ABSTRACT
A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 degrees C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K(m) and V(max) of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml(-1) and 85 mmol min(-1) mg(-1), respectively, and for chondroitin sulfate C, 0.5 mg ml(-1) and 103 mmol min(-1) mg(-1), respectively.
Subject(s)
Chondroitin Lyases/isolation & purification , Serratia marcescens/enzymology , Serratia marcescens/isolation & purification , Soil Microbiology , Chondroitin Lyases/biosynthesis , Chondroitin Lyases/chemistry , Chondroitin Sulfates/metabolism , Chromatography, Gel , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Dermatan Sulfate/metabolism , Dimerization , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Heparin/metabolism , Heparitin Sulfate/metabolism , Hyaluronic Acid/metabolism , Hydrogen-Ion Concentration , Isoelectric Point , Kinetics , Molecular Sequence Data , Molecular Weight , Protein Subunits/chemistry , Sequence Analysis, DNA , Serratia marcescens/classification , Serratia marcescens/cytology , Substrate Specificity , TemperatureABSTRACT
In order to develop the potential of chitinase-producing micro-organisms as biocontrol agents for insect pests, five chitinase-producing bacterial strains (C1, C2, C3, C4 and C5) previously isolated from soil samples were chosen to infect grassland locusts. The data showed that the mortality rate of locusts fed with strain C4 was significantly higher than that of other groups, and its pathogenicity was confirmed by Koch's law. Midgut tissues of locusts infected with C4 were examined with a light microscope. Apparent histopathologic changes in midgut cells partly explained the pathogenesis of locusts. Therefore, strain C4 was considered to be a potential biocontrol agent. To determine the taxonomic position of C4, physiological and biochemical characteristics were determined and molecular identification was performed. The 16S rDNA gene of C4 was amplified, cloned and sequenced. Comparative sequence analysis demonstrated that C4 corresponded to the genera Sanguibacter, Oerskovia and Cellulomonas. On the basis of phenotypic characterization and sequence similarity analysis, strain C4 was more closely related to the genus Sanguibacter. This chitinase-producing strain C4, which closely corresponds to the species of the genus Sanguibacter and is pathogenic to locusts, is here reported for the first time.