Innovative fermentation strategies for the production of extremophilic enzymes

A new type of microfiltration (MF) bioreactor. developed in our laboratory, was investigated for use in improving efficiency of the production of extr emophilic enzymes. In spite of the difficulties in cultivating hyperthermop hiles, we achieved, in 300 h fermentation, more than 38 g/l dry weight of S ulfolobus solfataricus using a MF technique, and we demonstrated that the a ctivity of alcohol dehydrogenase (ADH), as the reporter enzyme, was not aff ected by cell density. However, hyperthermophile cultivation is difficult t o scale up because of evaporation and the very low growth rate. Thus, to ac hieve high productivity we cultivated, in the MF bioreactor, recombinant me sophilic hosts engineered for the production of two thermophilic enzymes, n amely, trehalosyldextrin-forming enzyme (SsT-DFE) and trehalose-forming enz yme (SsTFE) from Sulfolobus solfataricus. The traditional Luria-Bertani bro th used for recombinant Escherichia coli growth was replaced with a semidef ined medium. The latter was used in both the batch and the MF experiments, and the ratio of complex components (e.g., yeast extract and tryptone) to a simple carbon source (glycerol) was decreased during the fed-batch phase t o further decrease the medium cost in view of industrial applications. The bioprocess developed was able to improve productivity 500 fold for rSsTFE a nd 60 fold for rSsTDFE with respect to the wild type cultivated in MF mode. Comparisons with another recombinant enzyme, alpha -glucosidase (rSs alpha gly), from Sulfolobus solfataricus produced in our MF bioreactor are repor ted.