Identification, purification and characterisation of a keratinolytic enzyme of chryseobacteriun carnipullorum
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Date
2018-02
Authors
Mwanza, Elebert Pauline
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
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.
Description
Keywords
Chryseobacterium carnipullorum, Degradation, Keratinase, Keratin, Feather waste, Proteins, Dissertation (M.Sc.Agric. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2018