Identification, purification and characterisation of a keratinolytic enzyme of chryseobacteriun carnipullorum

[摘要] Microbial enzymes are essential for sustainable technology and green chemistry coupledwith a wide scope of genetic manipulation. Chryseobacterium carnipullorum 9_R23581T,isolated from raw chicken is a potential keratin degrader. Feather degradation is a challengefor most conventional proteases due to the structure of keratin material which makes upmore than 90% of the feathers. Keratin is composed of tightly packed α–helix and β–sheetsthat are further assembled into supercoiled polypeptide chains. Furthermore, the presence ofhydrophobic interactions, disulphide bridges and hydrogen bonds in keratin, contribute to itsrecalcitrant property, resulting in an extremely stable structural protein. Keratinases havehuge potential applications in various industries that include the poultry processing industry,production of rare amino acids and semi-slow nitrogen release fertilizers in organic farmingamong others. This study focused on identifying, purifying, and characterising a proteolyticenzyme produced by C. carnipullorum. Growth studies were conducted to determine thestage of enzyme production and it was observed that secretory enzymes are producedduring the exponential growth phase of C. carnipullorum. The secretory proteins werevisualised using SDS-PAGE and identified using LC-MS/MS. Primers were designed onselected genes of interest, which were amplified from the genome of C. carnipullorum(accession number NZ-FRCD01000002.1). Peptidase M64 was identified as the most likelymain component of the keratinolytic enzymes produced by the strain used in this study. Thekeratinolytic enzyme (peptidase M64) was expressed in E. coli BL21[DE3] cells and purifiedusing Immobilised Metal Affinity Chromatography (IMAC). The molecular mass of thekeratinase was determined to be about 50 kDa while its optimum temperature and pH were50°C and 8.5, respectively. Different enzyme assays were conducted to test activity. Theenzyme activity was inhibited by PMSF and it was enhanced by the presence of divalentmetal ions such as MgSO4 and CaCl2.

[发布机构] University of the Free State