Estimation of the Optimum pH for Invertase

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Introduction

Invertase is a yeast-derived enzyme that is mainly used in the food (confections) industry where it helps prevent sugar crystallization in confections by hydrolysis of sucrose to yield glucose and fructose (http//www.invertase.net/00000.htm).

Sucrose + HO --- glucose + fructose

An enzyme is a catalytic protein that changes the rate of a reaction without itself being consumed by the reaction. Due to its proteinous nature, invertase is sensitive to its environment. In particular, changes in temperature and pH would affect rate of enzyme activity (Campbell & Reece 00). Under optimal pH and temperature, rate of hydrolysis of sucrose would be at its maximum. This would then increase the rate of industrial processes involving the enzyme.Custom writing service can write essays on Estimation of the Optimum pH for Invertase

The aim of this study was to estimate the optimum pH for invertase by observing the extent of reaction on sucrose when it is subjected to solutions of invertase maintained at different pH levels. The extent of reaction on sucrose could be measured qualitatively using the Benedicts test for reducing sugars.

Methods

1 millilitre (mL) each of invertase at pH , 4, 7, and 11 was drawn out of the conical flasks they were in by using a French pipette. This was then added to each of 5 test tubes labeled with the respective pH values. 1 mL of distilled water was added to a 6th test tube, which was used as a control for the experiment. 1 mL of sucrose solution at 50 g/mL was added to each of the 6 test tubes using the French pipette again. (Water was added to the control to ensure that the volume of solution in all test tubes, and hence, concentration of the sucrose solution, was constant throughout the 6 test tubes.) All 6 test tubes were simultaneously placed in a water bath at 7oC for 5 minutes. This allowed invertase to catalyze the hydrolysis of sucrose at its optimum temperature. The 6 test tubes were then taken out of the water bath. A drop of Benedicts solution was added to each of the 6 test tubes. The test tubes were shaken well to ensure homogeneity of the solution. The 6 test tubes were then placed in another water bath at 85oC for a further 5 minutes. Benedicts solution would only act on reducing sugars in the solution (if present) at such a temperature. The test tubes were then taken out of the water bath and observed for any precipitate formed in the solution and the colour of the precipitate, if it was formed. The entire procedure was repeated twice, to obtain accuracy for the experiment.

Results

Benedicts solution remained blue in all of the test tubes with the exception of the test tube containing invertase at pH 4. In that test tube, orange precipitate was formed. This indicated that reducing sugars, in this case glucose and fructose, were present only in the test tube containing invertase at pH 4. This indicates that pH 4 is the optimal pH for invertase amongst the pH levels given.

Table 1. Results of the sucrose hydrolysis test when invertase was subjected to a range of pH

pH of buffered solution containing invertaseBenedicts solutionPresence of Reducing Sugars

Remained blueNone

4Orange precipitate formedIn moderate quantity

7Remained blueNone

Remained blueNone

11Remained blueNone

ControlRemained blueNone

The results were similar for the replicates.

Conclusion

From the pH values tested, the optimal pH for invertase would be at pH 4. Benedicts solution registered a positive test at pH 4, i.e. precipitate was formed in the solution. This indicates that invertase had catalyzed the hydrolysis of sucrose to fructose and glucose, both of which are reducing sugars.

(Benedicts solution tests for free aldehydes and ketones, either of which can be found in reducing sugars. Disaccharides like sucrose do not have free aldehydes or ketones and hence would not form precipitates in Benedicts solution.) (Richard M. McCourt)

At pH , 4, 7, , 11, Benedicts solution remained blue. This indicates that invertase had not catalyzed the hydrolysis of sucrose to fructose and glucose.

Changes to the pH of the environment affect the ionization of an enzymes amino acid side chains. At pH , the environment was too acidic for invertase to function. Additional H+ reacted with the carboxyl groups of the amino acids of the protein. This disrupted the ionic bonding of the protein at the tertiary structure, causing the enzyme to unfold. At pH 7, and 11, the environment was too alkaline for invertase to function. Additional OH- reacted with the amino groups of the amino acids of the protein. This too disrupted the ionic bonding at the tertiary structure, causing the enzyme to denature. (Campbell & Reece 00)

This experiment did not attain its objective in estimating the optimal pH in which invertase functions. Of the 5 pH values tested, only 1 resulted in a change in Benedicts solution, i.e. invertase catalyzed the hydrolysis of sucrose in only 1 of the 5 test tubes. This is insufficient to show or even estimate what the optimal pH for invertase is. As such, more values between the ranges of pH to pH 7 should be taken.

(add graph for rxn vs pH. Explain range.)

It should be noted that the precipitate obtained in test tube containing invertase at pH 4 was orange. Benedicts solution turns brick red when the percentage of reducing sugars in the solution is very high. This further suggests that pH 4 is not the optimal pH for invertase and that the optimal pH is slightly higher.

In fact, contrary to most other enzymes, invertase exhibits relatively high activity over a broad range of pH (.5--5.5), with the optimum near pH=4.5. (http//www.glue.umd.edu/~nsw/ench485/lab14.htm) This correlates with the results of this experiment, where invertase catalyzed the hydrolysis of sucrose at pH 4 (within the range) while at pH , 7, and 11 (outside of range), catalysis did not seem to occur.

A colorimeter could be used to increase the accuracy of the experiment. Since this experiment essentially estimates the rate of reaction of invertase-catalyzed hydrolysis by means of the Benedicts test, much hinges on the colour difference in precipitates formed in the Benedicts solution. This is subjective and it might be hard to differentiate between shades of orange precipitates, for instance. A colorimeter would be able to differentiate the shades of a colour more objectively.

References

CAMPBELL, N.A. and REECE, J.B. 00, Biology, 6th Ed, Benjamin Cummings

McCOURT, R.M. 188, Lab Manual to Accompany Biology, 1st Ed, Random House Inc

NAM, S.W., Experiment No. 14 Enzyme Kinetics of Invertase via Initial Rate Determination. Retrieved April 4th, 00 from University of Maryland website

http//www.glue.umd.edu/~nsw/ench485/lab14.htm

Invertase 00 000. Retrieved April 4th, 00 from http//www.invertase.net/00000.htm

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