Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C 1 to C 8 ), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH 2 or NADPH 2 could act as an electron donor. Formate and NAD + (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD + and secondary alcohol dehydrogenase generated the NADH 2 required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.

Over 20 new cultures of methane-utilizing microbes, including obligate (types I and II) and facultative methylotrophic bacteria were isolated. In addition to their ability to oxidize methane to methanol, resting cell-suspensions of three distinct types of methane-grown bacteria (Methylosinus trichosporium OB3b [type II, obligate]; Methylococcus capsulatus CRL Ml NRRL B-11219 [type I, obligate]; and Methylobacterium organophilum CRL-26 NRRL B-11222 [facultative]) oxidize C2 to C4 n-alkenes to their corresponding 1,2-epoxides. The product 1,2-epoxides are not further metabolized and accumulate extracellularly. Methanol-grown cells do not have either the epoxidation or the hydroxylation activities. Among the substrate gaseous alkenes, propylene is oxidized at the highest rate. Methane inhibits the epoxidation of propylene. The stoichiometry of the consumption of propylene and oxygen and the production of propylene oxide is 1:1:1. The optimal conditions for in vivo epoxidation are described. Results from inhibition studies indicate that the same monooxygenase system catalyzes both the hydroxylation and the epoxidation reactions. Both the hydroxylation and epoxidation activities are located in the cell-free particulate fraction precipitated between 10,000 and 40,000 x g centrifugation. On the basis of 180 incorporation from 1802 into the cellular constituents of Pseudomonas methanica, Leadbetter and Foster (9) suggested that the initial oxidative attack on methane involves an oxygenase. Higgins and Quayle (8) isolated CH318OH as the product of methane oxidation when suspensions of P. methanica or Methanomonas methanooxidans were allowed to oxidize methane in "802-enriched atmospheres. The subsequent observation of methanestimulated reduced nicotinamide adenine dinucleotide (NADH) oxidation catalyzed by extracts of Methylococcus capsulatus (14, 15) or Methylomonas capsulatus (6) suggested that the enzyme responsible for this oxygenation is a monooxygenase. These workers relied on indirect enzyme assays, measuring methane-stimulated NADH disappearance spectrophotometrically or methane-stimulated 02 disappearance polarographically. Recently, methane monooxygenase systems were partially purified from Methylosinus trichosporium OB3b (16, 17) and Methylococcus capsulatus (Bath) (3, 4). The epoxidation of 1-alkenes was first demonstrated by Van der Linden (18), who detected the formation of 1,2-epoxyoctane from 1-octene by heptane-grown resting cells of P. aeruginosa. Cardini and Jurtshuk (2) found that a cell extract of a Corynebacterium sp. oxidized I-octene to 1,2-epoxyoctane in addition to hydroxylating octane to octanol. Coon and his co-workers (5) isolated an enzyme system from P. oleovorans that catalyzed the hydroxylation of alkanes and fatty acids. We have also demonstrated the expoxidation of 1-octene by whole cells and a pu

Cell‐free extracts derived from yeasts Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL‐Y‐11328 Torulopsis sp. strain A1 and Kloeckera sp. strain A2 catalyzed an NAD+‐dependent oxidation of secondary alcohols (2‐propanol, 2‐butanol, 2‐pentanol, 2‐hexanol) to the corresponding methyl ketones (acetone, 2‐butanone, 2‐pentanone, 2‐hexanone). We have purified a NAD+‐specific secondary alcohol dehydrogenase from methanol‐grown yeast, Pichia sp. The purified enzyme is homogenous as judged by polyacrylamide gel electro‐phoresis. The purified enzyme catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones in the presence of NAD+ as an electron acceptor. Primary alcohols were not oxidized by the purified enzyme. The optimum pH for oxidation of secondary alcohols by the purified enzyme is 8.0. The molecular weight of the purified enzyme as determined by gel filtration is 98000 and subunit size as determined by sodium dodecyl sulfate gel electrophoresis is 48 000. The activity of the purified secondary alcohol dehydrogenase was inhibited by sulfhydryl inhibitors and metal‐binding agents.