| Abstract [eng] |
Keratinases are enzymes capable of breaking down insoluble, fibrillar, keratin-containing substrates. These biocatalysts are important due to their wide range of applications in the textile, leather, detergent, cosmetic, medicine industry and prion elimination. The aim of this work was to create and optimize a heterologous expression system for enzymes with keratinolytic activity. Tasks that needed to be complete during this study: Streptomyces sp. SK-91 genomic analysis and identification of genes encoding hypothetical keratinases, creation of selected gene constructs suitable for heterologous expression, selection of a suitable Escherichia coli strain for target protein synthesis, determination of optimal inducer concentration and optimal induction duration. Lastly, purification of target proteins. During the work, seven hypothetical keratin hydrolyzing proteins from Streptomyces sp. SK-91 strain were identified. The pET21c(+) and pET28b(+) constructs were created with the genes of the selected enzymes. Expression of these constructs was performed in E. coli BL21 (DE3), Rosetta (DE3) and C41 (DE3) strains using 0.5 mM and 1 mM IPTG for induction. Cells were cultured for 24 hours after induction. Induced and uninduced cells were sampled hourly for the first three hours and at hour 24 post-induction. After protein synthesis, it was found that E. coli Rosetta (DE3) is a suitable strain for the synthesis of recombinant KER78 and KER1314 proteins. In order to synthesize the KER910 protein, E. coli C41 (DE3) is a suitable heterologous host. In the course of this work, it was established that the optimal concentration of the inducer is 0.5 mM IPTG to synthesize KER78, and to synthesize KER78 without a signal peptide. For KER910 and KER1314 without a signal sequence, 1 mM inducer (IPTG) should be used. From the results, we can conclude that the synthesis of KER78 recombinant protein without a signal peptide begins 2 hours after induction, while one hour is enough to synthesize KER78, KER910 and KER1314 without a signal peptide. KER78, KER910 and KER1314 recombinant proteins were successfully purified by Ni2+ affinity chromatography. In order to restore the activity of the recombinant proteins, dialysis was performed, but no activity was detected in the zymogram. In order to better understand the action of keratinases and expand the application of these enzymes, scientific research in this direction is necessary. |
