FEMS Microbiology Letters 124 (1994) 399-404 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier 399 FEMSLE 06309 Characterization of a chitinase gene (ch/A) from Serratia marcescens BJL200 and one-step purification of the gene product May B. Brurberg a,b,*, Vincent G.H. Eijsink a and Ingolf F. Nes a a Norwegian Agricultural University, Laboratory of Microbial Gene Technology, P.O. Box 5051, 1432 As, Norway, and b The o Norwegian State Agricultural Research Stations, 1432 As, Norway (Received 14 October 1994; accepted 19 October 1994) Abstract: The nucleotide sequence of the chiA gene from Serratia marcescens strain BJL200 was determined. The gene was found to encode a protein of 563 amino acid residues, with a typical N-terminal signal peptide of 23 residues, that is cleaved off during export. The gene exhibited striking differences with two previously characterized chiA genes of S. marcescens in the region corresponding to amino acid residues 410-467 of the gene product. Periplasmic fractions of an Escherichia coli strain harbouring the cloned gene were used as starting material for the development of a fast, one-step purification protocol for the chitinase that is based on hydrophobic interaction chromatography. Key words: Serratia marcescens; Chitinase; Hydrophobic interaction chromatography Introduction The Gram-negative enterobacterium Serratia marcescens produces several chitinolytic enzymes [1] and is one of the most efficient bacteria for the biological degradation of chitin [2]. Chiti- nolytic enzymes are of biotechnological interest, since their substrate, chitin, is a major structural component of fungal cell walls. Thus, chitinolytic enzymes could in principle be employed as natu- ral anti-fungal agents, for example by expressing their genes in crop plants or in bacteria used in * Corresponding author. Tel.: (+ 47) 64 94 94 61; Fax: (+ 47) 64 94 14 65; e-mail: mbruberg@bioslave.uio.no. fermentation processes prone to fungal attack [3,4]. Sundheim et al. [5] cloned two chromosomal fragments encoding chitinolytic (and antifungal) activity from S. marcescens BJL200. In the pre- sent study we have analysed one of these frag- ments and determined the nucleotide sequence of the chitinase gene located on it. Protein engineering could be used to improve the stability and activity of the naturally occurring Serratia chitinases, in order to improve their ap- plicability. Efficient protein engineering requires simple and fast procedures for purification, that permit the rapid processing of large numbers of mutant proteins. Therefore, we have developed a fast, one-step procedure for purification of the chitinase from periplasmic fractions of E. coli SSDI 0378-1097(94)00462-5