Construction of a β-Glucanase HyperproducingBacillus subtilisUsing the Cloned β-Glucanase Gene and a Multi-copy Plasmid

We have cloned the β-glucanase gene from a β-glucanase producing strain, Bacillus subtilis Y-25, to construct a β-glucanase hyperproducing strain. The cloned 1.9 Kb EcoRI-HpaI fragment containing the entire β-glucanase gene was inserted into the EcoRI and PvuII sites of a multi-copy vector plasmid, pUB110. The resulting plasmid, named pLB100, was introduced into B. subtilis Y-25 to construct B. subtilis HL-25. HL-25 produced 347 units/ml of β-glucanase in a culture supernatant, which was about 19-fold higher than the amount produced by the original strain, Y-25, and corresponded to approximately 2 g of β-glucanase per 1. Furthermore, pLB100 is so stable in HL-25 that the proportion of cells carrying pLB100 was almost 100 % after cultivation for 100 generations without a selective antibiotic.