Purification, substrate specificity, and N-terminal amino acid sequence analysis of a beta-lactamase-free penicillin amidase from Alcaligenes sp.

A beta-lactamase-free penicillin amidase from Alcaligenes sp. active against various beta-lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.

Microbial transformations of chalcones: hydroxylation, O-demethylation, and cyclization to flavanones.

Microorganisms were examined for their potential to catalyze biotransformation reactions that mimic plant biosynthetic processes. Specifically, microorganisms were screened for their abilities to transform selected chalcones to flavonoid and other products. Aspergillus alliaceus UI 315 efficiently transformed 3-(2' ',3' '-dimethoxyphenyl)-1-(2'-hydroxyphenyl)propenone (2'-hydroxy-2,3-dimethoxychalcone) (1) to several products, all of which were characterized by UV, NMR, and mass spectral analyses. A. alliaceus cyclized 1 to three flavanones and to O-demethylated and hydroxylated chalcones, some of which functioned as intermediates in the cyclization process. Inhibition studies using SKF525A, metyrapone, and phenylthiocarbamide with whole cell reactions showed that as many as three cytochrome P450 enzymes may be involved in these reactions. One enzyme catalyzed chalcone cyclization; another, O-demethylation; and a third, hydroxylation of chalcones. Flavonoid products are racemic, unlike the same products that are stereoselectively cyclized in plants. This work shows that microorganisms are capable of cyclizing chalcones to form flavonoid products, thus affording a mimic of plant biosynthetic processes.

Microbial transformations of S-naproxen by Aspergillus niger ATCC 9142.

Aspergillus niger ATCC 9142 was used to catalyze the biotransformation of S(-)-naproxen (1) to three major metabolites that were isolated by solvent extraction, purified chromatographically, and characterized by mass spectrometry and NMR spectroscopy. Metabolites were identified as O-desmethylnaproxen (2), 7-hydroxynaproxen (3) and 7-hydroxy-O-desmethyinaproxen (4). The kinetics of naproxen biotransformation to 2 and 4 was established over an 84 h period to show that naproxen was completely metabolized at 36 h, the major metabolite was O-desmethylnaproxen at 24 h, and the 7-hydroxy-O-desmethylnaproxen that was formed after 24 h.

Purification and characterization of a Galactomyces reessii hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid.

Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1). The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1). The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.