Organism and maintenance
Pleurotus tuber-regium culture was obtained from the Microbiology Research Unit of Pure and Applied Biology and maintained throughout the experiment on the potato dextrose agar slant. This P. tuber-regium strain was earlier characterized and identified using molecular techniques .
Chemicals and reagents
Citric acid, sodium citrate, ammonium sulfate (enzyme grade), potassium chloride, and ɛ-amino-n-caproic acid were from BDH Chemical Limited, Poole, England. Ethanol, 3,5-dinitrosalicylic acid, and soluble starch were Sigma-Aldrich Co., St Louis, USA, Coomassie Brilliant-Blue, and Bovine Serum Albumin from Sigma Chemical Company, St Louis, Mo., USA. CM-Sephadex C-25 was from Pharmacia Fine chemical, Uppsala, Sweden. Disodium Hydrogen phosphate and monosodium dihydrogen phosphate were products of Kermel Reagent Company Limited, Tianjin, China. Other chemicals, solvents, and media ingredients used for the experimental purpose were of analytical grade and were procured from reputed chemical companies.
Media preparation and submerged fermentation
Culture media was prepared by modifying the method of Bamigboye et al., optimized for the cultivation of P. tuber-regium . Precisely 0.38 g/l yeast extract agar, 0.1 g/l of potassium dihydrogen phosphate (KH2PO4), and 0.06 g/l of magnesium sulfate (MgSO4.7H2O) were weighed into distilled water in a 250 mL conical flask. Precisely 1.8 g/l of soluble cassava starch was added to the culture medium as the primary carbon source for amylase. The prepared solution was autoclaved for 15 min at 121 °C. Five agar plugs of a seven-day-old culture of P. tuber-regium were used to inoculate the prepared media, incubated at 35 °C for 21 days.
Enzyme extraction and characterization
All the analyses were performed at temperatures between 0 and 35°C. The mycelia were separated from the submerged culture using cheesecloth, with the filtrate serving as the crude amylase. After that, the filtrate was studied for protein concentration and amylase activity.
The amylase activity was measured using the method of Miller with minor modifications . The reaction mixture contained 0.1 mL of 1% viscous starch and 0.05 mL of crude enzyme extract. The mixture was incubated at 37 °C for 30 min. The control tubes (enzyme blank) contained an equal quantity of the substrate and 0.05 mL of distilled water. The control and experimental tubes were incubated at the same temperature and time. The reaction was stopped by dispensing 1.0 mL of 3,5-dinitrosalicylic acid reagent into the mixture. The mixture’s temperature was raised to 100 °C for 5 min, cooled, and optical density measurement was taken at 540 nm. A unit of alpha-amylase activity was described as the quantity of enzyme required to liberate reducing sugar equivalent to 1 μmol glucose per minute under the indicated analysis conditions. A standard calibration curve of glucose was plotted and used for the estimation .
Determination of protein concentration
Bovine serum albumin was used as the standard to estimate protein concentration, with the protein absorbance interpolated from a standard protein curve. The reaction mixture consisted of a Bradford reagent (1.0 mL) and the enzyme solution (10 μl); the absorbance was read at 595 nm .
Ammonium sulfate precipitation
The crude enzyme was brought to 80% ammonium sulfate saturation (560 g/L) by adding and stirring solid ammonium sulfate. This was done for 1 h with occasional stirring until all the salts were completely dissolved in the supernatant. The mixture was maintained at 4 °C for 12 h, centrifuged for 30 min at 4000 rpm. The supernatant was thrown out, and the precipitate was collected and re-suspended in a small amount of 0.2 M phosphate buffer (pH 7.5) .
Desalting by dialysis
The ammonium sulfate precipitate was dialyzed against several changes of 0.2 M phosphate buffer, pH 7.5 for 18 h. The dialysate was centrifuged at 4000 rpm for 30 min to remove insoluble components, and the supernatant was assayed for alpha-amylase activity and protein concentration.
Sephadex C-25 ion exchange chromatography
Sephadex C-25 cation exchanger was pretreated by soaking 20 g of resin in 1 L of distilled water for 24 h. The resin was washed with 15 volumes of 0.5 M NaOH for 30 min. A series of stirring and decantation followed this until effluent had attained a pH of 8.0. It was later washed with 15 volumes of 0.5 M HCl followed by continuous washing with distilled water until the effluent was at pH 7 and later liberated with 0.2 M phosphate buffer with pH 7.5. A column (1.5 × 10 cm) of Sephadex C-25 was packed and equilibrated with 0.2 M phosphate buffer, pH 7.5. The column was layered with the dialyzed protein from the preceding step and washed with 0.2 M phosphate buffer, pH 7.5 to remove unbound proteins, followed by elution with an 80 mL linear gradient of 0–1 M NaCl in 0.2 M phosphate buffer, pH 7.5. Fractions of 2.0 mL were collected from the column with a flow rate of 30 mL per hour. Protein was monitored by the Bradford method. The fractions were also assayed for alpha-amylase activity. The active fractions from the column were pooled and dialyzed against 50% glycerol in a 0.2 M solution of phosphate buffer (pH 7.5). The dialyzed fraction was assayed for alpha-amylase activity and protein concentration. The enzyme obtained was used for other studies.
Effect of pH on the enzyme activity
The effect of pH on the enzyme activity was determined by analyzing the enzyme activity using different buffers: 50 mM citrate buffer (pH 3–5); 50 mM phosphate buffer (pH 6.0–7.0), 50 mM Tris buffer (pH 8.0–9.0) and 50 mM borate buffer (pH 10.0). The alpha-amylase activity was assayed by preparing 1% starch in the different buffer solutions.
Amylase was assayed at temperatures between 30 °C and 100 °C to determine the effect of temperature on enzyme activity and establish the optimum temperature of the enzyme. The assay mixture was first incubated at the indicated temperature with an aliquot of the enzyme, equilibrated at the same temperature. The amylase activity was assayed routinely, as previously described.
The heat stability of the enzyme was determined between 30 °C and 60 °C. The enzyme was incubated at a particular temperature, and an aliquot was taken for enzyme assay at 15 min intervals for 1 h.
Effect of metal ions on the enzyme activity
The effect of metal ions on P. tuber-regium amylase was tested. The ions tested were Na+, K+, Ca2+, Mg2+, and Fe3+ at 0.1 mM, 0.5 mM, and 1.0 mM in a typical amylase assay mixture. The metal salts were dissolved in distilled water, and the reaction mixture without the salts was taken as control with 100% activity.
The enzyme’s substrate specificity was determined using different compounds, including pectin, starch, pineapple peel, and plantain peel at 1.0 % concentrations in a typical alpha-amylase assay mixture.
Potential industrial applications of crude amylase from P. tuber-regium
Removal of sand and ink stain from fabric
This was done by a modified method of Saini et al.; two clean white fabrics (8 cm–40 cm) were stained with wet sand and ink (equal amount), soaked in 15 mL of crude amylase, or water for the control, after which the fabric was washed and visually observed for 10 min .
Fresh orange (Citrus sinensis) juice was treated with crude enzymes 9.5:0.5 of juice to amylase. The control set-up contained distilled water in place of crude amylase . Sodium benzoate 0.001 g was added to the experimental and control samples to inhibit microbial growth. The sample was incubated at 25 ± 3 oC for 24 h, and clarification was monitored.
Precisely 10 g of cassava starch was weighed into a 100-mL conical flask in duplicate. To the test experiment and control, 0.5 mL of crude amylase and 0.5 mL of water was added respectively, incubated at room temperature, and monitored for any physical changes for 8 h. Glucose released was monitored spectrophotometrically by dissolving aqueous glucose into the enzyme reagent with buffer solution (Tris buffer (pH 7.4) 92 mol/l, phenol 0.3 mol/l, glucose oxidase 1500 μ/l, peroxidase 1000 μ/l, 4-Aminophenozone 2.6 mol/l, glucose aqueous 100 mg/dl). The solution was mixed gently to ensure that all the reagent has gone into the solution before use.
Data were presented as the mean of three experiments. SPSS software version 10.0 was used for the statistical analysis to determine the significant differences between the test variables using two-way ANOVA.