Protease HY-3 produced by Aranicola proteolyticus is a valuable extracellular protease which shows a high activity on various kinds of protein substrates even in low temperature, wide range of pH and high salt condition. In this study, protease HY-3 gene was cloned and expressed in a recombinant Bacillus subtilis DB104. B.subtilis has been characterized extensively both physiologically and genetically. This wealth of information provides a sound base for the genetic engineering of the strain to develop the industrial process for the production of metabolites and enzymes. Besides, B.subtilis is a GRAS-generally recognized as safe-strain and the safety in the food industry is well recognized.
When the histidine-tagged protease HY-3 was produced with a recombinant B.subtilis DB104 harboring plasmid pSMHYMh, the maximum activity of protease was 195U/L. Considering basal level of protease activity of a mutant B.subtilis DB104 itself, the amount of protease HY-3 produced by recombinant strain was estimated to be about 30㎎/L. In addition to the production of protease HY-3 in recombinant B.subtilis, the purification process has been investigated through Ni-NTA affinity chromatography.
