Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune.
A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases. The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.
Yano
Shigekazu
S
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu 525-8577, Japan.
Wakayama
Mamoru
M
Tachiki
Takashi
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism*; D002688:Chitinases / Q000378:metabolism; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D025282:Mycelium / Q000502:physiology; D011523:Protoplasts / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012567:Schizophyllum / Q000502:physiology*; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D002688:Chitinases; C021780:exo-1,3-alpha-glucanase