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Appl Environ Microbiol, July 1998, p. 2644-2651, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Development of a Lipase Fermentation Process That Uses a Recombinant Pseudomonas alcaligenes Strain

Gijs Gerritse,1 Ronald W. J. Hommes,1 and Wim J. Quax1,2,*

Genencor International B.V., 2600 AP Delft,1 and Laboratory of Pharmaceutical Biology, University of Groningen, 9713 AV Groningen,2 The Netherlands

Received 8 January 1998/Accepted 15 April 1998

Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from P. alcaligenes was developed. Due to the inability of P. alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alcaligenes was isolated and characterized. Amplification of lipase gene copies in P. alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (>20) of the lipase structural gene, lipA. In 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 LipA only. During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.

* Corresponding author. Mailing address: Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Phone: 31503632558. Fax: 31503636908. E-mail: w.j.quax{at}farm.rug.nl.

Appl Environ Microbiol, July 1998, p. 2644-2651, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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