Selection of a subtilisin-hyperproducing Bacillus in a highly structured environment

Mutants that secrete increased amounts of enzyme into a selection medium can be efficiently enriched from large populations of mutagenized microorganisms during growth in hollow fibers. Under these conditions, each colony grows in its own microenvironment and cross-feeding between neighboring colonies is limited. We applied the technique to B. subtilis carrying a plasmid-encoded protease gene. The plasmid was subjected to random mutagenesis and clones secreting up to fivefold-increased amounts of enzyme were selected using a medium containing bovine serum albumin as the sole nitrogen source.