title: Gene cloning, expression, and characterization of mutanase from Paenibacillus curdlanolyticus MP-1.
abstract: Mutanases hydrolyze d-glucosidic linkages of α-1,3-linked polysaccharides which are important components of dental plaque. Therefore, these enzymes can be useful in preventive oral hygiene. A gene encoding mutanase was cloned from soil-isolated Paenibacillus curdlanolyticus MP-1 and expressed in Escherichia coli, and the resulting recombinant enzyme was characterized. The nucleotide sequence of the mutanase gene consisted of 3786 nucleotides encoding a protein of 1261 amino acids with a theoretical molecular weight of 131.62kDa. The deduced amino acid sequence exhibited a high degree of similarity with mutanases of Paenibacillus sp. KSM-M126 and Paenibacillus humicus NA1123, with 84% and 80% identity, respectively. The recombinant enzyme was purified 17.5-fold to homogeneity with a recovery of 37%. The purified mutanase showed optimal activity at pH 6.0 and 45°C, and was completely stable at pH 4.0-9.5 and up to 45°C. The enzyme was specific for α-1,3-glucosidic linkages and effectively solubilized fungal α-1,3-glucans and streptococcal mutans, releasing nigerooligosaccharides. The mutanase did not hydrolyze a synthetic substrate readily hydrolyzed by exoglucanases and the enzyme activity was not suppressed in the presence of deoxynojirimycin, an inhibitor of exo-type enzymes. These results suggest an endohydrolytic mode of action.
authors.affiliation: Department of Industrial Microbiology, Maria Curie-Skłodowska University, Akademicka 19, 20-031 Lublin, Poland. mplesz@poczta.onet.pl
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Pleszczyńska
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Boguszewska
authors.forename: Marek
authors.initials: M
authors.lastname: Tchórzewski
authors.forename: Adrian
authors.initials: A
authors.lastname: Wiater
authors.forename: Janusz
authors.initials: J
authors.lastname: Szczodrak
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005936:Glucans; D011993:Recombinant Fusion Proteins; C045788:alpha-1,3-glucan; D006026:Glycoside Hydrolases; C021780:exo-1,3-alpha-glucanase