Biotechnologically relevant enzymes in strains of Pseudomonas genus: Identification and recombinant expression of laccases and lipases

Bakterije roda Pseudomonas imaju sposobnost produkcije i razgradnje širokog spektra jedinjenja. Cilj ove doktorske teze bio je da se u kolekciji sojeva roda Pseudomonas identifikuju i rekombinantno eksprimiraju nove lakaze i lipaze, i evaluira njihov aplikativni potencijal. Sedam sojeva obuhvaćeno je analizom oksidativne i hidrolitičke aktivnosti prema pet različitih supstrata. Kod sojeva Pseudomonas putida F6, P. putida KT2440 i P. putida CA-3, utvrđeno je prisustvo gena koji kodiraju lakaze. Proteini McoCA3, McoKT, Cbp i CopA heterologo su eksprimirani u E. coli domaćinu, prečišćeni i okarakterisani. Pokazana je njihova aktivnost u širokom pH i temperaturnom opsegu kao i visoka termostabilnost. Ispitana je i sposobnost enzima da razgrade boje koje se koriste u tekstilnoj industriji, potencijalne zagađivače voda. Ćelijski ekstrakt koji je sadržao CopA enzim iz P. putida F6 pokazao je najznačajniju aktivnost razgradivši pet od sedam testiranih boja. Kod sojeva P. aeruginosa PAO1 i P. chlororaphis B-561, detektovana je lipolitička aktivnost i utvrđen potencijal za biorazgradnju polimera kao što su polihidroksialkanoati srednjeg lanca i polikaprolakton, a degradacioni potencijal soja B-561 potvrđen je i u modelu komposta. Homologo su eksprimirana tri proteina – LipA, PlcB i LipA, ali je zaključeno da nije došlo do njihove sekrecije. U ovom radu je pokazano da su Pseudomonas sojevi dobar izvor biotehnološki značajnih enzima lakaza i lipaza. Rekombinantna ekspresija lakaza dala je funkcionalne enzime pogodne za primenu u razgradnji sintetičkih boja iz otpadnih voda. Za primenu rekombinantnih lipaza u biorazgradnji polimernih materijala neophodna je optimizacija sekrecije, ali divlji sojevi koji eksprimiraju ove enzime su dovoljno dobri biokatalizatori za njihovu razgradnju.Pseudomonas strains have the ability to produce and degrade a wide range of compounds. The main objective of this thesis was to identify, recombinantly express and characterize novel laccases and lipases from Pseudomonas spp. collection, and to evaluate their application potential. Seven strains were analyzed for oxidative and hydrolytic activity towards five different substrates. Genes encoding laccases were detected in P. putida F6, P. putida KT2440 and P. putida CA-3 strains. Proteins McoCA3, McoKT, Cbp and CopA were heterologously expressed in E. coli, purified and characterized, exhibiting broad temperature and pH range and high thermal stability. The ability of enzymes to degrade textile dyes was also examined. All enzymes proved to be very efficient in degradation of synthetic dyes, with the CopA enzyme from P. putida F6 showing the most significant activity, degrading five out of seven tested dyes. Lipolytic activity was detected in two strains: P. aeruginosa PAO1 and P. chlororaphis B-561. The potential for biodegradation of polymeric materials such as medium chain length polyhydroxyalkanoates and polycaprolactone was demonstrated, while degradation potential of B-561 was confirmed in the compost system as well. Three lipases were homologously expressed – LipA, PlcB and LipA, but they were not secreted. This study showed that Pseudomonas genus is a good source of biotechnologically relevant enzymes. Recombinant laccase expression yielded enzymes suitable for application in the degradation of persistent synthetic dyes from wastewaters. For the application of recombinant lipases in the biodegradation of polymers, optimization of the secretion is necessary, however wild type strains expressing these enzymes are good enough biocatalysts for biodegradation purpose

Biotechnologically relevant enzymes in strains of Pseudomonas genus: Identification and recombinant expression of laccases and lipases

Bakterije roda Pseudomonas imaju sposobnost produkcije i razgradnje širokog spektra jedinjenja. Cilj ove doktorske teze bio je da se u kolekciji sojeva roda Pseudomonas identifikuju i rekombinantno eksprimiraju nove lakaze i lipaze, i evaluira njihov aplikativni potencijal. Sedam sojeva obuhvaćeno je analizom oksidativne i hidrolitičke aktivnosti prema pet različitih supstrata. Kod sojeva Pseudomonas putida F6, P. putida KT2440 i P. putida CA-3, utvrđeno je prisustvo gena koji kodiraju lakaze. Proteini McoCA3, McoKT, Cbp i CopA heterologo su eksprimirani u E. coli domaćinu, prečišćeni i okarakterisani. Pokazana je njihova aktivnost u širokom pH i temperaturnom opsegu kao i visoka termostabilnost. Ispitana je i sposobnost enzima da razgrade boje koje se koriste u tekstilnoj industriji, potencijalne zagađivače voda. Ćelijski ekstrakt koji je sadržao CopA enzim iz P. putida F6 pokazao je najznačajniju aktivnost razgradivši pet od sedam testiranih boja. Kod sojeva P. aeruginosa PAO1 i P. chlororaphis B-561, detektovana je lipolitička aktivnost i utvrđen potencijal za biorazgradnju polimera kao što su polihidroksialkanoati srednjeg lanca i polikaprolakton, a degradacioni potencijal soja B-561 potvrđen je i u modelu komposta. Homologo su eksprimirana tri proteina – LipA, PlcB i LipA, ali je zaključeno da nije došlo do njihove sekrecije. U ovom radu je pokazano da su Pseudomonas sojevi dobar izvor biotehnološki značajnih enzima lakaza i lipaza. Rekombinantna ekspresija lakaza dala je funkcionalne enzime pogodne za primenu u razgradnji sintetičkih boja iz otpadnih voda. Za primenu rekombinantnih lipaza u biorazgradnji polimernih materijala neophodna je optimizacija sekrecije, ali divlji sojevi koji eksprimiraju ove enzime su dovoljno dobri biokatalizatori za njihovu razgradnju.Pseudomonas strains have the ability to produce and degrade a wide range of compounds. The main objective of this thesis was to identify, recombinantly express and characterize novel laccases and lipases from Pseudomonas spp. collection, and to evaluate their application potential. Seven strains were analyzed for oxidative and hydrolytic activity towards five different substrates. Genes encoding laccases were detected in P. putida F6, P. putida KT2440 and P. putida CA-3 strains. Proteins McoCA3, McoKT, Cbp and CopA were heterologously expressed in E. coli, purified and characterized, exhibiting broad temperature and pH range and high thermal stability. The ability of enzymes to degrade textile dyes was also examined. All enzymes proved to be very efficient in degradation of synthetic dyes, with the CopA enzyme from P. putida F6 showing the most significant activity, degrading five out of seven tested dyes. Lipolytic activity was detected in two strains: P. aeruginosa PAO1 and P. chlororaphis B-561. The potential for biodegradation of polymeric materials such as medium chain length polyhydroxyalkanoates and polycaprolactone was demonstrated, while degradation potential of B-561 was confirmed in the compost system as well. Three lipases were homologously expressed – LipA, PlcB and LipA, but they were not secreted. This study showed that Pseudomonas genus is a good source of biotechnologically relevant enzymes. Recombinant laccase expression yielded enzymes suitable for application in the degradation of persistent synthetic dyes from wastewaters. For the application of recombinant lipases in the biodegradation of polymers, optimization of the secretion is necessary, however wild type strains expressing these enzymes are good enough biocatalysts for biodegradation purpose

Antimicrobial and anti-biofilm activity and biological decontamination efficiency of ED-1 emulsion

Antimicrobial and antibiofilm activity, as well as biological decontamination potential of emulsion ED-1, highly efficient in radiological decontamination of metal surfaces contaminated with uranium isotopes, was assessed. The antimicrobial potency of ED-1 was evaluated against 10 different microorganisms including four Gram-negative bacteria (Acinetobacter baumannii ATCC 19606, Escherichia coli NCTC 9001, Klebsiella pneumoniae ATCC 13803 and Pseudomonas aeruginosa NCTC 10662), four Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 6057, Listeria monocytogenes NCTC 11994, Staphylococcus aureus NCTC 6571) and two fungi (Candida albicans ATCC 10231 and Candida parapsilosis ATCC 22019). Although without strong bactericidal and fungicidal properties in standard agar diffusion assays, ED-1 effectively inhibited the growth of P. aeruginosa cells in liquid culture and more importantly, showed high potential to disperse P. aeruginosa biofilms. ED-1 was also capable to efficiently remove Bacillus subtilis ATCC 6633 spores in quantitative and a semi-quantitative biological decontamination tests on metal surfaces. Antimicrobial and antibiofilm activity and biological decontamination efficiency of ED-1 was comparable to and better than that of calcium hypochlorite solution or commercial decontaminant BX-24. This study highlighted the possibility to use ED-1, with up to 5-fold reduced amounts of calcium hypochlorite in comparison to currently used methodology, for both biological and radiological decontamination, resulting in both environmental and financial benefits

Limited aromatic pathway genes diversity amongst aromatic compound degrading soil bacterial isolates

Identifikacija i karakterizacija novih gena koji pripadaju putevima mikrobiološke razgradnje aromatičnih jedinjenja je od velikog značaja, jer su se pokazali kao izuzetno dobri biokatalizatori. U ovoj studiji, korišćenjem PCR metodologije, analizirano je prisustvo pet različitih gena iz biodegradativnog puta aromatičnih jedinjenja među 19 sredinskih izolata sa sposobnošću razgradnje širokog spektra aromatičnih jedinjenja. U slučaju 4-oksalokrotonat tautomeraze i toluen dioksigenaze, koji su detektovani kod većine sredinskih izolata, sekvence fragmenata su ukazivale na veoma ograničen diverzitet ova dva gena i visoku homologiju sa već poznatim sekvencama opisanim kod vrsta roda Pseudomonas. Korišćenjem degenerisanih prajmera konstruisanih na osnovu poznatih katehol-i naftalendioksigenaznih gena vrlo mali broj fragmenata je amplifikovan kod sredinskih izolata. Samo dve katehol 2,3-dioksigenaze iz dva izolata roda Bacillus su sekvenciranjem ukazale na različitost u odnosu na poznate sekvence, a pokazale međusobnu sličnost od 80-90%. Potencijalno tri nove katehol 1,2-dioksigenaze su identifikovane kod Bacillus sp. TN102, Gordonia sp. TN103 i Rhodococcus sp. TN112. Visok stepen homologije tautomeraza i toluen dioksigenaza među sredinskim izolatima izolovanim iz zagađene sredine ukazuje na horizontalni transfer gena, dok je ograničen uspeh u detektovanju preostala tri gena ukazao na potencijal da se među ovim izolatima mogu naći nove varijante gena iz puteva razgradnje aromatičnih jedinjenja.Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterized corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes

Biocatalytic potential of Streptomyces spp. isolates from rhizosphere of plants and mycorrhizosphere of fungi

Biocatalytic potential of Streptomyces strains isolated from the rhizosphere of plants and from mycorrhizosphere of fungi has been investigated. A total of 118 Streptomyces isolates were selected and functionally screened for 10 different biotechnologically important enzymatic activities: hydrolase (cellulase, cutinase, gelatinase, lipase, protease, polyhydroxyalkanoate (PHA) depolymerase), phenol oxidase and peroxidase (laccase, tyrosinase, and lignin peroxidase), and aminotransferase. Out of 118 tested Streptomyces spp., 90% showed at least one enzymatic activity. The most abundant were enzymes involved in the biomass degradation, as the production of cutinase, cellulase, and lignin peroxidase were detected in 31%, 40%, and 48% of the isolates, respectively. The improved specific activities of lipase (isolates BV315 and BV100) and tyrosinase (isolates BV87 and BV88) were shown in comparison with the industrially relevant activities of Pseudomonas strains. Plant rhizosphere soils were more prolific source of Streptomyces strains with biocatalytic potential in comparison with mycorrhizosphere soils. Overall, 284 enzyme activities among 118 Streptomyces isolates have been detected. This is the first comprehensive screening of Streptomyces isolates from rhizosphere and mycorrhizosphere soils for novel biocatalysts, showing that specific environmental habitats, such as rhizosphere soils, are treasure troves of Streptomyces with biocatalytic potential.This is the peer reviewed version of the paper: Biocatalytic potential of Streptomyces spp. Isolates from rhizosphere of plants and mycorrhizosphere of fungi—Spasic—2018—Biotechnology and Applied Biochemistry—Wiley Online Library. (без датума). Преузето 08. Децембар 2022., од [https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bab.1664

Supplementary data for the article: Spasic, J.; Mandic, M.; Djokic, L.; Nikodinovic-Runic, J. Streptomyces Spp. in the Biocatalysis Toolbox. Appl. Microbiol. Biotechnol. 2018, 102 (8), 3513–3536. https://doi.org/10.1007/s00253-018-8884-x

Supplementary material for: [https://doi.org/10.1007/s00253-018-8884-x]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2115

Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes

Laccases are multicopper-oxidases with variety of biotechnological applications. While predominantly used, fungal laccases have limitations such as narrow pH and temperature range and their production via heterologous protein expression is more complex due to posttranslational modifications. In comparison, bacterial enzymes, including laccases, usually possess higher thermal and pH stability, and are more suitable for expression and genetic manipulations in bacterial expression hosts. Therefore, the aim of this study was to identify, recombinantly express, and characterize novel laccases from Pseudomonas spp. A combination of approaches including DNA sequence analysis, N-terminal protein sequencing, and genome sequencing data analysis for laccase amplification, cloning, and overexpression have been used. Four active recombinant laccases were obtained, one each from P. putida KT2440 and P. putida CA-3, and two from P. putida F6. The new laccases exhibited broad temperature and pH range and high thermal stability, as well as the potential to degrade selection of synthetic textile dyes. The best performing laccase was CopA from P. putida F6 which degraded five out of seven tested dyes, including Amido Black 10B, Brom Cresol Purple, Evans Blue, Reactive Black 5, and Remazol Brilliant Blue. This work highlighted species of Pseudomonas genus as still being good sources of biocatalytically relevant enzymes

Povezanost kliničkih karakteristika i morfoloških parametara s rupturom aneurizme prednje komunikacijske arterije

We analyzed aneurysm morphology, demographic and clinical characteristics in patients with anterior communicating artery (ACoA) aneurysms to investigate the risk factors contributing to aneurysm rupture. A total of 219 patients with ACoA aneurysms were admitted to our hospital between January 2016 and December 2020, and morphological and clinical characteristics were analyzed retrospectively in 153 patients (112 ruptured and 41 unruptured). Medical records were reviewed to obtain demographic and clinical data on age, gender, presence of hemorrhage, history of hypertension, diabetes, heart disease, and kidney disease. Morphological parameters examined on 3-dimensional digital subtraction angiography included aneurysm size, neck diameter, aspect ratio, size ratio, bottleneck ratio, height/width ratio, aneurysm angle, (in)flow angle, branching angle, number of aneurysms per patient, shape of the aneurysm, aneurysm wall morphology, variation of the A1 segment, and direction of the aneurysm. Male gender, aspect ratio, height/width ratio, non-spherical and irregular shape were associated with higher odds of rupture, whilst controlled hypertension was associated with lower odds of rupture, when tested using univariate logistic regression model. In multivariate model, controlled hypertension, presence of multiple aneurysms, and larger neck diameter reduced the odds of rupture, while irregular wall morphology increased the risk of rupture. Regulated hypertension represented a significant protective factor from ACoA aneurysm rupture. We found that ACoA aneurysms in male patients and those with greater aspect ratios and height/width ratios, larger aneurysm angles, presence of daughter sacs and irregular and non-spherical shapes were at a higher risk of rupture.Analizirali smo morfologiju aneurizme, demografske i kliničke karakteristike u bolesnika s aneurizmom prednje komunikacijske arterije (ACoA) kako bismo istražili čimbenike rizika koji doprinose rupturi aneurizme. Ukupno je 219 bolesnika s aneurizmom ACoA primljeno u našu bolnicu u razdoblju od siječnja 2016. do prosinca 2020. godine, a morfološke i kliničke karakteristike analizirane su retrospektivno u 153 bolesnika (112 puknutih i 41 neprekinuta). Pregledani su medicinski zapisi kako bi se dobili demografski i klinički podaci za dob, spol, prisutnost krvarenja, povijest hipertenzije, dijabetes, srčane bolesti i bolesti bubrega. Morfološki parametri ispitani na trodimenzionalnoj digitalnoj subtrakcijskoj angiografiji uključivali su veličinu aneurizme, promjer vrata, odnos između normalne visine aneurizme i širine vrata aneurizme (aspect ratio), odnos između visine aneurizme i prosječnog promjera svih krvnih žila povezanih s aneurizmom (size ratio), odnos između širine fundusa aneurizme i širine vrata aneurizme (bottleneck ratio), odnos između najveće normalne visine aneurizme i širine aneurizme (height/width ratio), kut aneurizme, ugao ulaska tijeka krvne struje u fundus aneurizme (inflow angle), kut grananja, broj aneurizma po bolesniku, oblik aneurizme, morfologiju stijenke aneurizme, varijaciju segmenta A1 i smjer aneurizme. Muški spol, odnos između normalne visine aneurizme i širine vrata aneurizme, odnos između najveće normalne visine aneurizme i širine aneurizme, nesferičan i nepravilan oblik bili su povezani s većim izgledima za puknuće, dok je kontrolirana hipertenzija bila povezana s manjom vjerojatnosti puknuća kada je testirano primjenom modela s univarijatnom logističkom regresijom. U multivarijatnom modelu su kontrolirana hipertenzija, prisutnost više aneurizma i veći promjer vrata smanjili izglede za puknuće, dok je nepravilna morfologija stijenke povećala rizik od puknuća. Regulirana hipertenzija predstavlja značajan zaštitni čimbenik od pucanja aneurizme ACoA. Utvrdili smo da su aneurizme ACoA u muških bolesnika i one s većim odnosom između normalne visine aneurizme i širine vrata aneurizme te one s većim odnosom između najveće normalne visine aneurizme i širine aneurizme, većim kutovima aneurizme, prisutnošću kćeri vrećica te nepravilnim i nesferičnim oblicima u većem riziku od puknuća

Limited Aromatic Pathway Genes Diversity Amongst Aromatic Compound Degrading Soil Bacterial Isolates

Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterised corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13