Microbial serine proteases, particularly those from the subtilisin family of Bacillus origin, are commercially important enzymes that have been extensively studies and developed [1]. They are used in commercial products, e.g., detergents, skin cares, and cleaners, as well as in industry, e.g., leather processing and chemical synthesis. The physicochemical properties of many commercially used subtilisins have been improved through genetic engineering, making them more resistant to inactivation by detergents, high temperatures, and other harsh conditions. Nevertheless, more recent studies have identified a number of natural subtilisin homologues with unique properties, e.g., active at high pH, high temperatures, and/or high NaCl concentrations [[2], [3], [4]].

Recently, we identified a new variant of serine protease from Bacillus subtilis ZMS-2, which was isolated from a desert soil sample in Pakistan and evaluated it as a potential dehairing and bating agent in eco-friendly leather processing [5,6]. This enzyme (ZMS-2 protease) is a metal ion-, surfactant-, and denaturant-compatible alkaline serine protease having a molecular mass of 36.1 kDa. The enzyme was active at a wide pH range (5–12), with an optimum activity observed when the reaction was performed in Tris-HCl pH 8.5 at 60 °C. Interestingly, the proteolytic activity of the enzyme increased in the presence of certain metal ions, i.e., Zn2+ and Ag+ (up to 5 mM), and decreased in the presence of Co2+, Mn2+, Fe3+, Ca2+, Na+, Cu2+, Ni2+, Mg2+, K+, or Hg2+. The proteolytic activity of the enzyme also increased in the presence of H2O2 (1%), Triton X-100 (1%), and a number of organic solvents (1% v/v). These properties are highly relevant to the industrial applications of the enzyme, as metal ions and other chemicals are common contaminants in industrial processes. However, the molecular basis underlying this interesting trait is unknown.

Here, we report a comparative study between ZMS-2 protease and B. subtilis ATCC 6051 subtilisin E (SubE), both were recombinantly produced in Escherichia coli. Biochemical characterization of both enzymes under various conditions revealed their opposite catalytic activities in the presence of Zn2+, Ag+, or H2O2. In silico and modeling studies as well as point mutations of the enzymes revealed molecular features that are responsible for the increased activity of ZMS-2 protease under those conditions.