Enzyme Kinetics of ADH and Purification and Activity of ADH lab report

This lab study is focused on two experimental experiments: alcohol dehydrogenase enzyme kinetics and purification activities of purified alcohol dehydrogenase. The goals of these two experiment protocols are not the same. The aim of protocol four of the experiment is to ascertain the degree to which ethanol concentration influences ADH behaviors as well as to investigate the kinetic constants (Zanon, Peres, and Gattás, 2015). The experiment in Protocol 5 was designed to assess the concentration of pure enzymes as well as to assess the actions of the purified enzymes. There were two sources of ADH; one is the purchased enzymes from Sigma Aldrich, a biotechnology corporation that deals with biochemistry chemicals and other materials. The second source was from the baker’s yeast(Zanon, Peres and Gattás, 2015). ADH from yeast extracted through fermentation process using various chemical processes.

To determine the kinetic parameters for ADH, an experiment was set up by first turning spectrometer on(Zanon, Peres and Gattás, 2015). Data was collected in kinetic mode. Assay solution was then transferred into cuvette. The blank rate of reaction was determined by observing the change absorbance at 340nm vs time. At zero seconds, 0.1 ml of ADH enzyme was then added to the cuvette so as to initiate the reaction.

Enzyme

Parameters

Km (mmol L−1)

Vmax (μmol NADH min−1 mg−1)

Kcat (s−1)

Kcat/Km (M−1 s−1)

ADH

Soluble

18.2 ± 0.1

69.4 ± 0.1

172.6 ± 0.1

9500 ± 10

Immobilized

17.9 ± 0.1

0.45 ± 0.01

1.07 ± 0.02

59 ± 1

AldDH

Soluble

17 × 10−3 ± 1

24.1 ± 0.2

80.3 ± 0.2

4.7 × 106 ± 2

Immobilized

16 × 10−3 ± 1

0.13 ± 0.001

0.43 ± 0.001

26.8 × 103 ± 0.1

Results

Michaelis-Menten plot (initial rate (Δ[NADH]/sec) vs [CH3CH2OH])

Tube 

ΔA 340 nm/min

1

0.0170

2

0.0389

3

0.0257

4

0.0471

5

0.1506

6

0.1838

7

0.2182

8

0.2780

9

0.2941

10

0.3914

11

0.4616

Initial Velocity 0.0170s 0.0257s 0.038s 0.047s 0.15s 0.18 s 0.21s

0.27s 0.2941 s

0.006

0.3914s

0.4616s

Time

The graphs show the amount of the product formed at different substrate concentration. It is usually plotted as a function of time.

Blank: 0.0006 ΔA/min

Tempature of ADH

Trial 1

Trial 2

Trial 3

37 oC

0.1831

0.1527

0.1874

50oC

0.0795

0.0819

0.0903

70 oC

0.0006

- 0.0028

- 0.0024

Substrate activity profile 

Tube

Substrates

Blank

Trial 1

Trial 2

Trial 3

2

Ethanol

-0.009

0.1896

0.2136

0.2275

3

n-propanol

-0.0209

0.0359

0.0086

0.0395

4

2-propanol

0.0004

0.0016

0.0009

- 0.0005

5

butanol

- 0.0013

0.0080

0.0076

0.0061

Discussion

The kinetic results that were obtained from the process showed that the enzymes displayed their enzymatic activity only in cycle one. That is to say that the enzymes did not have any significant activity after their first kinetic assay. This can be attributed to the presence dendrimers that was combined with enzymes and NAD+ species. This might have limited the diffusion process and thus hindering the flow of reduced form of coenzymes from dendrimers into the bulk solution.

ADH kinetic behavior

Before the performance of the experiment, the influence of the ADH on the kinetic of the enzymes was evaluated. The result showed that there is the linear influence of ADH on the reaction rate.

Protocol 5

Collected 5 fractions of protein. Each fraction was diluted and measured absorbance

ADH fractions collected

Absorbance

Dilution factor

Fraction 1

0.033

1:2

Fraction 2

0.044

1:2

Fraction 3

0.041

1:2

Fraction 4

0.017

1:2

Fraction 5

0.007

1:2

Lysate

0.481

1:100

Lysate after adding PEG

0.056

1:50

Activity assay

Blank: -0.0016 ΔA/min

Tube

ΔA/min

1 ( containing fraction 1)

-0.0020

2

0.0315

3

0.1059

4

0.0023

5

-0.0003

6 ( containing lysate)

1.0698

7 ( containing PEG)

0.1152

Discussion

Purified enzymes preparations are considered to be homogeneous as tested in the experiment in protocol five by PAGE and gel filtration.

Fraction one and two are the most active fraction this is because they have high Absorbance factor as compared to the rest (Morgan and Moir, 1996). Proteins do not elute in a single fraction as shown in the table above.

For each fraction tested, the one with the highest protein is fraction two which also have the highest absorbance factor whereas the fraction with the least protein concentration is fraction five with the absorbance of 0.007. Also, the fraction with the highest protein concentration was the most active as observed from the experiment.

The initial rates obtained from enzymes that were purchased from Sigma Aldrich was not comparable to the one obtained from the purified yeast which was seen to have the highest initial rates.

Conclusion

From the experiment, it was expected that ADH could be purified using affinity chromatography and also the concentration of the purified enzymes was expected to be high on the fractions that most active proteins.

References

Morgan, C., & Moir, N. (1996). Rapid microscale isolation and purification of yeast alcohol dehydrogenase using Cibacron Blue Affinity chromatography. J. Chem. Educ, 73(11), 1040.

Zanon, J. P., Peres, M. F. S., & Gattás, E. A. L. Colorimetric assay of ethanol using alcohol dehydrogenase from dry baker’s yeast. 2015. Enz. Microbial Technol, 69, 1-5.