The G1 family of glutamic proteases is a novel family, first described in 2004. G1 proteases are characterized by high temperature optima (around 55°C) and acidic pH optima. Only G1 proteases from fungi and a single G1 protease from bacteria have been characterized. The enzymatic activity of G1 proteases is dependent on a highly conserved catalytic dyad consisting of a glutamine and a glutamate residue. It has not been confirmed whether these residues are essential for the enzymatic activity of the bacterial G1 protease PepG1 from Alicyclobacillus sp. DSM 15716. AsaG1 and CmaG1, from the archaeal species Acidilobus saccharovorans and Caldivirga maquilingensis, respectively, have been annotated as putative G1 proteases. AsaG1 and CmaG1 have never been characterized to confirm their possible classification as G1 proteases. In this study, expression of AsaG1 and CmaG1 were attempted in Bacillus subtilis, Aspergillus oryzae and E. coli. Yet, expression could not be confirmed in any of the applied host organisms. PepG1 was point mutated by site-directed mutagenesis at Q117 and Q117+E199 that form the putative catalytic dyad, followed by expression in B. subtilis and characterization. Mutation of Q117 and Q117+E199 led to loss of enzymatic activity. It is therefore suggested that Q117 and E199 are essential for the catalytic activity, and thereby form a catalytic dyad similar to that of formerly characterized fungal proteases. This gives strong evidence that PepG1 is correctly classified as G1 protease.
|Uddannelser||Molekylærbiologi, (Bachelor/kandidatuddannelse) Bachelor el. kandidat|
|Udgivelsesdato||22 jan. 2013|
|Vejledere||Tove Atlung & Kenneth Jensen|
- Glutamic Proteases
- heterologous expression