CLONING OF THE 52-KDA CHITINASE GENE FROM SERRATIA-MARCESCENS KCTC2172 AND ITS PROTEOLYTIC CLEAVAGE INTO AN ACTIVE 35-KDA ENZYME

A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated f rom a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We p urified both 52-kDa and 35-kDa chitinases using a chitin affinity colu mn and Sephacryl-S-300 gel filtration chromatography. We determined th at the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. mar cescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitina se derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45 degrees C and th e optimal pH of 5.5 were identical for both enzymes. The specific acti vities of the 52-kDa and 35-kDa chitinases on natural swollen chitin w ere 67 mu mol min(-1) mg(-1) and 60 mu mol min(-1) mg(-1), respectivel y. (C) 1998 Federation of European Microbiological Societies. Publishe d by Elsevier Science B.V.