Isolation of a chymotrypsinogen B-like enzyme from the archaeon Natronomonas pharaonis and other halobacteria

A protease of a molecular mass of approximately 30 kDa was isolated and pur ified from the haloalkaliphilic archaeon Natronomonas (formerly Natronobact erium) pharaonis. The enzyme hydrolyzed synthetic peptides, preferentially at the carboxyl terminus of phenylalanine or leucine, as well as large prot eins. Hydrolysis occurred over the range of pH from 6 to 12, with an optimu m at pH 10. The temperature optimum was 61 degrees C. The enzyme was nearly equally active over the range of salt concentration from 0.5 to 4 M (NaCl or KCl). A strong cross-reaction with a polyclonal antiserum against human chymotrypsin was observed. Enzymatic activity was inhibited by typical seri ne protease inhibitors. There was significant homology between N-terminal a nd internal sequences from autolytic fragments and the sequence of bovine c hymotrypsinogen B; the overall amino acid composition was similar to that o f vertebrate chymotrypsinogens. Evidence for a zymogen-like processing of t he protease was obtained. Cell extracts from other halobacteria exhibited s imilar proteolytic activity and immunoreactivity. The data suggested a wide spread distribution of a chymotrypsinogen B-like protease among halo- and h aloalkaliphilic Archaea.