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Emissions from aircraft have adverse effects on the air quality in and around airports, contributing to public health concerns within neighbouring communities. AVIATOR will adopt a multi-level measurement, modelling and assessment approach to develop an improved description and quantification of the relevant aircraft engine emissions, and their impact on air quality under different climatic conditions. Particulate and gaseous emissions in a test cell and on-wing from an in-service aircraft will be measured to determine pollutant plume evolution from the engine and APU exhaust. This will provide an enhanced understanding of primary emitted pollutants, specifically the nvPM and vPM (down to 10nm), and the scalability between the regulatory test cell and real environments. AVIATOR will develop and deploy a proof-of-concept low cost sensor network for monitoring UFP, PM and gaseous species across multiple airports and surrounding communities. Campaigns will be complemented by high-fidelity modelling of aircraft exhaust dynamics, microphysical and chemical processes within the plume. CFD, box, and airport air quality models will be applied, providing validated parameterisations of the relevant processes, applicable to standard dispersion modelling on the local scale. Working with the regulatory community, AVIATOR will develop improved guidance on measuring and modelling the impact of aircraft emissions, and will provide airports and regulators with tools and guidance to improve the assessment of air quality in and around airports.

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus . These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.

Chitinolytic enzymes are capable to catalyze the chitin hydrolysis. Due to their biomedical and biotechnological applications, nowadays chitinolytic enzymes have attracted worldwide attention. Chitinolytic enzymes have provided numerous useful materials in many different industries, such as food, pharmaceutical, cosmetic, or biomedical industry. Marine enzymes are commonly employed in industry because they display better operational properties than animal, plant, or bacterial homologs. In this mini-review, we want to describe marine chitinolytic enzymes as versatile enzymes in different biotechnological fields. In this regard, interesting comments about their biological role, reaction mechanism, production, functional characterization, immobilization, and biotechnological application are shown in this work.

Cloning and biochemical characterization of a novel extracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase from Streptomyces exfoliatus K10 DSMZ 41693 are described. The primary structure of the depolymerase (PhaZSex2) includes the lipase consensus sequence (serine-histidine-aspartic acid) which is known for serine hydrolases. Secondary structure analysis shows 7.9 % α-helix, 43.9 % β-sheet, 19.4 % β-turns, and 31.2 % random coil, suggesting that this enzyme belongs to the α/β hydrolase fold family, in agreement with other PHA depolymerases and lipases. The enzyme was efficiently produced as an extracellular active form in Rhodococcus and purified by two consecutive hydrophobic chromatographic steps. Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis of the purified enzyme revealed a monomer of 27.6 kDa with a midpoint transition temperature of 44.2 °C. Remarkably, the activity is significantly enhanced by low concentrations of nonionic and anionic detergents and thermal stability is improved by the presence of 10 % glycerol. PhaZSex2 is an endo-exohydrolase that cleaves both large and small PHA molecules, producing (R)-3-hydroxyoctanoic acid monomers as the main reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3-hydroxyalkanoic acids.

The pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (alpha-subunit) and 60.09 kDA (beta-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobicpocket involved in catalytic activity, including Serbeta1, Hisbeta23, Valbeta70, and Asnbeta272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of pva in S. lividans led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of SlPVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production.

Here, we describe the draft genome sequence of Actinoplanes utahensis NRRL 12052, a filamentous bacterium that encodes an aculeacin A acylase and a putative N-acyl-homoserine lactone acylase of biotechnological interest. Moreover, several nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) clusters and antibiotic resistance genes have been identified., (Copyright © 2015 Velasco-Bucheli et al.)

Here we report the draft genome sequence of Streptomyces exfoliatus DSMZ 41693, which includes a gene encoding a poly(3-hydroxyoctanoate) depolymerase, an enzyme which can be used for the industrial synthesis of chiral (R)-3-hydroxyalkanoic acids. In addition, the genome carries numerous genes involved in the biosynthesis of secondary metabolites, including polyketides and terpenes.

The ascomycin-producer strain Streptomyces ascomycinicus has been proven to be an extracellular poly(R)-3-hydroxybutyrate (PHB) degrader. The fkbU gene, encoding a PHB depolymerase (PhaZ Sa ), has been cloned in E. coli and Rhodococcus sp. T104 strains for gene expression. Gram-positive host Rhodococcus sp. T104 was able to produce and secrete to the extracellular medium an active protein form. PhaZ Sa was purified by two hydrophobic interaction chromatographic steps, and afterwards was biochemically as well as structurally characterized. The enzyme was found to be a monomer with a molecular mass of 48.4 kDa, and displayed highest activity at 45°C and pH 6, thus being the first PHB depolymerase from a gram-positive bacterium presenting an acidic pH optimum. The PHB depolymerase activity of PhaZ Sa was increased in the presence of divalent cations due to non-essential activation, and also in the presence of methyl-β-cyclodextrin and PEG 3350. Protein structure was analyzed, revealing a globular shape with an alpha-beta hydrolase fold. The amino acids comprising the catalytic triad, Ser(131)-Asp(209)-His(269), were identified by multiple sequence alignment, chemical modification of amino acids and site-directed mutagenesis. These structural results supported the proposal of a three-dimensional model for this depolymerase. PhaZ Sa was able to degrade PHB, but also demonstrated its ability to degrade films made of PHB, PHBV copolymers and a blend of PHB and starch (7∶3 proportion wt/wt). The features shown by PhaZ Sa make it an interesting candidate for industrial applications involving PHB degradation.

Cross-linked enzyme aggregates of poly-3-hydroxybutyrate (PHB) depolymerase from Streptomyces exfoliatus (PhaZ(Sex)-CLEAs) have been prepared. Acetone was used as the precipitating agent, while addition of bovine serum albumin (BSA) facilitated CLEAs formation. Conditions for enzyme precipitation and cross-linking have been optimized, and confocal scanning microscopy showed a homogeneous enzyme distribution in the biocatalyst. Obtained PhaZ(Sex)-CLEAs presented an average size of 50-300 μm, showing a high PHB depolymerase activity of 255 U/g wet biocatalyst at 40°C and pH 7.0. Temperature-activity profile of PhaZ(Sex)-CLEAs at pH 8.0 showed that the highest activity for pNPB hydrolysis was achieved at 60°C, whereas pH-activity profile at 40°C indicated that highest activity for PHB hydrolysis was achieved at pH 7.0. Additionally, immobilized biocatalyst could be recycled at least for 20 consecutive batch reactions without loss of catalytic activity, and showed higher pH and temperature stability, and better tolerance to several organic solvents than its soluble counterpart., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The phaZ ( Sex ) gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T (m)) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine-histidine-carboxylic acid in the active site. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.

Inhibition of recombinant D-amino acid oxidase from Trigonopsis variabilis (TvDAAO) activity in the presence of different sodium salts and potassium chloride is reported. A competitive inhibition pattern by sodium chloride was observed, and an inhibition constant value of K(i) = 85 mM was calculated. Direct connection of NaCl inhibition with FAD cofactor dissociation was confirmed by measuring the fluorescence of tryptophanyl residues of the holoenzyme.

Aculeacin A acylase from Actinoplanes utahensis (AuAAC), an amidohydrolase able to catalyze the acyl moieties of antifungal echinocandin antibiotics, has been also described to efficiently hydrolyze penicillin V and natural aliphatic penicillins to yield 6-aminopenicillanic acid (6-APA). Hence, taking into account its potential use in the synthesis of beta-lactam antibiotics as well as antifungal echinocandins, the recombinant enzyme was covalently immobilized onto several epoxy-activated supports in order to obtain a robust biocatalyst to be used in industrial bioreactors. The best biocatalyst was obtained by attaching the enzyme on Sepabeads EC-EP5 where immobilized AuAAC was homogeneously distributed over the surface of this support as shown by confocal scanning microscopy. The obtained biocatalyst showed a specific enzymatic activity of 35.2 IU/g wet carrier in the hydrolysis of penicillin V at pH 8.0 and 45 degrees C. Temperature-activity profile of immobilized AuAAC at pH 8.0 showed that the highest activity for the hydrolysis of penicillin V was achieved at 75 degrees C, whereas pH-activity profile at 40 degrees C indicated the highest activity for the hydrolysis of penicillin V was achieved at pH 8.5. The immobilized enzyme was highly thermostable since it suffered no loss of activity at 65 degrees C and pH 8.0 during 360 min, and it could be recycled for at least 30 consecutive batch reactions at pH 8.0 and 45 degrees C without loss of catalytic activity. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF besides its own substrate aculeacin A. Such interesting properties of this immobilized biocatalyst could allow its exploitation in industrial preparation of beta-lactam antibiotics and echinocandins., ((c) 2010 Elsevier Ltd. All rights reserved.)

Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM(-1) s(-1). Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5 degrees C.

d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) is a flavoenzyme with high biotechnological and industrial interest. The overexpression and purification of the apoprotein form of a recombinant His-tagged TvDAAO allowed us to go deep into the structural differences between apoenzyme and holoenzyme, and on the cofactor binding and its contribution to enzyme stability. A significant decrease in intrinsic fluorescence emission took place upon FAD binding, associated to cofactor induced conformational transitions or subunit dimerization that could affect the local environment of protein tryptophan residues. Furthermore, acrylamide-quenching experiments indicated that one of the five tryptophan residues of TvDAAO became less accessible upon FAD binding. A K(d)=1.5+/-0.1x10(-7) M for the dissociation of FAD from TvDAAO was calculated from binding experiments based on both quenching of FAD fluorescence and activity titration curves. Secondary structure prediction indicated that TvDAAO is a mixed alpha/beta protein with 8 alpha-helices and 14 beta-sheets connected by loops. Prediction results were in good agreement with the estimates obtained by circular dichroism which indicated that both the apoenzyme and the holoenzyme had the same structural component ratios: 34% alpha-helix content, 20% beta-structure content (14% antiparallel and 6% parallel beta-sheet), 15% beta-turns and 31% of random structure. Circular dichroism thermal-transition curves suggested single-step denaturation processes with apparent midpoint transition temperatures (T(m)) of 37.9 degrees C and 41.4 degrees C for the apoenzyme and the holoenzyme, respectively. A three-dimensional model of TvDAAO built by homology modelling and consistent with the spectroscopic studies is shown. Comparing our results with those reported for pig kidney (pkDAAO) and Rhodotorula gracilis (RgDAAO) d-amino acid oxidases, a "head-to-head" interaction between subunits in the TvDAAO dimer might be expected.