A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular beta-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g center dot L-1. After optimization of the medium, 297.71U center dot ml(-1) of beta-glucanase activity in the medium and 352350U center dot g(-1) of fl-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.