Purified glycerol oxidase from Aspergillus faponicus AT 008 was homogeneous by ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be 400,000 by sedimentation equilibrium, and the isoelectric point was found to be 4.9 by isoelectric focusing. The enzyme showed spectral characteristics of a heme protein. The reduced form possessed absorption maxima at 557 and 430 nm and the oxidized one at 557, 530, 420, 280, and 238 rim. The heme in the enzyme was identified as protoheme IX (one mol per mol of enzyme protein). Glycerol was the best substrate for the enzyme, and the Km value for glycerol was determined to be 10.4 mal. Dihydroxyacetone was oxidized at 59 % of that for glycerol, but glycerol 3-phosphate, dihydroxyacetone phosphate, methanol, and ethanol were not oxidized at all. The enzyme had an optimal pH at 7.0 with glycerol as substrate, and the enzymatic activity increased by treatment in alkaline pH. The enzyme was also activated by addition of several divalent metal ions including Zn2+, Nit+, and Mgt+.
With the use of crystalline preparations of tyramine oxidase of Sarcina lutea, substrate and inhibitor specificities of the enzyme were investigated. The enzyme oxidized tyramine and dopamine at almost the same rates. Other monoamines, diamines, polyamines and amino acids were not oxidized at all. The oxidation of tyramine proceeded as follows: Tyramine+O2+H2O•¨p-Hydroxyphenylacetaldehyde+NH3+H2O2. Ammonia and hydrogen peroxide were formed in stoichiometric amounts. The enzyme was not inhibited by carbonyl reagents, such as hydroxylamine, hydrazine, semicarbazide and isoniazid, but was inhibited by p-CMB and iproniazid.
The hydroxylation of 2-phenylpropionic acid (PPH) to 2-(/>-hydroxyphenyl)propionic acid (HPPH) was investigated using various microorganisms. The highest hydroxylating activity was found in Streptomyces rimosus ATCC 10907. The reaction product was isolated and identified as 2-(/?-hydroxyphenyl)propionic acid, which had no specific rotation, indicating that the reaction was not stereoselective at the benzylic position. In a 5-1 jar fermentor containing a medium consisting of 2% sorbitol, 2% soybean meal, and 1% malt extract (pH 7.2), the conversion rate from 0.2% of PPH reached 60% using a PPH-resistant mutant, KY-661-18. 2-(/?-Hydroxyphenyl)propionic acid (HPPH) is a very important chemical used mainly as the intermediate for many non-steroidal analgesics. Syntheses of HPPHhave already been reported, using the reduction of aryl acetophenones,1} and 1 ,2-aryl migration ofaryl propiophenones,2) but all of them require many steps such as the introduction of protective groups and their removal.
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