The Effects of Temperature and pH on the Activity of Fungal Amylase

The Effects of Temperature and pH on the Activity of Fungal Amylase

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1. Introduction

Amylases are a family of enzymes that break down starch into polymers that consists of glucose units (Naidu and Saranraj, 2013). The enzymes can be classified into three categories:  β-amylase, α-amylase, and γ-amylase. The first category, β-amylase, is formed from plants and microbes – fungi and bacteria. It speeds up the hydrolysis of the second -1, 4 glycosidic bonds from the non-reducing end. The second type, α-amylase, works only in the presence of calcium and breaks down amylose into maltose and maltotriose and amylopectin into “limit dextrin” and glucose. The last class, γ-amylase cleaves (1-6) glycosidic bonds and it optimally works in acidic environments with a pH of 3.0 (Naidu and Saranraj, 2013). For this experiment, α-amylase was used with an optimum pH and temperature of 6.7–7.0 and 63–70 °C, respectively. This paper investigates if temperature and pH affect the activity of fungal amylase. In the experimental group, crude amylase enzyme was subjected to varying temperature and pH while in the control group, the enzyme was subjected to a constant pH and temperature. The temperature and pH, in this case, were independent variables while enzyme activity was a depended variable.

2. Materials and Equipment

• NaOH
• HCL
• Thermometer
• Test tubes
• A cell-free supernatant
• Soluble starch
• Phosphate buffer ( pH 7 )
• Incubator
• DNS reagent
• Spectrophotometer

3. Methods
4. Enzyme Assay

At the beginning of the experiment, 1.0 mL of crude enzyme from a cell-free supernatant and 1mL of 1% soluble starch in phosphate buffer (pH 7) was put in a test tube. The tubes were enclosed and kept warm for 10 minutes at 30°C.  To stop the reaction, 1.0 mL DNS reagent was added in every tube to impede the reaction and reserved in a hot water bath for 10 minutes. The tubes were then cooled at room temperature, and the final volume made to 10 mL with distilled water. Reading of the absorbance by a spectrometer at 540 nm was done. The standard curve was used to determine the amylase activity. A unit of the activity of the enzyme was classified as the quantity of enzyme that released 1 μmol of glucose for every minute.

1. Effect of pH

The crude amylase enzyme provided by a cell-free supernatant was added to a soluble starch solution and subjected to varying pH from 4 to 9.  1N HCL and 1 N NaOH were used to alter the pH.

1. Effect of Temperature

The crude amylase enzyme provided by a cell-free supernatant was added to a soluble starch solution and subjected to varying temperature. The temperature was changed for three days as follows: 0°C, 25°C, 37°C, 45°C, 67°C and 85°C

4. Results
5. Formulae Used

A unit of the activity of the enzyme was classified as the quantity of enzyme that released 1 μmol of glucose for every minute.

1. Tabulated Data
2. Effect of Temperature

1. Effect of pH

• Maltose Standard Curve Data

1. Graphs
2. Maltose Standard Curve
3. Temperature vs Enzyme Activity

• pH and Enzyme Activity

1. Summary of Results

• An increase in maltose concentration increased the activity of fungal amylase, as shown in the first graph.
• An increase in temperature increased the activity of fungal amylase up to 45⁰ Beyond 45⁰ C, the activity of the enzyme began to decrease.
• An increase in pH increased the activity of fungal amylase up to 5. Beyond a pH of 5, the enzyme activity began to reduce.

5. Conclusions

The results indicate that temperature and pH have a significant effect on the activity of fungal amylase. The enzyme activity increases with an increase in pH and temperature up to optimum levels. Beyond the optimum level, the enzyme activity decreases. It is, however, essential to note that the optimum levels are below the expected values due to errors that might have occurred during the extraction and preparation of the enzyme. The amount of the reagents used could as well account for the given errors.

References

Alva, S., Anupama, J., Savla, J., Chiu, Y. Y., Vyshali, P., Shruti, M., … & Kumudini, B. S. (2007). Production and characterization of fungal amylase enzyme isolated from Aspergillus sp. JGI 12 in robust state culture. African Journal of Biotechnology, 6(5), 576.

Kundu, A. K., & Das, S. (1970). Production of amylase in liquid culture by a strain of Aspergillus oryzae. Appl. Environ. Microbial., 19(4), 598-603.

Naidu, M. A., & Saranraj, P. (2013). Bacterial amylase: a review. Int J Pharm Biol Arch, 4(2), 274-287.

Saranraj, P., & Stella, D. (2013). Fungal amylase—a review. Int J Microbial Res, 4(2), 203-211.