Abstract
Abstract 1. The inducible p-hydroxybenzoate hydroxylase of Pseudomonas putida which catalyzes the hydroxylation of p-hydroxybenzoate to protocatechuate has been obtained in crystalline form as a protein homogeneous upon ultracentrifugation and electrophoresis. The molecular weight is estimated to be 83,600. 2. The enzyme contains approximately 1 mole of flavin adenine dinucleotide per mole of protein. Reduced nicotinamide adenine dinucleotide phosphate, but not reduced nicotinamide adenine dinucleotide, serves as the hydrogen donor. 3. The enzyme is highly specific for p-hydroxybenzoate. Only four other aromatic compounds could be attacked at rates of less than 5% of the rate with p-hydroxybenzoate in all cases. 4. Under anaerobic conditions, the enzyme can catalyze the reduction of FAD by NADPH. This reaction requires the presence of the aromatic substrate to proceed at a significant rate. 5. The absorption spectrum of the enzyme in the visible region is slightly modified by the presence of the aromatic substrate.
Highlights
Cultures were prepared in batches of 10 liters in a New Brunswick fermenter and subjected to vigorous aeration during growth
Each culture was harvested with the use of an air-driven Sharples centrifuge before the population entered the stationary phase
Its specific activity remains unchanged for 3 months at -15”; stability decreases rapidly as it is purified, and the crystalline material loses about f5% of its activity if it is recrystallized in the unprotected state
Summary
The strain employed was Pseudomonas putida A.3.12 (ATCC 12633). The bacterium was cultivated at 30” in a mineral medium [16] which contained 20mM p-hydroxybenzoate.Cultures were prepared in batches of 10 liters in a New Brunswick fermenter and subjected to vigorous aeration during growth. The wet cell past,e was stored at - 15” until extraction.
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