In order to obtain particulate @entity577 monooxygenase (pMMO)-enriched membranes from @entity34170 ) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the @entity920 ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of @entity920 ion concentration in the growth medium on the @entity920 content in the membranes, as well as the specific activity of the enzyme. The optimal @entity920 concentration in the growth medium was found to be 30 to 35 micro M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of @entity21806 /min/mg of protein with @entity2125 as the reductant. The @entity920 stoichiometry is approximately 13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of @entity219 , and only traces of @entity68 were present in the pMMO-enriched membranes. Further purification by membrane solubilization in @entity10940 followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an alphabetagamma protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a @entity920 protein containing 13.6 mol of @entity920 /mol of pMMO and essentially no @entity68 (ratio of @entity920 to @entity68 , 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to @entity2125 and @entity24537 , the two common reductants used to assay the enzyme.

Production of high-quality particulate @entity577 monooxygenase in high yields from XXXX ) with a hollow-fiber membrane bioreactor.

@entity2125 :: ('MESH:D009243', 'Chemical') :: ['NADH'] @entity21806 :: ('MESH:C009068', 'Chemical') :: ['propylene oxide'] @entity24537 :: ('MESH:C016421', 'Chemical') :: ['duroquinol'] @entity219 :: ('MESH:D015032', 'Chemical') :: ['zinc'] @entity577 :: ('MESH:D008697', 'Chemical') :: ['methane'] @entity920 :: ('MESH:D003300', 'Chemical') :: ['copper'] @entity34170 :: ('243233', 'Species') :: ['Methylococcus capsulatus (Bath'] @entity10940 :: ('MESH:C040358', 'Chemical') :: ['dodecyl beta-D-maltoside'] @entity68 :: ('MESH:D007501', 'Chemical') :: ['iron']
@entity34170 :: (243233,Species) :: ['Methylococcus capsulatus (bath']
