Scientific paper on amylase enzyme use for laundry detergent
α-Amylase from A. oryzae more effective than B. subtilis at laundry temperatures.
1. INTRODUCTION
1. INTRODUCTION
Laundry detergent is a common household product that many people use daily. Unfortunately, there are some stubborn types of molecules that detergents can have difficulty eliminating alone. Washing clothes at a higher temperature can improve detergent effectiveness, but can also damage the clothes. Most washing machines run their cold cycle below 30°C, and their warm cycle between 30°C -40°C (Textile Industry Affairs 2010). Hot washes can vary between 40°C-95°C.
Detergents mostly work by using surfactants: molecules with both hydrophobic and hydrophilic parts. The hydrophobic parts of these surfactants bind to other hydrophobic stain molecules such as fats, sugars, and proteins. The hydrophilic parts stay on the outside of the stain molecule where it can bind with water, creating a sphere of water around the stain molecule and lifting it free of the clothing to be washed away with the dirty water.
Biofilms are sticky colonies of microorganisms that adhere to surfaces. Bacteria, fungi and protists can create biofilms by excreting extracellular polymeric substances (EPS) to produce the biofilm extracellular matrix. EPS include carbohydrates, proteins, and nucleic acids. The biofilm matrix not only adheres these colonies to surfaces, but also protects the microorganisms within by acting as a barrier against drugs or chemicals. This quality makes biofilms especially hard to clean effectively and therefore potentially dangerous to human health, as many types of bacteria living in these biofilms cause sicknesses. This is especially an issue in hospital laundries.
Enzymes are specific types of proteins that work as catalysts for chemical reactions, and are commonly used in many types of industrial applications. In laundry detergent they improve effectiveness in breaking down certain types of stains, while still being safe for the environment (Bhange et al. 2016). Common enzymes that are used in detergent include protease (for proteins such as blood), lipase (for fats such as grease), and amylase (for carbohydrates such as starch).
α-Amylase is a common type of glycoside hydrolase, which works by breaking apart linkages in large polysaccharides (such as starch), thereby breaking them down into smaller molecules (such as glucose) (Dey and Banerjee 2015; Maalej et al. 2013). For amylase to be used in laundry detergent, it must be effective at a broad range of alkaline pH and temperatures (Hammami et al. 2017; Maalej et al. 2013; Nasri et al. 2015; Vojcic et al. 2015). The characteristics of amylase depends upon its specific source, as natural environments differ in many variables (Dey and Banerjee 2015). For this reason, we tested amylase from two different sources, Bacillus subtilis (bacterial) and Aspergillus oryzae (fungal), to see which would perform better at varying temperatures.
The average optimum pH and temperature for B. subtilis (depending on the specific strain) is between 6-10, and 37°C-135°C, respectively (Desouza et al. 2010). For A. oryzae, the average optimum pH varies between 5-9, and the optimum temperature varies between 25°C-65°C (Desouza et al. 2010; Dey and Banerjee 2015). Since B. subtilis has a wider overall range of optimum temperatures, and higher average optimum pH, it seems logical that it would perform well in laundry detergent. Therefore, we hypothesize that B. subtilis will perform more effectively in breaking down starch than A. oryzae at a range of common washer temperatures.
2. MATERIALS AND METHODS
2.1 Materials
The A. oryzae amylase was from Carolina Biological Supply, and the food grade B. subtilis amylase was from Brewcraft, USA. The 6.8pH buffer solution was made from dibasic and monobasic potassium phosphate (reagent grade, Fisher Scientific). Lugol’s iodine was lab grade and from Ward’s Science. The starch solution was made using soluble potato starch powder from ChemProducts.
2.2 Enzyme Concentration
Five test tubes were filled with 5 mL of distilled water. Into the first test tube was added 5 mL of 1% amylase solution from A. oryzae. This solution was mixed, and 5 mL of the solution from the first test tube was transferred to the second test tube, thereby becoming diluted. This process was repeated for all 5 test tubes, wherein the previous test tube donated 5mL to the next test tube. Therefore the last test tube had a final concentration of 0.031% amylase. Then this entire process was repeated with five fresh test tubes using the amylase from B. subtilis, so the final concentrations of both amylase solutions were the same.
2.3 Temperature Experiments
Six test tubes were labeled 1 through 6, and 2 mL of 1% starch solution was pipetted into each. Then 4 mL of distilled water was added to each tube. A disposable pipette was used to add 1 mL of 6.8 pH buffer to each tube. Tubes 1 and 4 were then placed in a 55°C water bath, tubes 2 and 5 were placed in a 37°C water bath, and tubes 3 and 6 were left in a test-tube rack at room temperature (22°C).
A second set of six test tubes were labeled 1A through 3A, and 4B-6B. Disposable pipettes were used to add 1 mL of A. oryzae 0.031% amylase solution to test tubes 1A-3A, and 1 mL of B. subtilis 0.031% amylase solution to test tubes 4B-6B. As with the first set, tubes 1A and 4B were then placed in a 55°C water bath, tubes 2A and 5B were placed in a 37°C water bath, and tubes 3A and 6B were left in a test-tube rack at room temperature (22°C).
All test tubes (2 starch and 2 amylase per temperature area) were left for over 10 minutes in order to reach temperature, while two test plates were prepared with 1 drop of I2Kl solution per compartment.
After about 10 minutes, test tubes 1 and 1A were then mixed together and a timer was started. Every 20 seconds, 1 drop of the mixture was added to a test plate compartment containing I2Kl, until a blue color was no longer produced and the I2Kl solution remained yellow-amber (indicating all the starch was digested). If within 15 minutes there was no color change, the experiment with that particular reaction mixture was terminated. This process was then repeated 5 more times (with tubes 2 and 2A, 3 and 3A, 4 and 4B, etc.).
It is important to note that with the final testing of 5 and 5B, since we expected it would take longer than 9 minutes 20 seconds, we did not start adding drops of the mixture to I2Kl until after 8 minutes had elapsed. This decision was made due to having limited resources of I2Kl.
3. RESULTS
Tube
|
Temp.
(°C)
|
Time of Starch Disappearance (in minutes)
|
|
A. oryzae
|
1&1A
|
55°
|
0:30
|
2&2A
|
37°
|
0:40
|
|
3&3A
|
22°
|
4:50
|
|
B. subtilis
|
4&4B
|
55°
|
9:20
|
5&5B
|
37°
|
>15 minutes
|
|
6&6B
|
22°
|
>15 minutes
|
Table 1: Time of starch disappearance using 0.0031% amylase Figure 1: Time of starch disappearance compared to temperature
4. DISCUSSION
Laundry machines run cycles at varying temperatures, from cold to hot wash and rinse cycles. Therefore it is important to take temperature into account when determining which amylase would work best as a detergent additive. Our room temperature (22°C) results were used to approximate cool wash conditions, our 37°C water bath was used to approximate warm wash conditions, and our 55°C water bath was used to approximate hot wash conditions in home laundry machines.
Although amylase from different strains of B. subtilis have been found to have a wide range of optimum temperatures, it seems to have poor performance at low temperatures. This is supported by the range of active temperatures found to be 37°C-135°C in previous studies (Desouza et al. 2010). It can therefore be hypothesized that amylase from B. subtilis would perform better at higher temperatures than those tested under the conditions in this experiment.
The amylase from A. oryzae was found to perform much more efficiently at the conditions tested than the enzyme from B. subtilis. This contradicts our hypothesis that B. subtilis amylase would break down starch the fastest. However, it is not altogether surprising considering other research has found A. oryzae amylase has optimum temperatures that vary between 25°C-65°C (Desouza et al. 2010, Dey and Banerjee 2015). It certainly performed well at low temperatures (22°C to 37°C) in this experiment. However it should be noted that there was a significant difference between 37°C and 55°C, so it would be worth comparing the two amylases at higher temperatures as well.
It is important to note that due to time and materials restraints, we were unable to repeat the experiment to confirm our results. It would be beneficial to repeat this experiment with an additional high temperature to extrapolate more extreme washing conditions, such as hospital hot wash conditions (around 90°C).
In conclusion, amylase from A. oryzae was much more efficient than amylase from B. subtilis in breaking down starch within the temperature range of 22°C to 55°C. However, pH is also important for laundry detergent additives, and since B. subtilis has been tested to be active at a wider range of alkaline pHs (6.0-10.0) than A. oryzae (5-9), it would be worth testing the combined effects of pH and temperature to find the best amylase for use as a detergent additive (Bhange et al. 2016; Desouza et al. 2010; Dey and Banerjee 2015).
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