Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium<jats:named-content content-type="genus-species">Rhodococcus opacus</jats:named-content>1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg<jats:sup>−1</jats:sup>represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process.</jats:p>

Co-metabolic formation of substituted phenylacetic acids by styrene-degrading bacteria

“…But strain ST is known to produce this compound as an intermediate [4]. These results strongly confirm former studies [34], [35] and indicate that these strains mentioned degrade styrene via the route of site-chain oxygenation.

Fig. 2(a) Chromatographic and (b) spectral identification of the metabolite phenylacetic acid during styrene degradation of strains Kp5.2, CWB2, and 1CP.Cell suspensions of Sphingopyxis sp.

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“…A modification of this pathway by deletion or substitution of enzymes is possible as mentioned by Hartmans et al [20] or Toda and Itoh [47] . Previous studies have elucidated enzymes involved in the side-chain oxygenation of styrene in, for example, representatives of the genera Corynebacterium , Rhodococcus , Pseudomonas , Sphingopyxis , or Xanthobacter [5], [20], [22], [33], [34], [35], [47].

Fig. 1Biotransformation of styrene and substituted analogs to phenylacetic acid(s).Styrene (R1, R2 = H) is transformed to phenylacetic acid through enzymes of side-chain oxygenation by the following steps: (a) initial epoxidation to styrene oxide by styrene monooxygenase (SMO), (b) isomerization to phenylacetaldehyde by styrene oxide isomerase (SOI), (c) oxidation by phenylacetaldehyde dehydrogenase (PAD) (reviewed by [32], [36]).

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On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2

“…However, previous reports, as well as this study, indicate that this is rather unlikely, since we detected only minor SOR and PAR activity in the crude extract of strain CWB2. Both assumptions would not explain the fast degradation of styrene found in these strains (3,52,53). However, strain ST-10 accumulated epoxide when incubated with styrene, and thus it remains to be shown whether the rest of the genes are also homologs to the styrene degradation cluster in strain CWB2.…”

Gene redundancy of two-component (chloro)phenol hydroxylases inRhodococcus opacus1CP

“…Cell disruption was achieved as described by means of a French press (Oelschlägel et al ., ) or with a swing mill (Moiseeva et al ., ), and cell debris was removed by centrifugation (100 000 g , 1 h, 4 °C). The clear supernatant served as cell‐free crude extract.…”