DEVELOPMENT OF A LIPASE FERMENTATION PROCESS THAT USES A RECOMBINANT PSEUDOMONAS-ALCALIGENES STRAIN

Pseudomonas alcaligenes M-l secretes an alkaline lipase, which has exc ellent characteristics for the removal of fatty stains under modern wa shing conditions. A fed-batch fermentation process based on the secret ion of the alkaline lipase from P. alcaligenes was developed. Due to t he inability of P. alcaligenes to grow on glucose, citric acid and soy bean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alca ligenes was isolated and characterized. Amplification of lipase gene c opies in P. alcaligenes with the aid of low- and high-copy-number plas mids resulted in an increase of lipase expression that was apparently colinear,vith the gene copy number, It was found that overexpression o f the lipase helper gene, lipB, produced a stimulating effect in strai ns with high copy numbers (>20) of the lipase structural gene, lipA. I n strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to L ipA only, During scaling up of the fermentation process to 100 m(3), s evere losses in lipase productivity were observed. Simulations have id entified an increased level of dissolved carbon dioxide as the most pr obable cause for the scale-up losses. A large-scale fermentation proto col with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.