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The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

The present invention is directed to a process for the production of alkyl alkanoate wherein a polypeptide that has alcohol acetyl transferase activity (AAT) and has in its three-dimensional structure an alpha-beta hydrolase fold and an active site comprising a serine, histidine, and optionally aspartic acid is, used to convert: - C3-C10 alkanol and acetyl coenzyme A or, - C1-C10 alkanol with C3-C10 acyl coenzyme A, or - Methanol with acetyl coenzyme A, into an alkyl alkanoate. The process is further directed to the use of a recombinant expression vector or plasmid or host cell comprising the nucleotide sequence encoding the polypeptide. The polypeptides according to the invention are genes isolated from Kluyveromyces marxianus, Kluyveromyces lactis, Wickerhamomyces ciferrii, Cyberlindnera jadinii, Hanseniaspora uvarum, Eremothecium cymbalarie and Saccharomyces cerevisiae.

The present invention relates to a process for the production of ethyl acetate wherein a polypeptide having alcohol acetyl transferase activity converts ethanol and acetyl coenzyme A into ethyl acetate. The polypeptide has an alpha-beta hydrolase fold and an active site comprising a serine, histidine, and optionally aspartic acid in its three-dimensional structure. The polypeptides according to the invention are genes isolated from Kluyveromyces marxianus, Kluyveromyces lactis, Wickerhamomyces ciferrii, Cyberlindnera jadinii, Hanseniaspora uvarum, Eremothecium cymbalarie and Saccharomyces cerevisiae.

Thermostable Cas9 nucleases. The present invention relates to the field of genetic engineering and more particularly to nucleic acid editing and genome modification. The present invention provides an isolated Cas protein or polypeptide fragment thereof having an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% identity therewith, wherein the Cas protein or polypeptide is capable of DNA cleavage at a temperature in the range 50°C and 100°C inclusive. The invention further provides isolated nucleic acid molecules encoding said Cas9 nucleases, expression vectors and host cells. The Cas9 nucleases disclosed herein provide novel tools for genetic engineering at elevated temperatures and are of particular value in the genetic manipulation of thermophilic organisms; particularly microorganisms.

The kinetic activity of individual enzyme molecules was determined in aqueous droplets generated in a nano- and microfluidic device. To avoid high background noise, the enzyme and substrate solution was confined into femtolitre carriers to achieve single-molecule encapsulation. The tiny droplets (f~2.5-3 μm) generated from this fluidic system were highly monodisperse, beneficial for an analysis of single enzyme activity. Single-enzyme kinetics has previously been demonstrated in the microfluidic format in PDMS containers [1], surface-immobilized droplets [2], or liposomes [3]. Single- enzyme analysis in the droplet-based microfluidics was reported before [4] by encapsulating highly-diluted enzyme solution (110 fM) into large droplets (f~40 μm). But to our knowledge this is the first demonstration of the direct method of single enzyme encapsulation and analysis at high enzyme concentration in tiny droplets in a microfluidic system.

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-ß-D-xylopyranoside monoacetates as substrates in a ß-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic a/ß-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction

In 1776 voer Allessandro Volta met een klein bootje langs de oever van het Lago Maggiore. Toen hij met een stok in de modder stak, zag hij gasbellen, die hij opving in een omgekeerd glazen vat. Hij constateerde dat hij het opgevangen gas kon aansteken en dat het brandde met een blauwe vlam. De brieven van Volta over wat hij aria infiammabile of “brandbare lucht” noemde, vormen de eerste beschrijvingen van een gas dat we nu beter kennen als methaan (CH4). Wat weten we nu, bijna 250 jaar later, over die “brandbare lucht”?

Een duik nemen in de Noordzee op nieuwjaarsdag vinden we behoorlijk stoer. De Russen doen er een schepje bovenop door het begin van de winter te vieren met een duik in ijswater van nul graden. Dat valt best wel mee, zeker wanneer je dit afwisselt met een bezoek aan de sauna met een luchttemperatuur van 90 graden. Hiermee hebben we de extremen die we als mens kunnen trotseren echter wel gehad. Wat te denken van een duik in kokend water, zwemmen in een zwembad gevuld met azijn of ammonia, of kokend zwavelzuur? Wat voor ons onmogelijk is, is voor veel micro-organismen de normaalste zaak van de wereld

The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 ± 2 mM) and relatively low ATP levels (0.43 ± 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus

Bioethanol is one of the main biofuels capable of substituting petroleum usage in vehicles, and has had one of the largest impacts to date. The first generation of bioethanol production has had a worldwide impact, though it is restricted by energy intensive grain-to-bioethanol production technology, and land usage pressures (food vs. fuel). The development of second generation production technology, producing a wider range of bioalcohols from lignocellulosic biomass, removes many of the obstacles and expenses that first generation production faces. By utilising a wider base of feedstocks (energy crops, cellulosic residues, waste materials) and by employing highly-efficient biochemical production techniques, the efficiency and environmental impact of bioalcohol production are significantly improved. This book provides a comprehensive and timely reference on the biochemical conversion of lignocellulosic biomass for the production of fuel alcohols, expertly reviewing the development of the entire second-generation bioalcohol production chain. The book covers the process engineering, technology, modelling and integration of the entire production chain, from feedstock pretreatment on to hydrolysis, to fermentation, and on to purification. The book primarily covers the production of bioethanol, but extends into coverage of the production of longer-chain bioalcohols that will be elemental to future biofuel utilisation

A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4¿C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 °C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 °C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an ¿/ß-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family.

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden-Meyerhof and the Entner-Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

A Gram-negative, facultatively anaerobic, rod-shaped, dissimilatory chlorate-reducing bacterium, strain AW-1(T), was isolated from biomass of an anaerobic chlorate-reducing bioreactor. Phylogenetic analysis of the 16S rDNA sequence showed 100␜equence similarity to Pseudomonas stutzeri DSM 50227 and 98.6␜equence similarity to the type strain of P. stutzeri (DSM 5190(T)). The species P. stutzeri possesses a high degree of genotypic and phenotypic heterogeneity. Therefore, eight genomic groups, termed genomovars, have been proposed based upon DeltaT(m) values, which were used to evaluate the quality of the pairing within heteroduplexes formed by DNA--DNA hybridization. In this study, DNA--DNA hybridization between strain AW-1(T) and P. stutzeri strains DSM 50227 and DSM 5190(T) revealed respectively 80.5 and 56.5␜imilarity. DNA--DNA hybridization between P. stutzeri strains DSM 50227 and DSM 5190(T) revealed 48.4␜imilarity. DNA--DNA hybridization indicated that strain AW-1(T) is not related at the species level to the type strain of P. stutzeri. However, strain AW-1(T) and P. stutzeri DSM 50227 are related at the species level. The physiological and biochemical properties of strain AW-1(T) and the two P. stutzeri strains were compared. A common characteristic of P. stutzeri strains is the ability to denitrify. However, in growth experiments, strain AW-1(T) could use only chlorate or oxygen as an electron acceptor and not nitrate, perchlorate or bromate. Strain AW-1(T) is the first chlorate-reducing bacterium described that does not possess another oxyanion-reduction pathway. Cell extracts of strain AW-1(T) showed chlorate and bromate reductase activities but not nitrate reductase activity. P. stutzeri strains DSM 50227 and DSM 5190(T) could use nitrate or oxygen as an electron acceptor, but not chlorate. Chlorate reductase activity, in addition to nitrate reductase activity, was detected in cell extracts of both P. stutzeri strains. Chlorite dismutase activity was absent in extracts of both P. stutzeri strains but was present in extracts of strain AW-1(T). Based on the hybridization experiments and the physiological and biochemical data, it is proposed that strain AW-1(T) be classified as a novel species of Pseudomonas, Pseudomonas chloritidismutans sp. nov. The type strain is strain AW-1(T) (=DSM 13592(T)=ATCC BAA-443(T)).

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the -glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U?mg1) and pyruvaldehyde (32 U?mg1) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 °C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.

A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of protoheme IX as a prosthetic group (ferric heme), in a stoichiometry of 0.25 heme per subunit. Electron paramagnetic resonance analysis confirmed the presence of ferric heme and identified the proximal axial ligand as a histidine. The enzyme showed both catalase and peroxidase activity with pH optima of 6.0 and 4.5, respectively. Optimal temperatures of 70°C and 80°C were found for the catalase and peroxidase activity, respectively. The catalase activity strongly exceeded the peroxidase activity, with Vmax values of 9600 and 36 U mg-1, respectively. Km values for H2O2 of 8.6 and 0.85 mM were found for catalase and peroxidase, respectively. Common heme inhibitors such as cyanide, azide, and hydroxylamine inhibited peroxidase activity. However, unlike all other catalase-peroxidases, the enzyme was also inhibited by 3-amino-1,2,4-triazole. Although the enzyme exhibited a high thermostability, rapid inactivation occurred in the presence of H2O2, with half-life values of less than 1 min. This is the first catalase-peroxidase characterized from a hyperthermophilic microorganism.

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The kcat/Km values for alanine and pyruvate formation were 41 and 33 s1 mM1, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

Contains fulltext : mmubn000001_111343151.pdf (Publisher’s version ) (Open Access) Promotores : G. Vogels, J. Keltjens en C. van der Drift 171 p.