Effective detection of biocatalysts with specified activity by using a hydrogel-based colourimetric assay - [beta]-galactosidase case study

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 pa...

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Bibliographic Details
Published in:PloS one 2018-10, Vol.13 (10), p.e0205532
Main Author: Labus, Karolina
Format: Article
Language:English
Subjects:
Beta galactosidases
Biocatalysts
Biological products
Case studies
Online Access:Get full text
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Summary: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-[beta]-D-galactopyranoside (ONPG) was used as the immobilized substrate and [beta]-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 [beta]-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.sup.. L.sup.-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).
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0205532