Collagenase Activity of Bromelain Immobilized at Gold Nanoparticle Interfaces for Therapeutic Applications.

Adrianne M M Brito,Iseli L Nantes-Cardoso,Marcelo Y Icimoto,Vitor Oliveira

doi:10.3390/pharmaceutics13081143

Adrianne M M Brito, Iseli L Nantes-Cardoso + Show 2 more

Open Access

https://doi.org/10.3390/pharmaceutics13081143

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Abstract

Bromelain (Bro) is a multiprotein complex extracted from the pineapple plant Ananas comosus, composed of at least eight cysteine proteases. Bro has a wide range of applications in medicine and industry, where the stability of its active proteases is always a major concern. The present study describes the improvement of stability and gain of specific activity in the enzymatic content of Bro immobilized on gold nanoparticles (GNPs). GNPs were synthesized in situ using Bro as the reducing and stabilizing agents and characterized by surface plasmon resonance and transmission electron microscopy. Consistent with the structural changes observed by circular dichroism analysis, the association with GNPs affected enzyme activity. The active Bro immobilized on GNPs (NanoBro) remained stable under storage and gained thermal stability consistent with a thermophilic enzyme. Proteolytic assays were performed on type I collagen membranes using fluorescence spectroscopy of O-phthaldialdehyde (OPA), changes in the membrane superficial structure, and topography by scanning electron microscopy, FTIR, and scanning laser confocal microscopy. Another characteristic of the NanoBro observed was the significant increase in susceptibility to the inhibitory effect of E-64, indicating a gain in cysteine protease activity. The higher stability and specific activity of NanoBro contributed to the broadening and improvement of Bro applications.

Highlights

The present study describes the synthesis and characterization of gold nanoparticles (GNPs) produced using Bro as a reducing and stabilizing agent
NanoBro was synthesized in situ using Bro as a reducer agent and a biotemplate for the growth of gold nanocrystals, and the nanostructures were characterized and applied for collagenolytic activity
During GNP formation, the SPR band presented thesized only with HEPES buffer lost its reddish coloration, which could be attributed to a progressive blueshift, probably because of the deaggregation promoted by protein capgold oxidation or extensive GNP aggregation that shifted the spectra to the infrared specping [25]

Summary

Introduction

Several enzymes have been purified partially or completely for therapeutic, biotechnological, and industrial applications, and proteolytic enzymes are extensively used for these purposes [1]. Proteolytic enzymes catalyze the hydrolysis of peptide bonds in a variety of biological processes. Proteases are classified according to the primary amino acid residue acting on catalysis and are divided into nine classes: cysteine, serine, aspartic, metallo, glutamic, threonine, and asparagine proteases, as well as mixed and unknown types [2]. Cysteine proteases ( called thiol proteases) are present in all living organisms and share a common catalytic mechanism involving a nucleophilic cysteine in the catalytic unit, which is composed of a triad of amino acids. The first isolated and characterized cysteine protease was papain obtained from papaya latex (Carica papaya)

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