Identification, purification and characterisation of a keratinolytic enzyme of chryseobacteriun carnipullorum
Mwanza, Elebert Pauline
MetadataShow full item record
Microbial enzymes are essential for sustainable technology and green chemistry coupled with a wide scope of genetic manipulation. Chryseobacterium carnipullorum 9_R23581T, isolated from raw chicken is a potential keratin degrader. Feather degradation is a challenge for most conventional proteases due to the structure of keratin material which makes up more than 90% of the feathers. Keratin is composed of tightly packed α–helix and β–sheets that are further assembled into supercoiled polypeptide chains. Furthermore, the presence of hydrophobic interactions, disulphide bridges and hydrogen bonds in keratin, contribute to its recalcitrant property, resulting in an extremely stable structural protein. Keratinases have huge potential applications in various industries that include the poultry processing industry, production of rare amino acids and semi-slow nitrogen release fertilizers in organic farming among others. This study focused on identifying, purifying, and characterising a proteolytic enzyme produced by C. carnipullorum. Growth studies were conducted to determine the stage of enzyme production and it was observed that secretory enzymes are produced during the exponential growth phase of C. carnipullorum. The secretory proteins were visualised using SDS-PAGE and identified using LC-MS/MS. Primers were designed on selected genes of interest, which were amplified from the genome of C. carnipullorum (accession number NZ-FRCD01000002.1). Peptidase M64 was identified as the most likely main component of the keratinolytic enzymes produced by the strain used in this study. The keratinolytic enzyme (peptidase M64) was expressed in E. coli BL21[DE3] cells and purified using Immobilised Metal Affinity Chromatography (IMAC). The molecular mass of the keratinase was determined to be about 50 kDa while its optimum temperature and pH were 50°C and 8.5, respectively. Different enzyme assays were conducted to test activity. The enzyme activity was inhibited by PMSF and it was enhanced by the presence of divalent metal ions such as MgSO4 and CaCl2.