# Enzyme Technology Questions and Answers – Recent Advances – Stabilization of Enzymes in Biphasic Aqueous-Organic Systems

This set of Enzyme Technology Question Paper focuses on “Recent Advances – Stabilization of Enzymes in Biphasic Aqueous-Organic Systems”.

1. One major factor that must be addressed for the use of biphasic systems in many enzyme-catalyzed reactions is ____________
a) solvent polarity
b) inactivation of the enzyme
c) stability of the enzyme
d) partition coefficient

Explanation: One major factor that must be addressed for the use of biphasic systems in many enzyme-catalyzed reactions is stability of the enzyme. There should be a distinction made between the more hydrophilic enzymes and the more hydrophobic enzymes which is often associated with lipid and membranes. The stability of hydrophilic enzymes depends on the presence of a thin layer of water within the microenvironment. This amount of water is minimal and can be said that the enzyme is operating in an almost anhydrous state effectively. Hydrophobic lipases retain its activity even if fewer molecules of water are remaining, assuming that it is just sufficient to stabilize the conformation of the active site.

2. In biphasic systems, the limited amount of water available considerably reduces the rate of thermoinactivation.
a) True
b) False

Explanation: In biphasic systems, there is a pH of minute pools of water which contain no free hydrogen ions. Hence, it is impossible to measure or control the pH directly. But, the enzyme is said to ‘remember’ the pH of its last aqueous solution and functions as though it maintaining the same pH. If the enzyme-bound water is diluted by the use of the more water-soluble organic solvents, then the enzyme is usually inactivated. Under the conditions where this does not occur, the limited amount of water available, and the associated reduced water activity reduces the rate of thermoinactivation. Hence the above statement is true.

3. Due to organic phase in biphasic system, the most important factor in the balance between stabilization and inactivation is ______________
a) solvent polarity
b) inactivation of the enzyme
c) stability of the enzyme
d) partition coefficient

Explanation: Due to the organic phase in biphasic system, the most important factor in the balance between stabilization and inactivation is solvent polarity. Lower solvent polarity has lesser capacity to disrupt the structure of the necessary tightly bound water molecules. The best measure of solvent polarity is the logarithm of the partition coefficient. For the use of biphasic systems in many enzyme-catalyzed reactions, the important factor to be addressed is stability of the enzyme. Inactivation of enzyme may occur when the enzyme-bound water is diluted by the use of the more water-soluble organic solvents.

4. What does the following equation represent?
$$Log \,P=Log_{10} (\frac{[Material_{octanol}]}{[Material_{water}]})$$
a) Solvent polarity
b) Km
c) pH
d) Ionic strength

Explanation: Solvent polarity is the most important factor in the balance between stabilization and inactivation and is measured in terms of Log of partition co-efficient of the organic liquid between n-octanol and water. The equation is given by,
$$Log \,P=Log_{10} (\frac{[Material_{octanol}]}{[Material_{water}]})$$
The higher the Log P, the more hydrophobic is the solvent.

5. What is represented in the following diagram?

a) Effect of pH on activity of free enzyme
b) Dependence of the activity of immobilized enzymes in biphasic systems
c) Miachelis Menten plot
d) Bacterial growth curve

Explanation: The plot represents schematic diagram showing the dependence of the activity of immobilised enzymes in biphasic systems. It shows a clear correlation between the activity of biocatalysts in two-phase systems and the Log P as a S-shaped curve. This plot suggests that enzymes are generally inactivated by solvents with log P < 2, but are little affected by solvents with Log P > 4. There is some variation in the effects between different enzymes and different solvents which makes activity prediction rather imprecise between 2 – 4 of log P. The range 2 – 4 includes some of the most utilized organic phases which may be suitable for some and harmful in others. The solubility of the reactants and products may considerably reduce the range of log P that are available for a particular application because many basically nonpolar molecules possessing some polar structural regions cause lack of solubility in strongly hydrophobic solvents.

6. Which of the following factor is not important for making the choice of organic solvents in biphasic system?
a) Solvent polarity
b) Ease of recovery
c) Reproducible and linear
d) Specific inhibitory effects

Explanation: The choice of organic phase depends on the following factors
* Solvent polarity
* Cost
* ease of recovery
* fire and fume hazards
* specific inhibitory effects
The response should be accurate, precise, reproducible and linear over the useful analytical range, without dilution or concentration. It should also be free from electrical noise. This is a feature that a biosensor should possess.

7. Biphasic systems may be stabilized by the use of __________
a) deuterated water
b) sugar
c) co-factors
d) co-enzymes

Explanation: Biphasic systems may be stabilized by the use of deuterated water (D2O) as it reduces the rate of thermal inactivation. But it does cause an increase in the pKas of ionizing groups by about 0.4 with the associated changes in the pH-activity and pH-stability relationships. Though, the cost of deuterated water is high, the small amount necessary and the ease with which it may be recovered at the end of a catalytic process makes it useful to be used in biphasic systems.

8. S-shaped curve may be shifted to the left by immobilizing the enzyme within highly hydrophilic support.
a) True
b) False

Explanation: S-shaped curve showing dependence of the activity of immobilised enzymes may be shifted to the left by immobilizing the enzyme within a highly hydrophilic support. This may be achieved by impregnating a beaded hydrophilic polymer with the enzyme followed by suspension of the wet beads directly in the organic phase. These shifts are very important as they greatly increase the choice of suitable organic phase. Hence the above statement is true.

9. Which of the following is not an advantage of impregnated wet beads?
a) Protection of the contained enzyme
b) More facile recovery of the biocatalyst
c) Solvent polarity
d) Allowing efficient and continuous use of biphasic PBRs

* Protection of the contained enzyme at higher rates of stirring.
* Enabling more facile recovery of the biocatalyst.
* Allows to be used with low molecular weight hydrophilic coenzymes with the assistance of a coenzyme regenerating process.
* Allowing efficient and continuous use of biphasic PBRs as long as the moving organic phase is saturated with water.
Solvent polarity is one of the factors for the choice of the organic phase.

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