Novel xylanases from Simplicillium obclavatum MTCC 9604: comparative analysis of production, purification and characterization of enzyme from submerged and solid state fermentation
© Roy et al.; licensee Springer. 2013
Received: 24 May 2013
Accepted: 8 August 2013
Published: 14 August 2013
The production of extracellular xylanase by a newly isolated fungus Simplicillium obclavatum MTCC 9604 was studied in solid-state and submerged fermentation. Multiple xylanases and endoglucanases were produced by the strain during growth on wheat bran in solid state fermentation (SSF). A single xylanase isoform was found to be produced by the same fungus under submerged fermentation (SF) using wheat bran as sole carbon source. Enzyme activity, stability and the protein yield were much higher in SSF than SF. The two dimensional zymogram of the crude enzyme indicated the presence of six isoforms with different pI values starting from pH 3–10. The optimum temperature and pH for the partially purified xylanase activity were 50°C and pH 5.0 respectively; xylanase enzymes exhibited remarkable stability over a broad pH range and the temperature range of 30-60°C which has great potential to be used in biofuels, animal feed and food industry applications.
Xylanases (endo-1, 4-β-D-xylanohydrolase; EC 188.8.131.52) are glycosidases involved in depolymerization of xylan, the major renewable hemicellulosic polysaccharide of plant cell wall. The hemicelluloses represent an enormous reserve of utilizable biomass as hardwoods consist of 25-32% hemicelluloses whereas softwood contains only 15-25% hemicelluloses. Undoubtedly, hemicelluloses are the second most abundant renewable resource, only exceeded by cellulose. Xylanase is produced by bacteria (Gilbert and Hazlewood 1993; Kiddinamoorthy et al. 2008; Sanghi et al. 2008; Sunna and Antranikian 1997), fungi (Chavez et al. 2006; Chavez et al. 2002a, b; Liao et al. 2012; Liu et al. 2006; Nair et al. 2008; Okafor et al. 2007; Sunna and Antranikian 1997), actinomycetes (Ninawe et al. 2008) and yeast (Liu et al. 1998). Recently, interest in xylanase has markedly increased due to its wide variety of biotechnological applications such as pre-bleaching of pulp, improving the digestibility of the animal feed stocks, modification of cereal based stuffs, bioconversion of lignocellulosic material and agro waste to fermentable products, clarification of fruit juices and degumming of plant fibers (Kapoor et al. 2001; Kuhad and Singh 1993; Ratanakhanokchai et al. 1999) etc. The cellulase free xylanase active at high temperature and pH are gaining importance in pulp and paper industry as they reduce the need for toxic chlorinated compounds making the bleaching process environment friendly (Srinivasan and Rele 1999; Viikari et al. 1994).
Industrial purification of enzymes on large scale is associated mainly with substrate. The use of agriculture residues as low cost substrates for the production of industrial enzymes is a significant way to reduce production cost. The technique of fermentation using solid substrate has the great advantage over submerged fermentation (SF) due to absence or near absence of aqueous phase that provides natural habitat for growth of microorganisms. Other advantages are as follows: economy of the space, simplicity of the media, simple machinery, greater compactness of the fermentation vessel owing to a low water volume, greater product yields, reduced energy demand, lower capital and recurring expenditure in industries, easier scale up processes, lesser volume of solvent needed for product recovery, absence of foam build – up and easier control of contamination due to the low moisture level in the system (Archana and Satyanarayana 1997; Ayyachamy and Vatsala 2007).
The majority of xylanases utilized in biotechnology are still derived from well characterized bacteria and fungi. In this study, xylanase producing fungus Simplicillium obclavatum MTCC 9602 was isolated from soil. The main objective of this study is the comparative analysis of production, purification and characterization of xylanase enzyme from submerged and solid state fermentation. In order to investigate the biotechnological applications of this enzyme, it would be desirable to purify and characterize it.
This paper reports for the first time about the production and properties of multiple forms of xylanases from the S. obclavatum MTCC 9602. An attempt was made in optimizing a low cost solid state fermentation medium that could allow enhanced product recovery as well as high enzyme productivity. Production of xylanase was also compared with the submerged fermentations (SF). In the present study, optimum culture condition, isozyme pattern, pH optimum, temperature optimum, pH stability, thermal stability, two dimensional gel electrophoresis followed by zymogram analysis and the action of several chemicals on the partially purified enzyme from SSF culture and enzyme from SF culture have also been characterized.
Results and discussion
Optimization of culture conditions and production of xylanase in SSF and SF
SDS-PAGE and zymogram analysis of the expression multiplicity of xylanases
Partial purification of xylanase obtained from SSF
Partial Purification profile of xylanase of S. obclavatum from SSF
Total protein (mg)
Xylanase total activity (μ mol/min/ml)
Specific activity (μ mol/min/mg)
Ammonium sulfate precipitation
Superose 12 FPLC
Effect of pH on xylanase activity and stability
Effect of temperature on xylanase activity and stability
Effect of metal ions and other compounds on xylanase from SSF culture condition
Effect of metal ions and other compounds on xylanase of S. obclavatum from SSF
Relative activity of Xylanase at 3 mM
Relative activity of Xylanase at 6 mM
S. obclavatum MTCC 9604 produces multiple xylanolytic enzymes in solid state fermentation using wheat bran as inducer. Apart from the production of industrial enzyme in SSF, the studies on this particular microorganism may also have mycological significance. This study clearly showed that environmental factors dictate extracellular protein secretion and isoform variation. Nonetheless, xylan degrading enzymes produced in solid state culture were remarkably stable towards a wide range of pH and temperature which make them potentially suitable for applications in different industries.
Material and methods
All the reagents were analytical grade and purchased from Sigma Chemical Co (St Louis, MO, USA) unless specified.
White, long, obclavate xylanase producing fungi was isolated from soil in our laboratory. The strain was sent to Institute of Microbial Technology (IMTECH) for identification. IMTECH identified the culture as Simplicillium obclavatum MTCC 9604 which was used for the production of xylanase. The fungal colonies were reached up to 30–35 mm in diameter in potato dextrose agar (PDA) plates after seven days of incubation at 30°C. The microorganism was maintained over PDA and it was cultivated for the xylanase production in both submerged and solid state fermentation described below.
Production of xylanase in submerged fermentation
Spores were collected from PDA slants in Mandels mineral salt solution (Mandels et al. 1974). The composition of Mandels mineral salt solution was as follows: H2O 495 ml, concentrated HCl 5 ml, FeSO4, 7H2O, 4.6, MnSO4, H2O, 0.89, ZnSO4, H2O, 1.78, CoCl2, 1.25. For submerged fermentation, 104 -106 spores of S. obclavatum was grown in 50 ml liquid medium containing wheat bran as sole source of carbon. Medium composition (g L-1) was as follows: 1.4 (NH4)2 SO4, 0.3 Urea, 2 KH2PO4, 0.3 MgSO4 7H2O, 0.3 CaCl2, 0.4% Trace Metal (g ml-1). The submerged culture was incubated at 30°C for 3 days at 150 rpm shaking condition. After 3 days, culture filtrate was collected by filtration. Extracellular xylanase activity was measured in the culture filtrate of the submerged fermentation.
Partial purification of xylanase from submerged fermentation
All purification steps were performed at 4°C unless otherwise stated. The culture filtrate was subjected to 0-80% (NH4)2SO4 saturation. At 80% salt saturation, no enzyme activity remained in the supernatant as all had been recovered in the precipitate. The precipitated protein collected by centrifugation at 10, 000 rpm was dissolved in minimum volume of 50 mM potassium phosphate buffer pH 7.2 and then it was dialyzed against the same buffer. The dialysate was used for xylanase assay.
Production of xylanase in solid state fermentation
Spores were collected from PDA slants in Mandels mineral salt solution and spread over the solid bed (104 -106 spores per 5.0 g of wheat bran) uniformly maintaining the moisture content of the solid bed of about 50% with Mandel’s salt medium. Mandels salt medium was homogeneously mixed with active inoculum and incubated at 30°C for 7 days. Fermented substrates were extracted at different time interval to optimize the culture incubation time in the solid bed. After proper incubation, the enzyme was extracted from wheat bran by agitating it in 20 ml of 50 mM potassium phosphate buffer, pH-7.2 for 1 h in a shaker with 150 rpm. Fermented substrates were separated from the extraction buffer by filtration through cheese- cloth. Supernatant was collected from the agitated mixture after a centrifugation at 12000 rpm for 30 min at 4°C. After that the collected supernatant was subjected to partial purification.
Partial purification of xylanase from SSF
All purification steps were performed at 4°C unless otherwise stated. Finely powdered (NH4)2SO4 was added to the enzyme extract to 80% saturation. After 2 h on ice, the precipitate formed was collected by centrifugation at 10, 000 rpm, dissolved in 50 mM potassium phosphate buffer pH 7.2 and dialyzed against the same buffer. At 80% salt saturation, almost no enzyme activity remained in the supernatant as all had been recovered in the precipitate. After dialysis, the non-dissolved protein was removed by centrifugation at 10,000 rpm for 10 min at 4°C and the clear supernatant was applied to a FPLC Superose 12 10/300 GL (Amersham Pharmacia, Uppsala, Sweden) pre equilibrated with 50 mM potassium phosphate buffer pH 7.2. The enzyme was eluted in the same buffer with a constant flow rate. The elution of the protein was monitored at 280 nm. Protein concentration was determined according to the method of Bradford (Bradford 1976). The active fractions were collected and kept at −80°C and used further for xylanase assay and zymogram analysis.
All xylanase assays were performed with 50 mM potassium phosphate buffer pH −7.2 unless otherwise specified. Xylanase activity was determined as per the International Union of Pure and Applied Chemistry recommendations, with 10% xylan as the substrate. The xylanase assay was carried out by incubating appropriately diluted enzyme in 50 mM potassium phosphate buffer pH −7.2 with 10% birchwood xylan (w/v) at 50°C for 30 min. The released reducing sugar was determined by dinitrosalicylic acid (DNS) method using D-xylose as standard (Miller 1959). Soluble xylan was prepared by suspending 4 g of birchwood xylan in 100 ml of 50 mM K2HPO4/KOH buffer, pH-7.0, and stirring the suspension for 4 h followed by centrifugation. The soluble fraction corresponds to 50% (w/v) of the total xylan (Yang et al. 1995). One unit of xylanase activity was defined as the amount of enzyme that produced 1 μmol of xylose equivalent per min from xylan under assay conditions.
SDS polyacrylamide gel electrophoresis and zymogram analysis
SDS polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed using 10% (w/v) polyacrylamide gel with a 4% stacking gel and the Mini-Protean II system (BioRad) according to the method described by Laemmli, with some modifications (Laemmli 1970). Protein bands were visualized by staining by Coomassie Brilliant Blue. The zymogram analysis of xylanase from SSF culture was performed according to Tseng et al. (Tseng et al. 2002). After the separation of the enzyme samples by SDS-PAGE containing 2% birchwood xylan, the gel was divided into two parts. One part was processed for protein staining with Coomassie Brilliant Blue and the other part was used for zymogram analysis. For zymogram analysis, the gel was washed first with 50 mM potassium phosphate buffer pH −7.2 containing 25% isopropanol and the gel was kept in the same solution for 1 h at room temperature. The gel was again washed with 50 mM potassium phosphate buffer pH −7.2 without isopropanol and it was kept in the same buffer for 30 min at room temperature. After that the gel was incubated at 37°C for 10 min. Finally the gel was stained with 0.1% Congo red solution followed by destaining with 1 M NaCl solution until pale-red hydrolysis zones appeared against a red background. The molecular weights of the isoforms were determined by correlating it with identical SDS –PAGE profile of protein molecular weight standard.
2D gel electrophoresis followed by zymogram analysis of xylanase
2D gel electrophoresis of xylanase was performed with the Ettan IPGphor II system (GE healthcare limited, Buckinghamshire UK). For the first dimension 7 cm NL, pH 3–10, immobilized strips were used where 250 μg of protein was loaded together with the rehydration buffer containing 8 M urea, 2% (v/v) triton x 100, 0.28% (w/v) DTT, 1% (v/v) IPG buffer containing ampholytes and 0.002% (w/v) bromophenol blue (all final concentrations). For the preparation of sample, 250 μg proteins were directly solubilized in the above rehydration buffer and subjected to two consecutive freeze thaws for complete lysis in presence of protease inhibitor cocktail. IPG strips were rehydrated with samples at 50 μA/strip for 12 h at 20°C and then isoelectric focusing was performed at 25°C by a linear increase to 500 V for 2 h followed by a gradient increase to 4500 V over 1 h and then held at 4500 V until a total of 14 KVH was reached. For the second dimension the IPG strips were first equilibrated for 15 min in SDS equilibrium buffer containing 50 mM Tris–HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT and 0.002% (w/v) bromophenol blue and then in 50 mM Tris–HCl (pH 8.8), 6 M urea (w/v), 30% glycerol (v/v), 2% SDS (w/v), 2% iodoacetamide (w/v) and 0.002% (w/v) bromophenol blue for another 15 min. The strips were then embedded in 0.5% (W/V) agarose on top of a 10% acrylamide gel with 2% xylan as a substrate containing 3% stacking gel. Zymogram analysis of xylanase was performed according to Tseng et al.
Effect of pH and temperature on xylanase
To determine the optimum pH of xylanase, 10% xylan was taken in buffers of different pH for the experimental purpose. The partially purified enzyme either from SSF or SF was then added to the buffer of different pH containing substrate and the reaction mixture was incubated for 30 min at 50°C. Liberated reducing sugar was then estimated by 3, 5-Dinitrosalicylic acid (DNS) reagent.
For pH stability experiment, the enzyme was exposed for 1 h at 25°C to the corresponding buffer. The residual activity of the enzyme was measured in 50 mM potassium phosphate buffer, pH 7.2. The following buffer solutions were employed to determine the pH optima and pH stability curve: 50 mM sodium acetate buffer (pH 4–5), 50 mM potassium phosphate buffer (pH 6–8), and 50 mM Glycine-NaOH buffer (pH 9–10).
To determine the optimum temperature of xylanase, partially purified enzyme either from SSF or SF was incubated with 10% of the substrate at different temperature for 30 min in 50 mM potassium phosphate buffer, pH 7.2. The liberated sugar was then estimated by 3, 5-Dinitrosalicylic acid (DNS) reagent.
Thermostability of xylanase was determined by incubating the partially purified enzyme either from SSF or SF in 50 mM potassium phosphate buffer, pH 7.2 at different temperature for 1 h. After incubation it was subjected to centrifugation to precipitate the denatured protein which may generate due to thermal denaturation. The enzyme was diluted appropriately before the assay. The residual activity of the enzyme was measured in 50 mM potassium phosphate buffer, pH 7.2 by Dinitrosalicylic acid (DNS) reagent.
Effect of metal ions and other compounds on xylanase from SSF condition
To investigate the effect of metal ions, EDTA, SDS, triton-X100, DTT, β-mercaptoethanol (BME), NaCl, L-tryptophan and L-cysteine on the enzyme activity, 3 mM and 6 mM concentration of those compounds were used in the assay mixture and the % residual enzyme activities were measured by DNS reagent.
The authors would like to acknowledge DST-FIST, UGC-CAS and UPE programme of Dept. of Biochemistry, DBT- C.U.- IPLS programme Govt. of India, Centre for Research in Nano Science and Nanotechnology (CRNN), University of Calcutta for equipment and infrastructural support.
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