Purification of toxic protease from Brevibacterium otitidis KB76 with both metal and hydrosulfuryl at the active site

An extracellular toxic protease, KB76 from Brevibacterium otitidis was successfully purified to 31.3-fold by anion-exchange and gel filtration chromatography. The molecular mass was determined to be 47 kDa using SDS-PAGE. The optimum temperature and pH of the protease were 7.4 and 40A degrees C, respectively. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibited the activity of the enzyme but soybean trypsin inhibitor and aprotinin had no obvious inhibition, which suggested the presence of both metal and hydrosulfuryl at or near the active site. Additionally, the isoelectric point of this protein was 5.5 +/- 0.2. Its apparent K-m and V-max for the synthetic substrate N-succinyl-L-phenylalanine p-nitroanilide were 2.41 mM and 21.74 mu M/min, respectively. Further, studying the lethality of the protease on mice by intraperitoneal injection, it exhibited 48-h LD50 value of 9.6 mg/kg body weight. Gross and electron microscopic study in mice revealed that purified protease was capable of eliciting a variety of tissue responses resulted in liver necrosis. In conclusion, this protease produced by B. otitidis represents a potential toxic agent.