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)

Colourimetric Plate Assays Based on Functionalized Gelatine Hydrogel Useful for Various Screening Purposes in Enzymology

Hydrogels are intensively investigated biomaterials due to their useful physicochemical and biological properties in bioengineering. In particular, naturally occurring hydrogels are being deployed as carriers for bio-compounds. We used two approaches to develop a plate colourimetric test by immobilising (1) ABTS or (2) laccase from Trametes versicolor in the gelatine-based hydrogel. The first system (1) was applied to detect laccase in aqueous samples. We investigated the detection level of the enzyme between 0.05 and 100 µg/mL and pH ranging between 3 and 9; the stability of ABTS in the solution and the immobilised form, as well as the retention functional property of the hydrogel in 4 °C for 30 days. The test can detect laccase within 20 min in the concentration range of 2.5–100 µg/mL; is effective at pH 3–6; preserves high stability and functionality under storage and can be also successfully applied for testing samples from a microbial culture. The second system with the immobilised laccase (2) was tested in terms of substrate specificity (ABTS, syringaldazine, guaiacol) and inhibitor (NaN3) screening. ABTS appeared the most proper substrate for laccase with detection sensitivity CABTS > 0.5 mg/mL. The NaN3 tested in the range of 0.5–100 µg/mL showed a distinct inhibition effect in 20 min for 0.5 µg/mL and total inhibition for ≥75 µg/mL

Functional Properties of Two-Component Hydrogel Systems Based on Gelatin and Polyvinyl Alcohol—Experimental Studies Supported by Computational Analysis

The presented research is focused on an investigation of the effect of the addition of polyvinyl alcohol (PVA) to a gelatin-based hydrogel on the functional properties of the resulting material. The main purpose was to experimentally determine and compare the properties of hydrogels differing from the content of PVA in the blend. Subsequently, the utility of these matrices for the production of an immobilized invertase preparation with improved operational stability was examined. We also propose a useful computational tool to predict the properties of the final material depending on the proportions of both components in order to design the feature range of the hydrogel blend desired for a strictly specified immobilization system (of enzyme/carrier type). Based on experimental research, it was found that an increase in the PVA content in gelatin hydrogels contributes to obtaining materials with a visibly higher packaging density, degree of swelling, and water absorption capacity. In the case of hydrolytic degradation and compressive strength, the opposite tendency was observed. The functionality studies of gelatin and gelatin/PVA hydrogels for enzyme immobilization indicate the very promising potential of invertase entrapped in a gelatin/PVA hydrogel matrix as a stable biocatalyst for industrial use. The molecular modeling analysis performed in this work provides qualitative information about the tendencies of the macroscopic parameters observed with the increase in the PVA and insight into the chemical nature of these dependencies

Comparative Study on Enzyme Immobilization Using Natural Hydrogel Matrices—Experimental Studies Supported by Molecular Models Analysis

Currently, great attention is focused on conducting manufacture processes using clean and eco-friendly technologies. This research trend also relates to the production of immobilized biocatalysts of industrial importance using matrices and methods that fulfill specified operational and environmental requirements. For that reason, hydrogels of natural origin and the entrapment method become increasingly popular in terms of enzyme immobilization. The presented work is the comparative research on invertase immobilization using two natural hydrogel matrices—alginate and gelatin. During the study, we provided the molecular insight into the structural characteristics of both materials regarding their applicability as effective enzyme carriers. In order to confirm our predictions of using these hydrogels for invertase immobilization, we performed the typical experimental studies. In this case, the appropriate conditions of enzyme entrapment were selected for both types of carrier. Next, the characterization of received invertase preparations was made. As a final experimental result, the gelatin-based hydrogel was selected as an effective carrier for invertase immobilization. Hereby, using mild conditions and a pro-ecological, biodegradable matrix, it was possible to obtain very stable and reactive biocatalyst. The choice of gelatin-immobilized invertase preparation was compatible with our predictions based on the molecular models of hydrogel matrices and enzyme used

Development and Validation of a Virtual Gelatin Model Using Molecular Modeling Computational Tools

To successfully design and optimize the application of hydrogel matrices one has to effectively combine computational design tools with experimental methods. In this context, one of the most promising techniques is molecular modeling, which requires however accurate molecular models representing the investigated material. Although this method has been successfully used over the years for predicting the properties of polymers, its application to biopolymers, including gelatin, is limited. In this paper we provide a method for creating an atomistic representation of gelatin based on the modified FASTA codes of natural collagen. We show that the model created in this manner reproduces known experimental values of gelatin properties like density, glass-rubber transition temperature, WAXS profile and isobaric thermal expansion coefficient. We also present that molecular dynamics using the INTERFACE force field provides enough accuracy to track changes of density, fractional free volume and Hansen solubility coefficient over a narrow temperature regime (273–318 K) with 1 K accuracy. Thus we depict that using molecular dynamics one can predict properties of gelatin biopolymer as an efficient matrix for immobilization of various bioactive compounds, including enzymes

Biodegradable polylactide and thermoplastic starch blends as drug release device – mass transfer study

Four different compositions of polylactide/thermoplastic starch blends (PLA/TPS blends) for application as drug carriers were examined. Initially, using cyanocobalamin (1.355 kDa) as a model compound, the blend with the highest starch content (wt. 60%) was selected for further research of mass transfer phenomenon. In this case, different concentrations of acetaminophen (0.151 kDa), doxorubicin hydrochloride (0.580 kDa) and cyanocobalamin (1.355 kDa) were used for determination of particular releasing profiles. Besides from the comparative analysis of obtained results, the values of the overall mass transfer coefficient (K) were calculated for each of tested drug molecules. Depending on the size and properties of used compound, determined values of the coefficient range from 10−11 to 10−13 m/s. Based on these outcomes, it could be stated that PLA/TPS blend selected in preliminary research, seems to be preferred material for fabrication of long-term drug delivery systems, which could be successfully applied for example in anti-cancer therapy

Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

<div><p>Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH₂), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH₂. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH₂ complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.</p></div

Stability of Tyrosinase in Presence of Reaction Mixture Components with Borate Buffer.

<p>Relative activity of native (black) and immobilized enzyme (gray) after 1 h incubation at 30°C in 0.5 M borate buffer, pH 9 (Control) containing 1 mM L-tyrosine (L-tyr), 1 mM L-DOPA, 2 mM ascorbic acid (AH₂), or 6.7 mM hydroxylamine (HA).</p

Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Reaction Systems with Borate Buffer (BB; 0.5 M), and/or Ascorbic Acid (AH₂; 2 mM), and/or Hydroxylamine (HA; 6.7 mM).

<p>Selected Parameters of Processes of 1 mM L-tyrosine Hydroxylation Using Native or Immobilized Tyrosinase in Reaction Systems with Borate Buffer (BB; 0.5 M), and/or Ascorbic Acid (AH₂; 2 mM), and/or Hydroxylamine (HA; 6.7 mM).</p

Simplified Schematic Representation of L-tyrosine Hydroxylation to L-DOPA by Tyrosinase in the Reaction System with Boron Buffer.

<p>Boron buffer was applied to create complexes with L-DOPA and ascorbic acid (AH₂) that minimize suicide inactivation caused by both compounds; AH₂ was added to reduce DOPA-quinone back to L-DOPA; hydroxylamine (HA) was added to reduce <i>met</i>-Tyr to <i>deoxy</i>-Tyr in reduced accessibility of L-DOPA and AH₂. The scheme was designed according to that described by Marin-Zamora et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164213#pone.0164213.ref028" target="_blank">28</a>]. The black arrows denote the reaction scheme without additives.</p