7 research outputs found
Steady state kinetic analyses of nitroalkane oxidase mutants
Nitroalkane oxidase (NAO) catalyzes the oxidation of neutral
nitroalkanes to aldehydes and ketones with oxygen consumption and the production of
hydrogen peroxide and nitrite. The enzyme is a flavoprotein from the fungus Fusarium
oxysporum. The active site base, Asp402, abstracts one proton from the substrate to give
a carbanion which then attacks the flavin adenine dinucleotide (FAD). The three
dimensional crystal structure of NAO shows that Arg409 is 3.6 Å from Asp402. When
Arg409 is mutated to Lys, the rate constant for proton abstraction decreases 100-fold.
The three-dimensional structure of NAO also reveals the existence of a tunnel which
extends from the protein exterior and terminates at the FAD N5 atom and the residues
Asp402 and Phe401. We mutated amino acids in the tunnel into tryptophan,
phenylalanine and leucine. The L99W, S276W and S276A enzymes showed the biggest
decreases in both kcat and kcat/Km; these amino acids are closest to the FAD molecule and
the active site. Mutation of amino acids farther away from the active site showed very
small changes in the kinetic parameters. Ser276 is hydrogen bonded to Asp402 in the
wild-type enzyme. When this amino acid is mutated to alanine or tryptophan, k3, the rate constant for proton abstraction, decreases around 35 fold. Asp402, Arg409 and Ser276
constitute a catalytic triad in the active site of nitroalkane oxidase, and both Arg409 and
Ser276 are important for positioning Asp402 and catalysis
Biomanufacturing and testbed development for the continuous production of monoclonal antibodies
Please click Additional Files below to see the full abstract
Metaproteogenomic analysis of a sulfate-reducing enrichment culture reveals genomic organization of key enzymes in the m-xylene degradation pathway and metabolic activity of proteobacteria
Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase
Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array
Online monitoring of monoclonal antibody product titers
throughout
biologics process development and production enables rapid bioprocess
decision-making and process optimization. Conventional analytical
methods, including high-performance liquid chromatography and turbidimetry,
typically require interfacing with an automated sampling system capable
of online sampling and fractionation, which suffers from increased
cost, a higher risk of failure, and a higher mechanical complexity
of the system. In this study, a novel nanofluidic system for continuous
direct (no sample preparation) IgG titer measurements was investigated.
Tumor necrosis factor α (TNF-α), conjugated with fluorophores,
was utilized as a selective binder for adalimumab in the unprocessed
cell culture supernatant. The nanofluidic device can separate the
bound complex from unbound TNF-α and selectively concentrate
the bound complex for high-sensitivity detection. Based on the fluorescence
intensity from the concentrated bound complex, a fluorescence intensity
versus titer curve can be generated, which was used to determine the
titer of samples from filtered, unpurified Chinese hamster ovary cell
cultures continuously. The system performed direct monitoring of IgG
titers with nanomolar resolution and showed a good correlation with
the biolayer interferometry assays. Furthermore, by variation of the
concentration of the indicator (TNF-α), the dynamic range of
the system can be tuned and further expanded