Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

276 Appendix 3
Preparation of orange peel extract
Peel the outer layer (the ‘zest’) of one or two oranges using a knife. Add the peel
to 50–60 ml of 50 mm sodium citrate buVer (pH 5.5) containing 2.3% NaCl, and
homogenise in a blender for 2 min until homogeneous. If you have access to a
centrifuge, then spin at 12 000 g for 10 min, and decant the clear orange supernatant
into a beaker for use in the enzyme assays. The extract is stable for at
least 24 h if kept on ice.
Assays for enzyme-catalysed hydrolysis
Devise a suitable thin-layer chromatography system to separate the diester (4)
from hydroxy-ester (2) and acylated acid (3). Typically 1:1 ethyl acetate/petroleum
ether (60–80 8 fraction); dichloromethane; or dichloromethane/10% methanol
are useful eluents. Dissolve 0.1 g of your diester (4) in 5 ml acetone, and set
up the following incubations in screw-topped vials:
(a) 0.1 ml diester (4) solution þ 0.9 ml 50 mm sodium citrate buVer (pH 5.5);
(b) 0.1 ml diester (4) solution þ 0.9 ml orange peel extract;
(c) 0.1 ml diester (4) solution þ 0.9 ml pig liver esterase stock (1.0 unit ml
1 in
50 mm potassium phosphate buVer (pH 7.0) );
(d) 0.1 ml diester (4) solution þ 0.9 ml porcine pancreatic lipase stock (1.0 unit
ml
1 in 50 mm potassium phosphate buVer (pH 7.0) )
Analyse the four incubations by thin-layer chromatography at 30 min, 1 h,
2 h and 24 h time points, and you should see enzymatic hydrolysis to (2) or (3).
In all cases that we have examined some enzymatic hydrolysis was observed
with at least one of the three enzymes, and in nearly all cases the enzymatic
hydrolysis was speciWc for production of either hydroxy-ester (2) or acyl
acid (3).
Further reading
T.D.H. Bugg, A.M. Lewin & E.R. Catlin (1997) RegiospeciWc ester hydrolysis by orange
peel esterase – an undergraduate experiment. J. Chem. Educ., 74, 105–7.
A.I. Vogel (1989) Vogel’s Textbook of Practical Organic Chemistry, 5th edn. Longman,
Harlow.