Stephanie G. Burton

The search for the ideal biocatalyst

While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.

Biocatalysis with polyphenol oxidase: a review

Abstract A comprehensive review of the properties of polyphenol oxidase (EC 1.14.18.1) is given, including information relevant to the application of the enzyme as a biocatalyst, in aqueous and organic media. Literature reporting the use of the enzyme as a biocatalyst, and as a model enzyme in general studies of biocatalysis, is summarised.

A capillary membrane bioreactor using immobilized polyphenol oxidase for the removal of phenols from industrial effluents

Abstract A capillary membrane bioreactor has been developed and tested for the removal of phenolic compounds from synthetic and industrial effluents. Polyphenol oxidase was immobilized on single capillary membranes in a small-scale bioreactor using two morphologically different polymeric membranes. One has a novel structure with no external supporting skin layer. This membrane allows greater flux and was shown to facilitate high efficiency in removal of reaction products from the reactor. Using this high flux membrane, 949 μmol phenolics were removed from a solution containing 4 m m total phenolics using 45 U polyphenol oxidase in 8 h as compared with 120 μmol removed using non-immobilized enzyme which was inactivated (due to product inhibition) after 7 h. No precipitated melanoid products were observed in the permeate from the capillary membrane bioreactor, and in order to remove the colored quinone-type products of the reaction, a packed column containing chitosan was integrated into the system. Almost complete removal of the colored quinones and associated polymers from the permeate was observed.

A novel application for Neurospora crassa: Progress from batch culture to a membrane bioreactor for the bioremediation of phenols

Abstract The capacity of a Neurospora crassa fungal system to produce oxidative enzymes, and their application in the biodegradation of phenolic compounds, was demonstrated in static and shaken non-immobilized batch cultures, and by capillary membrane-immobilized biofilms. Extracellular laccase activity was produced at 10–12 U/mL (800 U/g wet mass) in static flask cultures, and 1.5 U/mL (130 U/g wet mass) in shaken batch cultures, respectively, over 8–15 days. Polyphenol oxidase was shown to be produced as an intracellular enzyme, at levels of 374 U/g wet mass. The production of laccase in a capillary membrane bioreactor was sustained at a level of 10 U/mL of permeate (1080 U/g wet biomass), typically over 30–40 days. Two phenolic substrates, phenol and p-cresol, both common components of industrial effluent streams, were chosen as model pollutants for bioremediation studies using the N.crassa enzyme system. In flask cultures, 18 mg p-cresol and 23 mg phenol respectively were removed from 5 mM solutions/g wet biomass, over a 6 day period. Over the same time period, immobilized cultures were found to convert 10 mg p-cresol or 8 mg phenol/g biomass. The immobilized biomass in a continuous reactor was found to have the capacity to sustain this removal efficiency continuously for a 4-month period, whereas the batch liquid culture systems remained active for approximately 8–15 days, after which cultures were no longer viable. This is the first demonstration of the use of immobilized N.crassa biofilms and their continuous application for bioremediation of phenols.

Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation

Abstract Internally skinned polysulphone capillary membranes were coated with a viscous chitosan gel and used as an immobilization matrix for polyphenol oxidase. Bench-scale, single-capillary membrane bioreactors then were used to determine the influence of the chitosan coating on product removal after substrate conversion by immobilized polyphenol oxidase during the treatment of industrial phenolic effluents. The results indicate that greater efficiency was achieved in the removal of polyphenol oxidase-generated products by the chitosan membrane coating, as compared with chitosan flakes. This facilitated an increase in the productivity of the immobilized enzyme.

Fungal Bioremediation of Phenolic Wastewaters in an Airlift Reactor

Of the various types of industry‐generated effluents, those containing organic pollutants such as phenols are generally difficult to remediate. There is a need to develop new technologies that emphasize the destruction of these pollutants rather than their disposal. In this work the white rot fungus, Trametes pubescens, was demonstrated to be an effective bioremediation agent for the treatment of phenolic wastewaters. An airlift loop reactor was optimized, in terms of volumetric oxygen transfer rate (KLa = 0.45 s‐1), to provide an environment suited to rapid growth of T. pubescens (μ = 0.25 day‐1) and a particularly efficient growth yield on glucose of 0.87 g biomass·g glucose‐1. The phenolic effluent was shown to be a paramorphogen, influencing fungal pellet morphology in the reactor, as well as increasing laccase enzyme activity by a factor of 5 over the control, to a maximum of 11.8 U·mL‐1. This increased activity was aided by the feeding of nonrepressing amounts (0.5 g·L‐1) of glucose to the reactor culture. To our knowledge the degradation results represent the highest rate of removal (0.033 g phenol·g biomass‐1·day‐1) of phenolic compounds from water reported for white rot fungi.

Phenoxazinone synthase: what's in a name?

The name phenoxazinone synthase (PHS, 2-aminophenol:oxygen oxidoreductase, EC 1.10.3.4) is used for the enzyme catalysing the oxidative coupling of substituted o-aminophenols to produce phenoxazinones. This review reveals that the traditional classification of PHS conflicts with recent sequence-based information that shows its relationship with two distinct copper protein groups. Different PHS roles, namely spore pigmentation in Streptomyces antibioticus (phsA) and biosynthesis of the antibiotic grixazone in Streptomyces griseus subsp. griseus (GriF), indicate an example of convergent evolution. Here, we review the classification, distribution and roles of PHSs, comparing them with copper oxidases at genetic and structural levels and exploring their potential application in the production of new antibiotics.

Development of bioreactors for application of biocatalysts in biotransformations and bioremediation

Biotransformation systems, whether used for environmentally benign biocatalysis of synthetic reactions, or bioremediation of pollutants, require suitable biocatalysts and suitable bioreactor systems with particular characteristics. Our research focuses on the bioconversion of organic compounds, many of which are industrial residues, such as phenols, poly-aromatic hydrocarbons, heterocyclic compounds, and polychlorinated biphenyls. The purpose of such biotransformations can be twofold: firstly, to remove them from effluents and convert them to less toxic forms, and secondly, to convert them into products with economic value. We conduct research in utilizing various isolated-enzyme and whole-cell biological agents; bioreactors, including novel membrane bioreactors, are used as a means of supporting/immobilizing, and hence applying, these biocatalysts in continuous systems. In addition, the enzyme systems are characterized biochemically, to provide information which is required in modification, adaptation, and scale-up of the bioreactors. The paper summarizes research on application of biofilms of fungal and bacterial cells and their enzymes, including hydrolases, polyphenol oxidase, peroxidase and laccase, in bioreactor systems including continuously operating membrane bioreactors.

Biotransformation of phenols using immobilised polyphenol oxidase

Polyphenol oxidase (PPO), obtained from Agaricus bisporus, can be used in hydroxylating a range of phenolic substrates to yield catechols which are then oxidised by the enzyme to give o-quinone products. The objective of this study was to develop systems whereby phenols could be transformed by PPO, and the products of the biotransformation could be isolated and characterised. By comparing the product mixtures obtained using soluble PPO and various forms of immobilised PPO, in aqueous and non-aqueous media, we have found significant differences in reaction rates and in the proportions of catechol and quinone produced. PPO in solution is inactivated by the reaction products, but when it is immobilised, the separation of products from the enzyme reduces this inhibition. Immobilisation also leads to increased stability, and allows continuous use of the enzyme. In bioreactors containing customised novel asymmetric capillary membranes as the enzyme support, high concentrations of phenolic substrates were converted. The addition of a chitosan-containing column downstream from the capillary membrane bioreactor facilitated the removal of the coloured quinone products from the permeate, and recycling of the substrate solution.

Production of D-amino acids from D,L-5-substituted hydantoins by an Agrobacterium tumefaciens strain and isolation of a mutant with inducer-independent expression of hydantoin-hydrolysing activity

Conversion of D,L-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine by Agrobacterium tumefaciens RU-OR involved a racemase, an hydantoinase and an unusual D-selective N-carbamylamino acid amidohydrolase which was active at alkaline pH and was not inhibited by N-carbamyl-L-amino acids. Enzyme activity was induced by growth in media containing 2-thiouracil. A mutant strain (RU-ORL5) was isolated, which expressed both the hydantoinase and N-carbamylamino acid amidohydrolase enzymes in the absence of inducer.