Toward the development of a biocatalytic system for oxidation of p-xylene to terephthalic acid: oxidation of 1,4-benzenedimethanol

2006

Inactivation mechanisms of chloroperoxidase (CPO) from Caldariomyces fumago have been investigated with the aim of improving the practical utility of CPO for hydrocarbon oxidation. Deactivation studies in the presence of oxidants (i.e., hydrogen peroxide and t-butyl hydroperoxide) indicated that CPO lost oxidation activity toward hydrocarbon substrates during dismutation of hydrogen peroxide. The loss of enzyme activity was accompanied by the apparent destruction of the heme rather than aggregation or denaturation of the apo-protein. The decrease of enzyme activity was significantly retarded by adding the radical scavenger t-butyl alcohol at pH 4.1, or by optimizing the reaction pH. CPO retained greatest oxidation activity at pH 5-6, which may produce a more favorable ionization state of the key amino acid (Glu-183) and thus reduce radical formation. As a result of higher activity at pH 5-6, the total turnover numbers (TTN, defined as the amount of product produced over the catalytic lifetime of the enzyme) for the oxidation of toluene and o-, m-, p-xylenes in substrate/aqueous emulsion systems ranged from ca. 10% to 110% higher at pH 5.5 (20,000 to 45,000 mol product/mol enzyme) compared to pH 4.1. Furthermore, TTNs of CPO increased with increasing turnover frequencies, indicating that higher activity toward reducing substrates reduces radical formation and stabilizes CPO toward inactivation by H(2)O(2). These findings demonstrate the important relationship between CPO stability and activity, and illustrate that large improvements in CPO activity and stability can be achieved through solvent engineering.

Section: Methodsmentioning

confidence: 99%

Deactivation mechanisms of chloroperoxidase during biotransformations

Park

2006

“…The concentrations of benzoic acid, o-m-p-toluic acids, and phenylacetic acid were determined by HPLC using a YMC-Pack ODS-A column (Waters, Milford, MA) based on a previously published method (Morgan et al, 2002). The sample was prepared as follows: after separation of the aqueous phase from the organic phase by centrifugation at 3,800g for 20 s, the aqueous phase was mixed with pure methanol (1:9) and filtered through a 0.45 mm membrane.…”

Section: Methodsmentioning

confidence: 99%

New reaction system for hydrocarbon oxidation by chloroperoxidase

Park

2005

A novel reaction system was developed to maximize the catalytic efficiency of chloroperoxidase (CPO, from Caldariomyces fumago) toward the oxidation of hydrocarbons. The reaction system consisted of an organic/aqueous emulsion comprising pure substrate and aqueous buffer supplemented with the surfactant dioctyl sulfosuccinate. The emulsion system attenuated not only the destabilizing effects of the substrate and product on the enzyme by emulsifying the compounds, but also oxidant toxicity (oxidative stress) by increasing substrate availability. As a result, CPO exhibited total turnover numbers (TTNs, defined as the amount of product produced over the catalytic lifetime of the enzyme) of ca. 20,000 mol product/mol enzyme for the oxidation of styrene, toluene, and o-, m-, p-xylenes. The TTNs are over 10-fold higher than those previously reported for the oxidation of benzylic hydrocarbons by CPO. This study represents a significant step toward the development of CPO as a practical catalyst for large-scale organic syntheses.

“…The samples were shaken (300 RPM) and incubated at 30jC. The initial rate of substrate disappearance was monitored by HPLC using a previously published method (Morgan et al, 2002).…”

Section: Oxidation Of Terephthaldicarboxaldehyde (Da) By Galactose Oxmentioning

confidence: 99%

Salt‐activation of nonhydrolase enzymes for use in organic solvents

Morgan

2003

Enzymatic reactions are important for the synthesis of chiral molecules. One factor limiting synthetic applications of enzymes is the poor aqueous solubility of numerous substrates. To overcome this limitation, enzymes can be used directly in organic solvents; however, in nonaqueous media enzymes usually exhibit only a fraction of their aqueous-level activity. Salt-activation, a technique previously demonstrated to substantially increase the transesterification activity of hydrolytic enzymes in organic solvents, was applied to horse liver alcohol dehydrogenase, soybean peroxidase, galactose oxidase, and xanthine oxidase, which are oxidoreductase and oxygenase enzymes. Assays of the lyophilized enzyme preparations demonstrated that the presence of salt protected enzymes from irreversible inactivation. In organic solvents, there were significant increases in activity for the salt-activated enzymes compared to nonsalt-activated controls for every enzyme tested. The increased enzymatic activity in organic solvents was shown to result from a combination of protection against inactivation during the freeze-drying process and other as-yet undetermined factors.