Biology subjects

Golyshin, P. N.

Publications and source records attributed to Golyshin, P. N..

4 recordsLinked to original sources

bioRxiv2022

Thermophilic carboxylesterases from hydrothermal vents of the volcanic island of Ischia active on synthetic and biobased polymers and mycotoxins

Hydrothermal vents have a widespread geographical distribution and are of high interest for investigating microbial communities and robust enzymes for various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes (Geobacillus and Brevibacillus spp.), Proteobacteria and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate (PHB) and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus spp., and to some extent, Fontimonas and Schleiferia spp. The screening at 37-70{o}C of metagenomic fosmid library from above enrichment cultures resulted in identification and successful production in Escherichia coli of three hydrolases (IS10, IS11 and IS12), all derived from yet uncultured Chloroflexota and showing low sequence identity (33-56%) to characterized enzymes. Enzymes exhibited maximal esterase activity at temperatures 70-90{o}C, with IS11 showing the highest thermostability (90% activity after 20 min incubation at 80{o}C). IS10 and IS12 were highly substrate-promiscuous and hydrolysed all 51 monoester substrates tested. Enzymes were active with polyesters (PLA and polyethylene terephthalate model substrate, 3PET) and mycotoxin T-2 (IS12). IS10 and IS12 had a classical /{beta} hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in the hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 [A] revealed the presence of the N-terminal {beta}-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 includes catalytic residues Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain encloses the catalytic cleft like a lid contributing to substrate binding. Thus, this study has identified novel thermotolerant carboxylesterases with a broad substrate range including polyesters and mycotoxins for potential applications in biotechnology. IMPORTANCEHigh-temperature-active microbial enzymes are important biocatalysts for many industrial applications including recycling of synthetic and biobased polyesters increasingly used in textiles, fibres, coatings and adhesives. Here, we have discovered three novel thermotolerant carboxylesterases (IS10, IS11 and IS12) from high-temperature enrichment cultures from the Ischia hydrothermal vents incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest "effective volumes" among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous compared to IS10 and IS12, IS11 had a higher thermostability with high temperature optimum (80-90 {o}C) for activity, hydrolysed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity in these enzymes makes them attractive candidates for further optimisation and potential application in plastics recycling.

microbiology↗

bioRxiv2022

The mobility of the cap domain is essential for the substrate promiscuity of a family IV esterase from sorghum rhizosphere microbiome

Metagenomics offers the possibility to screen for versatile biocatalysts. In this study, the microbial community of the Sorghum bicolor rhizosphere was spiked with technical cashew nut shell liquid, and after incubation, the eDNA was extracted and subsequently used to build a metagenomic library. We report the biochemical features and crystal structure of a novel esterase from the family IV, EH0, retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH0 (Topt, 50 {degrees}C; Tm, 55.7 {degrees}C; pHopt, 9.5) was stable in the presence of 10-20% v/v organic solvents and exhibited hydrolytic activity against p-nitrophenyl esters from acetate to palmitate, preferably butyrate (496 U mg-1), and a large battery of 69 structurally different esters (up to 30.2 U mg-1), including bis(2-hydroxyethyl)-terephthalate (0.16 {+/-} 0.06 U mg-1). This broad substrate specificity contrasts with the fact that EH0 showed a long and narrow catalytic tunnel, whose access appears to be hindered by a thigth folding of its cap domain. We propose that this cap domain is a highly flexible structure whose opening is mediated by unique structural elements, one of which is the presence of two contiguous proline residues likely acting as possible hinges, that altogether allow for the entrance of the substrates. Therefore, this work provides a new role for the cap domain, which until now was thought to be immobile elements that contain hydrophobic patches involved in substrate pre-recognition and in turn substrate specificity within family IV esterases. IMPORTANCEA better understanding of structure-function relationships of enzymes allows revealing key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH0, a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH0 revealed the potential of the sorghum rhizosphere microbiome as a source of enzymes with interesting properties, such as pH and solvent tolerance and remarkably broad substrate promiscuity. Its structure resembled those of homologous proteins from mesophilic Parvibaculum and Erythrobacter spp. and hyperthermophilic Pyrobaculum and Sulfolobus spp. and had a very narrow, single-entry access tunnel to the active site, access which is controlled by a capping domain that includes a number of not conserved proline residues. These structural markers, distinct from those of other substrate promiscuous esterases, can help tuning substrate profiles beyond tunnel and active site engineering.

biochemistry↗

bioRxiv2020

Multidrug Resistance and High Prevalence of class 1 Integrons in Escherichia coli Isolated from Irrigation Water and Vegetables in Parts of Nsukka and Enugu, Nigeria

In spite of treated wastewater presenting itself as an attractive alternative to scarce quality water in the developing countries, the associated contamination of fresh produce by irrigation waters leading to outbreak of foodborne illnesses is on the rise. Horizontal transfer of integrons play important role in the spread and maintenance of antimicrobial resistance among strains of Escherichia coli. This study assessed the effluents from the University of Nigeria, Nsukka Wastewater Treatment Plant (UNN-WWTP) as well as vegetables irrigated with the effluent, and vegetables sold in selected markets from Nsukka and Enugu cities for the presence of E. coli and determined the prevalence integrons in multidrug-resistant isolates. Isolation of E. coli was done using eosin methylene blue agar and isolates subjected to Gram staining for identification of presumptive colonies. Confirmation of E. coli was achieved by polymerase chain reaction (PCR) technique, targeting beta-glucuronidase (uidA). Resistance to antibiotics was determined using the Bauer-Kirby disk diffusion assay and the Clinical and Laboratory Standard Institute criteria. Integrons were detected by multiplex PCR using primers specific for class 1 and 2 integrons. A total of 178 E. coli isolates were obtained from WWTP effluent (41), and vegetables from greenhouse (46), farms (55) and market (36). Multi-drug resistance was detected in all the isolates, ranging from five-drug resistance in a single isolate to 16-drug resistance patterns in two different isolates. Of the total isolates, class 1 integrons were abundantly detected in 175 (98.3%) and class 2 in 5 (2.8%). All the class 2 integrons were found in isolates that were positive for class 1. The high detection of E. coli in the studied effluent and vegetables pose potential public health hazards heightened by observed multidrug resistance in all the isolates and the high prevalence of class 1 integron. It is concluded that the vegetable samples are significant reservoirs for potentially pathogenic E. coli. Therefore, vegetable irrigation farming with unsafe water should be discontinued, while appropriate improvement strategies to ensure compliance should be facilitated without further delay.

microbiology↗

bioRxiv2019

Differential polysaccharide utilization is the basis for a nanohaloarchaeon : haloarchaeon symbiosis.

Nanohaloarchaeota, a clade of diminutive archaea, with small genomes and limited metabolic capabilities, are ubiquitous in hypersaline habitats, which they share with the extremely halophilic and phylogenetically distant euryarchaea. Some of these nanohaloarchaeota and euryarchaea appear to interact with each other. In this study, we investigate the genetic and physiological nature of their relationship. We isolated the nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh and the haloarchaeon Halomicrobium sp. LC1Hm from a solar saltern, reproducibly co-cultivated these species, sequenced their genomes, and characterized their metabolic/trophic interactions. The nanohaloarchaeon is a magnesium-dependent aerotolerant heterotrophic anaerobe of the DPANN superphylum; it lacks respiratory complexes and its energy production relies on fermentative metabolism of sugar derivatives, obtained by depolymerizing alpha-glucans or by acquiring the chitin monomer N-acetylglucosamine from the chitinolytic haloarchaeal host. Halomicrobium is a member of the class Halobacteria and a chitinotrophic aerobe. The nanohaloarchaeon lacks key biosynthetic pathways and is likely to be provided with amino acids, lipids, nucleotides and cofactors via physical contact with its host Halomicrobium. In turn, the ability of Ca. Nanohalobium to hydrolyse alpha-glucans boosts the hosts growth in the absence of a chitin substrate. These findings suggest that at least some members of the nanohaloarchaea, previously considered ecologically unimportant given their limited metabolic potential, in fact may play significant roles in the microbial carbon turnover, food chains, and ecosystem function. The behaviour of Halomicrobium, which accommodates the colonization by Ca. Nanohalobium, can be interpreted as a bet-hedging strategy, maximizing its long-term fitness in a habitat where the availability of carbon substrates can vary both spatially and temporarily.

microbiology↗