A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.
Background Biosimilars must meet stringent regulatory requirements, both at the time of authorization and during their lifecycle. Yet it has been suggested that divergence in quality attributes over time may lead to clinically meaningful differences between two versions of a biologic. Therefore, this study investigated the batch-to-batch consistency across a range of parameters for released batches of the etanercept biosimilar (SB4) and infliximab biosimilar (SB2). Methods SB4 (Benepali ® ) and SB2 (Flixabi ® ) were both developed by Samsung Bioepis and are manufactured in Europe by Biogen at their facility in Hillerød, Denmark. A total of 120 batches of SB4 and 25 batches of SB2 were assessed for consistency and compliance with specified release parameters, including purity, post-translational glycosylation (SB4 only), protein concentration, and biological activity. Results The protein concentration, purity, tumor necrosis factor-α (TNF-α) binding, and TNF-α neutralization of all batches of SB4 and SB2 were within the strict specification limits set by regulatory agencies, as was the total sialic acid (TSA) content of all batches of SB4. Conclusions Quality attributes of SB4 and SB2 batches showed little variation and were consistently within the rigorous specifications defined by regulatory agencies.As of August 2019, the Biogen Hillerød facility (Denmark) became Fujifilm Diosynth Biotechnologies Denmark ApS, a contract development and manufacturing organization (CDMO) for the manufacture of biosimilars developed by Samsung Bioepis. Key PointsBiosimilars are held to the same rigorous quality standards as any other biologic. SB4 and SB2 biosimilars demonstrated a high degree of batch-to-batch consistency.Quality attributes including purity, percentage of high molecular weight species, tumor necrosis factor-α (TNFα) binding, and TNF-α neutralization remained well within acceptance limits. 2
The chromosomal locus NP_636946 of Xanthomonas campestris DSM 3586 (ATCC 33913) which was earlier presumed to encode a quinoprotein glucose dehydrogenase has been cloned, expressed in Escherichia coli and the recombinant enzyme has been characterised. It was found to have no glucose dehydrogenase activity but to be active on many different polyols and diols, aliphatic alcohols, certain aldonic acids and amino-sugars. The product of D: -gluconic acid oxidation was 5-keto-D: -gluconic acid. The enzyme differs from polyol/gluconate dehydrogenases found in Gluconobacter by its single-chain architecture, different substrate specificity and much higher (20- to 30-fold) expression level in E.coli.
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