Optimization of the hydroxylation of 2-cyclopentylbenzoxazole with Cunninghamella blakesleeana DSMZ 1906.

Biohydroxylation of 2-cyclopentyl-1,3-benzoxazole with the filamentous fungus Cunninghamella blakesleeana DSMZ 1906 was studied in a 15-l stirred tank reactor. The aim of the work was to avoid substrate limitation through sub-optimal mixing by formation of pellets with a uniform pellet size distribution of 250-500 microm, obtained at an inoculum concentration of 10(7) spores ml(-1) and an agitation rate of 390 rpm. Due to the high toxicity of the educt, 2-cyclopentyl-1,3-benzoxazole, on the fungus, the medium composition, the time of educt addition, and the educt starting concentration were optimized to reach high educt tolerance and hydroxylation activity. A good maintenance of biotransformation capacity was obtained without excessive loss of activity of the biocatalyst by addition of 30 mg 2-cyclopentyl-1,3-benzoxazole/g biomass (cell dry mass) during the stationary phase in a medium which was optimized in batch fermentations with experimental designs. An increase in product yield and quality (enantiomeric excess) was achieved by developing feeding strategies combining the educt and medium components. The resulting fermentation broth contained 450 mg l(-1) of the product (1S,3S)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol with an enantiomeric excess of 95%, which represents a 48% increase over former reported results.