| The feather-degrading bacterium Bacillus licheniformis PWD-1 can hydrolyze and grow on chicken feathers. It was hypothesized that feather degradation was executed by an extracellular proteolytic enzyme, or, keratinase. The study was initiated to isolate keratinase from PWD-1 culture medium. Keratinase was purified by using ultrafiltration, CM-cellulose ion-exchange chromatography, and Sephadex G-75 molecular permeation. The keratinase was found to be a potent serine protease, with a molecular weight of 33 kDa, optimum pH at 7.5 toward feather keratin, and pI at 7.25. It is also a thermostable protease with maximum activity at 50;Subsequently, efforts were made to isolate kerA, the gene which encodes the keratinase. Two methods, namely, PCR (polymerase chain reaction)-walking and PCR-screening of genomic library were developed. By using these two methods, a 1.4 kb kerA containing the promoter, ribosomal binding site, pre-and pro-regions, the structure gene, and a transcriptional terminator was isolated and sequenced.;To enhance production of the keratinase, kerA was inserted into plasmids pUB18 and pUB18-P43. Three new plasmid vectors, pLB3, pLB29 and pLB36, with kerA insertions were constructed. Transformation of a protease-deficient strain Bacillus subtilis DB104 with these vectors resulted in three new strains, FDB-3, FDB-29 and FDB-36. All three transformants were able to produce active keratinase in both Luria-Bertani (LB) and feather media. FDB-29 produced the highest proteolytic activity among the three transformants grown at 37;During this series of studies, an additional project was carried out to evaluate the possibility for immobilizing the keratinase on glass beads. The report in short form is appended to this thesis. |
