PURIFICATION AND CHARACTERIZATION OF TRYPSIN INHIBITOR PROTEIN FROM SEEDS OF MOMORDICA DIOICA

Abstract

Objective: To purify the trypsin inhibitor protein from the seeds of Momordica dioica and characterize the protein for its stability and effect on trypsin
activity.
Methods: The total protein was extracted from the seeds of M. dioica, and the purification of the protein was performed by ion exchange
chromatography and ultrafiltration technique. The antitrypsin activity assay of the purified inhibitor protein was carried out using N-benzoylDL-arginine-p-nitroanilide
(BAPNA)
as the chromogenic
substrate
at
various
pH
and
temperature
ranges
to
determine
the
stability of
the
protein.

The
inhibitory
effect
of
purified protein
on trypsin activity
was
characterized
by
enzyme kinetic study.
Sodium dodecyl
sulfate-polyacrylamide
gel
electrophoresis
analysis
was
performed
under
the
non-reducing
condition to
determine
the
presence
of
inhibitor
protein
in
various
fractions
of elutes

and
its molecular mass.
Results: The purified protein from M. dioica seeds showed almost 96.17±0.034% trypsin activity inhibition and 0.96±0.00 U trypsin inhibitory activity
(TIA) at 30°C and pH 8. This trypsin inhibitory protein from M. dioica (MdTi) was found to be stable over a temperature range of 30-100°C and the pH
range of 3-10 retaining the antitrypsin activity. The molecular mass of MdTi was found to be Ì´12 kDa. From the enzyme kinetics, it was found that Km
value remains unaffected with decrease in the Vmax of trypsin in presence of inhibitor.
Conclusion: M. dioica seeds were found to posses serine protease inhibitor protein. The protein was purified and characterized as thermostable as
well as pH tolerant trypsin inhibitor with high TIA. It can be well explored for its use in the agriculture industry for pest management as well as the
therapeutic applications.
Keywords: Serine proteases, Trypsin inhibitor, Momordica dioica, Trypsin inhibitory protein from Momordica dioica, Non-competitive inhibition.