The aim of this study was to investigate and understand factors which influence an activity of alcohol dehydrogenase (ADH). The factors included temperature, alcohol oxidation, and type and concentration of a substrate. The study attempted to determine an effect of oxidized ethanol reactions on initial ADH activity. Besides, the efficacy of ADH was examined under different temperatures, specifically the temperatures of 37, 50 and 70 °C were studied. It was predicted that there is a specific temperature at which ADH activity has its optimum level. Moreover, the effect of four substrates, namely, n-propanol, 2-propanol, butanol and ethanol, on ADH activity was carried out.
The Sigma-Aldrich commercial yeast ADH sample had a Km value of 2.1*10-2 M in ethanol. In the study with a purified ADH sample, recorded Km value was higher (1.00* 100) as compared with the standard (2.1*10-2 M) given alcohol as the substrate. The rationale might be due to low enzyme concentration of the purified sample compared with the standard (2.1*10-2 M). The initial velocity for the purified ADH sample was 1.6667E-8M (calculated) compared with the Sigma-Aldrich standard ADH sample of 2.00E-08M . The difference might be due to relatively low concentration of the ADH from the purified sample compared with the standard. Other studies concluded that the initial velocity for ADH was 1.9mmol/L (Neto, Forti, Zucolotto, Ciancaglini, & De Andrade, 2011); whereas, Ying et al. (2014) found the initial velocity of ADH as 1.44 mmol/L.
It was speculated that a certain substrate might have a greater effect on ADH activity than others might. Finally, Bradford assay was utilized to measure protein concentrations and to determine the ADH activity for different elution fractions. The study found out that change in temperatures, type of substrate and concentration of the substrate/enzyme determines the rate of ADH activity. ADH was more effective on ethanol substrate followed by 1-propanaol, butanol, and 2-propanol with the order of reducing activity. Besides, ADH activity had its optimum levels at 37°C (average body temperature). At 50°C, the enzyme activity was reduced, and at 70°C, it had almost no activity.
General objectives of this study were:
i. To determine the initial rate of alcohol dehydrogenase (ADH) as catalyzed through ethanol oxidation.
ii. To determine the impact of a temperature change on the ADH enzyme activity.
iii. To examine the influence of substrate on the ADH enzymatic activity; whereby, the substrates included ethanol, 2-propanol, n-propanol, and butanol.
iv. To determine the protein concentration and ADH activity in every elution fractions.
Materials and Methods
ADH Purification Protocol
The experiment included preparation of yeast cells for protein precipitation by use of polyethylene glycol (PEG). The isolated protein sample was then purified using the affinity chromatography. The efficacy of the purified ADH from yeast cells, Saccharomyces cerevisiae, was compared with the commercially (Sigma-Aldrich) available ADH with reference to the laboratory.
The Influence of Ethanol Concentration on ADH Activity
For the investigation of the influence of ethanol concentration, 11 blank tubes were prepared. Volume measurements and setup were done in accordance with the information contained in the lab manual. The spectrometer was set in the kinetics mode whereby wavelength was set at 340nm for a total time of 30 sec in 31 cycles, and the lag/rate was 0/30sec. However, ADH was not added at this point. The absorbance values of the blanks were measured and recorded. Then, at a time of zero (0), 0.1mL of ADH was added into the blanks followed by mixing, then absorbance values taken immediately (ΔA340nm/sec).
The Influence of Temperature Changes on ADH Enzymatic Activity
For the determination of temperature effect on ADH activity, different sets of ADH was incubated in various water baths set at 37, 50 and 70 °C. Two sets of three EtOH tubes were made for three temperature setups (37, 50 and 70 °C). However, ADH was not added at this point. The assay solutions were placed in the cuvette and the absorbance rates of the blanks were recorded. Then, 0.1mL of heated ADH (37, 50 and 70 °C) was added into respective tubes, mixture followed by absorbance readings (at ΔA340nm/sec).
The Substrate Activity Profile
Three blank tubes were filled as follows: tube 1 with ethanol, tube 2 with n-propanol, tube 3 with 2-propanol, and tube 4 with butanol. Three trials were prepared for each substrate. Then the solution was transferred into the respectively labeled cuvettes. Thereby, 0.1mL of ADH was added into blanks, mixed well followed by a recording of absorbance values at ΔA340nm/sec.
Bradford Assay (Protein Concentration)
Seven tubes were set according to the lab procedure. In three trials, the absorbance (595nm) of the bovine serum albumin (BSA) samples was determined. After this, the 3.0mL of Bradford reagent (Coomassie Brilliant Blue) were added to the BSA tubes and incubated for 10 minutes. Absorbance values were measured and recorded. Then, 200μL of the isolation buffer was added into the 100μL of the lysate supernatant. Subsequently, 100μL of the polyethylene glycol (PEG) was added to each solution. Then, the solution was added until the value of 300μL in each tube. After this, the elution fractions were diluted following the manual. The Bradford reagent (3mL) was then added to tubes E1 to E5 and incubated for 5minutes, then, 3.0mL of the Bradford reagent was added to the lysate and the PEG supernatant, then, incubated for 10 minutes. Absorbance values were then read (595nm) and recorded.
ADH Activity in Every Elution Fractions
The blanks were prepared for each elution fractions. All components, except the elution fraction solution, were added and blanked. The spectrometer was set at the kinetic mode (340nm 30sec). The solution (1μL) was then transferred to the cuvette and absorbance for blanks recorded. At zero time, 0.1mL of the elution fractions were added into the blanks, mixed and absorbance values (ΔA340nm/sec) taken.
All the collected elution fraction samples were active but varied in initial rates. The ADH were bind tightly with Cibacron Blue F3G-A, inside the affinity chromatography column. After the application of the elution buffer (contains NAD+ buffer), ADH from the elution fraction sample was slowly detached from Cibacron Blue F3G-A, and bonded to NAD+, because of the competition of ADH binding between Cibacron Blue F3G-A & NAD+. Each elution fraction was still containing some enzyme (ADH) (Uthoff & Steinbuchel, 2012). The Sigma-Aldrich referred NAD+ acted as a coenzyme to ADH. Protein eluted in all elution fractions, even in lysate supernatant and PEG precipitation supernatant.
The Lineweaver-Burk curve , demonstrated that the maximum reaction velocity (Vmax) for ADH was 5.00E-08; whereas, the Michaelis-Menten constant (Km) was 1.00E+00M. The low Km implied that ADH had a weak binding power to provided alcohol substrate. Comparatively, the Sigma-Aldrich commercial yeast ADH sample had a Km value of 2.1*10-2 M in ethanol and the purified ADH sample had a higher Km value (1.00* 100) compared with the standard (2.1*10-2 M) given alcohol as the substrate. The rationale might be due to low enzyme concentration of the purified sample compared with the standard. The purified ADH was found to have an initial velocity of 2.00*107.
The lysate supernatant had the highest protein concentration. However, it had the least concentration of ADH recovered since it contained a various amount of unbounded, inactive proteins, and enzyme. Moreover, the ADH concentration in lysate supernatant solution was not high since it did not undergo any enzyme purification processes.
E1 had the highest protein concentration, which contained only pure ADH (enzyme) inside of a solution. All the purified solutions had first eluted all the unbounded proteins by washing the column with the isolation buffer, and then eluted ADH off the column by adding the elution buffer. E5 contained the least protein concentration and had the second smallest initial rate of enzymatic activity.
The rate of an enzyme-catalyzed reaction depends on the concentrations of enzyme and substrate. The increase in the concentration of enzyme or substrate raises the rate of reaction increases, although, to a certain optimum level were the effect ceases. Second, Enzyme is very specific and works only with a certain substrate. As a result, the increasing substrate concentration level is going to raise the enzymatic reaction rate; moreover, ethanol is the optimal substrate molecules to perform the NAD+ reduction reaction. Third, enzyme activity is extremely temperature-sensitive; raising the temperature by 10ºC can greatly affect the overall enzymatic activity, and extreme heat will make most of the enzyme denatures and enzymatic activity will stop. The effect of four substrates, namely, n-propanol, 2-propanol, butanol and ethanol on ADH activity is carried out. ADH specificity tends to be limited to basic alcohols with straight-chain aliphatic groups; ethanol remains the most suitable substrate for ADH (Neto, et al., 2011).
ADH is most active at 37ºC since it has the largest initial rate, however, after raising the enzyme temperature to 70ºC, the initial enzyme rate is low and close to 0, which means most of the enzymes has denatured during the water bath process . Most enzymes perform at the optimum level of 37°C (Bisswanger, 2014).
The purified ADH sample recorded a higher Km value (1.00M) of compared with the provided Sigma-Aldrich standard AHD (2.1*10-2 M) given ethanol as the substrate. Higher Km values indicated a weaker enzyme binding power to its substrate and vice versa. The initial velocity for the purified ADH sample was 1.6667E-8M (calculated) compared with the Sigma-Aldrich standard ADH sample of 2.00E-08. The difference might occur due to relatively low concentration of the ADH from the purified sample compared with the standard. ADH demonstrated the highest rate of activity at 37°C, a moderate effect at 50°C, and almost no activity at 70°C. Higher temperatures seemed to denature ADH and hence inhibit its activity. Similarly, ADH was found to have had the greatest level of activity on ethanol substrate, followed by 1-propanol, butanol, and the least effect was on 2-propanol substrate with the order of reducing enzyme activity. ADH from different sources were both active in catalyzing reactions; however, differences in the rate of enzyme activity might occur due to differences in enzyme concentrations or availability of coenzyme NAD+ given similar substrate concentrations.