A novel thermostable inhibitor of trypsin and subtilisin from the seeds of Brassica nigra: amino acid sequence, inhibitory and spectroscopic properties and thermostability

Genov, Nicolay; Goshev, Ivan; Nikolova, D.; Georgieva, Dessislava; Filippi, Bruno; Svendsen, Ib

doi:10.1016/s0167-4838(97)00049-6

Cited by 26 publications

(12 citation statements)

References 22 publications

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“…On the other hand, the two-subunit TIs from S. arvensis (23) and B. nigra (24) have sequence homology with mature napin. The allergen protein from S. alba (40) also has sequence homology with BjTI.…”

Section: Discussionmentioning

confidence: 99%

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Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor

Mandal

Kundu

Roy

et al. 2002

Journal of Biological Chemistry

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A number of trypsin inhibitor (TI) genes have been used to generate insect-resistant plants. Here we report a novel trypsin inhibitor from Indian mustard Brassica juncea (BjTI) that is unique in being the precursor of a 2S seed storage protein. The inhibitory activity is lost upon processing. The predicted amino acid sequence of the precursor based on the B. juncea 2S albumin (Bj2S) gene cloned and sequenced in this laboratory (Bj2Sc; GenBank TM accession number X65972) showed a soybean-TI active site-like motif GPFRI at the expected processing site. The BjTI was found to be a thermostable Kunitz type TI that inhibits trypsin at a molar ratio of 1:1. The 20-kDa BjTI was purified from midmature seeds and found to be processed in vitro to 9-and 4-kDa subunits upon incubation with seed extract. The Bj2Sc sequence was expressed in Escherichia coli pET systems as the inhibitor precursor. The radiolabeled gene product was expressed in vitro in a coupled transcriptiontranslation system and showed the expected processing into subunits. Two in vitro expressed pre-2S proteins, mutated at Gly and Asp residues, were processed normally to mature subunits, showing thereby no absolute requirement of Gly and Asp residues for processing. Finally, the 2S gene was introduced into tobacco and tomato plants. Third generation transgenics expressing BjTI at 0.28 -0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm, Spodoptera litura. This novel TI can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption.

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Section: Discussionmentioning

confidence: 99%

“…On the other hand, TIs from Spergula arvensis (23) and Brassica nigra (24) are larger proteins composed of 9-and 4-kDa subunits, lack chymotrypsin inhibitor activity, and are similar to the napin family 2S albumin in amino acid sequence. In all of these crucifers, the processed protein is the inhibitor.…”

mentioning

confidence: 99%

Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor

Mandal

Kundu

Roy

et al. 2002

Journal of Biological Chemistry

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“…Porcentaje de cada fracción proteica a partir de la concentración de proteínas solubles totales obtenidas de la curva de BSA (g) por cada 100 g de muestra desgrasada en Plukenetia volubilis y Plukenetia huayllabambana. biológica como inhibidores de proteasas y como proteínas de defensa (Svendsen et al 1994, Genov et al 1998 (Lea & Leegood 1999). Esta fracción es la más difícil de definir y entender dado que son insolubles en agua, etanol, soluciones salinas y sólo se solubilizan en medios ácidos (pH 2) o álcalis (pH 12).…”

Section: Caracterización Bioquímica De Las Fracciones De Osborne: Eleunclassified

PERFIL DE PROTEÍNAS DE LAS SEMILLAS DE “SACHA INCHI” (PLUKENETIA VOLUBILIS L. Y PLUKENETIA HUAYLLABAMBANA BUSSMANN, TÉLLEZ & GLENN)

Lopez¹,

Cruz²,

Gutiérrez³

2016

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Keywords: albumin -gliadin -globulin -glutelin -Plukenetia volubilis -Plukenetia huayllabambanaIn Peru, Plukenetia it is a genus of tropical plants of the family Euforbiacea, called inchi, sacha inchi, sacha peanut, peanut of the Inca or rustic peanut, which are in use as food for the tribes of the Amazonia since ancient times. The most cultivated species is Plukenetia volubilis, while Plukenetia huayllabambana is a species recently discovered in the Province of Rodriguez de Mendoza (Amazonas-Peru). The aim of our research was to obtain, quantify and characterize protein profile of the two species mentioned above. The seeds were dried, ground and had fat extracted using the method of Folch (chloroform: methanol 2: 1), and then proteins fractionated using the method of Osborne: albumins (bidistilled water), globulins (NaCl 10.5 M), gliadin (Ethanol 70%) and glutelins (0.1 M NaOH). The proximate analysis for each species was performed following the techniques proposed by the AOAC. Quantitation of total protein was by the Kjeldah method and soluble proteins by the method of Gornall (Biuret), the fractions being separated by 15% SDS-PAGE. The results show that the total protein content in P. volubilis is higher than in P. huayllabambana (30.67% vs. 27.91%). However, in the latter the fat content is higher (52.11% vs. 45.49%). The majority protein fraction of P. volubilis were globulins and for P. huayllabambana, albumins. Electrophoretic patterns show no qualitative differences but there are quantitative differences (p <0.05) between the two species.

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“…These include napins from kohlrabi (Brassica napus var rapifera), charlock (Sinapis arverse) and black mustard (B. nigra) , all of which are inhibitors of serine proteinases (Svendsen et al 1989(Svendsen et al , 1994Genov et al 1997) and may therefore playa role in defence. Similarly, nap ins from radish (Raphanus sativus) inhibit the growth of a range of plant pathogenic (Rico et al 1996).…”

Section: Salbuminsmentioning

confidence: 99%

The Biochemistry and Molecular Biology of Seed Storage Proteins

Autran¹,

Halford²,

Shewry³

2001

Plant Nitrogen

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A novel thermostable inhibitor of trypsin and subtilisin from the seeds of Brassica nigra: amino acid sequence, inhibitory and spectroscopic properties and thermostability

Cited by 26 publications

References 22 publications

Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor

Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor

PERFIL DE PROTEÍNAS DE LAS SEMILLAS DE “SACHA INCHI” (PLUKENETIA VOLUBILIS L. Y PLUKENETIA HUAYLLABAMBANA BUSSMANN, TÉLLEZ & GLENN)

The Biochemistry and Molecular Biology of Seed Storage Proteins

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