High performance affinity chromatography of Bacillus neutral proteases

Bacillus neutral proteases were purified using bacitracin‐silica as an affinity medium. Several chromatographic procedures were investigated, including high speed runs on columns with 40‐ to 60‐microns silica particles. The high speed procedure enabled the purification of 4.9 mg of B. subtilis neutral protease directly from 165‐ml culture supernatant within 1.5 h. The neutral proteases of B. polymyxa and B. stearothermophilus were also purified. The latter enzyme was further concentrated by a second affinity chromatography step, using Sepharose with glycyl‐D‐phenylalanine as a ligand. During the purification procedures isopropanol was used to prevent autodigestion of the enzymes.