Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9604490 | Journal of Biotechnology | 2005 | 10 Pages |
Abstract
The chemoenzymatic route to 2-deoxy-2-propionamido-d-mannose (1b), 2-butyramido-2-deoxy-d-mannose (2b) and 2-deoxy-2-phenylacetamido-d-mannose (3b) involved N-acylation of 2-amino-2-deoxy-d-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-d-glucose (1a), 2-butyramido-2-deoxy-d-glucose (2a) and 2-deoxy-2-phenylacetamido-d-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-d-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-d-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 °C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-d-glucose and 2-acetamido-2-deoxy-6-O-sulfo-d-glucose but not 2-acetamido-2-deoxy-1-O-phospho-d-glucose or 2-acetamido-2-deoxy-d-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 °C in the presence of 0.1 M ammonium sulfate.
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Physical Sciences and Engineering
Chemical Engineering
Bioengineering
Authors
Veronika Kristová, Ludmila MartÃnková, Lucie HuÅ¡áková, Marek Kuzma, Jana Rauvolfová, Daniel Kavan, Petr Pompach, Karel BezouÅ¡ka, VladimÃr KÅen,