Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10

<p>Abstract</p> <p>Background</p> <p>Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.</p> <p>Results</p> <p>A nitrilase gene was amplified by PCR from the cDNA library of <it>Aspergillus niger </it>K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in <it>Escherichia coli </it>BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val₃₂₇- Asn₃₅₆) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp₂₉₈-Val₃₁₃peptide was shortened to Asp₂₉₈-Arg₃₁₀in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.</p> <p>Conclusions</p> <p>The nitrilase from <it>Aspergillus niger </it>K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in <it>Aspergillus </it>and <it>Penicillium </it>genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by <it>in vitro </it>protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.</p

Syngas Production, Storage, Compression and Use in Gas Turbines

This chapter analyses syngas production through pyrolysis and gasification, its compression and its use in gas turbines. Syngas compression can be performed during or after thermal treatment processes. Important points are discussed related to syngas ignition, syngas explosion limit at high temperatures and high pressures and syngas combustion kinetics. Kinetic aspects influence ignition and final emissions which are obtained at the completion of the combustion process. The chapter is organized into four subsections, dealing with (1) innovative syngas production plants, (2) syngas compressors and compression process, (3) syngas ignition in both heterogeneous and homogeneous systems and (4) syngas combustion kinetics and experimental methods. Particular attention is given to ignition regions that affect the kinetics, namely systems that operate at temperatures higher than 1000 K can have strong ignition, whereas those operating at lower temperatures have weak ignition. Keywords: Pyrogas Pyrolysis Ignition Syngas Compression GasificationacceptedVersio

Epoxide hydrolase and its application in organic synthesis

International audienceOrganic chemists have become interested in enzymes as catalysts due to their high efficiencies and specificities. Moreover, recent progress in molecular biology and enzyme-related research areas enabled and simplified the production and purification of recombinant enzymes in large quantities and their engineering towards tailor-made biocatalysts using straightforward mutagenesis and screening techniques. This is also true for epoxide hydrolases (EHs), as evidenced by the many published research papers about the synthetic applications of naturally occurring or engineered EHs

Enantioselective Bio-Hydrolysis of Various Racemic and meso Aromatic Epoxides Using the Recombinant Epoxide Hydrolase Kau2

International audienceEpoxide hydrolase Kau2 overexpressed in Escherichia coli RE3 has been tested with ten different racemic and meso,-disubstituted aromatic epoxides. Some of the tested substrates were bi-functional, and most of them are very useful building blocks in synthetic chemistry applications. As a general trend Kau2 proved to be an extremely enantioselective biocatalyst, the diol products and remaining epoxides of the bioconversions being obtained – with two exceptions – in nearly enantiomerically pure form. Furthermore, the reaction times were usually very short (around 1 h, except when stilbene oxides were used), and the use of organic co-solvent was well tolerated, enabling very high substrate concentrations (up to 75 g/L) to be reached. Even extremely sterically demanding epoxides such as cis-and trans-stilbene oxides were transformed on a reasonable time scale. All reactions were successfully conducted on a 1-g preparative scale, generating diol-and epoxide-based chiral synthons with very high enantiomeric excesses and isolated yields close to the theoretical maximum. Thus we have here demonstrated the usefulness and versatility of lyophilizedEscherichia coli cells expressing Kau2 epoxide hydrolase as a highly enantioselective biocatalyst for accessing very valuable optically pure aromatic epoxides and diols through kinetic resolution of racemates or desymmetrization of meso epoxides

Bioproduction of Quercetin and Rutinose Catalyzed by Rutinosidase: Novel Concept of “Solid State Biocatalysis”

Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This &ldquo;solid-state&rdquo; enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of &ldquo;bio-quality.&rdquo; Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the &ldquo;Solid-State-Catalysis&rdquo; concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility

Mutation Hotspot for Changing the Substrate Specificity of &beta;-N-Acetylhexosaminidase: A Library of GlcNAcases

&beta;-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal &beta;-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers &ndash; they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases

Enzyme-mediated transglycosylation of rutinose (6-O-α-L-rhamnosyl-D-glucose) to phenolic compounds by a diglycosidase from Acremonium sp. DSM 24697

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-β-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-L-rhamnopyranosylβ-D-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values 6.0. The structure of 4-hydroxyphenyl-β-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-β-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 ◦C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.Fil: Mazzaferro, Laura. Universidad Nacional de La Pampa; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; ArgentinaFil: Weiz, Gisela. Universidad Nacional de La Pampa; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; ArgentinaFil: Braun, Lucas Ezequiel. Universidad Nacional de La Pampa; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; ArgentinaFil: Kotik, Michael. Czech Academy of Sciences. Institute of Organic Chemistry and Biochemistry; República ChecaFil: Pelantová, Helena. Czech Academy of Sciences. Institute of Organic Chemistry and Biochemistry; República ChecaFil: Kren, Vladimír. Czech Academy of Sciences. Institute of Organic Chemistry and Biochemistry; República ChecaFil: Breccia, Javier Dario. Universidad Nacional de La Pampa; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; Argentin

Bacteria as source of diglycosidase activity: actinoplanes missouriensis produces 6-O-α-l-rhamnosyl-β-d-glucosidase active on flavonoids

Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-β-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-β-D-glucosidase was identified in the genome sequence of A. missouriensis 431T (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.Fil: Neher, Bárbara Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; ArgentinaFil: Mazzaferro, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; ArgentinaFil: Kotik, Michael. Academy of Sciences of the Czech Republic. Institute of Microbiology; República ChecaFil: Oyhenart, Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; ArgentinaFil: Halada, Petr. Academy of Sciences of the Czech Republic. Institute of Microbiology; República ChecaFil: Breccia, Javier Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; ArgentinaFil: Křen, Vladimír. Academy of Sciences of the Czech Republic. Institute of Microbiology; República Checa; República Chec

The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities

Diglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-β-glucosidase I and II (αRβG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-β-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRβG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRβG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRβG I. On the other hand, αRβG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRβG I showed exclusive specificity toward 7-O-β-rutinosyl flavonoids, whereas αRβG II hydrolyzed both 7-O-β-rutinosyl- and 3-O-β-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-β-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRβG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.Fil: Weiz, Gisela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; Argentina. Universidad Nacional de La Pampa; ArgentinaFil: Mazzaferro, Laura. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; Argentina. Universidad Nacional de La Pampa; ArgentinaFil: Kotik, Michael. Biology Centre of the Academy of Sciences of the Czech Republic; República ChecaFil: Neher, Bárbara Daniela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; Argentina. Universidad Nacional de La Pampa; ArgentinaFil: Halada, Petr. Biology Centre of the Academy of Sciences of the Czech Republic; República ChecaFil: Křen, Vladimír. Biology Centre of the Academy of Sciences of the Czech Republic; República ChecaFil: Breccia, Javier Dario. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Confluencia. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas | Universidad Nacional de la Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de la Pampa. Grupo Vinculado Fundacion Centro de Salud E Investigaciones Medicas.; Argentina. Universidad Nacional de La Pampa; Argentin