138 2 Enzymes
2.6.1 SubstrateDetermination..................................
2.6.1.1 Principles..............................................
Qualitative and quantitative analysis of food con-
stituents using enzymes can be rapid, highly sen-
sitive, selective and accurate (examples in Ta-
ble 2.16). Prior purification and separation steps,
as a rule, are not necessary in the enzymatic ana-
lysis of food.
In an enzymatic assay, spectrophotometric or
electrochemical determination of the reactant or
the product is the preferred approach. When this
is not applicable, the determination is performed
by a coupled enzyme assay. The coupled reaction
includes an auxiliary reaction in which the food
constituent is the reactant to be converted to
product, and an indicator reaction which involves
an indicator enzyme and its reactant or product,
the formation or breakdown of which can be
readily followed analytically. In most cases, the
indicator reaction follows the auxiliary reaction:
(2.105)(2.106)Reactant A is the food constituent which is being
analyzed. C or R or S is measured. The equlib-
rium state of the coupledindicator reaction is con-
centration dependent. The reaction has to be ad-
justed in some way in order to remove, for exam-
ple, P from the auxiliary reaction before an equi-
librium is achieved. By using several sequential
auxiliary reactions with one indicator reaction, it
is possible to simultaneously determine several
constituents in one assay. An example is the anal-
ysis of glucose, lactose and saccharose (cf. Reac-
tion 2.105).
First, glucose is phosphorylated with ATP
in an auxiliary reaction (a). The product,
glucose-6-phosphate, is the substrate of the
NADP-dependent indicator reaction (b). Add-
Fig. 2.42.Enzymatic determination of glucose, saccha-
rose and lactose in one run. After adding cosubstrates,
ATP and NADP, the enzymes are added in the order:
hexokinase (HK), glucose-6-phosphate dehydrogenase
(G6P-DH),β-galactosidase (β-Ga) andβ-fructosidase
(β-F)ition of β-galactosidase starts the lactose
analysis (c) in which the released glucose, after
phosphorylation, is again measured through
the indicator reaction [(b) of Reaction 2.105
and also Fig. 2.42]. Finally, after addition of
β-fructosidase, saccharose is cleaved (d) and
the released glucose is again measured through
reactions (a) and (b) as illustrated in Fig. 2.42.2.6.1.2 End-PointMethod.......................................
This procedure is reliable when the reaction
proceeds virtually to completion. If the substrate
is only partly consumed, the equilibrium is
displaced in favor of the products by increasing
the concentration of reactant or by removing
one of the products of the reaction. If it is not
possible to achieve this, a standard curve must