Isolation, Partial Purification and Characterization of a Protease from Ginger Zingiberaceae officinale Roscoe
Isolation, Partial Purification and Characterization of a Protease from Ginger Zingiberaceae officinale Roscoe
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Date
2011-07
Authors
BASSA, OBED YAKUBU
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Abstract
Protease from Zingiberaceae officinale Roscoe (cv. Haliya indang) was
isolated, purified to homogeneity in a three step procedure involving
ammonium sulphate fractionation, gel filtration, and ion-exchange
chromatography. The partially purified enzyme was characterized. The
enzyme was found to be made up of two sub-units designated ‘ginger protease
I and II (GPI & GPII); the subunits had specific activity of 173.88±5.6 and
131.54±3.7mg gelatin/min/mg protein respectively, with purification folds of
3.40 and 2.57 over crude extract. The molecular weights of 23.97 and 25.05
kDa were obtained by SDS-PAGE. The enzyme subunits had similar optimum
temperature of 40oC, while retaining high activity at 46oC for GPI and 58oC
for GPII suggesting GPI to be more heat labile. The subunits incubated at
different temperatures showed highest stability at 40oC, with an optimum pH
of 6.0 and 5.5, retaining significant activity within a pH range of 5.5-7.5 for
GPI, and 5.0-7.5 for GPII. Mn2+, Zn2+ inhibited the subunits mildly while Hg2+
had a severe inhibition on the subunits. The subunits were also severely
inhibited by Iodoacetate (IA), a cysteine protease inhibitor. IA caused a mixed
inhibition with KI of 7.8mM and 18.8mM and Ki of 5.6mM and 13.5mM
respectively. Time course of activity remained high for about 160 hours of
storage at 4oC. The enzyme subunits demonstrated gelatin as its natural
substrate over other examined substrates with Km of 0.8475 and 1.163mg/ml
and Vmax of 555.56 and 454.55mg gelatin/ml respectively. The properties of
this enzyme meet the requirement needed for industrial application of the
enzyme.
Description
A project submitted to the Postgraduate School,
Ahmadu Bello University in partial fulfillment
for the award of M. Sc degree.
Department of Biochemistry
Ahmadu Bello University Zaria
Nigeria
July 2011
Keywords
Isolation,, Partial,, Purification, Characterization,, Protease,, Ginger Zingiberaceae,, officinale Roscoe