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 thetaio taomicron strain 217 was studied in Escherichia coli strain HB101 (pBT 4). First, two sets of complete 2(4) factorial designs were used to ev aluate 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 magnes ium were found to be the prevalent factors, the presence of tetracycli ne, 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 lya se respectively to be increased from 0.2 g l(-1) to 1.9 g l(-1) (dry w eight) and from 10 units ml(-1) to 210 units ml(-1) within 24 h at 30 degrees C in shake flasks.