An ericoid mycorrhizal fungus, L. incrustata (Isolate code ChemRU330/Genbank accession no: MF374380/South African National Collection of Fungi accession no: PPRI 17268), was obtained from the Mycorrhizal Research Laboratory, Rhodes University, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa. The isolate was maintained on potato dextrose agar (PDA). Incubation was performed at 28 °C for 21 days. The mycelia were kept on PDA, stored at 4 °C and regularly subcultured throughout the study period.
Production of amylase
Soluble Starch (1% w/v, Merck, Cas # 9005-84-9) was added to a 1000 mL−1 Erlenmeyer flask in an modified Melin–Norkrans (MMN) broth with the composition: (g L−1): glucose 1.0; yeast extract 3.0; malt extract 1.0; (NH4)2HPO4 0.25; KH2PO4 0.50; MgSO4·7H2O 0.15; CaCl2 0.05; NaCl 0.025; thiamine-HCl 100 µg L−1, ZnSO4·7H2O 0.003, and 1.2 mL of FeCl3 (1% w/v). Production medium was thoroughly mixed, and a 250 mL was placed in a 500 mL shake flask. The medium was sterilized at 121 °C for 15 min and was inoculated with five discs of 5 mm mycelial plugs of the fungus. The uninoculated medium was used as a control. Growth was allowed to proceed at 28 °C in the dark for 3 weeks on a rotary incubator shaker at 150 rpm. After 42 days of growth, the cultures were homogenised using IKA’s ULTRA-TURRAX homogeniser (20,000 rpm), and centrifugation was performed at 10,000×g for 15 min to obtain crude enzyme filtrates (supernatant) using a Beckman Coulter Avanti-J high-speed centrifuge.
Assay for amylase
A dinitrosalicylic acid (DNS) assay (Miller 1959) was conducted by adding 1% (w/v) soluble starch to a volume of 10 mL sodium acetate buffer (pH 5.0) in a Schott bottle and boiled for 30 s. A volume of 100 μL of crude enzyme and uninoculated control was added to 300 μL of the soluble starch-containing medium in triplicate, while the blank contained 400 μL buffer. All samples were incubated at 37 °C for 1 h, followed by centrifugation at 6000×g for 2 min. A 300 μL aliquot of DNS was added to 150 μL of each supernatant sample. This was followed by boiling on a heating block at 100 °C for 5 min after which it was cooled on ice for 5 min. A volume of 250 μL of each sample was placed into each well of a 96-well plate and read with the aid of a spectrophotometer (BioTek’s Synergy Mx) at a wavelength of 540 nm. The supernatant was taken to determine the reducing sugar using DNS assay with glucose as a standard.
Protein content was determined with Bradford reagent assay kit using bovine serum albumin as standard (Bradford 1976).
Ammonium sulphate precipitation
A 120 mL volume of the crude enzyme was brought to 80% saturation with solid ammonium sulphate according to the method of Kusuda et al. (2004). The mixture was left overnight at 4 °C on a magnetic stirrer. The pellet obtained from the centrifuged mixture was re-dissolved in 20 mM sodium acetate buffer (pH 5.0) to make up a volume of 10 mL.
Dialysis of the partially purified enzyme
A pre-treated dialysis bag was used for the dialysis (10 kDa cut-off) of the enzyme collected after ammonium sulphate precipitation. The partially purified enzyme (10 mL) was dialyzed against 20 mM sodium acetate buffer (pH 5.0) at 4 °C with three changes of a buffer according to the method described by Kusuda et al. (2004).
A vertical glass chromatographic column (1.5 × 50 cm) was packed using Sephadex G-100. The dialyzed enzyme solution (1.5 mL) was used after it was concentrated by ultrafiltration (Amicon Ultra-100 centrifugal filter device; cut-off 10 kDa). Gel filtration chromatography was carried out using sodium acetate buffer (20 mM, pH 5.0), at a flow rate of 1.2 mL min−1. All fractions collected were subjected to analysis by measuring the absorbance at 280 nm, followed by the activity assay, the active fractions (fractions no. 36–40; 5 mL) were pooled together afterwards.
Identification of hydrolytic products using thin layer chromatography (TLC)
Soluble starch was used as the substrate. The mixture was incubated in a dry heating block at 45 °C for 1 min up to 24 h and samples were removed at various time intervals for analysis. Samples were then boiled for 5 min at 100 °C to denature the proteins and then centrifuged at 6000×g for 5 min. The supernatant (80 µL) was used for TLC analysis. Identical volumes (5 µL) of the supernatant were applied to Silica Gel 60G F254 HPTLC plates (Merck, Darmstadt, Germany). Plates were developed twice with n-butanol: acetic acid: water (2:1:1, v/v/v). Then, to detect carbohydrates, plates were briefly submerged in methanol containing 5% (v/v) sulfuric acid and 0.3% α-naphthol. Plates were then air dried, heated at 120 °C for 10 min and observed for the bands formed by various hydrolytic products.
AMG activity staining (zymography)
The enzyme from crude extract was analyzed by its native protein pattern using the copolymerization method of Martinez et al. (2000). Sample was diluted in 1:1 in sample buffer (0.125 M Tris; 20% v/v glycerol; 0.04% v/v bromophenol blue) and boiled at 95 °C for 5 min. The resolving gel (12%) consisted of:- polyacrylamide 4.0 mL, 1.5 M Tris–HCl buffer (pH 8.8) 2.5 mL, distilled water 2.25 mL, 2% w/v soluble starch 1.25 mL, 10% w/v sodium dodecyl sulfate (SDS) 100 µL, 10% w/v ammonium persulfate 50 µL, and N,N,N′,N′-tetramethylethylenediamine (TEMED) 5 µL. The stacking gel (4%) consisted of: polyacrylamide 0.65 mL, 0.5 M Tris–HCl buffer (pH 6.8) 1.25 mL, distilled water 3.05 mL, 10% w/v SDS 50 µL, 10% w/v ammonium persulfate 25 µL, and TEMED 5 µL. Starch-SDS-PAGE (PAGE—polyacrylamide gel electrophoresis) was carried out in two phases viz. gels were first subjected to a constant voltage of 30 V for 30 min to ensure the tracking dye (bromophenol blue) enters the separating gel. After this, gels were finally subjected to a constant voltage of 100 V for 75 min. Electrophoresis was carried at a temperature of 0–2 °C to allow the enzyme to migrate inactively without hydrolyzing the starch. After electrophoresis, the gel was washed in distilled water and then incubated in 0.1 M phosphate-citrate and 0.05 M NaCl buffer (pH 6.0) for 3 h at 39 °C. Again, gels were washed and fixed in 12% trichloroacetic acid (TCA) for 10 min, followed by another washing and gently shaking (50 rpm) in 2.5% w/v Triton X-100 for 1 h at 4 °C to remove SDS and restore activity. Finally, the gels were stained with Lugol solution (6.7 mg mL−1 KI and 3.3 mg mL−1 I2), and photographs were taken.
Molecular weight determination by SDS-PAGE electrophoresis
SDS-PAGE was performed using 8 × 10 × 0.75 cm gels in a Mini-Protean II (Bio-Rad) gel apparatus. Samples were treated with reducing (containing 2-mercaptoethanol) sample buffer and boiled for 5 min before loading the gel. After electrophoresis, proteins in the gel were visualized by staining with Coomassie Blue R-250 (Laemmli 1970).
Effect of pH on activity and stability of the AMG
The optimum pH for the enzyme was determined by incubating enzyme with the substrate (1%, w/v) prepared in 0.1 M buffer having pH values of 1.0, 2.0 (hydrochloric acid–potassium chloride); 3.0 (citrate); 4.0, 5.0 (acetate); 6.0, 7.0 (phosphate); 8.0 and 9.0 (Tris–HCl) at a temperature of 45 °C for 1 h, after which the enzyme activity was measured. The pH stability was determined by pre-incubating the enzyme in buffers of different pH (3.0, 4.0, 5.0, 6.0, 7.0 and 8.0) at 37 °C for 6, 12, 18, 24 and 30 h before determining the residual activity by the standard procedure.
Effect of temperature on activity and stability of the AMG
The optimum pH for the enzyme was determined by incubating enzyme with the substrate (1% w/v) prepared in 0.1 acetate buffer (pH 5.0). The enzyme was incubated at different temperatures of 4, 20, 30, 40, 50, 60, 70 and 80 °C for 1 h, after which, the enzyme activity was measured. The temperature stability was investigated by pre-incubating the enzyme at different temperatures (37, 45, 50, 60, and 70 °C) for 6, 12, 18, 24 and 30 min before determination of the residual activity by the standard procedure.
Effect of metal ions and other additives on the activity of the AMG
Metal ions such as K+, Na+, Ca2+, Mn2+, Fe2+, Mg2+, Al3+, Hg2+, and Cd2+ (all supplied in chloride form) were each applied to determine the effect on the activity of the enzyme. Each metal ion was used at a concentration of 1, 5, and 10 mM, while incubation was performed at 45 °C for 1 h. The following compounds—sodium azide (NaN3), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), Tween 20, SDS, and indole-3-acetic acid (IAA) were tested for their inhibitory effect on AMG activity. The purified enzyme was incubated at 45 °C for 1 h in 20 mM sodium acetate buffer (pH 5.0), containing each inhibitor or denaturing agent at final concentrations of 1, 5, and 10 mM, respectively. The relative enzyme activity was determined under standard assay conditions.
The Michaelis–Menten substrate saturation curve was used to determine the Km and Vmax value of the AMG by measuring the rate of soluble hydrolysis under standard assay conditions. The reaction mixture was 20 mM acetate buffer (pH 5.0), with the soluble starch substrate at concentrations ranging from 0.2 to 10 mg mL−1. The values for Km and Vmax were then determined using KaleidaGraph.
All experiments were carried out in triplicate. Data were analysed on Microsoft® Excel software, and all values were presented as the standard errors of the means (± SEM).