2009_Book_FoodChemistry

96 2 Enzymes
2.2.4 Isolation and Purification
Most of the enzyme properties are clearly and reliably
revealed only with purified enzymes. As
noted under enzyme isolation, prerequisites for
the isolation of a pure enzyme are selected protein
chemical separation methods carried out at 0–
4 ◦ C since enzymes are often not stable at higher
temperatures.
Tissue Disintegration and Extraction. Disintegration
and homogenization of biological tissue requires
special precautions: procedures should be
designed to rupture the majority of the cells in
order to release their contents so that they become
accessible for extraction. The tissue is usually
homogenized in the presence of an extraction
buffer which often contains an ingredient to protect
the enzymes from oxidation and from traces
of heavy metal ions. Particular difficulty is encountered
during the isolation of enzymes which
are bound tenaciously to membranes which are
not readily solubilized. Extraction in the presence
of tensides may help to isolate such enzymes. As
a rule, large amounts of tissue have to be homogenized
because the enzyme content in proportion
to the total protein isolated is low and is usually
further diminished by the additional purification
of the crude enzyme isolate (cf. example in Table
2.3).
Enzyme Purification. Removal of protein impurities,
usually by a stepwise process, is essentially
the main approach in enzyme purification. As
a first step, fractional precipitation, e. g. by
ammonium sulfate saturation, is often used or the
extracted proteins are fractionated by molecular
weight e. g., column gel chromatography.
The fractions containing the desired enzyme
activity are collected and purified further, e. g.,
by ion-exchange chromatography. Additional
options are also available, such as various forms
of preparative electrophoresis, e. g. disc gel
electrophoresis or isoelectric focusing. The
purification procedure can be substantially shortened
by using affinity column chromatography.
In this case, the column is packed with a stationary
phase to which is attached the substrate or
a specific inhibitor of the enzyme. The enzyme is
then selectively and reversibly bound and, thus,
in contrast to the other inert proteins, its elution
is delayed.
Control of Purity. Previously, the complete removal
of protein impurities was confirmed by
crystallization of the enzyme. This “proof” of pu-
Table 2.3. Isolation of a glucosidase from beans (Phaseolus vidissimus)
No. Isolation step Protein α-Glucosidase
(mg) Activity (µcat) Specific activity Enrichment Yield (%)
(µcat/mg) (-fold)
1. Extraction with 0.01 mol/L
acetate buffer of pH 5.3
2. Saturation to 90% with 44,200 3840 0.087 1 100
ammonium sulfate followed
by solubilization in buffer
of step 1
3. Precipitation with 7610 3590 0.47 5.4 93
polyethylene glycol (20%).
Precipitate is then solubilized
in 0.025 mol/L Tris-HCl buffer
of pH 7.4
4. Chromatography on DEAE- 1980 1650 0.83 9.5 43
cellulose column, an
anion exchanger
5. Chromatography on 130 845 6.5 75 22
SP-Sephadex C-50,
a cation exchanger
6. Preparative isoelectric focusing 30 565 18.8 216 15