title: Production of alpha-amylase in fed-batch cultures of vgb+ and vgb- recombinant Escherichia coli: some observations.
abstract: Synthesis and excretion of Bacillus stearothermophilus alpha-amylase is analyzed in fed-batch cultivations of Escherichia coli JM103[pMK79] and E. coli JM103[pMK57], the former strain containing the plasmid-encoded Vitreoscilla hemoglobin (VHb) gene (vgb) and the latter strain being devoid of this gene. Fed-batch operation is observed to be substantially superior to batch operation as concerns the alpha-amylase production rate and the extent of excretion of the enzyme. Faster feeding of a nutrient medium (LB or M9) discourages synthesis of alpha-amylase. While synthesis of alpha-amylase in the vgb(-) strain is discouraged when oxygen availability is reduced, the reverse is the case with the vgb(+) strain, the promotion of alpha-amylase synthesis in the latter strain being linked to the synthesis of VHb. Increased availability of the principal carbon source (glucose) in a defined medium leads to overproduction of both alpha-amylase and VHb under oxygen limitation, which may be responsible for the segregational instability observed with the vgb(+) strain. The very high extents of excretion of alpha-amylase attained in fed-batch cultures are encouraging for downstream processing of the recombinant protein.
authors.affiliation: Departments of Chemical and Environmental Engineering and of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616, USA.
authors.forename: N
authors.initials: N
authors.lastname: Enayati
authors.forename: C
authors.initials: C
authors.lastname: Tari
authors.forename: S J
authors.initials: SJ
authors.lastname: Parulekar
authors.forename: B C
authors.initials: BC
authors.lastname: Stark
authors.forename: D A
authors.initials: DA
authors.lastname: Webster
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D006088:Gram-Negative Aerobic Bacteria / Q000235:genetics; D006454:Hemoglobins / Q000096:biosynthesis / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003470:Culture Media; D006454:Hemoglobins; D011994:Recombinant Proteins; D000516:alpha-Amylases; D010100:Oxygen