02058nas a2200229 4500008004100000022001400041245012000055210006900175260010600244300001200350490000700362520113700369653001301506653004101519653002801560653003201588100001901620700002001639700002401659700002101683856012401704 2005 eng d a0022-191000aCharacterization of two midgut proteinases of helicoverpa armigera and their interaction with proteinase inhibitors0 aCharacterization of two midgut proteinases of helicoverpa armige aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLANDbPERGAMON-ELSEVIER SCIENCE LTDcMAY a513-5220 v513 a

Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0 kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants. (C) 2005 Elsevier Ltd. All rights reserved.

10aelastase10aHelicoverpa armigera gut proteinases10ainsect-resistant plants10aplant proteinase inhibitors1 aTelang, M., A.1 aGiri, Ashok, P.1 aSainani, Mohini, N.1 aGupta, Vidya, S. uhttps://library.ncl.res.in/content/characterization-two-midgut-proteinases-helicoverpa-armigera-and-their-interaction-002134nas a2200265 4500008004100000022001400041245013000055210006900185260007900254300001200333490000800345520113900353653002701492653001401519653001601533653002601549653003201575653001601607100002501623700002801648700002301676700002001699700002001719856012901739 2005 eng d a1096-495900aUnraveling biochemical properties of cockroach (Periplaneta americana) proteinases with a gel X-ray film contact print method0 aUnraveling biochemical properties of cockroach Periplaneta ameri a360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USAbELSEVIER SCIENCE INCcJUL a261-2660 v1413 a

Eleven proteinase activity bands were detected in American cockroach (Periplaneta americana) gut. These were partially purified and characterized using a gel X-ray film contact print method. Cockroach gut proteinases (CGPs) show activity over a broad range of pH with maximum activity between pH 6 and 10, and optimal activity at 50-70 degrees C. CGPs were partially purified by preparative gel electrophoresis and analyzed using synthetic substrates and inhibitors. Four of the proteases exhibited chymotrypsin-like (C1 to C4) activity and seven trypsin-like (T I to T7) activity. Accuracy of the gel X-ray film contact print method is confirmed by including bovine chymotrypsin in CGP analysis. Inhibition of CGPs with different plant proteinaceous proteinase inhibitors allowed identification of potential CGP inhibitors. Our results imply that presence of several CGP activity bands, and their stability and activity over a broad pH and temperature range might contribute to adaptation of P americana to extreme environmental conditions and the polyphagous nature of the species. (c) 2005 Elsevier Inc. All rights reserved.

10aactivity visualization10acockroach10agut enzymes10aPeriplaneta americana10aplant proteinase inhibitors10aProteinases1 aHivrale, Vandana, K.1 aChougule, Nanasaheb, P.1 aChhabda, Pavan, J.1 aGiri, Ashok, P.1 aKachole, M., S. uhttps://library.ncl.res.in/content/unraveling-biochemical-properties-cockroach-periplaneta-americana-proteinases-gel-x-ray-002460nas a2200241 4500008004100000022001400041245014600055210006900201260000900270300001100279490000800290520157900298653001901877653002901896653002301925653003201948653001401980653002701994100002402021700002202045700002002067856013102087 2024 eng d a0006-291X00aEngineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis0 aEngineering inhibitory repeat domains of PinII type proteinase i cNOV  a1508080 v7353 a

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

10aCapsicum annum10aInhibitory repeat domain10amolecular modeling10aplant proteinase inhibitors10aProteases10aSaturation mutagenesis1 aBarbole, Ranjit, S.1 aJoshi, Rakesh, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/engineering-inhibitory-repeat-domains-pin-ii-type-proteinase-inhibitors-indicate-their-high