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 α-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity.
The gene of α-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 α-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of α-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (α-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.
The recombinant α-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant α-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-β-D-thiogalactopyranoside. The recombinant α-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.

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