Mutanase from a Paenibacillus isolate: nucleotide sequence of the gene and properties of recombinant enzymes.
A mutanase (alpha-1,3-glucanase)-producing microorganism was isolated from a soil sample and was identified as a relative of Paenibacillus sp. The mutanase was purified to homogeneity from culture, and its molecular mass was around 57 kDa. The gene for the mutanase was cloned by PCR using primers based on the N-terminal amino acid sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of 3651-bp open reading frame that encoded a predicted 1217-amino acid polypeptide including a 43-amino acid signal peptide. The mature enzyme showed similarity to mutanases RM1 of Bacillus sp. strain RM1 and KA-304 of Bacillus circulans with 65.6% and 62.7% identity, respectively. The predicted molecular mass of the mutanase was 123 kDa. Thus, the enzyme purified from the isolate appears to be truncated by proteolysis. The genes for the full-length and truncated mutanases were expressed in Bacillus subtilis cells, and the corresponding recombinant enzymes were purified to homogeneity. The molecular masses of the two enzymes were 116 and 57 kDa, respectively. The specific activity was 10-fold higher for the full-length enzyme than for the truncated enzyme. The optimal pH and temperature for both recombinant enzymes was pH 6.4 in citrate buffer and 45 degrees C to 50 degrees C. Amongst several tested polysaccharides, the recombinant full-length enzyme specifically hydrolyzed mutan.
Sumitomo
Nobuyuki
N
Tochigi Research Laboratories of Kao Corporation 2606 Akabane, Ichikai, Haga, Tochigi, 321-3497, Japan.
Saeki
Katsuhisa
K
Ozaki
Katsuya
K
Ito
Susumu
S
Kobayashi
Tohru
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D011134:Polysaccharides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C021780:exo-1,3-alpha-glucanase