Resumo

Proteases are the main group of commercialized enzymes. Keratinases are proteases that hydrolyze keratin, a protein hardly degraded in the environment. Keratinases have important applications in tannery and other industries as depilatory agent. The keratinase KerS14, produced by Bacillus subtilis S14 strain, has a great potential in tannery since it is able to hydrolyze keratin without damaging collagen. In this work, KerS14 ORF was cloned in a suitable vector and the recombinant protein expressed in an active form. Primers were design with sites for restriction enzymes NheI (forward) and BamHI (reverse). Reverse primer was also design with codons for a His-tag tail. KerS14 ORF was amplified, the amplicon was linked to vector pET5a and the identity of construction was confirmed by PCR, cleavage by restriction enzymes and DNA sequencing. Escherichia coli was transfected by heat-shocking with the plasmid. Cells were plated in Luria-Bertani (LB) agar with ampicillin. Small-scale tests were performed in order to find the best set of expression conditions yielding soluble protein. E. coli was grown at 37°C and 180 rpm in LB medium in containing ampicillin until an optical density of 0.6–1 at 600 nm. Expression levels were analyzed by SDS-PAGE and Western-blot using an anti-histidine tag antibody. The KerS14 fusion construct was expressed in E. coli BL21DE3 grown in LB medium at 25ºC after induction with 1 mM IPTG. Cells were ressuspended in lysis buffer (50 mM tris-HCl pH 8) and disrupted by addition of Lisozyme at a final concentration of 1 mg/mL and incubation at 30 °C during 30 minutes. Lysates cells were centrifuged and the supernatant collected. Recombinated KerS14 (rKerS14) was assayed, using N-suc-Ala-Ala-Pro-Phe-pNA as substrate. The enzyme activity was 5.8 enzymatic units.mL^-1.h^-1 and specific activity per milligram of total protein was 10.2 enzymatic units.mL^-1.mg^-1. The protein purification protocol is being further optimized to increase the protein yield. Also, site directed mutagenesis are in progress in order to improve rKerS14 thermal stability.