Production of recombinant keratinase for poultry feather degradation

The keratinase gene (kerA) of Bacillus licheniformis ATCC®53757 was PCR amplified and subsequently cloned into Bacillus megaterium expression vector; pHIS1525.SPlipA and transformed in Bacillus megaterium ATCC®14945. The kerA gene carrying the recombinant plasmid pKERHIS1525.SPlipA was expressed in Bacillus megaterium under xylose inducible promoter, purified using Ni-NTA affinity column chromatography and consequently produced an extracellular keratinase activity of 29 U ml-1 after 18 h of incubation. The recombinant strain was further examined for feather degradation of intact chicken feathers. The chopped chicken feathers were partially degraded by the recombinant strain after 3days and the total macroscopic digestion was ultimately observed after seven days resulting in a yellowish peptide rich fermentation broth.


1. Introduction
2. Background
2.1 Keratinase
2.2 Keratin Structure
2.3 Polymerase chain reaction
2.3.1 Polymerase chain reaction
2.3.2 Designing the oligonucleotide primers for PCR
2.3.3 Accurate PCR-temperature
2.4 Digestion with restriction enzymes
2.4.1 Restriction digestion
2.4.2 Vector, pHIS1525.SPlipA
2.5 Bacillus megaterium as a host cell
2.6 Protoplast formation
2.7 Enzyme purification
2.8 Sodium dodecyl sulphate polyacrylamide gel electrophoresis
3. Materials and methods
3.1 Overnight culture of Bacillus licheniformis
3.2 DNA extraction
3.3 PCR
3.3.1 Keratinase gene (Ker gene)
3.3.2 PCR program
3.3.3 PCR procedure
3.4 DNA gel electrophoresis
3.5 Gene isolation from the agarose gel
3.6 Digestion with restriction enzymes
3.6.1 Vector
3.6.2 Restriction digestion
3.7 Ligation
3.8 Transformation
3.9 Protease assay
3.10 Enzyme purification
3.11 Enzyme assay
3.13 Degradation of keratinous waste by recombinant B.megaterium in Erlenmeyer flasks
4. Results
4.1 Cloning of KerA gene from Bacillus licheniformis into expression vector
4.2 Expression of kerA gene in Bacillus megaterium
4.3 Protease activity
4.4 Enzyme purification
4.6 Enzyme activity
4.7 Degradation of chicken feathers by recombinant strain
5. Discussion
6. Conclusions

Author: Alinezhad, Saeid,Mirabdollah, Amir

Source: University of Boras

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