Two-step purification of a highly thermostable alkaline protease from salt-tolerant alkaliphilic Streptomyces clavuligerus strain Mit-1.

An alkaline protease from a salt-tolerant alkaliphilic Streptomyces clavuligerus was purified to homogeneity by 141-fold with a yield of 12% using two-step method of salt precipitation and ion exchange chromatography on DEAE cellulose. The apparent molecular mass was 49+/-2 kDa and the enzyme appeared as monomer based on SDS and Native-PAGE. The temperature optimum was 70 degrees C with significant stability at 60-80 degrees C for more than 60 min. The enzyme was active over the pH range of 8.5-11, with an optimum at 10-11. The serine nature of the protease was confirmed by PMSF inhibition. The enzyme was highly resistant against chemical denaturation and displayed varied effects towards metal ions. The results are significant as extremozymes are difficult to purify and therefore, a two-step purification of alkaline protease from relatively less explored group of actinomycetes is quite appealing.