The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Uwe T. Bornscheuer - One of the best experts on this subject based on the ideXlab platform.
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engineering the amine transaminase from Vibrio fluvialis towards branched chain substrates
Chemcatchem, 2016Co-Authors: Maika Genz, Okke Melse, Sandy Schmidt, Clare Vickers, Mark Dörr, Tom Van Den Bergh, Henk-jan Joosten, Uwe T. BornscheuerAbstract:Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results.
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Engineering the Amine Transaminase from Vibrio fluvialis towards Branched‐Chain Substrates
ChemCatChem, 2016Co-Authors: Maika Genz, Okke Melse, Sandy Schmidt, Clare Vickers, Mark Dörr, Tom Van Den Bergh, Henk-jan Joosten, Uwe T. BornscheuerAbstract:Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results.
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Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis.
International journal of molecular sciences, 2015Co-Authors: Maika Genz, Clare Vickers, Mark Dörr, Tom Van Den Bergh, Henk-jan Joosten, Matthias Höhne, Uwe T. BornscheuerAbstract:To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5′-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.
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alteration of the donor acceptor spectrum of the s amine transaminase from Vibrio fluvialis
International Journal of Molecular Sciences, 2015Co-Authors: Maika Genz, Clare Vickers, Mark Dörr, Tom Van Den Bergh, Henk-jan Joosten, Matthias Höhne, Uwe T. BornscheuerAbstract:To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5′-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.
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engineering the active site of the amine transaminase from Vibrio fluvialis for the asymmetric synthesis of aryl alkyl amines and amino alcohols
Chemcatchem, 2015Co-Authors: Alberto Nobili, Matthias Höhne, Hannes Kohls, Ivan Trentin, Carola Schulzke, Fabian Steffenmunsberg, Uwe T. BornscheuerAbstract:Although the amine transaminase from Vibrio fluvialis has often been applied as a catalyst for the biocatalytic preparation of various chiral primary amines, it is not suitable for the transamination of a-hydroxy ketones and aryl-alkyl ketones bearing an alkyl substituent larger than a methyl group. We addressed this problem through a systematic mutagenesis study of active site residues to expand its substrate scope towards two bulky ketones. We identified two mutants (F85L/V153A and Y150F/V153A) showing 30-fold increased activity in the conversion of (S)-phenylbutylamine and (R)-phenylglycinol, respectively. Notably, they facilitated asymmetric synthesis of these amines with excellent enantiomeric purities of 98% ee.
Alan Villalobos - One of the best experts on this subject based on the ideXlab platform.
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redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin
Protein Engineering Design & Selection, 2013Co-Authors: Katarina S. Midelfort, Rajesh Kumar, Seungil Han, Michael J. Karmilowicz, Kevin Mcconnell, Daniel K. Gehlhaar, Anil Mistry, Jeanne S. Chang, Marie Anderson, Alan VillalobosAbstract:Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this, <450 variants were screened, which allowed accurate assessment of enzyme performance using a low-throughput ultra performance liquid chromatography assay. During the course of this work, crystal structures of Vfat wild type and an improved variant (Vfat variant r414) were solved and they are reported here for the first time. This work also provides insight into the critical residues for substrate specificity for the transamination of (R)-ethyl 5-methyl 3-oxooctanoate and structurally related β-ketoesters.
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Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin
Protein engineering design & selection : PEDS, 2012Co-Authors: Katarina S. Midelfort, Rajesh Kumar, Seungil Han, Michael J. Karmilowicz, Kevin Mcconnell, Daniel K. Gehlhaar, Anil Mistry, Jeanne S. Chang, Marie Anderson, Alan VillalobosAbstract:Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this,
Li-li Chen - One of the best experts on this subject based on the ideXlab platform.
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Survival of Psychrotrophic Vibrio mimicus , Vibrio fluvialis and Vibrio parahaemolyticus in Culture Broth at Low Temperatures.
Journal of food protection, 1994Co-Authors: Hin-chung Wong, Li-li ChenAbstract:Pathogenic Vibrios are important etiologic agents in tropical regions and have been frequently recovered from seafoods and aquacultured foods. A number of psychrotrophic Vibrios were isolated and selected from frozen seafoods and their survivals in tryptic soy broth (TSB) at 4°C and -30°C were studied. These psychrotrophic strains showed good survival at low temperatures and could probably enhance the risk of Vibrios in frozen foods. Vibrio mimicus 70 and 198, Vibrio fluvialis 52 and Vibrio parahaemolyticus 205 survived well at 10°C, 4°C and -30°C, while the non-cold fitter V. fluvialis 28 was completely inactivated in the test periods. These strains were not heat resistant and could be easily inactivated by heat treatment. Effect of phosphates may be different for. various Vibrio species at low temperatures. Survival of V. parahaemolyticus 205 was significantly protected by the heated metaphosphate at 4°C by lowering the lethality of cold injured cells but not by increasing the level of uninjured viable cells. Pyrophosphate was inhibitory to this V. parahaemolyticus strain at -30°C.
Katarina S. Midelfort - One of the best experts on this subject based on the ideXlab platform.
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redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin
Protein Engineering Design & Selection, 2013Co-Authors: Katarina S. Midelfort, Rajesh Kumar, Seungil Han, Michael J. Karmilowicz, Kevin Mcconnell, Daniel K. Gehlhaar, Anil Mistry, Jeanne S. Chang, Marie Anderson, Alan VillalobosAbstract:Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this, <450 variants were screened, which allowed accurate assessment of enzyme performance using a low-throughput ultra performance liquid chromatography assay. During the course of this work, crystal structures of Vfat wild type and an improved variant (Vfat variant r414) were solved and they are reported here for the first time. This work also provides insight into the critical residues for substrate specificity for the transamination of (R)-ethyl 5-methyl 3-oxooctanoate and structurally related β-ketoesters.
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Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin
Protein engineering design & selection : PEDS, 2012Co-Authors: Katarina S. Midelfort, Rajesh Kumar, Seungil Han, Michael J. Karmilowicz, Kevin Mcconnell, Daniel K. Gehlhaar, Anil Mistry, Jeanne S. Chang, Marie Anderson, Alan VillalobosAbstract:Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this,
M. Mosihuzzaman - One of the best experts on this subject based on the ideXlab platform.
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Structural studies of Vibrio fluvialis M-940 O-antigen polysaccharide.
Carbohydrate Research, 1993Co-Authors: Lennart Kenne, M.mahbubur Rahman, Bengt Lindberg, M. MosihuzzamanAbstract:Abstract The structure of the Vibrio fluvialis M-940 O-antigen polysaccharide has been investigated by sugar and methylation analyses, specific degradations, NMR spectroscopy, and mass spectrometry. It is proposed that it consists of a heptasaccharide unit having the following structure. α- l -Rha p -(1 → 2)-α- l -Fuc p -(1 → 2)-α- d -Gal p -(1 → 2)-α- l -Fuc p -(1 → 3)-β- d -Glc p A-(1 → 4)-α- l -Rha p (1 → 3)-β- d -Glc p NAc-(1 → The heptasaccharide is most probably linked to the 3-position of an α- d -galactopyranosyl residue in the core.
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Lipopolysaccharide Composition and Virulence Properties of Clinical and Environmental Strains of Vibrio fluvialis and Vibrio mimicus
Microbiology and immunology, 1992Co-Authors: Mohammed Mahbubur Rahman, Firdausi Qadri, M. J. Albert, Anwar Hossain, M. MosihuzzamanAbstract:Vibrio mimicus strains W-26768 (stool isolate) and N-1301 (environmental isolate) and Vibrio fluvialis strains AA-18239 (stool isolate) and M-940 (environmental isolate) were studied for virulence properties and lipopolysaccharide composition. All four strains were hydrophobic, produced cytotoxin, adhered to HeLa cells and showed mannose-sensitive agglutination of guinea pig erythrocyte. The strains were negative for enterotoxin production and were mostly susceptible to the common antibiotics. The environmental and clinical isolates of both species were anti-genically unrelated to each other. Lipopolysaccharide antigen analysis showed that O-antigen polysaccharides of two strains of V. fluvialis and two strains of V. mimicus differed with respect to the sugar components. Only LPS from V. mimicus W-26768 showed the presence of an unusual sugar, 3,6-dideoxy-3-acetamido-hexose. The sugar compositions of these V. fluvialis and V. mimicus strains differed from those of previously reported Japanese isolates. These differences probably reflect differences in the serogroup of strains.
