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 BBa_K2497999 was based on a KerP sequence combined with an pBAD promotor which originates from E.coli and that is induced by L-arabinose. The utilized promotor can be also found in the iGEM registry (BBa_K206000). 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 first. From there it was cloned into the pSB1C3 backbone and submitted to the iGEM registry afterwards. KerP gene sequence was taken from http://www.uniprot.org/. Gene sequence was optimized to eliminate illegal restriction sites. The construct was synthezied by IDT.
|pSB1C3 - pBAD - KerP||BBa_K2497999|