Isoelectric focusing, effect of reducing agents and inhibitors: partial characterization of proteases extracted from Bromelia karatas

María De Lourdes García-Magaña,Efigenia Montalvo-González,Enrique Rudiño-Piñera,Julián González-Borrayo,Sonia G Sáyago-Ayerdi,Jesús Aarón Salazar-Leyva

doi:10.1007/s13765-018-0380-6

María De Lourdes García-Magaña, Efigenia Montalvo-González + Show 4 more

Open Access

https://doi.org/10.1007/s13765-018-0380-6

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Abstract

The aim of this research is the partial characterization of proteases extracted from B. karatas; the isolation and purification of proteases from B. karatas fruits were achieved using precipitation, separation by size exclusion chromatography and anion-exchange chromatography; molecular mass (MM) was determined, and the effect of inhibitors, reducing agents and heat on enzyme activity was analyzed. These proteases were compared with proteases from Bromelia pinguin (B. pinguin) and evaluated under similar conditions. The isolation procedure was adequate; only a few protein bands are present in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Furthermore, zymogram analysis showed protein bands with enzyme activity. Inhibitors, reducing agents and heat were unable to inactivate the proteases extracted from B. karatas and B. pinguin. The semi-purified extracts are a set of proteases with a MM of 66 kDa, but different isoelectric points (3.5–6.5 for B. karatas and 5–9 for B. pinguin), which are found in quaternary structures with proteolytic activity. When denatured, they segment into fragments of approximately 20 and 10 kDa. The data indicate that these plants could be used as sources of proteases since they present good proteolytic activity (21.93 UT for proteases from B. karatas and 43.58 UT for proteases from B. pinguin) and that B. Karatas has potential applications comparable to B. pinguin in the food and health industries.

Highlights

Proteases can be classified according to the characteristics of their active sites; this includes serine proteases, aspartic proteases, metalloproteinases and cysteine proteases [1]
The proteolytic activity of the concentrated proteases was 21.93 UT for proteases from B. karatas and 43.58 UT for proteases from the B. pinguin. This means that the extraction process did not affect the proteolytic activity, it was clear that proteases from B. pinguin had higher enzymatic activity than proteases from B. karatas
isoelectric focusing (IEF) (Fig. 2B) shows the presence of several bands corresponding to proteins with several different isoelectric points (IPs)

Summary

Introduction

Proteases can be classified according to the characteristics of their active sites; this includes serine proteases, aspartic proteases, metalloproteinases and cysteine proteases [1]. Plant proteases have been used since ancient times They are mainly applied in the food industry (brewing, baking, tenderizing and production of protein hydrolysates) and as a therapeutic alternative (anthelmintic, immune response modulators, wound healing and antitumor agents). Examples of these enzymes are bromelain and papain from pineapple (Ananas comosus) and papaya (Carica papaya), respectively. Within the Bromeliaceae family there are other fruits such as Aguama or Guamara (Bromelia pinguin or B. pinguin) and Cocuixtle (Bromelia karatas or B. karatas) [3] that are considered rich in proteases and have hardly been studied. Their use has origins in preColumbian cultures dating back thousands of years [4]

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