A novel cucumisin-like serine protease from leaf of legume Canavalia ensiformis
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ORIGINAL ARTICLE
A novel cucumisin-like serine protease from leaf of legume Canavalia ensiformis Rayane Natashe Gonçalves1 • Dario Eluan Kalume2 • Maria Antonieta Ferrara1 • Raquel Elisa da Silva-Lo´pez1 Received: 29 January 2020 / Accepted: 19 July 2020 Ó Society for Plant Biochemistry and Biotechnology 2020
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. Keywords Canavalia ensiformis Leaf aqueous extract Serine proteases Cucumisin Isolation Characterization 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-(4guanidino) butane EC Enzyme classification EDTA Ethylenediaminetetraacetic acid SBTI Soy bean trypsin inhibitor PEP Pepstatin SDS-PAGE Sodium dodecylsulfate-polyacrylamide gel electrophoresis & Raquel Elisa da Silva-Lo´pez [email protected] 1
Departament of Natural Products, Farmanguinhos, Av. Brasil 4365, Rio de Janeiro, RJ, CEP 21045-900, Brazil
2
Interdisciplinary Laboratory of Medical Research, IOC Oswaldo Cruz Institute (FIOCRUZ), Av. Brasil 4365, Rio de Janeiro, RJ, CEP 21045-900, Brazil
L-TAME BTEE L-BAME L-BAPNA TPCK
N-a-Tosyl-L-arginine methyl ester N-Benzoyl-L-tyrosine ethyl ester N-benzoyl L-alanine methyl ester Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride N-Tosyl-L-phenylalanine chloromethylketone
Introduction Nowadays, proteases represent a very significant group of industrial enzymes, accounting for 60% of the total enzyme market, ranging from detergent additives to effective therapeutics (Li and Yi 2013). In addition, plant proteases are part of a growing class of therapeutic
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