Styrene oxide isomerase of Rhodococcus opacus 1CP, a highly stable and considerably active enzyme

Abstract

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 Rhodococcus opacus 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(-1) 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.

Fig 1

Upper pathway of styrene degradation via side chain oxygenation. Styrene is converted by styrene monooxygenase (SMO) to styrene oxide, which undergoes isomerization by styrene oxide isomerase (SOI) to phenylacetaldehyde. The latter compound is oxidized by phenylacetaldehyde dehydrogenase (PAD) to phenylacetic acid.

Fig 2

Detection and size determination of the 1CP SOI by disc SDS-PAGE applying SOI active gel slugs (A) and membrane fractions of differently induced biomasses of strain 1CP and P. fluorescens ST (B). (Lanes A1 to A3) SOI active gel slices obtained from disc SDS-PAGE of an 11-fold SOI enrichment using nondenaturing sample treatment (0.4% SDS) were denatured (4% SDS, 10% mercaptoethanol, 45 min, 40°C) and subjected to a second disc SDS-PAGE. (Lane A4) A 42-fold enrichment of the 1CP SOI obtained from CHAPS/GdnHCl treatment was preincubated with TFA and subjected to denatured SDS-PAGE. Gels were consecutively stained with Coomassie blue and silver. (B) SDS-PAGE of enriched integral membrane fractions of styrene-grown strain 1CP (1CP+; specific SOI activity, 99.6 U mg⁻¹), phenylacetic acid-grown strain 1CP (1CP−; no measurable SOI activity), and styrene-grown P. fluorescens ST (ST+; specific SOI activity, 28.4 U mg⁻¹). Membrane samples were denatured (4% SDS, 10% mercaptoethanol, 45 min, 40°C), and the gel was finally stained with Coomassie blue. Arrowheads indicate an SOI-related protein band. Lanes M, unstained protein molecular mass markers (Fermentas).

Fig 3

Temperature dependence of SOI stability and activity. The stability (solid line) of SOI activity is expressed as percentage of initial activity (100% = 91.7 U mg⁻¹) and was assessed by determination after heat treatment at 21°C to 70°C of the partially purified SOI preparation for 30 min. Isomerase activity (broken line) was determined in a similar temperature range and is relative to the specific activity at room temperature (100% = 84.6 U mg⁻¹). Data points represent the means of duplicate measurements, and standard error bars are given.

Fig 4

pH dependence of SOI stability and activity. Partially purified SOI was incubated at a certain pH for 15 h, and the stability (solid line) of SOI is expressed as percentage of activity relative to a 15-h incubation at pH 7.0 (100% = 93.1 U mg⁻¹). pH dependence of activity (broken line) was determined by means of the standard assay and phosphate buffer in a pH range of 4.6 to 9.1. Obtained activities are relative to the activity at pH 7.3 (100% = 88.9 U mg⁻¹). Data points represent the means of duplicate measurements, and standard error bars are given.

Fig 5

Preparation of phenylacetaldehyde from styrene oxide by enriched SOI. Each assay mixture contained 1 mmol of racemic styrene oxide and various amounts of enriched SOI in 10 ml phosphate buffer (pH 7.0). Quenched samples were analyzed for phenylacetaldehyde by RP-HPLC. Data points represent the means of duplicate measurements, and standard error bars are given.

Fig 6

Irreversible inhibition of SOI activity by phenylacetaldehyde. Equal amounts of SOI were incubated in the presence of 0 to 90 mM phenylacetaldehyde for 15 min under constant shaking. Incubation mixtures were centrifuged, the SOI-containing pellet was washed several times with phosphate buffer, and residual SOI activity was determined and referred to the initial activity (100% = 16.0 U mg⁻¹). Data points represent the means of quadruplicate measurements, and standard error bars are given.