Purification and Characterization of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707

다국어 초록 (Multilingual Abstract)

Protocatechuate 3,4-dioxygenase was isolated and characterized from Pseudomonas pseudoalcaligenes KF707 for the purpose of developing a new anti-browning agent. The protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707 was purified 296.8-fold, and showed specific activity of 121.7 U/mg. Based on the SDS-polyacrylamide and gel permeation chromatography, the molecular weight of protocatechuate 3,4-dioxygenase was 189.9 kDa and was composed of 3 αβ protomers,with molecular weights of 29.0 kDa of α subunit and 34.3 kDa of â subunit. The optimal pH and temperature were 7.5 and 38oC,respectively. Km values of catechol, protocatechuate, gallate, pcresol,caffeic acid, catechin, L-DOPA, 4-methylcatechol and pyrogallol were 14, 17, 2, 10, 12, 20, 30, 21 and 3 μM, and the Vmax/Km (mim−1) values were 0.052, 3.06, 0.35, 0.01, 0.03, 0.02,0.006, 0.008 and 0.11, respectively. This indicates that the enzyme is active on a wide range of phenyl compounds, in contrast to the high specificity of similar enzymes from other sources. Our data also show that the turnover number of protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707 is 68s−1, which is much higher than the known values from other sources.

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