Date Published: October 11, 2018
Publisher: Public Library of Science
Author(s): Karolina Labus, Bing Xu.
The main aim of this study was to prepare gelatine-based hydrogels containing entrapped substrate and to examine the applicability of these matrices for detection of enzymes with a specified catalytic activity. The general research concept assumed the use of a substrate that, in the presence of a particular enzyme, will quickly undergo conversion to a coloured product. ortho-Nitrophenyl-β-D-galactopyranoside (ONPG) was used as the immobilized substrate and β-galactosidase from Kluyveromyces lactis as the biocatalyst to be determined. Among other factors, the range of detectable concentrations of galactosidase, the operational pH range, the time necessary to achieve a visible response and the preferred storage conditions for the test were determined. As a result, an effective colourimetric test for β-galactosidase detection was obtained. Its main advantages include (i) the effective detection of the enzyme at concentrations greater than or equal to 0.6 mg.L-1, (ii) the ability to perform initial quantification of the enzyme on the basis of the intensity of the obtained colour (iii) applicability in a wide pH range (from 4.0 to 9.0), (iv) a relatively short response time (from 1 to a maximum of 30 minutes) and (v) stability in long-term storage at 4°C (90 days without loss of specific properties).
The detection of biocatalysts of interest in multi-component mixtures, such as plant extracts or microbial post-culture fluids, is a notable challenge for modern biotechnology. The essential key to facilitating this task is to find a rapid, easy, and highly selective method for determining the catalytic activity. One possibility is the use of a substrate specific to one particular biocatalyst with a given activity. The most favourable would be an assay in which the product of the catalysed reaction provides an intense colour in a relatively short time.
The colourimetric test for β-galactosidase detection proposed in this study is based on the use of a synthetic substrate specific for this enzyme, ONPG, immobilized by entrapment inside the crosslinked structure of a hydrogel matrix. This compound is well known and is commonly used to determine the catalytic activity of β-galactosidase [33–35]. The presence of this biocatalyst is indicated by the appearance of a yellow colour as a result of the enzymatic hydrolysis of ONPG. In this case, o-nitrophenol (a product of ONPG decomposition catalysed by β-galactosidase) is responsible for the colour (Fig 1).
A tangible result of the research performed in this study is a new, simple and rapid test for the detection of β-galactosidase–an effective biocatalyst for lactose hydrolysis. The proposed solution is based on the use of a specific substrate (ONPG) entrapped in a crosslinked hydrogel matrix. Among other factors, the range of β-galactosidase concentrations that could be effectively detected, the operational pH range, the time necessary to achieve a visible response and the preferred storage conditions of the test were determined in this study. In particular, the colourimetric test for β-galactosidase obtained as a result of these studies is distinguished by (i) the ease of making a gelatine hydrogel matrix, (ii) high selectivity and sensitivity, (iii) operation over a wide pH range, (iv) a relatively short response time and (v) the possibility of long-term storage without loss of its specific properties.