Abstract: Amylases are enzymes that act on starch, converting it into sugars by their catalytic activities. They are highly rated amongst enzymes because of their numerous applications in industries. Microorganisms are the best source of amylase and they are advantageous because they can easily be exploited to produce the enzyme, α-amylase. This research work therefore, employed a strain of Rhizopus arrhizus fisher which was capable of growth on 1% flour medium for the production of α-amylase within ten days of incubation. α-Amylase activity was optimum on the 8th day of inoculation and was expressed as 933 U. The crude enzyme was partially purified by a combination of ammonium sulphate precipitation and dialysis. The activity of the partially purified enzyme was optimum at 30°C and pH 6.0. The enzyme was capable of degrading starch with an optimum activity expressed at 1.2 mg mL–1 concentration. It was stimulated by NaCl and CaCl2 but inhibited by P-chloromercuric benzoate and Ethylene Diamine Tetraacetic Acid (EDTA). The enzyme was affected by heat with complete loss of activity within 10 min of heating at 70°C. Rhizopus arrhizus can therefore be suitably genetically modified and employed for the commercial production of α-amylase.
INTRODUCTION
Amylases are hydrolytic enzymes with their beneficial uses dating back to several decades (Sundarram and Murthy, 2014). Amylases are enzymes catalyzing the conversion of starch to sugar (Raul et al., 2014). They had been isolated from different microbial and plant sources and Bacillus species are the most widely used for commercial production of amylase (Sundarram and Murthy, 2014). Bacillus subtilis serves as a good source of α-amylase for industrial purposes (Sani et al., 2014). Alkaliphilic α-amylase from B. subtilis (A10) was isolated from orchard soils of Kahramanmaras in Turkey. The α-amylase was found to be suitable for waste-paper, starch and bioethanol industries (Aygan et al., 2014). The storage of stability of five α-amylase from miswak, (Salvadora persica) a medicinal plant which serves as a natural toothbrush was reported. It was discovered that the enzyme in the toothpaste retained 55% of its original activity after ten months of storage at room temperature (Mohamed et al., 2014). Adejuwon et al. (2013) in an earlier research reported that strains of Candida albicans and Fusarium sp. were involved in the production of α-amylase. Immobilization can improve the activity of α-amylase and its stability when compared with free enzyme in solution without hampering the enzymatic reaction rate (Singh et al., 2014). This research was therefore, carried out with a view of obtaining α-amylase with appreciable activity from R. arrhizus fisher for industrial purposes.
MATERIALS AND METHODS
Source and identification of isolate: The isolate of R. arrhizus used for this research was isolated from mouldy bread and identified using techniques contained in the illustrated Handbook of Fungi (Hanlin, 1990; Cannon and Kirk, 2007). The identification was done by observing cultural and morphological characteristics. The isolate was cultured on Potato Dextrose agar. The nature of growth, rate of growth, colony colour and sporulation patterns were carefully observed. Sporulating mature cultures were used in microscopic examination. Fungal samples were taken from advancing margins and centres of the growth regions with the aid of sterile inoculating needle. The samples were smeared on glass slides and stained with lactophenol cotton blue. Macroscopic and microscopic morphological characteristics like arrangement and shape of spores, type of sporangia, type of hyphae and presence or absence of septa on hyphae were examined under the high power objective of a compound binocular microscope.
Culture conditions and preparation of inocula: The isolate was subcultured and maintained on Potato Dextrose agar plates and slants. The fungus was further subcultured into test tubes of the same medium and incubated at 25°C. Ninety-six hours-old cultures of Rhizopus arrhizus were used as the inoculum. According to the modified method of Olutiola and Ayres (1973), cultures were grown on 1% flour medium. Conical flasks (250 mL) containing 100 mL flour medium were inoculated with 1 mL of an aqueous spore suspension containing approximately 5×105 spores mL-1 of isolate. Experimental flasks contained the inoculated sterilized medium while control flasks contained only the sterilized uninoculated medium. Spores were counted using the Neubauer counting chamber (Olutiola et al., 1991). Experimental and control flasks were incubated without shaking at 25°C (Olutiola and Nwaogwugwu, 1982).
Flour as a source of carbon: The flour used in this study was obtained for Eagle Flour Mill Company, Ibadan, Nigeria. One gram of the flour was weighed into conical flask containing 100 mL distilled water. The contents of the flask were mixed by swirling and then sterilized at 15 Ib in‾2 for 20 min. The flour medium were allowed to cool and then used as the growth medium and carbon source.
Enzyme and protein assays
α-amylase: α-amylase activity was determined using the method of Pfueller and Elliott (1969). The reaction mixtures consisted of 2 mL of 0.2% (w/v) starch in 0.2 M citrate phosphate buffer, pH 6.0 as substrate and 0.5 mL of enzyme. Controls consisted of only 2 mL of the prepared substrate. The contents of both experimental and control tubes were incubated at 35°C for 20 min. The reaction in each tube was terminated with 3 mL of 1 N HCl. Enzyme (0.5 mL) was then added to the control tube. Two millilitre of the mixture from each of the sets of experimental and controls were transferred into new sets of clean test tubes. Three millilitre of 0.1 N HCl were added into the contents of each test tube after which 0.1 mL of iodine solution was added. Optical density readings were taken at 670 nm. One unit of enzyme activity was defined as the amount of enzyme which produced 0.1% reduction in the intensity of the blue colour of starch-iodine complex under conditions of the assay. Specific activity was calculated as enzyme units per milligram protein.
Protein concentration determination: Protein concentration was routinely determined by the method of Lowry et al. (1951). The Lowry assay, a colometric protein assay, is based on the reaction of the protein with copper in alkali and the reduction of the phosphomolybdic-phosphotungstic reagent (Folin’s reagent) by the copper treated protein. Absorbances of treated samples were taken at 600 nm. Serial dilutions of Bovine serum albumin treated likewise were used to plot a standard graph. The unknown protein value in each test sample was interpolated from the standard calibration graph.
Ammonium sulphate fractionation: The crude enzyme, on the day of optimum activity during samplings, was treated with ammonium sulphate (analytical grade) at 90% saturation (662 g L-1). Precipitation was allowed to continue at 4°C for 24 h. The mixtures were centrifuged at 4,000 rpm for 30 min at 4°C using a high speed cold centrifuge (Optima LE-80K Ultracentrifuge, Beckman, USA) at the Central Laboratory, Obafemi Awolowo University, Ile-Ife, Nigeria. The supernatant was discarded. The precipitate was re-constituted in 0.2 M citrate phosphate buffer, pH 6.0. The protein contents were determined using the Lowry et al. (1951) method while α-amylase activity was determined using the method of Pfueller and Elliott (1969).
Dialysis: Using acetylated dialysis tubings (Visking dialysis tubings, Sigma) (Whitaker et al., 1963) and a multiple dialyser, the enzyme preparation was dialysed under several changes of 0.2 M citrate phosphate buffer, pH 6.0 at 4°C for 24 h. The protein content of the dialysed enzyme was determined using the Lowry et al. (1951) method while α-amylase activity was determined using the method of Pfueller and Elliott (1969).
Properties of the partially purified enzyme: The effects of temperature, pH, salts and some chemicals on the activity of the partially purified α-amylase from R. arrhizus were investigated.
Effect of temperature: The substrate used was 0.2% (w/v) starch in 0.2 M citrate phosphate buffer, pH 6.0. The reaction mixture was 2 mL of substrate and 0.5 mL of enzyme. Incubation was at a range of 20-40°C for 1 h. α-amylase activity was there after determined.
Stability test at 70°C: The effect of heat (70°C) on the stability of the partially purified enzymes at different periods, 0, 2, 5, 10, 20, 25 and 30 min was examined. The activities of the heated enzymes were determined by incubating 0.5 mL of each enzyme with 2 mL of the citrate phosphate buffered 0.2% starch substrate (pH 6.0) at 35°C for 1 h, α-amylase activity was there after determined.
Effect of pH: The substrate used was 0.2% (w/v) starch in 0.2 M citrate phosphate buffer at different pH values ranging from pH 3.0-8.0. The reaction mixture was 2 mL of substrate and 0.5 mL of enzyme. Incubation was at 35°C for 1 h, α-amylase activity was then determined as described.
Effect of substrate concentrations: Different concentrations, 0.2-1.4 mg mL-1 starch (Sigma) in 0.2 M citrate phosphate buffer, pH 6.0 were used as substrates. The reaction mixture was 2 mL of substrate and 0.5 mL of enzyme, incubated at 35°C for 1 h, α-amylase activity was afterwards determined.
Effect of salts and some chemicals: The effects of NaCl and CaCl2 at varying concentrations (0, 5, 10, 15, 20 and 30 mM) on the activities of the purified α-amylase were examined. Each salt was prepared in 0.2% starch in citrate phosphate buffer pH 6.0. The reaction mixture, 2 mL of substrate and 0.5 mL of enzyme, was incubated at 35°C for 1 h. α-amylase activity was then determined.
Different concentrations (0, 2, 4, 6 and 10 mM) each of p-chloromercuric benzoate and EDTA were prepared in 0.2% starch in citrate phosphate buffer, pH 6.0. These were used as substrates. α-amylase activity was determined as described.
RESULTS
The results of this investigation revealed that Rhizopus arrhizus fisher grew on 1% flour medium. Culture filtrate obtained after eight days of incubation exhibited appreciable α-amylase activity (Fig. 1).
Effect of temperature: α-amylase activity was affected by the temperature of the reaction medium. Optimum activity of the enzyme was observed at 30°C after which there was a gradual decline in the activity of the enzyme (Fig. 2).
Effect of pH: α-amylase activity increased slightly from pH 3.5 and it gradually increased to pH 6.0 after which there was a decline in the enzymes activity (Fig. 3).
| Fig. 1: | Effect of days of incubation on α-amylase activity produced by Rhizopus arrhizus fisher in 1% flour medium |
| Fig. 2: | Effect of temperature on α-amylase activity produced by Rhizopus arrhizus fisher |
| Fig. 3: | Effect of pH on α-amylase activity produced by Rhizopus arrhizus fisher |
| Fig. 4: | Effect of p-chloromercuric benzoate on α-amylase activity produced by Rhizopus arrhizus fisher |
| Fig. 5: | Effect of EDTA on α-amylase activity produced by Rhizopus arrhizus fisher |
Effects of chemicals: Enzymes activity decreased gradually with increasing concentrations of p-chloromercuric benzoate and EDTA (Fig. 4 and 5).
Effect of salts: α-amylase activity steadily increased with increasing concentrations of NaCl and CaCl2 (Fig. 6 and 7).
Effect of substrate concentration: There was a gradual increase in amylase activity as the concentrations of substrate (starch) increased. Enzyme activity reached an optimum at 1.2 mg mL-1 starch concentrations after which there was a decline (Fig. 8).
| Fig. 6: | Effect of NaCl on α-amylase activity produced by Rhizopus arrhizus fisher |
| Fig. 7: | Effect of CaCl2 on α-amylase activity produced by Rhizopus arrhizus fisher |
| Fig. 8: | Effect of starch concentration on α-amylase activity produced by Rhizopus arrhizus fisher |
Effect of time of heating: Enzyme activity gradually decreased with increase in the time of heating the enzyme at 70°C. The reaction mixture at 0 min exhibited the highest enzyme activity after which there was a gradual decline. The enzyme was completely denatured after 5 min of heating at 70°C (Fig. 9).
DISCUSSION
The result of this investigation revealed that the fungal strain of Rhizopus arrhizus fisher used for this research was capable of growth on flour medium with the production of α-amylase within ten days of incubation. α-amylase activity expressed as 933 units was optimum on the eighth day of inoculation with an optimum temperature of 30°C and pH 6.0.
| Fig. 9: | Effect of heat (70°C) on α-amylase activity produced by Rhizopus arrhizus fisher |
The enzyme activity was stimulated by NaCl and CaCl2 but inhibited by P-chloromercuric benzoate and ethylene diamine tetraacetic acid. According to Pandey et al. (2000), most amylases have been produced from soil fungi such as Aspergillus, Penicillum and Rhizopus. Suganthi et al. (2011) using A. niger reported that the amylase activity was as high as 22.5 U mg-1 on the substrate, gingely oil cake at four days of incubation. The specific activity was 311 U mg-1 for the enzyme at six days of incubation in groundnut oil cake substrate. The specific activity was 86 U mg-1 for the enzyme at five days of incubation in Black gram bran substrate. For six days of incubation the specific activity was 311 U mg-1 which was higher than in any other substrates. In 7 days of incubation in Rice bran the specific activity was 65 U mg-1 and for eight days of incubation in coconut oil cake the specific activity was 112 U mg-1. Aspergillus fumigates NTCC1222, showed high amylase activity (341.7 U mL-1) at 6 days incubation (Singh et al., 2014). Rani (2012) also supported the finding on optimum temperature of amylase enzyme to be 30°C. Maximum amylase activity was recorded at 30°C and further increase in temperature resulted in decrease in activity of amylase (Alva et al., 2007). The pH is one of the important factors that determine the growth and morphology of microorganisms as they are sensitive to the concentration of hydrogen ions present in the medium. The pH is known to affect the secretion of α-amylase and its stability (Fogarty, 1983). The effect of initial pH values in the range of 4-7.5 on enzyme production was investigated and initial pH medium of 6.2±0.1 resulted in enhanced α-amylase production (Esfahanibolandbalaie et al., 2008). The optimum pH for amylase production was 6.0 (Fig. 3) which is similar to the findings of Patel et al. (2005) who reported in A. oryzae. The biomass yield was found to be higher in case of pH 5.0 contradictory to the findings of Olama and Sabry (1989) where the amylase activity and the biomass yield was maximum at pH 7.0 in Aspergillus flavus and Penicillium purpurescence. Aspergillus oryzae, A. ficuum and A. niger were found to give significant yields of α-amylase between pH 5.0-6.0 (Bhimba et al., 2011). In this research, the effect of pH on the enzyme activity indicates that the amylase was optimally active in the pH range of 5.5-6.5 with its peak, at 6.0. This suggests that the enzyme would be useful in processes requiring an acidic pH condition.
From the results of the effect of metal ions on total α-amylase activity, it was deduced that NaCl and CaCl increased the total α-amylase activity in comparison with the control, confirming previous reports which indicated that amylases are mostly metalloenymes and require calcium and manganese ions for its activity, structural integrity and stability (Gangadharan et al., 2006; Varalakshmi et al., 2009; Michelin et al., 2010). Irshad et al. (2012) reported that CaCl2 was found to enhance the activity of α-amylase up to 92 U mL-1, while EDTA, TEMED compounds had inhibitory action to α-amylase activity with AgNO3 being the strongest inhibitor. Rani (2012) also confirmed that NaCl act as activators to enhance the amylase activity in a reaction medium. According to Chung et al. (1995), the enzyme activity of amylase from Thermococcus profundus DT5432 was slightly stimulated in the presence of dithiothreitol (DTT), β-mercaptoethanol and 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB), indicating that cysteine residue (s) do not take part in catalysis. Also, the enzyme activation by dithiothreitol (DTT) β-mercaptoethanol and 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB) could be attributed to the reduction in aggregate size by destroying the intermolecular disulfide linkages and/or by the protection of thiol groups that stabilize the three dimensional structure of enzyme (Khedher et al., 2008).