title: [Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme].
abstract: In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036, China.
authors.forename: Xue-Ming
authors.initials: XM
authors.lastname: Tang
authors.forename: Zheng-Xiang
authors.initials: ZX
authors.lastname: Wang
authors.forename: Wei-Lan
authors.initials: WL
authors.lastname: Shao
authors.forename: Ji-Quan
authors.initials: JQ
authors.lastname: Liu
authors.forename: Hui-Ying
authors.initials: HY
authors.lastname: Fang
authors.forename: Jian
authors.initials: J
authors.lastname: Zhuge
mesh_terms: D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005285:Fermentation; D005818:Genetic Engineering; D008670:Metals / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008670:Metals; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C032923:microbial serine proteinases