Allen I. Laskin

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Abstract Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C 1 to C 6 ), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O 2 → aldehyde + H 2 O 2 Formaldehyde + O 2 → formate + H 2 O 2 . Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K m values for methanol and formaldehyde are 0.5 and 3.5 m m , respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents ( p -chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.

NAD-linked formate dehydrogenase from methanol-grown Pichia pastoris NRRL-Y-7556

Abstract An NAD-linked formate dehydrogenase (EC 1.2.1.2.) from methanol-grown Pichia pastoris NRRL Y-7556 has been purified. The purification procedure involved ammonium sulfate fractionation, hollow-fiber H1P10 filtration, ion-exchange chromatography, and gel filtration. Both dithiothreitol (10 m m ) and glycerol (10%) were required for stability of the enzyme during purification. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and by sedimentation pattern in an ultracentrifuge. The enzyme has a molecular weight of 94,000 and consists of two subunits of identical molecular weight. Formate dehydrogenase catalyzes specifically the oxidation of formate. No other compounds tested can replace NAD as the electron acceptor. The Michaelis constants were 0.14 m m for NAD and 16 m m for formate (pH 7.0, 25 °C). Optimum pH and temperature for formate dehydrogenase activity were around 6.5–7.5 and 20–25 °C, respectively. Amino acid composition of the enzyme was also studied. Antisera prepared against the purified enzyme from P. pastoris NRRL Y-7556 form precipitin bands with isofunctional enzymes from different strains of methanol-grown yeasts, but not bacteria, on immunodiffusion plates. Immunoglobulin fraction prepared against the enzyme from yeast strain Y-7556 inhibits the catalytic activity of the isofunctional enzymes from different strains of methanol-grown yeasts.

Identification and purification of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from C1-utilizing microbes

Phenazine methosulfate (PMS)-dependent methanol dehydrogenase has been reported from many methylotrophic bacteria [l-4]. This enzyme oxidizes primary alcohols from Cr-C,, but does not oxidize secondary alcohols. Nicotinamide adenine dinucleotide (NAD)dependent alcohol dehydrogenases have been reported from liver and yeast [5]. These alcohol dehydrogenases oxidize primary alcohols and acetaldehyde but have no activity on methanol. In addition, the alcohol dehydrogenases from yeast and liver also oxidize some secondary alcohols at a very low rate (<l% of their ethanol activity). NAD(P)-dependent alcohol dehydrogenases were also reported inPseudomonas [6,7], Escherichiu coli [8] and Leuconostoc [9]. However, these enzymes were active only on long-chain primary alcohols or hydroxy fatty acids [7]. To our knowledge, no secondary alcohol-specific alcohol dehydrogenase (SADH) has been reported. We have recently identified an NAD-linked, secondary alcohol-specific, alcohol dehydrogenase in cell-free extracts of various gaseous hydrocarbon-utilizing microbes. This enzyme is also found in cells grown on methanol. It specifically and stoichiometrically oxidizes secondary alcohols to their corresponding methyl ketones. This enzyme has been purified 2600-fold and shows a single protein band on acrylamide gel electrophoresis.

Epoxidation and Ketone Formation by C1-Utilizing Microbes

Publisher Summary Epoxides are extremely valuable products because of their ability to undergo a variety of chemical reactions. The products of epoxidation are industrially important because of their ability to polymerize under thermal, ionic, and free radical catalysis to form epoxy homopolymers and copolymers. Ethylene oxide and propylene oxide constitute the two important commercial epoxides. Past studies of epoxide formation showed that the rate of propylene oxide production was linear for the first 120 minutes for strains CRL M1 and OB3b, and for the first 60 minutes for strain CRL 26. In the epoxidation of propylene by cell suspensions of methane-utilizing bacteria, no formation of 3-hydroxy-l-propene was detected. The first bacterial ketone formation from gaseous alkanes was demonstrated with methane-grown Pseudomonas methanica. This strain was able to oxidize but not assimilate propane and butane, in the presence of the growth substrate (methane). Products of this cooxidation of propane were n -propanol, propionic acid, and acetone.

Single Cell Protein

Publisher Summary This chapter provides an overview on the present status of the single-cell protein (SCP) field and some of its recent trends. The association of microorganisms with food is ancient, encompassing the use of yeast in baking and brewing, and the role of bacteria and other organisms in foods such as cheese, yogurt, sausage, and a wide variety of fermented foods. The SCP field includes many species of different microorganisms grown on wide variety of carbon substrates, including n-paraffins, methane, methanol, ethanol, acetate, CO 2 , and a plethora of carbohydrate and cellulosic materials from many different by-product and waste sources. Recommended criteria and procedures for production and testing of SCP materials with regard to both safety and nutritional value are found in a series of guidelines published by the Protein-Calorie Advisory Group of the United Nations. This chapter also discusses certain unique developments that have occurred in the recent past in the field of SCP.

Purification and properties of a NAD-linked 1,2-propanediol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-dependent 1,2-propanediol dehydrogenase (EC 1.1.1.4) activity was detected in cell-free crude extracts of various propane-grown bacteria. The enzyme activity was much lower in 1-propanol-grown cells than in propane-grown cells of Pseudomonas fluorescens NRRL B-1244, indicating that the enzyme may be inducible by metabolites of propane subterminal oxidation. 1,2-Propanediol dehydrogenase was purified from propane-grown Ps. fluorescens NRRL B-1244. The purified enzyme fraction shows a single-protein band upon acrylamide gel electrophoresis and has a molecular weight of 760,000. It consists of 10 subunits of identical molecular weight (77,600). It oxidizes diols that possess either two adjacent hydroxy groups, or a hydroxy group with an adjacent carbonyl group. Primary and secondary alcohols are not oxidized. The pH and temperature optima for 1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propan1,2-propanediol dehydrogenase are 8.5 and 20-25 degrees C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The Km values at 25 degrees C, pH 7.0, buffer solution for 1,2-propanediol and NAD are 2 X 10(-2) and 9 X 10(-5) M, respectively. The 1,2-propanediol dehydrogenase activity was inhibited by strong thiol reagents, but not by metal-chelating agents. The amino acid composition of the purified enzyme was determined. Antisera prepared against purified 1,2-propanediol dehydrogenase from propane-grown Ps. fluorescens NRRL B-1244 formed homologous precipitin bands with isofunctional enzymes derived from propane-grown Arthrobacter sp. NRRL B-11315, Nocardia paraffinica ATCC 21198, and Mycobacterium sp. P2y, but not from propane-grown Pseudomonas multivorans ATCC 17616 and Brevibacterium sp. ATCC 14649, or 1-propanol-grown Ps. fluorescens NRRL B-1244. Isofunctional enzymes derived from methane-grown methylotrophs also showed different immunological and catalytic properties.

Novel Enzymes from Methylotrophic Microorganisms

In the course of our studies on methylotrophs, microorganisms that grow on methane and on other C1 compounds, we discovered that cell suspensions of methane-grown bacteria have the ability to oxidize gaseous 1-alkenes to their corresponding 1,2-epoxyalkanes, as well as to hydroxylate not only methane, but also the other gaseous alkanes (1). Thus, ethylene, propylene, 1-butene, and butadiene are converted to expodies, and methand, ethane, propane, and butane are hydroxylated; both primary and secondary alcohol products were detected. The product epoxides are not further metabolized and they accumulate extracellularly.