Ilya V. Lyagin

Production of biofuels from pretreated microalgae biomass by anaerobic fermentation with immobilized Clostridium acetobutylicum cells.

The purpose of this work was to study the possible use of pretreated biomass of various microalgae and cyanobacteria as substrates for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum cells immobilized into poly(vinyl alcohol) cryogel. To this end, the biochemical composition of photosynthetic microorganisms cultivated under various conditions was studied. The most efficient technique for pretreating microalgal biomass for its subsequent conversion into biofuels appeared to be thermal decomposition at 108 °C. For the first time the maximum productivity of the ABE fermentation in terms of hydrogen (8.5 mmol/L medium/day) was obtained using pretreated biomass of Nannochloropsis sp. Maximum yields of butanol and ethanol were observed with Arthrospira platensis biomass used as the substrate. Immobilized Clostridium cells were demonstrated to be suitable for multiple reuses (for a minimum of five cycles) in ABE fermentation for producing biofuels from pretreated microalgal biomass.

A simple and highly effective catalytic nanozyme scavenger for organophosphorus neurotoxins

ABSTRACT A simple and highly efficient catalytic scavenger of poisonous organophosphorus compounds, based on organophosphorus hydrolase (OPH, EC 3.1.8.1), is produced in aqueous solution by electrostatic coupling of the hexahistidine tagged OPH (His6‐OPH) and poly(ethylene glycol)‐b‐poly(l‐glutamic acid) diblock copolymer. The resulting polyion complex, termed nano‐OPH, has a spherical morphology and a diameter from 25 nm to 100 nm. Incorporation of His6‐OPH in nano‐OPH preserves catalytic activity and increases stability of the enzyme allowing its storage in aqueous solution for over a year. It also decreases the immune and inflammatory responses to His6‐OPH in vivo as determined by anti‐OPH IgG and cytokines formation in Sprague Dawley rats and Balb/c mice, respectively. The nano‐OPH pharmacokinetic parameters are improved compared to the naked enzyme suggesting longer blood circulation after intravenous (iv) administrations in rats. Moreover, nano‐OPH is bioavailable after intramuscular (im), intraperitoneal (ip) and even transbuccal (tb) administration, and has shown ability to protect animals from exposure to a pesticide, paraoxon and a warfare agent, VX. In particular, a complete protection against the lethal doses of paraoxon was observed with nano‐OPH administered iv and ip as much as 17 h, im 5.5 h and tb 2 h before the intoxication. Further evaluation of nano‐OPH as a catalytic bioscavenger countermeasure against organophosphorus chemical warfare agents and pesticides is warranted.

Polyhistidine-containing organophosphorus hydrolase with outstanding properties

The catalytic and physical–chemical properties of organophosphorus hydrolase (OPH) modified by the addition of an N-terminal dodecahistidine tag (His12-OPH) have been investigated. Introduction of the His12-tag caused a 30- and 74-fold increase in catalytic efficiency of the enzyme with parathion and methyl parathion, respectively, compared to OPH. Concurrently, the His12-OPH had a more alkaline pH-optimum and extended temperature range than OPH and OPH modified with a hexahistidine tag. A study of His12-OPH thermostability showed that the enzyme had a tendency to oligomerise. This resulted in a decrease in the enzymatic activity of His12-OPH at temperatures <50°C, but provided the enzyme with much higher thermostability at temperatures >50°C, compared to OPH.

Immobilized biocatalysts for detoxification of neurotoxic organophosphorous compounds

New biocatalysts were developed using organophosphorus hydrolase (OPH, EC 3.1.8.1) with a polyhistidine tag at the N-terminus of the protein (His6-OPH). The use of His6-OPH together with previously developed approaches for the entrapment of cells into poly(vinyl alcohol) cryogels and covalent immobilization of enzymes into porous fabric materials, impregnated with chemically cross-linked chitosan sulphate gel, enabled dramatic improvement of catalytic characteristics against various organophosphorous compounds (OPCs; Paraoxon, Coumaphos, Methyl parathion, etc.). The polyhistidine tag of OPH was used to create a new immobilized biocatalyst using metal-chelating carriers, such as Ni2+-nitrilotriacetic acid-agarose and Co2+-iminodiacetic acid-polyacrylamide cryogel. The latter biocatalyst had high activity and stability for the continuous hydrolysis of OPCs.

Catalytic characteristics of enzyme-polyelectrolyte complexes based on hexahistidine-containing organophosphorus hydrolase

A number of covalent and non-covalent polyelectrolyte complexes based on enzyme such as hexahistidine-containing organophosphorus hydrolase was developed in this work. Polyanions and polycations were used in the form of block-copolymers with poly(ethylene glycol). To obtain covalent complexes of the enzyme, different coupling agents (glutaric aldehyde, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt) were tried. The best catalytic efficiency of the action of the covalently bound complex ((2.8 ± 0.3) × 108 M−1 s−1) was obtained for samples with EDC. The modification of the protein surface was undertaken as an approach to its further biomedical application.

Complex effect of lignocellulosic biomass pretreatment with 1-butyl-3-methylimidazolium chloride ionic liquid on various aspects of ethanol and fumaric acid production by immobilized cells within SSF

The pretreatment of softwood and hardwood samples (spruce and hornbeam wood) with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was undertaken for further simultaneous enzymatic saccharification of renewable non-food lignocellulosic biomass and microbial fermentation of obtained sugars to ethanol and fumaric acid. A multienzyme cocktail based on cellulases and yeast or fungus cells producing ethanol and fumaric acid were the main objects of [Bmim]Cl influence studies. A complex effect of lignocellulosic biomass pretreatment with [Bmim]Cl on various aspects of the process (both action of cellulases and microbial conversion of hydrolysates to target products) was revealed. Positive effects of the pretreatment with [Bmim]Cl included decreasing the lignin content in the biomass, and increasing the effectiveness of enzymatic hydrolysis and microbial transformation of pretreated biomass. Immobilized cells of both yeasts and fungi possessed improved productive characteristics in the biotransformation of biomass pretreated with [Bmim]Cl to ethanol and fumaric acid.

Antioxidants as stabilizers for His6-OPH: is this an unusual or regular role for them with enzymes?

The effect of 14 different antioxidants on the activity of a hexahistidine-tagged organophosphorus hydrolase (His6-OPH) has been studied in vitro. It has been found that antioxidants can have a positive, neutral or negative effect on the activity of His6-OPH in a native form or in the form of an enzyme-polyelectrolyte complex, while the enzyme itself does not affect their antioxidant activity. A significant stabilizing effect of a number of antioxidants on His6-OPH has been shown against its inhibiting with organic solvents (DMSO and isopropyl alcohol). The kinetics of the process has been studied. Based on molecular docking of all tested antioxidants to the surface of His6-OPH dimer, options of their localization have been identified. These data were used to explain the revealed stabilizing effect of the antioxidants on the enzyme as well as their negative influence on His6-OPH activity.

Nanozyme technology at Moscow State University. Achievements and development perspectives

The work describes novel functional bionanosystems for treatment and diagnostics on the basis of proteins, enzymes, polymeric coatings, and magnetic nanoparticles developed at Lomonosov Moscow State University Laboratory for Chemical Design of Bionanomaterials in collaboration with scientists from UNC Eshelman School of Pharmacy (USA). The properties of enzymes (superoxide dismutase, catalase, organophosphate hydrolase, and lysines of bacteriophages) and other drug molecules immobilized in polymeric complexes, as well as the methods for targeted drug delivery using cell-mediated systems and magnetic nanoparticles in in vitro and in vivo operating conditions, are discussed. Physical and chemical characteristics, including data on the functional properties of the nanoformulations, are obtained. The nanoformulations developed demonstrated high potential therapeutic efficacy for the treatment of central nervous system and brain diseases, inflammations (including inflammatory diseases of the eye), cancer and infectious diseases, neurotoxic injury, and others. The possibilities of remote control biochemical reactions using a nonheating low-frequency alternating magnetic field (AMF) for the controlled release of drugs are analyzed in the review. The experimental results of the AMF effects on bionanosystems containing magnetic nanoparticles, such as changing the catalytic activities of enzymes bound to magnetic nanoparticles and ‘disordering’ of the lipid bilayer in membranes, are considered.

Effect of dimerization on the catalytic properties of native and chimeric organophosphorus hydrolase determined by molecular modeling of the enzyme structure

The mechanisms of structural reorganization of a protein globule resulting in changes in the stability of enzyme depending on the pH of the medium were revealed by molecular dynamics modeling of the structure of the organophosphorus hydrolase (OPH) dimer. The same reorganization leads to changes in the substrate specificity of the enzyme depending on its genetic modification, which was experimentally confirmed. Based on the obtained theoretical data, it was concluded that the dimerization significantly affects the catalytic characteristics of the native form of OPH. An analysis of the whole set of theoretical and experimental data concerning the characteristics of the OPH chimeric forms suggests that their changes with respect to native OPH is a decrease in the level of dimerization of chimeric protein molecules.

Novel approach to quorum quenching: rational design of antibacterials in combination with hexahistidine-tagged organophosphorus hydrolase

Abstract N-acyl homoserine lactones (AHLs) are quorum sensing (QS) signal molecules used by most Gram-negative pathogenic bacteria. In this article the lactonase activity of the preparations based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) towards AHLs was studied. Initially, three of the most interesting β-lactam antibiotics were selected from seven that were trialed during molecular docking to His6-OPH. Combinations of antibiotics (meropenem, imipenem, ceftriaxone) and His6-OPH taken in the native form or in the form of non-covalent enzyme-polyelectrolyte complexes (EPCs) with poly(glutamic acid) or poly(aspartic acid) were obtained and investigated. The lactonase activity of the preparations was investigated under different physical-chemical conditions in the hydrolysis of AHLs [N-butyryl-D,L-homoserine lactone, N-(3-oxooctanoyl)-D,L-homoserine lactone, N-(3-oxododecanoyl)-L-homoserine lactone]. An increased efficiency of catalytic action and stability of the lactonase activity of His6-OPH was shown for its complexes with antibiotics and was confirmed in trials with bacterial strains. The broadening of the catalytic action of the enzyme against AHLs was revealed in the presence of the meropenem. Results of molecular docking of AHLs to the surface of the His6-OPH dimer in the presence of antibiotics allowed proposing the mechanism of such interference based on a steric repulsion of the carbon chain of hydrolyzed AHLs by the antibiotics bounded to the enzyme surface.