[Expression, purification and characterization of a thermostable pectate lyase from Thermotoga maritima].
The structure gene pelA from Thermotoga maritima MSB8 encoding pectate lyase was amplified and ligated into pHsh, resulting pHsh-pelA. Through structural optimization on pHsh-pelA, the ultimate plasmid, pHsh-pelC, which possessed the most appropriate structure and free energy of mRNA, was obtained. Pectate lyase C (PelC) was obtained after expressing pHsh-pelC in Escherichia coli JM109. The optimum activity of PelC was determined at pH 8.5 at 90 degrees C, with a half-life for almost 2 h at 95 degrees C. PelC was stable at the pH range of 8.2-9.8, and was dependent on Ca2+ for activity and stability. The enzyme kept stable for a long time and possessed a high level of activity at 60 degrees C. The kinetic assay using polygalacturonic acid (PGA) as substrate gave K(m) and V(max) of 0.11 mmol/L and 327 U per mg of protein. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant PelC was about 43 kD, which was exactly the size predicted. The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and cheap induction. Moreover, the superior stability of the recombinant enzyme laid the base for large-scale fermentation application.
Li
Ping
P
Department of Life Sciences, Nanjing Normal University, Nanjing 210046, China.
Jing
Qingqing
Q
Shao
Weilan
W
D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006358:Hot Temperature; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011995:Recombination, Genetic; D020124:Thermotoga maritima / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase