Abstract
BackgroundGlutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Previously, only glutamic peptidases derived from filamentous fungi have been characterized.ResultsWe report the first characterization of a bacterial glutamic peptidase (pepG1), derived from the thermoacidophilic bacteria Alicyclobacillus sp. DSM 15716. The amino acid sequence identity between pepG1 and known fungal glutamic peptidases is only 24-30% but homology modeling, the presence of the glutamate/glutamine catalytic dyad and a number of highly conserved motifs strongly support the inclusion of pepG1 as a glutamic peptidase. Phylogenetic analysis places pepG1 and other putative bacterial and archaeal glutamic peptidases in a cluster separate from the fungal glutamic peptidases, indicating a divergent and independent evolution of bacterial and fungal glutamic peptidases. Purification of pepG1, heterologously expressed in Bacillus subtilis, was performed using hydrophobic interaction chromatography and ion exchange chromatography. The purified peptidase was characterized with respect to its physical properties. Temperature and pH optimums were found to be 60°C and pH 3-4, in agreement with the values observed for the fungal members of family G1. In addition, pepG1 was found to be pepstatin-insensitive, a characteristic signature of glutamic peptidases.ConclusionsBased on the obtained results, we suggest that pepG1 can be added to the MEROPS family G1 as the first characterized bacterial member.
Highlights
Glutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin
The MEROPS peptidase database [2] has assigned sixty-six open reading frames (ORFs) to family G1 with the majority being derived from Ascomycetes
Low bootstrap values prevent deduction of the mutual relationship between the bacterial G1 peptidases from the generated maximum likelihood phylogenetic tree, except for the observation that bacterial G1 peptidases from Proteobacteria (Bin and Bvi) and Firmicutes (Ame, Cat, Ckl, Geo, pepG1 and Rsa 1+2) fall into two different clusters (Figure 1)
Summary
From the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Biotech industries are becoming more and more successful in providing enzymatic solutions to an ever increasing number of industrial processes. Besides being able to catalyze the enzymatic reaction in the industrial process, the enzymes must be able to survive the often harsh industrial conditions. One of the frequently required capabilities of an industrial enzyme is the ability to function at high temperatures in either an acidic or alkaline environment. Enzymes with such properties can either be designed in silico or by high-throughput screening of microorganisms.
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