COMPARATIVE-STUDIES OF CHITINASE-A AND CHITINASE-B FROM SERRATIA-MARCESCENS

Serratia marcescens produces several chitinolytic enzymes, including c hitinase A (ChiA) and chitinase B (ChiB). In this study, ChiB was puri fied to homogeneity using a newly developed protocol based on hydropho bic interaction chromatography. Subsequently, characteristics of ChiB and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and ChiB shared several characteristics such as a broad ph optimum around ph 5.0-6.0, and a temperature optimum between 50 and 60 degrees C. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 degrees C, ph 6.1. Analyses of the degradatio n of various IV-acetylglucosamine oligomers, fluorogenic substrates an d colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc )(2)] from (GlcNAc)(n) and thus possess an exo-N,N'-diacetylchitobiohy drolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc)(n) substrates, indicating that they also have an endochitinase (ChiA) or exo-N,N',N ''-triacetylchitotriohydrolase (Ch iB) activity. Kinetic analyses with thylumbelliferyl-beta-D-N,N'-diace tylchitobioside. an analogue of (GlcNAc)(3), showed cooperative kineti cs for ChiA, whereas for ChiB normal hyperbolic kinetics were observed . ChiA had a higher specific activity towards chitin than ChiB and syn ergistic effects on the chitin degradation rate were observed upon com bining the two enzymes. These results, together with the results of se quence comparisons and previous studies of the cellular localization o f the two chitinases in 5. marcescens indicate possible roles for ChiA and ChiB in chitin breakdown.