Stepan Shipovskov

Antifouling enzymes and the biochemistry of marine settlement.

Antifouling coatings are used extensively on marine vessels and constructions, but unfortunately they are found to pose a threat to the marine environment, notably due to content of metal-based biocides. Enzymes have repeatedly been proposed as an alternative to traditional antifouling compounds. In this review, the enzymes claimed to hold antifouling activity are classified according to catalytic functions. The enzyme functions are juxtaposed with the current knowledge about the chemistry of settlement and adhesion of fouling organisms. Specific focus will be on bacteria, microalgae, invertebrate larvae and macroalgae zoospores. Two main concepts in enzyme-based antifouling are identified: breakdown of adhesive components and catalytic production of repellent compounds in-situ. The validity of the various modes of action is evaluated and the groups of enzymes with the highest potential are highlighted.

Soft interactions at nanoparticles alter protein function and conformation in a size dependent manner.

Weak protein-nanoparticle (NP) interactions are studied in a low binding regime as a model for the soft protein corona around nanoparticles in complex biological fluids. Noncovalent, reversible interactions between Subtilisin Carlsberg (SC) and silica NPs shows significant alteration in conformation and enzymatic activity in a NP-size dependent manner. Very weak interactions between SC and silica NPs were revealed by centrifugation-based separations and further supported by small-angle X-ray scattering, while bovine serum albumin was used as a strongly interacting reference. Secondary and tertiary structure changes of SC were studied via circular dichroism and correlated to enzymatic activity where the enzyme kinetics showed a critical role for nanoparticle size.

Catalytic activity of laccases in aqueous solutions of ionic liquids

The ionic liquids, [bmim]Br and [bmim][N(CN)2] (where [bmim] = 1-butyl-3-methylimidazolium), stimulated laccase-catalysed oxidation of catechol when provided at concentrations between 10–20% and 50–60% (v/v) in water, respectively. However, activity was inhibited at higher and lower concentrations. [bmim][BF4] was inhibitory at all concentrations tested, but residual activity was still retained in [bmim][BF4] with ≤ 20% water.

Electrochemical DNA sandwich assay with a lipase label for attomole detection of DNA

A fast and sensitive electrochemical lipase-based sandwich hybridization assay for detection of attomole levels of DNA has been developed. A combination of magnetic beads, used for pre-concentration and bioseparation of the analyte with a lipase catalyst label allowed detection of DNA with a limit of 20 amol.

Peroxidase biocathodes for a biofuel cell development

In terms of sustainable energy production, enzymatic biofuel cells are attractive for a number of special applications, such as disposable implantable power suppliers for medical sensor-transmitters and drug delivery/activator systems and self-powered enzyme-based biosensors; they offer practical advantages of using abundant organic raw materials as biofuels for clean and sustainable energy production. In this paper we discuss what power densities can be expected from enzymatic biofuel cells and what are the possibilities and limits for their optimization. As example, in this work, the designed hybrid biofuel cell formed by the battery type Zn anode and the biocathode, comprising horseradish peroxidase (HRP) immobilized on graphite and utilizing H2O2 as an oxidizer, is considered. The cell yields the open circuit voltage Voc of 1.68 and 1.57 V and the short-circuit current density isc of 800 μA cm−2 at pH 6 and 580 μA cm−2 at pH 7.45 in quiescent solutions. The biofuel cell operated at 1.5 V for 6 days; t...

Quantitative Turbidimetric Assay of Enzymatic Gram-Negative Bacteria Lysis

In this Technical Note, the quantitative turbidimetric assay for determination of the bacteriolytic activity of enzymes with gram-negative bacteria is proposed. The reactivity of hen white-egg lysozyme toward gram-negative E. coli intact cells was studied. It was found that the highest lysis rate occurred at pH 8.9 in the system containing 0.03 M NaCl. The mechanism of the reaction is discussed and applied for the quantitative evaluation of the reaction rate. The proposed method enables fast, reliable, and reproducible analysis of bacteriolytic activity of lysozyme with gram-negative bacteria.

A ROBUST METHOD OF DETERMINATION OF HIGH CONCENTRATIONS OF PEPTIDES AND PROTEINS

In this paper, we pioneer application of a unique method of protein determination by coloring peptide bonds for analysis of a variety of biomolecules with different grades of purity (e.g., oligopeptides, membrane, and glycol proteins). We demonstrated that the calibration curve for all studied molecules is universal and linear within 0.1 to 1.2mg protein content range. The assay thus can be used to analyze peptides without preliminary dilutions and calibration in up to 1g/ml solutions of peptides, which is crucial for many biotechnological processes, such as development of coatings, scaffolds, and biocompatible materials.

Biocatalysis of theophylline oxidation by microbial theophylline oxidase in the presence of non-physiological electron acceptors

Microbial theophylline oxidase (ThOx) is a redox enzyme catalysing 8-hydroxylation of theophylline to form 1,3-dimethyluric acid. In this work, ThOx has been characterized as a fragile haem-containing protein complex composed of several non-covalently bound dynamic domains with molecular weights of around 60 and 210 kDa, and capable of formation of 1.5 MDa assemblies as well. The rate of theophylline oxidation by ThOx with the non-physiological electron acceptor ferricyanide was 0.17 s−1, approaching that with cytochrome c, 0.33 s−1. The apparent catalytic constant depended on the electron acceptor concentration. At concentrations lower than 0.2 mM the reaction did not fit the Michaelis–Menten scheme, and some non-catalytic processes dominated in the overall reaction. The kinetics of ThOx catalysis were also studied at electrodes modified with self-assembled monolayers (SAM) of hydroxyl- and amine-terminated alkanethiols. Different compositions of the SAM provide different orientations of ThOx on these layers. Depending on the orientation of ThOx onto the SAM-modified electrodes, the heterogeneous electron transfer (ET) constant, ks, which characterizes the ET reaction between the electrodes and the haem of ThOx (Eo/ of 87 mV (NHE)) was 0.4 s−1 and 3.2 s−1. Only the low-ET-rate orientation appeared to be productive for the electrocatalytic function of ThOx, giving a reaction similar to that with ferricyanide and cytochrome c. The apparent efficiency of ThOx bioelectrocatalysis in the absence of mediators was substantially lower than that mediated by ferricyanide or cytochrome c. This lower efficiency is consistent with a correspondingly lower amount of ThOx being in direct ET contact with the electrodes and thus involved in electrocatalysis.

Lysis of Escherichia coli cells by lysozyme: discrimination between adsorption and enzyme action.

The key factors of enzymatic lysis of cells are the interaction between the enzyme and the cell - catalytic and non-catalytic adsorption of enzyme on cell surface. Here, the studies of lysis of intact Escherichia coli cells by chicken egg white lysozyme were performed. It was found that the ionic strength has a dual effect onto the system. On the one hand, the desorption constant of the enzyme increases with the increase of the solution ionic strength, which results in a better enzyme performance. On the other hand, due to the higher osmosis, the cell lysis rate decreases with the increasing of ionic strength of the system. It was found that pH 8.6 and 30 mM NaCl are optimal conditions for lysis of E. coli cells by lysozyme.

Implementation of cross-linked enzyme aggregates of proteases for marine paint applications

Cross-linked enzyme aggregates (CLEAs) of proteases were tested in artificial seawater (ASW) both as it is and as a component of the paint. It is found that all CLEAs have tolerance to xylene and have great stability in dried paint. Moreover, CLEA Bacillus licheniformis shows 900% activation during the storage in ASW in dried paint.