A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

Abstract

BackgroundMicrobial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications.ResultsA gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX).ConclusionsThe stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.

Combined milk gel generated with a novel coagulating enzyme by Virgibacillus sp. SK37, a moderately halophilic bacterium

“…SK37 and constructed in E. coli DH10B (Phrommao et al . ). This bacterium was originally isolated from fermented Thai fish sauce and was found to produce several extracellular proteases with high activity.…”

“…SK37, which is a moderately halophilic bacterium isolated from Thai fish sauce (Phrommao et al . ). The enzyme has been cloned and successfully overproduced in an Escherichia coli system.…”

Characterization of a new oxidant-stable serine protease isolated by functional metagenomics

“…Subtilisins have been further classified into six subfamilies, namely true subtilisins, high-alkaline proteases, intracellular proteases, phylogenetically intermediate subtilisins between true subtilisins and high-alkaline proteases, high-molecular-mass subtilisins, and oxidant-stable proteases. The phylogenetic analysis shown here was done with SBcas3.3, the protease to which SBcas3.3 is the most similar (ZP_10168560: regulatory P domain of subtilisin-like proprotein convertases, Desulfobacter postgatei ) and representative members of each of the S8A groups mentioned above, as well as members of the AprX subfamily (Phrommao et al 2011) (a new group whose position inside the S8A protease family is not well defined). The Poisson correction method was used to compute evolutionary distances.…”

“…Several non-secreted proteins have been identified by functional metagenomics, probably as a result of membrane leakage after prolonged growth (Biver and Vandenbol 2013; Phrommao et al 2011). Its absence in the culture supernatant after one night does not mean, however, that SBcas3.3 is a membrane-anchored or intracellular protease when produced in its original host.…”

“…Though SBcas3.3 contains this conserved methionine, it resists H 2 O 2 concentrations much higher than 50 mM, and its activity is even enhanced up to 10 g/l H 2 O 2 (0.29 M), in contrast to other oxidant-stable proteases such as AprX from Virgibacillus sp. SK37 (Phrommao et al 2011). The ability to resist oxidizing agents is particularly sought in the detergent industry, which uses oxidants as bleaching agents in an increasing number of products.…”