A Novel Trypsin and α-Chymotrypsin Inhibitor from Maclura pomifera Seeds (original) (raw)
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Identification of plant extracts expressing trypsin inhibitor
Plant protease inhibitors (PPIs) are compounds which play a potent defensive role against pest and pathogens, thereby protecting the plants. In this work we tested different plant extracts to identify extracts containing inhibitor against trypsin activity. Plant extracts were made by homogenizing soaked leaves/seeds/root modification of plants in bicarbonate buffer, pH 9.0.The homogenates were centrifuged at 10,000 rpm at 4°C for 10 minutes. The supernatant containing the soluble proteins were used for protease inhibition assay. Protease inhibition assay was carried out in a total volume of 1 ml containing 67 µl trypsin (Bovine), 25 µl plant extract, 511 µl Tris buffer, 67µl NaCl and 330 µg Nα-Benzoyl-DL-Arginine-P-Nitro Anilide (BAPNA) as substrate. On screening 19 extracts from different plants, we found 9 of them inhibited trypsin activity greater than 40%. The percentage inhibition of these plants are Garcinia xanthochymus (96.7± 0.4 %), Datura stramonium (86.1±1.6%), Ricinus communis (74.05±0.45%), Phyllanthus amarus (72.05±2.15%), Santalum album (64.4±1.7 %), Plectranthus ambonicus (62.15±2.45% ), Croton hirtus ( 55.85±0.9% ), Zea mays (48±2.1% ) and Prunus cerasifera (45.38±0.125%). Of these, to our knowledge no trypsin inhibitor was reported from Garcinia xanthochymus, Datura stramonium, Plectranthus ambonicus, Prunus cerasifera, Phyllanthus amarus, Santalum album, and Croton hirtus. As trypsin is a serine protease, a predominant class of protease in the gut of lepidopteran larvae, this screening will be useful to identify extract containing protease inhibitor against the gut protease of lepidopteran pests. Protease inhibitors from these extracts can be exploited for control of lepidopteran pests. Keywords: trypsin, proteases, plant protease inhibitors, Garcinia xanthochymus, Datura stramonium
Planta, 2016
A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M(-1) cm(-1). MpBBI inhibits strongly tryp...
Frontiers in Physiology, 2016
Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors...
Protein Journal, 2004
Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves of being attacked by insects. In this work, a novel trypsin inhibitor (PPTI) was purified from the seeds of the native Brazilian tree Poecilanthe parviflora (Benth) (Papilioinodeae, Leguminosae) by gel filtration chromatography on a Sephadex G-100 followed by Superdex G75 chromatography (FPLC), Sepharose 4B-Trypsin column, and fractionated by reversed-phase HPLC on a C-18 column. SDS-PAGE showed that PPTI consisted of a single polypeptide chain with molecular mass of about 16 kDa. The dissociation constant of 1.0 × 10−7 M was obtained with bovine trypsin. PPTI was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the PPTI showed a high degree of homology with other Kunitz-type inhibitors. Trypsin-like activity in midguts of larval Diatraea saccharalis, Anagasta kuehniella, Spodoptera frugiperda, and Corcyra cephalonica were substantially inhibited by PPTI.