Study On Heterogenously Expression Of Keratinase From S.fradiae S-22l And Character Of The Keratinase

The aim of this study was to clone and express keratinase gene (sfks) which was from S. fradiae S-221, then sublimate the expressional keratinase and analyze enzyme activity and cut sites of the substrates.1. Expression of sfks gene in E.coli and S. lividans TK24. The keratinase propeptide gene which was cuted its exo-secerated peptide (38aa) was named sfk and it was expressed as inclusion body in E.coli. The mature keratinase (191aa) gene which has N-terminal leading peptide (78aa) and its exo-secerated peptide (38aa) was named sfks and it was successfully expressed in S. lividans TK24.2. The successfully expressed keratinase was purified using ammonium sulphate precipitation, negative ion exchange column and molecular sieve techniques (SuperdexTM 200 10/300 GL). The purified enzyme sfks has a molecular mass of about 19.1kDa, as determined by sodium dodecyl sulfate polyarylamide gel electrophoresis (SDS-PAGE) analysis. The concentration of the purification keratinase was 27.3μg/ml which was determined by BCA protein concentration testing kit.3. The optimal temperature in pH9.0 Tris-HCl Buffer was 37℃. The keratiase was inactivated by serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF) and its activity for keratin azure (Sigma-Aldrich, USA) was increased highly by Dithiothreitol (DDT). DDT could dissociate the disulfide bond between collective crystalloid proteins and bring facility to degrade keratin azure substrate. At the same time, for enzymatic activity study, casein, elastin and keratin azure were selected to be the substrates.4. In order to analyse the restriction enzyme cutting sites furtherly, after Green fluorescent protein (GFP) and casein degraded by sfks, degraded peptides of GFP and casein were analyzed by Agilent LC-MS QTOF techniques. The keratinase may have no specific enzyme cut sites and it cuts the substrates into peptides in different length. This showed a new enzyme cut style and it may have further foreground.