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A novel cucumisin-like serine protease from leaf of legume Canavalia ensiformis

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Abstract

Proteases are essential for plant physiology. Leguminosae species express high level of these enzymes, however, they were only reported in seeds. The present work isolated and characterized serine proteases of the aqueous extract from Canavalia ensiformis leaf (CE-A), a tropical legume. This extract was loaded on to benzamidine affinity column, and the serine protease fraction (CE-ABza) was purified 1.65-fold, yielding a total recovery of 62%. In a gelatin-SDS-PAGE, CE-ABza presented activity at 90 kDa under non-reducing, and 17, 32, and 90 kDa under reducing conditions. Peptidomimetic substrates for both trypsin and chymotrypsin as well as proteins with biotechnological relevance were digested, in distinctive levels, by CE-ABza. The maximal activity was at pH 8.5 and 9.5, and 40 °C. Protease activity was not affected at 70 °C for 24 h; however, it was completely inhibited by benzamidine and N-tosyl-L-phenylalanine chloromethylketone. Divalent cations had negative modulation on CE-ABza activity. Mass spectrometry experiments identified 11 orthologous proteases from this legume species, suggesting that CE-ABza shares similar and specific sequences especially with a serine protease, cucumisin. CE-ABza is a valuable source of very active and thermal stable serine proteases, which can be a potential candidate for biotechnological and therapeutical applications.

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Abbreviations

BSA:

Bovine serum albumin

Bza:

Benzamidine

CE-A:

Aqueous leaf extract from Canavalia ensiformis

CE-ABza:

Serine proteases rich fraction from Canavalia ensiformis leaf extract

E-64:

L-trans-epoxysuccinyl-leucylamido-(4-guanidino) butane

EC:

Enzyme classification

EDTA:

Ethylenediaminetetraacetic acid

SBTI:

Soy bean trypsin inhibitor

PEP:

Pepstatin

SDS-PAGE:

Sodium dodecylsulfate-polyacrylamide gel electrophoresis

L-TAME:

N-α-Tosyl-L-arginine methyl ester

BTEE:

N-Benzoyl-L-tyrosine ethyl ester

L-BAME:

N-benzoyl L-alanine methyl ester

L-BAPNA:

Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride

TPCK:

N-Tosyl-L-phenylalanine chloromethylketone

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Acknowledgments

The authors are grateful to Valério Morelli for supplying C. ensiformis; the Proteomics and Mass Spectrometry Unit platform (UEMP) at the Federal University of Rio de Janeiro (UFRJ) run by Dr. Russolina Zingali for support in the use of the license Mascot Server for protein identification searches; and Augusto Vieira and Ana Lúcia Carvalho for helping with mass spectrometric analysis. Financial support was provide by the Fundacão de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); the Fundação para o Desenvolvimento Científico e Tecnológico em Saúde (Fiotec); and the Farmanguinhos postgraduate program.

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Authors and Affiliations

  1. Departament of Natural Products, Farmanguinhos, Av. Brasil 4365, Rio de Janeiro, RJ, CEP, 21045-900, Brazil

    Rayane Natashe Gonçalves, Maria Antonieta Ferrara & Raquel Elisa da Silva-López

  2. Interdisciplinary Laboratory of Medical Research, IOC - Oswaldo Cruz Institute (FIOCRUZ), Av. Brasil 4365, Rio de Janeiro, RJ, CEP, 21045-900, Brazil

    Dario Eluan Kalume

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  1. Rayane Natashe Gonçalves
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  2. Dario Eluan Kalume
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  3. Maria Antonieta Ferrara
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  4. Raquel Elisa da Silva-López
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Correspondence to Raquel Elisa da Silva-López.

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Gonçalves, R.N., Kalume, D.E., Ferrara, M.A. et al. A novel cucumisin-like serine protease from leaf of legume Canavalia ensiformis. J. Plant Biochem. Biotechnol. 30, 147–159 (2021). https://doi.org/10.1007/s13562-020-00578-5

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