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

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).

<p>NDM-1 Carbapenemase-Producing Enterobacteriaceae are Highly Susceptible to Ceragenins CSA-13, CSA-44, and CSA-131</p>

“…The appearance of a yellow color as a result of the enzymatic hydrolysis of ONPG is used to monitor the permeabilization of the bacterial inner membrane. 27,28 According to our study, CSA-131 exerted the greatest disruption of the inner membrane integrity in E. coli BAA 2471 ( Figure 6A), K. pneumoniae subsp. pneumoniae BAA 2472 ( Figure 6C), and K. pneumoniae BAA 2473 ( Figure 6D).…”

“…However, in the case of damage to the outer membrane, enhanced uptake of NPN as well as increased fluorescence can be observed. 27 Data presented in Figure 5A-D indicate that all CSAs studied were able to permeabilize the outer bacterial membrane in a dose-dependent manner. It is notable that CSA-131 at doses of 1-10 μg/mL caused a 1.5-2-fold increase in the outer membrane disruption compared to the control ( Figure 5A-D).…”

Enzyme Bioink for the 3D Printing of Biocatalytic Materials

“…They represent a promising class of bioactive materials for applications in biocatalytic reactors, [25][26][27] sensors 23,[28][29][30] but also as an additive to aid tissue engineering. 18 They are ideal candidates for adding functionality to biocatalytic hydrogels, due to their biocompatibility and high substrate specificity and selectivity, respectively.…”

Χαρακτηρισμός των βιολογικών δράσεων και των βιοχημικών μηχανισμών δράσης κλασμάτων από ρητίνη και φύλλα του Pistacia lentiscus L. var. chia και η εμπλοκή τους στη σηματοδότηση γλυκοκορτικοειδών ορμονών

“…Η αντίδραση που λαμβάνει χώρα βασίζεται στην υδρόλυση του ONPG σε β-D-γαλακτόζη και ortho-nitrophenol (ONP) από το ένζυμο της β-γαλακτοσιδάσης. Το ONP παράγει κίτρινο χρώμα το οποίο φωτομετρείται στα 420nm (Εικόνα 16)[221]. Αντίδραση β-γαλακτοσιδάσης[221].…”

“…Το ONP παράγει κίτρινο χρώμα το οποίο φωτομετρείται στα 420nm (Εικόνα 16)[221]. Αντίδραση β-γαλακτοσιδάσης[221].…”