Ching-Tsang Hou

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.

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.

Microbial Oxidation of Gaseous Hydrocarbons

Publisher Summary Prior to 1970, most of the activity on microbial oxidation of gaseous hydrocarbons was concentrated in Fosters laboratory in Texas and in Quayles laboratory in Sheffield, England. The methylotrophic bacteria can be classified into two major classes, depending on whether they are obligate or facultative. The obligate methylotrophs have three subgroups: first—those with a type I membrane structure can utilize methane and have the ribulose monophosphate pathway of carbon assimilation, second—those with a type II membrane structure can grow on methane and have the Icl – -serine pathway, and third—those with no internal membrane structure are unable to use methane and have the ribulose monophosphate pathway.

Microbial oxidation of methane and methanol: purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora.

Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C1-C10 tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD+, NADP+, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552 nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.