Team:Stuttgart/Composite Part

Composite Part

KerP from Pseudomonas aeruginosa

Keratinases are proteolytic enzymes that are applied in agro-industrial, pharmaceutical and biomedicals fields(Satyanarayana et al. 2013). In our project we used keratinases for establishing a microbial tube cleaner. Hair mostly consists of alpha-keratin which is hydrolyzed by keratinases. By using the keratinases we want to avoid chemical compounds that are recently present in tube cleaners. Many different keratinases produced by different microorganisms (e.g. Pseudomonas aeruginosa,Bacillus subtilis) have been reported. These enzymes vary in their biochemical and biophysical properties e.g. temperature and pH activity range. KerP is a keratinase originating from Pseudomonas aeruginosa It was succesfully transformed and expressed as an extracellular protein in E.coli with a size of 33 kDa and a specific activity of 3,7 kU/mg. This keratinase belongs to the group of serine proteases with optimal activity at pH 9 and 50°C (Sharma and Gupta 2010). Our construct was based on a KerP sequence combined with a constitutive promotor, which clonation into E.coli has been already shown to be actively secreted (Sharma and Gupta 2010). The utilized promotor can be also found in the iGEM registry (BBa_J23119). Additionally a pelB sequence that provides for extracellular protein secretion was added. This pelB sequence is also a part which is available at the iGEM registry (BBa_K208004). Since the KerP sequence was not provided at other part we synthesized this construct by IDT (Integrated DNA Technologies, BVBA, Belgium) and cloned it into the pSB1K3 backbone. From there it was cloned into the pSB1C3 backbone and submitted to the iGEM registry afterwards.