ANALYTICAL APPLICATIONS OF ENZYMES

All living organisms from bacteria to man are built and maintained by biological catalysts called
enzymes. Some enzymes have been evolved over millions of years to perform very specific
biochemical tasks. Some enzymes have been designed by nature to build chemical compounds
up, while others are responsible for either breaking them down or modifying them.
ANALYTICAL METHOD:
Analytical method means a technique used qualitatively or quantitatively to determine the
composition of a sample or a microbial contamination of a sample.
ANALYTICAL ADVANTAGES OF ENZYMES:
 Selectivity – Find target in complex mixtures.
 Sensitivity – Low detection limits in complex mixtures.
 Specificity – False negatives and false positives are rare.
 Safety – For shipping, storage, handling and disposal.
Properties and selection of suitable enzymes for industrial and analytical applications
Like all other proteins, enzymes are sensitive to environmental factors such as temperature, pH, and ionic
strength, and under certain conditions, an enzyme can even lose its catalytic activity permanently through
"precipitation” or "denaturation”. Also, all enzymes exhibit different biochemical characteristics, such as variable
affinities for their substrate (the "Km value”), variable speed of reaction (the "Vmax value”), variable pH
optima, variable cofactor requirements, and variable optimal ionic strengths. Enzymatic reactions can also
be significantly affected by "inhibitors”, such as heavy metal ions, or other biological compounds, that
manipulate the biochemical properties of an enzyme, and slow the reaction down. Sometimes this inhibition is
intended by Nature as a control mechanism for the activity of a particular enzyme, while other times this
phenomenon results from the simple fact that a particular com- pound just happens to fit into the active-site of
the enzyme in question. Thus, when developing industrial or analytical applications, after selecting an enzyme
simply based on the reaction that it performs, research scientists must then care- fully check that the enzyme
will be adequately stable under the conditions that it will be used in, and that in those conditions, catalysis is
still performed efficiently. Furthermore, a final challenge must be overcome for analytical enzymes, in that a
way must also be found to accurately quantify the products of the reaction, and it is for this reason that a
spectrophotometer must be employed.
Example 1: determination of ammonia (NH4+)

The quantitative determination of ammonia is very important in medical diagnostics, and food and
beverage analysis. For instance, in the wine industry accurate ammonia determination is critical in both
checking optimal levels of yeast available nitrogen (YAN) is present for efficient fermentation, yielding
good quality wine, and in the prevention of over-supplementation with diammonium phosphate (DAP),
 that can lead to the formation of ethyl carbamate.

The enzyme catalysed reaction in the case of ammonia determination actually leads to a reduction in
absorbance, rather than an increase. Only a single enzyme, glutamate dehydrogenase, is required to catalyse
the reaction (equation 1)

During the reaction, NADPH which absorbs light strongly, is converted to NADP +, that does not absorb
light, and thus the absorbance . In this case, the urease produces two molecules of ammonia (as
ammonium NH4 +) and this is taken into account in the equation when calculating the results falls during
the reaction, until the end- point is reached. 2-Oxoglutarate is an additional substrate required by the
enzyme and is present in excess levels, to ensure the reaction proceeds as rapidly as possible.

When both an ammonia and urea determination is required, such as in the wine industry, the ammonia test kit
reaction can be extended by the subsequent addition of urease, according to equation 2. Practically, this is achieved
by performing two reactions, i.e. after the first "ammonia” reaction has reached its end-point, a second reaction
is then initiated by the addition of urease, and a further reduction in absorbance occurs.
Example 2: determination of D-glucose and D-fructose

In the food and beverage industries, the quantification of D-glucose and D-fructose is very important, as these
sugars are present in many food ingredients, or are added in the form of high-fructose syrup sweeteners.
In the wine industry, D-glucose and D-fructose are the principle fermentable sugars utilised by the yeast,
and represent approximately 25 % of fresh grape juice by weight. After fermentation is complete, the residual
levels of these sugars are also determined prior to supplementation.
Similarly to the urea and ammonia example above, test kits for D-glucose and D-fructose are also designed
to independently measure both analytes in the same cuvette, however, in this case, it is the pro- ducts of the
reactions that absorb light, and thus absorbance increases are measured. Also, in the case of D-glucose and D-
fructose analysis, four chemical reactions are actually involved in the final reaction scheme as follows :
The first reaction involves the conversion of D-glucose and D-fructose into D-glucose-6-phosphate (G-6- P) and D-
fructose-6-phosphate (F- 6-P), respectively, by the enzyme hexokinase (equations 3 and 4).

In the next step after an initial absorbance reading has been taken, the enzyme glucose-6-
phosphate dehydrogenase is added to the cuvette, and G-6-P is quickly converted into
gluconate-6-phosphate. While at the same time NADP + that does not absorb light strongly
(Equation 5). During this reaction nothing happens to the F-6-P, as Glucose-6-phosphate
dehydrogenases, as it is absolutely specific only for G-6-P.
IMMOBILIZED ENZYME IN ANALYTICAL CHEMISTRY:
The high specificity towards a given substrate has resulted in enzymes being used in analytical
chemistry for several years. The gradual replacement of classical wet chemistry techniques by
enzyme-catalyzed reactions is particularly evident in the clinical laboratory. Furthermore, it is
sensitive to heat and microbial attack.
Immobilized enzymes is the possibility of changing the chemical properties of the enzyme
catalyst.
ENZYME ELECTRODES:
Immobilized enzymes were coupled to electrochemical sensor to yield an analytical device that
incorporated the enzyme reagent and the sensor in the same unit.
Immobilized urease is used for the measurement of the physiologically important substrate,
urea.
Both ion selective and gas electrode rely upon the enzyme catalyzed hydrolysis of urea.
Both electrodes function by diffusion of the substrate urea into the matrix of the immobilized
urease, the products of the reaction affecting the sensor response in relation to concentration.