2009_Book_FoodChemistry

138 2 Enzymes
2.6.1 Substrate Determination
2.6.1.1 Principles
Qualitative and quantitative analysis of food constituents
using enzymes can be rapid, highly sensitive,
selective and accurate (examples in Table
2.16). Prior purification and separation steps,
as a rule, are not necessary in the enzymatic analysis
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:
Fig. 2.42. Enzymatic determination of glucose, saccharose
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) and β-fructosidase
(β-F)
(2.105)
(2.106)
Reactant A is the food constituent which is being
analyzed. C or R or S is measured. The equlibrium
state of the coupled indicator reaction is concentration
dependent. The reaction has to be adjusted
in some way in order to remove, for example,
P from the auxiliary reaction before an equilibrium
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 analysis
of glucose, lactose and saccharose (cf. Reaction
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-
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-Point Method
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