title: Efficient secretory expression of an alkaline pectate lyase gene from Bacillus subtilis in E. coli and the purification and characterization of the protein.
abstract: The gene encoding pectate lyase (PL) from Bacillus subtilis WSHB04-02 was amplified by PCR, fused with a periplasmic secretion signal peptide sequence, pelB, from pET22b(+), cloned and expressed in Escherichia coli cells using a temperature control vector, pHsh. The recombinant E. coil was grown in a 5 l fermentor. PL was secreted in broth at 22 U l(-1) after 20 h when temperature was increased from 30 degrees C to 42 degrees C. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. It was optimally active at pH 9.4 and 50 degrees C over 30 min. Analysis of polygalacturonic acid (PGA) degradation products by electrospray ionization (ESI)-mass spectrometry (MS) indicated that PL produced a mixture of unsaturated oligo-galacturonides including unsaturated tri-galacturonic acid and unsaturated bi-galacturonic acid but not unsaturated mono-galacturonic acid.
authors.affiliation: Laboratory of Environmental Biotechnology, School of Biotechnology, Southern Yangtze University, Wuxi, Jiangsu Province, PR China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhuge
authors.forename: Guo-Cheng
authors.initials: GC
authors.lastname: Du
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.forename: Jian
authors.initials: J
authors.lastname: Zhuge
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase