The contribution of two disulfide bonds in the trypsin binding domain of horsegram (Dolichos biflorus) Bowman-Birk inhibitor to thermal stability and functionality

Kumar, Vinod; Gowda, Lalitha R.

doi:10.1016/j.abb.2013.06.002

Cited by 17 publications

(10 citation statements)

References 57 publications

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“…6A and 7A,B). Such structural stability was reported in BBIs from horse gram (Kumar and Gowda, 2013) and black gram (Prasad et al, 2010b). Conversely, RsKI is sensitive to thermal treatment and showed a remarkable decrease in its ellipticity when heated at 80°C (Fig.…”

Section: Secondary Structural Analysis Of Rsbbi and Rskisupporting

confidence: 70%

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

et al. 2019

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Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100°C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100°C and retained > 60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75°C. Further, the significant inhibitory activity of RsBBI (IC 50 = 24 ng) and RsKI (IC 50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively. vegetative organs during biotic and abiotic stresses (Jamal et al., 2013; Yamchi et al., 2017). They also act as pseudosubstrates of proteases and stabilize them during desiccation. The PIs are rapidly degraded during seed germination to release essential amino acids and they reappear in cotyledons to protect them from invading pests and pathogens. They also take part in programmed cell death in plants. Bowman-Birk

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Section: Secondary Structural Analysis Of Rsbbi and Rskisupporting

confidence: 70%

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

et al. 2019

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“…TIs of these cultivars remain active even when being exposed to 90 °C for 60 min. For the case of adzuki beans, the TI was inactivated by using β‐mercaptoethanol prior to electrophoresis (Klomklao and others ; Kumar and Gowda ) so it would be of interest to study the application of higher temperatures during different times to determine the conditions at which these molecules remain active. This strategy would require the use of large amounts of energy so for the case of heat stable TIs, other treatments should be developed.…”

Section: Physical Processesmentioning

confidence: 99%

Inactivation Methods of Trypsin Inhibitor in Legumes: A Review

Avilés‐Gaxiola

Chuck‐Hernández

Saldivar

2017

Journal of Food Science

191

113

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Seed legumes have played a major role as a crop worldwide, being cultivated on about 12% to 15% of Earth's arable land; nevertheless, their use is limited by, among other things, the presence of several antinutritional factors (ANFsnaturally occurring metabolites that the plant produces to protect itself from pest attacks.) Trypsin inhibitors (TIs) are one of the most relevant ANFs because they reduce digestion and absorption of dietary proteins. Several methods have been developed in order to inactivate TIs, and of these, thermal treatments are the most commonly used. They cause loss of nutrients, affect functional properties, and require high amounts of energy. Given the above, new processes have emerged to improve the nutritional quality of legumes while trying to solve the problems caused by the use of thermal treatments. This review examines and discusses the methods developed by researchers to inactivate TI present in legumes and their effects over nutritional and functional properties.

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“…The studies of Hogg (2003) reported that disulphide bonds have been added to the PIs during evolution to enhance the protein stability, which in turn prevent their digestion by cellular proteases and thereby increase their half-life by molecular packing. Stability against high temperature and pH was also observed with other group of PIs from seeds of Dolichus biflorus (Kumar and Gowda, 2013), Pithecellobium dumosum (Rufino et al, 2013), and Putranjiva roxburghii (Chaudhary et al, 2008). …”

Section: Discussionmentioning

confidence: 62%

Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera

et al. 2016

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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 and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus.

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The contribution of two disulfide bonds in the trypsin binding domain of horsegram (Dolichos biflorus) Bowman-Birk inhibitor to thermal stability and functionality

Cited by 17 publications

References 57 publications

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

Inactivation Methods of Trypsin Inhibitor in Legumes: A Review

Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera

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