Effective detection of biocatalysts with specified activity by using a hydrogel-based colourimetric assay – β-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 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).本研究的核心目标为制备负载包埋底物的明胶基水凝胶，并考察此类基质在检测具有特定催化活性酶类中的应用潜力。本研究的整体研究思路预设采用一类底物：该底物在特定酶的作用下可快速转化为有色产物。本研究选用邻硝基苯基-β-D-半乳糖苷（ortho-Nitrophenyl-β-D-galactopyranoside，ONPG）作为固定化底物，以乳酸克鲁维酵母（Kluyveromyces lactis）来源的β-半乳糖苷酶作为待检测的生物催化剂。研究同时考察了多项关键参数，包括β-半乳糖苷酶的可检测浓度范围、反应适用pH范围、实现可见显色响应所需时长，以及该检测方法的最优储存条件。最终，本研究成功构建了可用于β-半乳糖苷酶检测的高效比色检测方法。该方法的核心优势如下：(1) 可有效检测浓度≥0.6 mg·L⁻¹的目标酶；(2) 可基于显色强度对酶进行初步定量；(3) 适用pH范围宽泛（4.0~9.0）；(4) 响应时长较短（1~30分钟）；(5) 在4℃条件下可长期稳定储存90天，且不丧失特异性检测性能。