An experimental 100 kilowatt wind turbine generator

Experimental generator consists of two blades mounted on 100 foot tower, driving transmission train and electric generator mounted on top of tower. Machine generates 100 kW of electricity at wind speeds from 18 to 60 miles per hour. Yaw control mechanism automatically orients machine into wind

The vast repertoire of carbohydrate oxidases:An overview

Carbohydrates are widely abundant molecules present in a variety of forms. For their biosynthesis and modification, nature has evolved a plethora of carbohydrate-acting enzymes. Many of these enzymes are of particular interest for biotechnological applications, where they can be used as biocatalysts or biosensors. Among the enzymes catalysing conversions of carbohydrates are the carbohydrate oxidases. These oxidative enzymes belong to different structural families and use different cofactors to perform the oxidation reaction of CH-OH bonds in carbohydrates. The variety of carbohydrate oxidases available in nature reflects their specificity towards different sugars and selectivity of the oxidation site. Thanks to their properties, carbohydrate oxidases have received a lot of attention in basic and applied research, such that nowadays their role in biotechnological processes is of paramount importance. In this review we provide an overview of the available knowledge concerning the known carbohydrate oxidases. The oxidases are first classified according to their structural features. After a description on their mechanism of action, substrate acceptance and characterisation, we report on the engineering of the different carbohydrate oxidases to enhance their employment in biocatalysis and biotechnology. In the last part of the review we highlight some practical applications for which such enzymes have been exploited

Mechanistic and Crystallographic Studies of Azoreductase AzoA from Bacillus wakoensis A01

The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100-400 s-1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases

Identifikacija podtipova protozoona Trypanosoma vivax izdvojenih iz goveda i koza pomoću mikrosatelitskih markera.

Microsatellite DNA polymorphisms can be utilised to assess intra-specific genetic diversity and hence are useful for characterisation of species and strains of trypanosomes. Here, we present four new microsatellite markers specific for T. vivax isolated from Ugandan cattle and goats. The GeneDB partial shotgun 5x coverage sequence of T. vivax available as of 1st August 2005 was used and targeted the genomic sequence of T. vivax that has no cross amplification with other livestock-infective trypanosomes. Only di-; tri-; tetra;- and pentanucleotide microsatellites not less than five units were selected. Although pentanucleotide repeats on screening appeared to have the desired variability, they gave poorer PCR products compared to di-, tri- and tetranucleotide repeats. Mononucleotide repeats presented difficulty in detecting visible bands on agarose gels from their amplification and were omitted from this study. Clear length polymorphism was obtained with guanine, thymine and adenine repeated 16 times (GTA)16 while cytosine, adenine, cytosine and thymine (CACT)15 gave size and length variability. Bands of similar size were obtained from thymine and adenine (TTA)24 microsatellite, approximately 150 base pairs long and 180-200 base pairs from the cytosine and adenine (CA)26 microsatellite. These findings suggest that different subtypes of T. vivax exist in Uganda; the polymorphic forms derived from microsatellite band size differences may suggest this parasite exhibits virulence differences as has been shown in T. Congolense subtypes.Polimorfizam mikrosatelitske DNA može se rabiti za procjenu unutarvrsne genetske raznolikosti pa tako i za karakterizaciju vrsta i sojeva tripanosoma. Prikazana su četiri nova mikrosatelitska markera specifična za vrstu T. vivax izdvojenu iz goveda i koza u Ugandi. GenDB kratka i specifična sekvencija T. vivax dostupna nakon 1. kolovoza 2005. bila je ciljano rabljena za određivanje genomskoga slijeda za protozoon T. vivax koji nije pokazivao križne reakcije s drugim tripanosomama zaraznima za stoku. Izabrani su bili samo di-, tri-, tetrai pentanukleotidni mikrosateliti. Premda se činilo da pentanukleotidne ponavljajuće sekvencije u probirnom testu imaju potrebnu varijabilnost, one su dale lošije PCR proizvode u odnosu na di-, tri- i tetranukleotidne ponavljajuće sekvencije. Mononukleotidne ponavljajuće sekvencije nisu dale jasno vidljive trake na agaroznom gelu pa nisu bile dalje istražene. Jasan polimorfizam postignut je upotrebom gvanina, timina i adenina sa šesnaesterostrukim ponavljanjem (GTA)16 dok je sekvencija citozin, adenin, citozin i timin (CACT)15 bila varijabilna u odnosu na veličinu i dužinu. Sekvencije slične veličine bile su dobivene od mikrosatelita koji su sadržavali timin i adenin (TTA)24, a one od 150 parova baza te 180 - 200 parova baza od mikrosatelita citozina i adenina (CA)26. Ovi nalazi govore u prilog postojanju različitih podtipova protozoona T. vivax u Ugandi, koji bi se mogli odlikovati i različitom virulencijom kao što je dokazano za podtipove T. congolense

Analysis of the structure and substrate scope of chitooligosaccharide oxidase reveals high affinity for C2-modified glucosamines

Chitooligosaccharide oxidase (ChitO) is a fungal carbohydrate oxidase containing a bicovalently bound FAD cofactor. The enzyme is known to catalyse the oxidation of chitooligosaccharides, oligomers of N-acetylated glucosamines derived from chitin degradation. In this study, the unique substrate acceptance was explored by testing a range of N-acetyl-D-glucosamine derivatives, revealing that ChitO preferentially accepts carbohydrates with a hydrophobic group attached to C2. The enzyme also accepts streptozotocin, a natural product used to treat tumours. Elucidation of the crystal structure provides an explanation for the high affinity towards C2-decorated glucosamines: the active site has a secondary binding pocket that accommodates groups attached at C2. Docking simulations are fully in line with the observed substrate preference. This work expands the knowledge on this versatile enzyme

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.</p

Kinetic and structural properties of a robust bacterial l‐ amino acid oxidase

L-Amino acid oxidase (LAAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme active on most proteinogenic L-amino acids, catalysing their conversion to α-keto acids by oxidative deamination of the substrate. For this oxidation reaction, molecular oxygen is used as the electron acceptor, generating hydrogen peroxide. LAAO can be used to detect L-amino acids, for the production of hydrogen peroxide as an oxidative agent or antimicrobial agent, and for the production of enantiopure amino acids from racemates. In this work, we characterised a previously reported LAAO from the bacterium Pseudoalteromonas luteoviolacea. The substrate scope and kinetic properties of the enzyme were determined, and the thermostability was evaluated. Additionally, we elucidated the crystal structure of this bacterial LAAO, enabling us to test the role of active site residues concerning their function in catalysis. The obtained insights and ease of expression of this thermostable LAAO provides a solid basis for the development of engineered LAAO variants tuned for biosensing and/or biocatalysis

Energy partitioning in N2 microwave discharges: integrated Fokker-Planck approach to vibrational kinetics and comparison with experiments

This work investigates energy transfers between electrons, vibrational and translational degrees of freedom and their effect on dissociation mechanisms in a N2 microwave plasma in the pressure range between 50 and 400 mbar. A novel self-consistent 0D plasma chemistry model describing vibrational kinetics via the vibrational energy equation and the Fokker–Planck approach is developed. It is used to simulate conditions achieved experimentally, providing good agreement with measured values of vibrational and gas temperature and electron density. Above 100 mbar, energy efficiency of dissociation increases with power density, due to the significant contribution of collisions between vibrationally excited N2 and electronically excited molecules. Energy transfer to vibrations is maximum at low power density and low pressure due to reduced gas heating

Biochemical and Structural Characterization of a Uronic Acid Oxidase from Citrus sinensis

Aldaric acids are attractive diacids that can be prepared by selective oxidation of carbohydrates. For this, effective biocatalysts are in demand. This work reports on the discovery, biochemical and structural characterization of a VAO-type flavin-containing carbohydrate oxidase from Citrus sinensis: URAOCs3. URAOCs3 could be overexpressed using prokaryotic and eukaryotic expression systems. Extensive biochemical characterization revealed that the enzyme displays a high thermostability and an exquisite selectivity for uronic acids, galacturonic acid and glucuronic acid. The enzyme was further investigated by determining the crystal structure. The selective oxidation of D-galacturonic acid in a complex mixture was demonstrated, showing how URAOCs3 was found to be highly effective in selectively producing galactaric acid while leaving other carbohydrates untouched. In addition to the specific discovery of URAOCs3, these findings suggest that plant proteomes can be an interesting source for new biocatalysts.</p