For enzymatic synthesis of octyl-beta-D-galactopyranoside (octyl-gal) from lactose and n-octanol. Escherichia coli beta-galactosidase (beta-Gal) was expressed and displayed on the surfaces of Bacillus subtilis spores. The spore-displayed beta-Gal was found to be stable when an amphiphilic 1,2-dimethoxyethane (DME) was used as a co-solvent; the transgalactosylation efficiency and octyl-gal conversion were optimal at 50% (v/v) DME. In addition, the product was maximally obtained from 100 mM lactose in a phosphate buffer/n-octanol/DME (25/25/50, v/v) mixture. By increasing the agitation speed and the amount of spores displaying beta-Gal, a yield of 33.7 mM octyl-gal was obtained over 24 h in a batch mode, which is much higher than in other octyl-gal bioconversion processes, such as those involving lipid-coating, reverse micelles, or whole cells. On the other hand, intermittent addition of spore-displayed beta-Gal and/or lactose in the reaction medium had no effect on the octyl-gal yield. The synthesized octyl-gal was hydrolyzed by the spore-displayed beta-Gal, and a high concentration of octyl-gal competitively inhibited the enzymes (K-i value of 10.8 mM). In summary, we demonstrate that octyl-gal synthesis by spore-displayed beta-Gal in non-aqueous medium can be significantly improved with the use of DME as a co-solvent. (c) 2010 Elsevier Inc. All rights reserved.