Identificación y clonación de secuencias codificantes de peroxidasas de granos inmaduros de fréjol (Phaseolus vulgaris)

Abstract

Peroxidases are hemoproteins whose catalytic activity is used in diagnostic tests, development of biosensors and bioremediation, the most used are Horseradish (Armoracia rusticana) peroxidase or HRP and Soybean (Glycine max) peroxidase or SBP. Currently these enzymes have a high cost, so it is necessary to find alternatives for obtaining them and have immediate availability in the country for application in the development of diagnostic tools. In the search for alternatives to commercial peroxidase, a literature review was carried out to identify previously described sequences of SBP, the localized sequences were used to identify orthologous coding sequences in Phaseolus vulgaris (Common bean), with the identified sequences were designed oligonucleotides for the generation and amplification of the respective cDNAs. After obtaining total RNA from immature grain cuticle, synthesis of the cDNA and generation of PCR products was carried out. PCR products sequencing results showed that it has been possible to obtain sequences encoding the orthologue peroxidase of Ep (NP_001238315.1, NM_001251386 .1) and Prx2 (NP_001237601.1, NM_001250672.2) from common bean. The sequences obtained in this work have a high identity with the reported sequences in Ph. vulgaris, Ep (PHAVU_006G129900g) with 99.08 % and 99.69 % for Prx2 (PHAVU_003G078600g). Cloned coding peroxidases sequences in pET15b vector were induced Escherichia coli Origami (DE3) and total protein extract obtained showed enzymatic activity on 3,3′- diaminobenzidine, 4-chloro-1-naphthol and syringaldazine peroxidases substrates. Finally, the results obtained allow us to conclude that it was possible to obtain two protein coding sequences with peroxidase activity from Phaseolus vulgaris.