Novel extracellular proteolytic activity inPediococcus acidilacticiATCC 8042

Abstract

Proteolytic systems are common in lactic acid bacteria, but there are few reports about proteases or peptidases in the genus Pediococcus. To evaluate the presence of these types of enzymes, Pediococcus acidilactici ATCC 8042 was cultured in MRS broth. Supernatants collected during the log phase showed proteolytic activity towards an elastin dispersion when assayed using a spectrophotometer. Zn2+ showed a stimulatory effect, and the proteolytic activity reached its maximum when 200 mmol/L NaCl was included in the reaction buffer. On the other hand, activity was reduced when 5 mmol/L EDTA, 10 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L 1,10-phenanthroline were used or when the sample was heat treated. Zymograms showed two different proteolytic bands when gelatin was used as a substrate (>200 and 107 kDa), but only the higher molecular mass band was detected when casein or elastin was used. The gelatinolytic activity was not detected with zymograms of the 107 kDa band, which was the one inactivated by heat treatment. The use of a renaturing SDS-PAGE gel with embedded Micrococcus lysodeikticus cells allowed for the detection of a band with peptidoglycan hydrolase activity migrating at about 110 kDa. This activity was lost when 10 mmol/L EDTA was added to the renaturing buffer. Therefore, Pediococcus showed at least three different extracellular enzymes that were produced during the logarithmic growth phase and acted on peptide substrates. Each showed different substrate specificity, ion requirements, and thermostability.