[Properties of alkaline pectate lyase from recombinant strain E. coli JM109 (pHsh PL)].
Alkaline pectate lyase (PL) from recombinant strain E. coli JM109 (pHsh PL) was purified by a three-step process including (NH4)2SO4 precipitation followed by dialysis and chromatography. The purified enzyme appeared homologous on SDS-PAGE. The specific activity of the purified enzyme reached 1079 U/mg. The optimal pH and temperature were in the ranges of pH 9.0 to 10.0 and 50 degrees C to 66 degrees C. The enzyme was preferable in optimal pH range in enzymatic retting of flax. Enzyme activity slightly increased in the presence of Mg2+ ion, whereas decreased in the presence of other ions, especially Fe2+. The K(m) of the purified enzyme for polygalacturonic acid was 20.93 mg/L, the V(max) for polygalacturonic acid hydrolysis was 105.3 micromol of unsaturated products per min and Ea was 21.74 kJ/mol. The results of the decay constant (k(d)) analysis on condition of PL bonding polygalacturonic acid (k(d) = 0.02 min(-1)) and PL without polygalacturonic acid (k(d) = 0.0342 min(-1)) showed the substrate was helpful to decrease thermal inactivation of PL. The products (unsaturated oligomers) from polygalacturonic acid degraded by PL were analyzed by electrospray ionization mass spectrometry(ESI-MS). The following data were obtained: ESI-MS m/z, 350.82 (unsaturated bigalacturonic acid, uG2), 527.04 (unsaturated trigalacturonic acid, uG3). However, m/z 175 (unsaturated galacturonic acid, uGI) was not found. These results indicate that the final PGA degradation products was a mixture of unsaturated oligo-galacturonides including uG3 and uG2 except for uG1. It suggests that the recombinant PL cannot degrade uG3 and uG2.
Zhuge
Bin
B
Lab Environ. Biotechnol., School of Biotechnology, Jiangnan University, The Key Laboratory of Industrial Biotechnology of Ministry of Education, Wuxi 214122, China. bzhuge@yahoo.com.cn
Du
Guocheng
G
Zhuge
Jian
J
Chen
Jian
J
D001407:Bacillus / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase