title: New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.
abstract: Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also determined on the fraction.
authors.affiliation: Centre de Recherche TEPRAL, Branche Boissons du Groupe Danone, Strasbourg, France. ilauer@tepral.fr
authors.forename: I
authors.initials: I
authors.lastname: Lauer
authors.forename: B
authors.initials: B
authors.lastname: Bonnewitz
authors.forename: A
authors.initials: A
authors.lastname: Meunier
authors.forename: M
authors.initials: M
authors.lastname: Beverini
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000516:alpha-Amylases; D008666:Metalloendopeptidases; D002118:Calcium