Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties

Two extracellular alkaline proteases produced by an alkalophilic Bacillus i solate were purified and characterized using acetone precipitation, DEAE- a nd CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chrom atographic techniques. Analysis of the purifred proteases by SDS-PAGE revea led that both proteases, AP-1 and AP-2 were homogenous with molecular weigh t estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 an d AP-2 were at temperatures of 50 and 55 degrees C and pHs of 11 and 12, re spectively. The enzymes were also stable in the pH range of 6.0-12.0 for a period of 4 h with and without Ca2+ (5 mM) and temperatures of up to 50 deg rees C. The half-lives of the enzymes recorded at 50 degrees C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native pro tein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA . The K-m values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, re spectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also in dicated that small aliphatic and aromatic amino acids were the preferred re sidues at the P-1 position. (C) 1999 Elsevier Science Ltd. All rights reser ved.