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. 2018 Oct 11;13(10):e0205532.
doi: 10.1371/journal.pone.0205532. eCollection 2018.

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

Karolina Labus  1
Affiliations

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

Karolina Labus. PLoS One. .

Abstract

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).

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Conflict of interest statement

The author has declared that no competing interests exist.

Figures

Fig 1
Fig 1. Reaction scheme of ortho-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis in the presence of β-galactosidase as a biocatalyst.
Fig 2
Fig 2. Response obtained in tests of β-galactosidase solutions at different concentrations; test time 10 min; enzyme concentrations 1.0–100 mg.L-1.
Fig 3
Fig 3. Visual response obtained after different times in the hydrogel-based test with immobilized ONPG performed for β-galactosidase in the concentration range of 5.1–323 mg.L-1.
Fig 4
Fig 4. Visual response obtained after different times of the hydrogel-based test with immobilized ONPG, performed to test for β-galactosidase in the concentration range of 0.6–5.1 mg.L-1.
Fig 5
Fig 5. Visual response obtained after different times in the hydrogel-based test with immobilized ONPG performed at a constant concentration of β-galactosidase (82 mg.L-1) prepared in a pH range from 4.0 to 9.0.
Fig 6
Fig 6. Storage stability of ortho-Nitrophenyl-β-D-galactopyranoside (ONPG) in native form and immobilized in the gelatine-based hydrogel matrix.
Conditions: 4°C, 1–20 days.
Fig 7
Fig 7. Storage stability of hydrogel-based test with immobilized ONPG.
Conditions: 4°C, 1–90 days. Test properties were examined by carrying out the β-galactosidase detection assay for 5 and 10 minutes.
See this image and copyright information in PMC

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Grants and funding

This research was supported by the Wrocław Centre of Biotechnology, programme The Leading National Research Centre (688/2017/KNOW to KL) for the years 2014 – 2018 (Poland). The funding sponsor had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.