03 0.05 0.10 0.09 0.09 0.02 0.01 0.02 0.03 0.03
Fig. 2. Influence of temperature on xylanase production by Aspergillus foetidus MTCC 4898 under submerged fermentation.
temperature not only affects growth rates of organisms but also exhibit marked influence on the levels of xylanase production. Another important factor significantly affecting the production of xylanases is the initial pH of medium. Xylanase production by this isolate was observed in the range 2.0–7.0 pH (Fig. 3). Growth and xylanase production completely ceased at 8.0 and 9.0 pH. Maximum xylanase production (80.5 U/ml) was found at pH 5.0. A marginal decrease in xylanase yield was seen at lower pH, i.e. at pH 3.0 and 4.0. Decline in xylanase production was remarkable at pH 6.0 and 7.0. The results clearly indicated acidophilic nature of the fungus. With rare exceptions, fungi reported to produce xylanases at initial pH lower than 7.0. There has been increased usage of xylanase preparations having an optimum pH around 5.0 invariably from fungi [17]. 3.3. Xylanase production on various substrates Among the various substrates tested, 1% birchwood xylan was found to be the most effective for xylanase production (Table 1). Maximum xylanase production (80.5 U/ml) was achieved in 4 days of incubation at
Fig. 3. Influence of pH of the medium on xylanase production by Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation.
30 8C (pH 5.0) with only 0.05 U/ml filter paper activity. By reducing the concentration of the xylan (0.5%) xylanase production was significantly reduced. Wood pulp (2.0%) was also found to be a good substrate, which yielded 51.2 U/ ml xylanase activity. It was striking that, even in presence of such a cellulose rich substrate also, cellulase production was negligible. Wheat straw (2.0%) and corncobs (2.0%), individually showed relatively lesser xylanase production 20.1 and 29.0 U/ml, respectively but again with negligible cellulase activity. In A. terreus, selective induction of xylanases occurred by employing xylan or xylobiose or Dxylose as the substrate whereas in presence of cellulose or cellobiose both are produced [18]. In order to increase the accessibility of the cellulosic and hemicellulosic substrates, mild alkali treatment with1 M NaOH is a widely accepted method for solubilizing lignin from lignocellulose. In this study, xylanase activity did not increased even after mild alkali treatment, rather, it was marginally reduced. Such negative effects of alkali treatment on xylanase production were also reported by studies on Aspergillus ochraceus. A possible explanation for this observation has been suggested that rapid consumption of carbon sources and concurrent release of monomeric sugars would lead to a repression in enzyme synthesis [19]. Use of soluble sugars can reduce certain problems during large-scale fermentation. In this study pure sugars gave good growth but xylanase production was very poor. With 1% glucose, 1% lactose and 1% xylose, xylanase production was 2.9, 3.5 and 25.6 U/ml, respectively. Glucose, xylose and lactose (1.0% (w/v)) proved to be ineffective as inducers for xylanase production in Fusarium oxysporum as reported by Christakopoulos et al. [20]. Smith and Wood [21] also reported very low titres of xylanases on glucose and lactose, while xylose gave slightly better production (25.0% production of the control). As reviewed by Bajpai [22], fungal xylanase appear to be inducible or under derepression control, which includes enzyme production on carbon sources that are used slowly. In some fungi, high xylanase production has been shown to be linked strictly to cellulase production [5]. But
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Aspergillus foetidus MTCC 4898 did not produce high amount cellulases in spite of using cellulose rich substrates like wood pulp, wheat bran and corncobs. This is one of the attractive features of this isolate.
Table 3 Effect of combination of nitrogen sources on xylanase production by Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation
3.4. Influence of nitrogen sources
Ammonium sulphate (1.4) + urea (0.3) + proteose peptone (1.0) Ammonium sulphate (1.4) + urea (0.3) Ammonium sulphate (1.4) + urea (0.3) + proteose peptone (0.5) Urea (0.3) + proteose peptone (1.0) Ammonium sulphate (1.4) + proteose peptone (1.0) Ammonium sulphate (1.4) Ammonium sulphate (2.8) Proteose peptone (1.0) Proteose peptone (2.0) Urea (0.3) Urea (0.6) Calcium nitrate (2.0) + urea (0.3) + proteose peptone (1.0) Sodium nitrate (1.8) +urea (0.3) + proteose peptone (1.0) Ammonium nitrate (1.2) +urea (0.3) + proteose peptone (1.0)
The mechanisms that govern the formation of extracellular enzymes are influenced by the availability of precursors for protein synthesis [2]. Furthermore, the nitrogen source can significantly affect the pH of the medium during the course of fermentation [23]. The effect of different organic nitrogen sources were also studied. Maximum xylanase activity (105.4 U/ml) was evident when 0.05% proteose peptone was added (Table 2). Organism could show significantly good activity even in the absence of any organic nitrogen source. These results are in contrast with many investigators, who have reported that fungi produce more xylanases on addition of complex organic nitrogen sources [24–27]. Although Kelley has shown that higher amounts of easily fermentable carbon in the complex nitrogen sources could enhance growth rather than enzyme production [28]. When different combinations of organic and inorganic nitrogen sources were incorporated in the Mandels and Sternburg’s medium (Table 3), maximum yield of xylanases (105.4 U/ml) was obtained using a combination of ammonium sulphate (1.4 g/l), urea (0.3 g/l) and proteose peptone (0.5 g/l). Excluding either urea or ammonium sulphate from the combination produced a significant decrease in the yield of the enzyme. It was also observed that ammonium sulphate could be replaced by calcium nitrate and sodium nitrate without much loss of the xylanase yield. 3.5. Influence of size of inoculum The inoculum size must be large enough to colonize all the substrate particles [29]. In the present investigation, four
Table 2 Effect of complex organic nitrogen sources on xylanase production by Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation Source of organic nitrogen
Rate of addition (g/l)
Xylanase activity (U/ml)
Proteose-peptone
0.0 0.2 0.5 1.0
102.6 104.6 105.4 81.5
Peptone (bacteriological)
0.5 1.0
84.2 75.6
Yeast extract
0.5 1.0
73.1 78.0
Tryptone
0.5 1.0
84.6 73.8
Combination of nitrogen sources (g/l)
Xylanase activity (U/ml) 81.7 102.6 105.4 65.2 71.8 50.6 64.5 27.8 69.7 65.9 56.9 96.4 95.3 70.1
different levels of inoculum were studied (Fig. 4). Maximum xylanase production (210 U/ml) was found using 1.5 108 spores/ml inoculum. Further increase in the size of inoculum was not beneficial for xylanase production. By increasing the size of inoculum from 1.5 104 to 1.5 108 spores/ml, the enzyme production increased to about two fold. It was also noticed that maximum enzyme production occurred in a shorter time (3 days), when 1.5 108 spores/ml inoculum was used. Studies on Aspergillus awamori by Smith and Wood [21] indicated optimum inoculum size 106 to 107 spores/ml for xylanase production. Lower than 106 spores/ml inoculum showed a significant lag in xylanase production. 3.6. Partial purification of xylanase Ammonium sulphate fractionation (40–70% saturation) of crude xylanase yielded 84.7% of the enzyme with 4.98fold purification (Table 4). Compared to many reports good yield and purification were achieved. Purification of xylanase from A. ochraceus NG-13 was done using 30– 60% ammonium sulphate fractionation, which yielded 56% xylanase with 2.6-fold purification [30]. Xylanases from two Aspergillus sp. were concentrated from 30 to 80% ammonium sulphate saturation with 62 and 67% yield [31]. 3.7. Detection of xylanase activity by zymogram staining Native-PAGE (incorporated with 0.1% xylan) of partially purified xylanases and subsequent activity staining revealed three clear and distinct bands, indicating the presence of multiple forms of xylanases (Fig. 5). Such multiple forms of xylanases have also been reported in different Aspergillus
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Fig. 4. Influence of size of inoculum on xylanase production by the Aspergillus foetidus MTCC 4898 at 30 8C under submerged fermentation. Table 4 Partial purification of xylanase produced under SmF by the Aspergillus foetidus MTCC 4898
Crude broth Dialyzed ammonium sulphate fraction (40–70%)
Volume (ml)
Total xylanase units
Total protein (mg)
Specific activity
Yield (%)
Purification
75.0 6.7
15068 12768
56.4 9.6
267.2 1330.0
100.0 84.7
1.00 4.98
strains [32,33] as well as in other organisms such as Clostridium, Aeromonas and Streptomyces sp. [34]. Gawande and Kamat [31] reported three and two forms of xylanases in two Aspergillus sp., respectively. Several factors could be responsible for the multiplicity of xylanases, viz. differential m-RNA processing, post secretional modification by proteolytic digestion and posttranslational modification such as glycosylation and autoaggregation [35]. Multiple xylanases can also be the product from different alleles of the same gene [34]. However, some of the multiple xylanases are the result of independent gene [36].
range (4.5–7.5), with optimum pH 6.0 was suitable for xylanase activity [30]. Stability of partially purified xylanase was studied by preincubating the enzyme in suitable buffers ranging from 4.0 to 8.0 at 50 8C for 30 min (Fig. 7). Minimum thermoinactivation was found at pH 5.0. A
3.8. Properties of partially purified xylanases 3.8.1. Effect of pH on enzyme activity and stability Enzyme activity is markedly affected by pH. This is because substrate binding and catalysis are often dependent on charge distribution on both, substrate and, in particular enzyme molecules. The favourable pH range for xylanase activity of A. foetidus MTCC 4898 was 4.6–5.6, with optimum pH 5.3 (Fig. 6). A significant decrease drop in enzyme activity was observed below 4.6 and above 5.6 pH. Sudden drop in the relative enzyme activity was seen at pH 7.0 (19.4%) and negligible at pH 8.0 (0.1%). Unlike neutral and alkaline pH, around 40% activity was retained at pH 3.0. The enzyme behaviour clearly indicates that it is more suitable for any application in the pH range of 3.0–6.0. Costa Ferreira et al. [37] also reported optimum pH around 5.0 for xylanases of A. niger isolate, with favourable pH range between 4.0 and 6.0. In another investigation on A. ochraceus NG-13 mutant strain, a relatively broader pH
Fig. 5. Zymogram staining on native PAGE of partially purified xylanases obtained from SmF on xylan based medium.
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Fig. 8. Influence of temperature on activity of partially purified xylanases of Aspergillus foetidus MTCC 4898. Fig. 6. Influence of pH on the activity of partially purified xylanases of Aspergillus foetidus MTCC 4898.
Marginal decrease (8.9%) in stability was found at pH 4.0 and about 17% decrease in stability was seen at pH 6.0, 7.0 and 8.0, compared to enzyme stability at pH 5.0. Thus, higher pH (5.0–8.0) caused little adverse effect on xylanase stability. 3.8.2. Effect of temperature on xylanase activity and stability Initial reaction rates for partially purified xylanases were determined at temperatures between 35 and 65 8C. For 10 min reactions, the optimum temperature was 50 8C (Fig. 8). Enzyme activity was reduced below 48 8C and above 55 8C. Drastic reduction in enzyme activity was found at 65 8C (18.4% residual activity). Three distinct xylanases from A. aculeatus showed optimum temperature between 50 and 70 8C [32]. Activation energy in the temperature range 35–50 8C was calculated to be 58 kJ/mol for the xylanase For A. ochraceus xylanase activation energy reported was 45 kJ/mol measured in the range of 30–45 8C [30].
Fig. 7. Influence of pH on stability (50 8C/30 min) of partially purified xylanases produced by Aspergillus foetidus MTCC 4898.
Utilization of enzymes in industrial processes often encounters the problem of thermal inactivation of the enzyme. Thermal stability studies were carried out by pre incubating the enzymes up to 3.0 h. in the range of 30– 70 8C. The xylanase retained 100% activity at 30 8C after 3.0 h, while at 40 8C only marginal decrease was found. The enzyme was sensitive to 50 8C, retaining 71% after 30 min exposure and only 36% activity after 3.0 h. At 60 8C the residual xylanase activity was less than 20% at the end of 0.5 h, while at 70 8C the enzyme was completely inactivated within 0.5 h (Fig. 9). The results clearly indicate that the suitable temperature range for industrial application for xylanase of A. foetidus MTCC 4898 is 30–50 8C. In another study, the xylanase from A. niger was found to be very stable at 40 8C with no decrease in activity up to 3.0 h and losing one-third of its activity at 50 8C. This lower activity was attributed to the presence of proteolytic enzymes [37].
Fig. 9. Themostability of partially purified xylanases of Aspergillus foetidus MTCC 4898.
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Substrate molecules have long been known to stabilize enzymes specifically by stabilizing their active site. Substrate provides conformational stability, which prevents unfolding [43]. 3.9. Storage stability For industrial applications, storage of the enzymes at room and/or refrigerated temperature, without appreciable loss of activity is one of the most important and desirable characteristics. The enzyme retained full activity when stored in deep freeze up to 6 months. At refrigeration temperature no loss of activity was found up to 2 weeks but after 4 weeks a marginal decrease (5–10%) was found. At room temperature decrease in activity was not observed for up to 2 days but at the end of a week 10% loss in enzyme activity was found. The present work has established the potential of locally isolated strain A. foetidus for production of xylanases. The properties of partially purified enzyme indicates its possible use in processes operated at moderate temperatures and pH which may include preparation of baked cereal food products and saccharification of agroresidues.
References
Fig. 10. (a) Influence of certain additives on heat stability (50 8C/30 min) of partially purified xylanases of Aspergillus foetidus MTCC 4898. (b) Influence of certain additives on heat stability (60 8C/15 min) of partially purified xylanases of Aspergillus foetidus MTCC 4898.
In the present study additives like KCl, CaCl2, glycerol and xylan showed remarkable improvement in thermostability of partially purified enzyme at 50 8C and at 60 8C (Fig. 10a and b). Polyethylene glycol (50 mM) did not give protection against thermoinactivation and Tween 80 a nonionic detergent showed only marginal improvement. Similar studies on T. lanuginosus indicated thermostabilization by glycerol, b-mercaptoethanol and polyethylene glycol [38]. Ghosh and Nanda [39] reported increase in enzyme stability of A. sydowii MG49 at 60 8C in presence of 10% glycerol. Bandivadekar and Deshpande [40] found increased half life of xylanase of Chainia sp. at 60 8C upon addition of Ca2+ (10 mM), polyethylene glycol (10 mM), cysteine (10 mM), Tween 80 and xylan (3%). Inorganic salts, one of these additives influence protein stability directly by preferential binding to the folded or unfolded protein, and/or indirectly by changing the properties of solvent water, mainly the water activity [41]. The protective effect of polyols like glycerol is the result of their capability to form hydrogen bonds with the solvent [42].
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