Cloning, heterologous gene expression and biochemical characterization of the alpha-1,3-glucanase from the filamentous fungus Penicillium purpurogenum.
There has been much recent interest in alpha-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for alpha-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 degrees C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.
Shalom
Gil
G
Division of Microbial Diseases, UCL Eastman Dental Institute, 256 Gray's Inn Road, London WC1X 8LD, UK.
Pratten
Jonathan
J
Wilson
Michael
M
Nair
Sean P
SP
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010407:Penicillium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases