Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family

Abstract

PurposeThis study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials.MethodsAllium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy.ResultsASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2–12 showing a decline in the activity around pH 4–5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10–80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH.ConclusionsTo conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.

Expression and characterization of a new serine protease inhibitory protein in Escherichia coli

“…The protein PI-QT exhibited inhibitory effects against trypsin and α-chymotrypsin with specific activities of 975 ± 26 and 417 ± 14 U/mg, respectively. The specific activity of the protein PI-QT was comparable with that of novel serpins reported in recent studies [21][22][23][24] . For example, Jiang et al 21 have cloned and expressed a novel serpin (Spi1C) from metagenomic library of uncultured marine microorganisms; the Spi1C protein exhibited inhibitory effects against trypsin and α-chymotrypsin with specific values of 6940 and 3640 U/mg, respectively.…”

“…Chan et al 22 have purified a thermostable trypsin inhibitor from small pinto beans and reported that the purified protein showed inhibitory activity against trypsin with specific value of 2398 U/mg. Shamsi et al 23 have isolated and purified a novel Kunitz trypsin inhibitor (ASPI) from garlic Allium sativum; the ASPI protein showed inhibitory activity against trypsin with specific activity of 30376 U/mg. In another study, Mohan et al 24 purified and characterized a protease inhibitor from Capsicum frutescens with specific activity against trypsin of 6749 U/mg.…”

Duplication and transcriptional divergence of three Kunitz protease inhibitor genes that modulate insect and pathogen defenses in tea plant (Camellia sinensis)

“…The three CsKPI proteins suppress the activity of trypsin and reduce the formation rate of the PNA reaction product (Fig. 3b, c), which mirrors the activities of KPIs from other plant species 40,41 . In addition, the inhibitory activities of the CsKPI2 and CsKPI3 mutant proteins were nearly abolished after two key residues (Arg and Lys) were replaced with alanine, suggesting that these two residues have essential roles in the inhibition of PIs (Supplementary Fig.…”

Soybean protease inhibitors increase Bacillus thuringiensis subs. israelensis toxicity against Hypothenemus hampei

“…The partially purified soybean meal extract unexpectedly processed DL-BApNA, indicating that it contained a measurable level of amidolytic activity (Shamsi et al, 2016). This finding suggests the presence of proteases in the soybean meal preparation, a feature that could be of benefit for the purposes of this work, given that Cry and Cyt proteins digested by insect endo-proteases may exhibit increased insecticidal activity (Vidal-Quist et al, 2010).…”