Purification and characterization of Rhizoctonia solani AG-4 strain ZB-34 α-amylase produced by solid-state fermentation using corn bran

Fungus and solid-state fermentation (SSF)

Rhizoctonia solani AG-4 strain ZB-34 was obtained from pods of common bean (Phaseolus vulgaris), grown on potato dextrose agar for 3 days at 25°C and stored at 4°C [10]. Corn bran, barley bran and wheat bran were obtained from a local feed store. Fermentations were done in 250 mL Erlenmeyer flasks containing 5 g of the substrate impregnated with 5 mL nutrient salt solution containing (w/v) 0.5% KH₂PO₄, 0.25% NaCl, 0.01% MgSO₄ · 7H₂O, 0.01% CaCl₂ and 0.005% streptomycin sulfate, pH 7.00. After the flasks were autoclaved, they were cooled to room temperature and inoculated aseptically with two disks of fungus (5 mm in diameter) grown on potato dextrose agar, thoroughly mixed and incubated at 28°C for 6 days [11, 12].

Enzyme extraction and determination of protein concentration

Enzyme was extracted in 50 mL phosphate buffer (50 mM, pH 7.00) on a rotary shaker at 250 rpm for 45 min. The slurry obtained was squeezed through four layers of muslin cloth and the extract was filtered through Whatman filter paper No. 1. After the centrifugation at 10,000 rpm for 10 min at 4°C, the supernatant was used as crude enzyme extract [13].

Protein concentration was determined by Bradford method, using BSA as standard [14].

Enzyme assay

Activity was assayed according to the procedure of Miller [15]. The reaction mixture containing an appropriate volume of 1% soluble starch, 50 mM buffer, and enzyme solution was incubated at 25°C for 15 min. The reaction was stopped by adding DNS reagent and incubated in a boiling water bath for 10 min followed by cooling. The absorbance was recorded at 540 nm and the liberated reducing sugar was calculated from a standard curve using glucose. One unit of enzyme activity was defined as the amount of enzyme producing 1 μmol reducing sugar per minute under the standard assay conditions.

Optimization of SSF process

Different cultural conditions were optimized such as solid substrate (corn bran, barley bran and wheat bran), initial medium pH (3.0, 5.0, 7.0, 9.0), moisture level (v/w, 40%, 60%, 80%, 100% and 120%), incubation temperature (20, 28 and 35°C), different incubation period (2, 4, 6 and 8 days), and the influence of supplementation of solid substrate with different carbon sources, nitrogen sources and detergents (1% w/w) [12].

Purification and characterization of the enzyme

Two gram insoluble corn-starch was added to 50 mL of crude enzyme extract and incubated at 4°C, with stirring at 140 rpm. After 60 min, the suspension was centrifuged (10,000 rpm, 10 min) at 4°C. The starch pellet was washed by using 50 mL of chilled phosphate buffer (50 mM, pH 7.00) for 5 min on ice and then the suspension was centrifuged again as mentioned above. Fifty milliliter phosphate buffer (50 mM, pH 7.00) preheated to 40°C was added to the starch pellet and incubated at 40°C for 1 h. Then, the suspension was centrifuged and the supernatant was checked for α-amylase activity and protein concentration [16].

Native polyacrylamide gel electrophoresis

Electrophoresis in non-denaturing conditions was done by using 8% resolving gel containing 1% starch. After the end of the run, the gel was washed in phosphate buffer (50 mM, pH 7.00) for 15 min. Then the gel was immersed in a solution of iodine (10 mM) and potassium iodide (14 mM) until the appearance of activity bands. Another gel prepared in the same manner was stained with Coomassie Brilliant Blue R-250 [17].

Enzyme kinetics

Enzyme kinetic parameters were obtained by measuring the hydrolysis of soluble starch at different concentrations in the standard reaction mixture. Maximum velocity (V_max) and K_0.5 values were determined from substrate-activity curve [18].

Effect of some metal ions and detergent on the enzyme activity

The effect of metal ions was investigated by adding chloride salts of Li⁺, Na⁺, Mn²⁺, Cu²⁺, Zn²⁺, Ni²⁺, Ca²⁺, Co²⁺ and Mg²⁺ directly to the standard reaction mixture at a final concentration of 1 mM. Also, the effect of 1 mM EDTA was investigated. Enzyme activity determined in the absence of metal ion or EDTA was defined as 100%.

To study the effect of some detergents on the α-amylase activity, Tween 20, SDS, Triton X-100 and Triton X-114 were separately added to the standard reaction mixture at the final concentration of 1%. The percentage residual activities were expressed by comparison with standard assay mixture with no chemical added.

Substrate specificity

In order to determine the substrate specificity of the enzyme soluble starch, maltose, glycogen, β-cyclodextrin, amilopectine and maltotriose were used as substrates and activity assays were performed in the standard reaction conditions.

Test of compatibility with commercial detergents and proteases

Potential of purified enzyme as a detergent additive was examined by using some commercial laundry and dish washing detergents available at a local market (Omo^®, Finish^®, Ariel^®, Persil^®, Alo^®, Fairy^®, Perwol^®, Woolite^® and Etimatik^®). Firstly, solutions were prepared from solid detergents at 7 mg/mL and liquid detergents as 10% (v/v) to simulate washing conditions and pre-heated at 100°C for 60 min to destroy the endogenous enzyme activity. α-Amylase assay was done in the presence of 1 mg/mL and 1% final concentration of solid and liquid detergents, respectively, at 50°C. The activity of the crude enzyme assayed in the absence of detergents was taken as 100% [19].

To test the stability of enzyme in the presence of protease, the purified amylase was mixed with a commercial protease preparation (proteinase K) (in the ratio of 1:1) and incubated for up to 150 min at room temperature. Thereafter, the residual activity was determined against the control (amylase without protease treatment) [20].

Wash performance analysis

To determine the efficacy of purified enzyme for use as a bio-detergent additive, wash performance was evaluated by determining the chocolate stain releasing capacity from cotton fabrics. Briefly, chocolate was liquefied at 70°C. Cotton fabrics (5 cm×5 cm) were stained with 300 μL of the liquefied chocolate and then dried overnight under in a hot air oven.

To test the wash performance, each piece of stained cloth was dipped in one of the following flasks containing: (a) 25 mL of tap water (control), (b) 20 mL of tap water and 5.0 mL of purified α-amylase (1 mg/mL), (c) 20 mL of tap water and 5 mL of commercial detergent (Persil^®, 1%), and (d) 20 mL of tap water and 5 mL of commercial detergent (Persil^®, 1%) containing purified enzyme (1 mg/mL).

Flasks were kept at 50°C for 60 min. Stain removal capabilities of the purified enzyme was examined visually by looking at the pieces of dried cloth. The untreated chocolate stained cloth piece was considered as a control [20].

Desizing of cotton fabric with purified α-amylase

To investigate the capacity of R. solani AG-4 strain ZB-34 α-amylase for desizing, cotton fabrics (5 cm×5 cm) were weighed and treated with 25 mL of soluble starch solution (1% w/v) at room temperature. After 15 min, they were dried and weighed again.

The cotton fabrics were desized in 25 mL acetate buffer (50 mM, pH 5.5) containing 1 mL purified α-amylase at 40 and 50°C for 1 h. The same procedure was applied by using tap water instead of buffer. Upon the completion of the reaction, the cotton fabrics were washed using tap water, dried at 105°C to a constant weight and weighed. The percent (%) removal of starch was calculated by applying the following formula [21]:

Desizing (%)= {Weight of starch removed by enzyme (g)Total starch present on the fabric (g)}×100

End-product analysis of starch hydrolysis

Purified enzyme was incubated with 1% (w/v) starch at optimum conditions. Twenty micromilliliter of aliquot (starch hydrolysis products) was withdrawn at 1, 5, 15 and 120 min. End products of hydrolysis were analyzed by using Kieselgel Silica gel (TLC-cards) in a solvent system of 2-propanol-ethylacetate-water 3:1:1 (v/v/v). Glucose, maltose, maltotriose and maltotetrose were used as standard. Spots were visualized by treating the cards with 20% (v/v) H₂SO₄ dissolved in methanol and heating to 110°C.

Treatment of raw apple juice with purified enzyme

Apples (Malus domestica cv. Golden Delicious) were cut into cubes and mashed in a mixer grinder and manually pressed using double layer cheesecloth to obtain raw or unclarified apple juice. After calcium chloride was added to the raw apple juice at a final concentration of 10 mM, aliquots were pasteurized (5 min at 90°C) and immediately cooled to 50°C. A mixture containing 4900 μL apple juice and 100 μL (1 mg/mL) enzyme was incubated at 50°C for 1 and 3 h. After centrifugation at 10,000 rpm for 10 min, absorbance (440 nm) of the supernatant was determined. Also, the total reducing sugar content was determined by the DNS method [22].

Statistical analysis

All experiments were carried out in triplicates. Data are represented by the mean±standard deviation. Statistical significance was calculated using oneway analysis of variance and Duncan’s test. A value of p<0.05 was taken as statistically significant.

Optimization of SSF process

The results of the optimization process are seen in Figure 1. Although all the solid substrates used supported both fungus growth and enzyme formation, the highest α-amylase activity was obtained with corn bran with nutrient solution adjusted to pH 7.0.

Figure 1:

Effect of solid substrate, initial pH of the medium, moisture level, fermentation temperature and fermentation period on the enzyme production.

Maximum enzyme production was determined at the moisture level of 100% (v/w) after 6 days incubation. When the fermentation period was prolonged after that time, the production of α-amylase declined. This may be due to low moisture content in the fermentation medium.

Enzyme production was also effected by the fermentation temperature. Although α-amylase was produced at all three temperatures, incubation temperature of 28°C, optimum temperature growth of R. solani AG-4 strain ZB-34, provided the highest α-amylase production.

To determine the effect of additives such as carbon sources (soluble starch and sucrose), organic nitrogen sources (yeast extract, peptone and urea) and detergents (SDS, Triton X-100 and Tween 20), they were separately added to the solid substrate at a final concentration of 1%. A significant improvement in the α-amylase production by R. solani AG-4 strain ZB-34 was obtained when the solid substrate was supplemented with 1% soluble starch (Figure 2). Addition of organic nitrogen sources such as urea, peptone and yeast extract decreased the α-amylase production by R. solani AG-4 strain ZB-34. It was previously reported that addition of nitrogen sources to the medium generally inhibited the production of α-amylase by microorganisms [23]. Also, addition of the SDS and Tween 20 decreased the enzyme production.

Figure 2:

Effect of some additives on α-amylase production by Rhizoctonia solani AG-4 strain ZB-34 under solid-state fermentation.

Purification and characterization of the enzyme

Results of the purification of α-amylase by starch affinity method are summarized in Table 1. The enzyme was purified 11.17-fold with specific activity of 35.64 U/mg protein. Preussia minima α-amylase was previously purified 7.44-fold with TCA concentration, gel filtration and anion exchange chromatography [24].

Native polyacrylamide gel electrophoresis

The electrophoresis in non-denaturing conditions was done. A single protein band observed in both of the gels indicated that an active α-amylase was successfully purified from R. solani AG-4 strain ZB-34 (Figure 3). It is clearly seen from the Figure 3 that although crude enzyme solution has several amylolitic enzyme activities, only one activity band was achieved after purification.

Figure 3:

Native PAGE and activity staining (A) Native-PAGE Coomassie Brillant Blue staining: line 1, crude enzyme extract; line 2, purified amylase, (B) Native-PAGE activity staining: line 1, purified amylase; line 2, crude enzyme extract.

Optimum pH and temperature of the purified enzyme

As shown in Figure 4A, maximal relative α-amylase activity was measured at pH 5.5. A similar result was reported previously for Penicillium camemberti PL21 [25]. It was reported that most fungal α-amylases had acidic and neutral range pH optimum and they were generally stable between pH 4.0 and pH 11.0 [26].

Figure 4:

pH-activity and temperature-activity profiles of Rhizoctonia solani AG-4 strain ZB-34 α-amylase.

(A) pH-activity profile of Rhizoctonia solani AG-4 strain ZB-34 α-amylase . Enzyme activity was assayed in the presence of soluble starch as a substrate and 50 mM buffer systems having different pH values. (B) Temperature-activity profile of R. solani AG-4 strain ZB-34 α-amylase. Optimum temperature of the enzyme was determined at optimum pH value by measuring the activity at 10–80°C.

The enzyme had a temperature optimum at 50°C (Figure 4B). Although the enzyme activity was also notably high between 40 and 60°C, it was negatively affected above 60°C. It was reported that α-amylases from Aspergillus niger and Penicillium camemberti had temperature optima at 30°C [25, 27].

Although R. solani AG-4 strain ZB-34 grows optimally at 28°C, the α-amylase produced from this organism was active between 40 and 60°C. This characteristic might be an advantage for the industry. Similar results were reported for α-amylase from Bacillus sp. [11].

Enzyme kinetics

The substrate saturation curve of the enzyme did not follow a hyperbolic relationship and plot of [S] versus V₀ produced a sigmoidal curve (Figure 5). The allosteric enzymes show such saturation curves [18] and R. solani AG-4 strain ZB-34 α-amylase might be allosteric. V_max and K_0.5 were determined as 238.8 U/mg protein and 0.03% from substrate-activity curve.

Figure 5:

Substrate-activity curve of Rhizoctonia solani AG-4 strain ZB-34 α-amylase.

Enzyme kinetic parameters were obtained by measuring the hydrolysis of soluble starch at different concentrations in the standard reaction mixture.

pH and thermal stability of the purified enzyme

Enzyme activity was maintained at about 100% in all of the tested pHs after 1 day incubation (Figure 6A). At the end of 3 days, the enzyme activity was conserved approximately 60%–70%.

Figure 6:

pH and thermal stability graphs.

(A) pH stability graph of Rhizoctonia solani AG-4 strain ZB-34 α-amylase. pH stability was determined by incubating the enzyme in the buffer solutions at 4°C for 24 h: acetate (pH 5.5) and phosphate (pH 7.0–8.0). At the end of the incubation, enzyme activity was assayed under standard reaction conditions. (B) Thermal stability graph of Rhizoctonia solani AG-4 strain ZB-34 α-amylase. To determine the thermal stability, the enzyme solutions were separately incubated at 4, 28, 40, 50 and 60°C. Aliquots were withdrawn at 15, 30, 60, 90 and 120 min, and α-amylase activity was determined at optimum conditions.

In one study, it was reported that an α-amylase purified from Anoxybacillus flavithermus conserved its original activity at pH 6.0, 7.0 and 8.0 for 1 h. After 3 h incubation it had a residual activity of around 80% at pH 6.0, and 60% at pH 7.0 and 8.0 [28]. It was also reported that α-amylase purified from Geobacillus thermoleovorans maintains its activity at a pH of 5.0 after 20 h of incubation. Its activity decreased approximately 50% after 24 h and lost at a later stage of incubation [29]. Therefore, it appears that R. solani AG-4 strain ZB-34 α-amylase is highly stable at the working pHs. It is clear that this feature will make a positive contribution to the availability of the enzyme in industrial areas.

The enzyme maintains full of its activity at 4°C (storage temperature) and 28°C (the growth temperature of fungus) for 120 min. It conserved its activity about 70% at 40°C, the optimum temperature, for 60 min. When this time increases, there is a further decrease in activity (Figure 6B).

Thermal stability of α-amylases obtained from a wide variety of sources may vary. α-amylase purified from Anoxybacillus flavithermus was stable at 40 and 45°C and lost full of its activity at 55°C after 2 h [28]. Bacillus licheniformis AI20 α-amylase conserved nearly 75% of its activity after 90 min at 70°C [30]. Aspergillus niger JGI 24 α-amylase has the highest stability at 30°C and maintains about 80% of its activity after 50 min [31]. The α-amylase produced by solid-state fermentation technique from Penicillium digitatum conserved its original activity at temperatures between 10 and 37°C after 30 min. But there was a dramatic decline in enzyme activity at 45°C [32].

In general, it can be seen that R. solani AG-4 strain ZB-34 α-amylase might be more advantageous than some other α-amylases in terms of thermal stability.

Effect of some metal ions and detergent on the enzyme activity

Rhizoctonia solani AG-4 strain ZB-34 α-amylase was activated at about 20% and 27% with Mn²⁺ and Ca²⁺, respectively in 1 mM final concentration (Figure 7). Nearly all α-amylases contain at least one conserved Ca²⁺ for structural integrity and enzymatic activity [33]. Increase in activity by the addition of this metal ion to the reaction medium may be due this reason.

Figure 7:

Effect of some metal ions and detergents on the enzyme activity.

Similar results can be found in the literature. CaCl₂ and MgCl₂ increased the total amylase activity produced by Preussia minima [24]. Bacillus sp. KSM-1378 α-amylase was inhibited 88%, 91%, 100% and 100% with Ni²⁺, Zn²⁺, Cd²⁺ and Hg²⁺ ions, respectively [34]. Geobacillus sp. LH8 α-amylase was inhibited at different ratios by Mg²⁺, Ni²⁺, Zn²⁺, Cu²⁺ and EDTA. Ca²⁺ almost activated the enzyme activity 2-fold at 10 mM concentration [35].

While Tween 20, Triton X-100 and Triton X-114 tested at 1% final concentration caused a slight increase in enzyme activity, a decrease of approximately 40% of activity occurred in the presence of SDS (Figure 7). The increase of α-amylase activity in the presence in some detergents suggests that these detergents may have positive effects on the enzyme conformation and on the hydrophobic interactions involved in stabilizing the tertiary structure of the protein molecule [36]. The decrease in enzyme activity in the presence of certain detergents can also be correlated with a possible deterioration of protein conformation.

The enzyme purified from Marinobacter sp. EMB8 was reported to retain its activity approximately 75% after 1 h treatment with 0.1% SDS and Triton X-100 [37].

The effect of detergents such as Triton X-100 and Tween-20 and surfactants such as SDS on the activity of α-amylase produced by Bacillus licheniformis AI20 was studied and found that detergents tested at 0.25% concentration did not alter enzyme activity. SDS at concentration of 1% reduced enzyme activity by 35% [30].

Bacillus licheniformis NH1 α-amylase was treated with Tween-20 and Triton X-100 at 1% concentration at 40°C for 1 h and there was no loss of activity [20]. Alicyclobacillus acidocaldarius α-amylase was determined to be stable in the presence of SDS, Triton X100 and Tween-20 [38].