Citation
Seow, Teck Keong
(1992)
Isolation and Characterisation of Cocoa Protease.
Masters thesis, Universiti Pertanian Malaysia.
Abstract
This project was initiated with the intent ion of isolating
and characterising protease from cocoa beans,Theobroma cacao
Linneaus because a greater knowledge of the proteases present in cocoa beans would lead to a better understanding of the
problem of inferior cocoa flavour in Malaysian cocoa beans .
The ammonium sulphate fractional precipitation method was used
to isolate the cocoa protease while the partial purification of
the enzyme was achieved by gel filtrat ion through Sephadex G-200. Four fractions precipitated with 0-20%, 20-40%, 40-60% and 60-80% saturations of ammonium sulphate were found to
be proteo lytically active against casein . Further studies were
conducted on the fractions precipitated with a -20% and 20-40% saturations of ammonium sulphate.Studies on the isolat ion
procedure showed that the add it ion of sodiumdodecyl sulphate
(SDS )or Triton X-lOa detergents did not enhance the efficiency of the isolation process. Temperature studies showed that both
the 0-20% and 20- 40 % fractions have temperature optima of
45-50°C but were unstable at those temperatures. Both
fractions possess more than one pH optima against both case in
and bovine serum albumin (BSA). The pH optima are in the
strong acidic pH and strong alkaline pH ranges. Inhibit or
studies showed that both the 0-20% and 20-40% fractions are
likely to contain cysteine proteases while not ruling out the
presence of a spartic proteases. The passage of the 0-20%
fraction through Sephadex G-200 produced two proteolytically
active protein peaks designated as Pl and P2 while that of the
20-40% fraction produced five activity peaks which were not
well separated. Studies with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SOS-PAGE) showed that the
0-20% fraction was relatively pure and that the Pl peak was
likely to be a protein aggregate. The characteristics of the
proteases in both fractions strongly indicate that these enzymes do play a role in the production of cocoa flavour and
its precursors during cocoa fermentation.
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