Abstract: The cell-envelope proteinase (CEP) was a key-enzyme in proteolytic system of Lactobacillus helveticus. In this paper, the calcium-free method was used to purify the CEP, and the best method of purify CEP was found in this article. Cells were washed three times in 50 mmol/L Tris-HCl (pH7.10) containing 30 mmol/L CaCl2. The proteolytic activity was released from the washed cells by incubation of 50 mmol/L Tris-HCl (pH7.10), containing 45mmol/L EDTA-Na2 at 43 ℃. After 90 min of incubation, the cells were removed by centrifugation(4500r/min×20min, 4 ℃), CEP was in the supernatant. Specific activity of CEP was improved 1.8 times purified by DEAE Sepharose CL-6B.

Key words: Lactobacillus helveticus, cell-envelope proteinase (CEP), proteinase activity