Efficient secretory expression of an alkaline pectate lyase gene from Bacillus subtilis in E. coli and the purification and characterization of the protein.
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.
Zhuge
Bin
B
Laboratory of Environmental Biotechnology, School of Biotechnology, Southern Yangtze University, Wuxi, Jiangsu Province, PR China.
Du
Guo-Cheng
GC
Shen
Wei
W
Zhuge
Jian
J
Chen
Jian
J
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
D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase