Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli.
The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2(4) factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 gl-1 to 1.9 gl-1 (dry weight) and from 10 units ml-1 to 210 units ml-1 within 24 h at 30 degrees C in shake flasks.
Hounsa
C G
CG
Laboratoire de Microbiologie Fondamentale et Appliquée, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Aubry
J M
JM
Dubourguier
H C
HC
Hornez
J P
JP
D000596:Amino Acids / Q000494:pharmacology; D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001439:Bacteroides / Q000235:genetics*; D002118:Calcium / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008957:Models, Genetic; D010406:Penicillins / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000235:genetics*; D011995:Recombination, Genetic; D013752:Tetracycline / Q000494:pharmacology
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D010406:Penicillins; C017721:casamino acids; D000667:Ampicillin; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D013752:Tetracycline; D008274:Magnesium; D005947:Glucose; D002118:Calcium