ABSTRACT
A Bowman-Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman-Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1-10) and temperatures (10-100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains ß sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10-8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.
Subject(s)
Seeds/chemistry , Solanum/chemistry , Trypsin Inhibitor, Bowman-Birk Soybean/chemistry , Trypsin Inhibitors/chemistry , Trypsin/chemistry , Amino Acid Sequence , Chymotrypsin/chemistry , Circular Dichroism/methods , Fabaceae/chemistry , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , TemperatureABSTRACT
BACKGROUND: Identification and characterisation of plant defensive molecules enrich our resources to design crop protection strategies. In particular, plant-derived proteinaceous inhibitor(s) of insect digestive enzymes appear to be a safe, sustainable and attractive option. RESULTS: A glycoprotein having non-competitive α-amylase inhibitory activity with a molecular weight of 8.3 kDa was isolated and purified from seeds of Withania somnifera α-amylase inhibitor (WSAI). Its mass spectrometry analysis revealed 59% sequence coverage with Wrightide II-type α-amylase inhibitor from Wrightia religiosa. A dose-dependent inhibition of α-amylases from Aspergillus oryzae, Bacillus subtilis, Helicoverpa armigera and Tribolium castaneum was recorded. Interestingly, WSAI did not inhibit human salivary α-amylase significantly. When adults of T. castaneum were fed with WSAI (1.6 mg g-1 ), decrease in consumption, growth and efficiency of conversion of ingested food was evident, along with over fourfold increases in feeding deterrence index. A decline in larval residual α-amylase activity after feeding of WSAI resulted in a reduction in longevity of T. castaneum. CONCLUSION: The study reflects the significance of WSAI in affecting the overall growth and development of T. castaneum. Pre- and post-harvest pest resistive capability makes WSAI a potential candidate for insect pest management. Further, the effectiveness of this inhibitor could be explored either in formulations or through a transgenic approach. © 2016 Society of Chemical Industry.
