Studies on an alkaline protease and a lipase of pseudomonas sp. KFCC 10818

Abstract

A gene, aprP, encoding an extracellular alkaline protease from Pseudomonas sp. KFCC 10818, was expressed in E. coli and the protein product, AprP, was purified to near homogeneity. The molecular weight of the alkaline protease was determined as 29 kDa by SDS-PAGE. The N-terminus of the processed and secreted AprP was identified as Ala-140. The purified AprP was most active at pH 11, which is 0.5-2 pH units higher than those of the known subtilisins. The AprP was stable in the pH range of 6 to 11. The stability of the protease was maintained below 53℃ in the presence of 2 mM $Ca^{2+}$. The optimum temperature of AprP was 60℃, which was shifted to ＞ 70℃ in the presence of 2 mM $Ca^{2+}$. The AprP showed above 50 % of the maximal activity at a low temperature, 15℃. Considering its alkalophilicity and high activity at low temperatures, AprP possesses good characteristics as a candidate for detergent formulation. Moreover, the $k_{cat}/K_m$ value ($9.2 \times 10^3 S^{-1}mM^{-1}$) of AprP for the hydrolysis of Suc-AAPF-pNA was 40 - 1400 times higher than those of subtilisins. Therefore, the AprP is the most efficient protease among previously described alkaline proteases. Furthermore, the AprP was more active toward azocasein than Savinase 4.0 T (Novo) in the presence of 0.1 % surfactants. In the cleaning activity for polluted clothes, AprP and Savinase 4.0 T showed almost equal activity at 25℃. And, the specific activity of AprP was about 2-fold higher than that of Savinase 6.0 T (Novo). The AprP studied in this work, therefore, can be an alternative to the existing proteases. An additional gene, limK, located immediately downstream of the lipase structural gene, lipK, of Pseudomonas sp. KFCC 10818, was cloned and sequenced and its function was studied. The limK gene could encode a polypeptide, LimK (lipase modulator), of 279 amino acids. When compared to the known modulator proteins, the LimK showed low sequence identities (17.1 - 19.9 %). The lipK gene could en...