Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

Abstract

Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N‐terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.

Fully Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydrolase Family 18 Chitinase Inhibitors

“…After the addition of 60 l of 0.5 M sodium carbonate, the amount of 4-nitrophenylate released was determined by the absorbance at 405 nm. IC 50 values of fully deacetylated chitooligosaccharides, (GlcN) [2][3][4][5][6][7] , were determined in the presence of different concentrations of inhibitor. For SmChiA and SmChiB, the K m values for pNP-(GlcNAc) 2 were determined with increasing concentrations of the substrate from 0.02 to 0.5 mM, and the K i values for (GlcN) 5,6 were determined with concentrations of the substrate from 0.075 to 0.3 mM.…”

“…For the experiments with (GlcN) [2][3][4][5][6][7] , OfChtI, at a concentration of 0.1-0.14 mM in 50 mM HEPES buffer, pH 8.0, was placed in the reaction cell with a volume of 202 l, and (GlcN) [2][3][4][5][6][7] , at a concentration of 0.75-4.2 mM in 50 mM HEPES buffer, pH 8.0, was placed in the ITC syringe. Aliquots of 2-3 l from the syringe were injected into the reaction cell at 120-s intervals with a stirring speed of 1000 rpm.…”

“…In Vivo Bioevaluation by Injection of Mixed (GlcN) [2][3][4][5][6][7] into the Insect O. furnacalis-O. furnacalis larvae were reared using an artificial diet under a photo period of 16 h light and 8 h darkness and a relative humidity of 70 -90% at 26 -28°C as described previously (41).…”

“…For example, the well studied bacterium Serratia marcescens contains chitinases to degrade chitin for the purpose of nutrition (1)(2)(3)(4). Insects secrete chitinases from epithelial cells to digest chitin during development (5,6).…”

Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing

“…Chitinolytic enzymes are normally found in the culture medium, suggesting that they are secreted. However, the chitinolytic enzymes were isolated from the culture medium after prolonged culturing times (6-8 d) (Monreal & Reese, 1969;Roberts & Cabib, 1982;Fuchs et al, 1986;Jones et al, 1986). Conceivably the presence of these enzymes in the culture medium may have occurred as a result of cell lysis.…”