Purification and Some Properties of Alcohol Oxidase from Alkane-Grown Candida Tropicalis

Biochem. J. (1992) 282, 325-331 (Printed in Great Britain) 325 Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis Francis M. DICKINSON and Catherine WADFORTH Department of Applied Biology, University of Hull, Cottingham Road, Hull HU6 7RX, U.K. Alcohol oxidase was purified to homogeneity from membrane fractions obtained from alkane-grown Candida tropicalis. The enzyme appears to be a dimer ofequal-sized subunits of Mr 70000. The purified enzyme is photosensitive and contains flavin-type material which is released by a combination of boiling and proteolytic digestion. The identity of the flavin material is not yet known, but it is not FMN, FAD or riboflavin. The enzyme is most active with dodecan-l-ol, but other long-chain alcohols are also attacked. The enzyme shows a weak, but significant activity towards long-chain aldehydes. Detailed kinetic studies with decan-1-ol as substrate suggest a group-transfer (Ping-Pong)-type mechanism of catalysis. INTRODUCTION portions of this culture were inoculated into each of six 3-litre conical flasks containing 500 ml of minimal salts Work from this laboratory has shown that growth of the yeast medium, which contained (g/l) KH2PO4, 7.0; Na2HPO4, 2.0; MgSO4, 1.5; yeast Candida tropicalis on long-chain alkane substrates induces the extract, 1.5; CaCI2, 0.1; FeCl2, 0.008; ZnSO4, 0.0001; synthesis of a long-chain-specific alcohol oxidase (Kemp et al., diammonium tartrate, 15; in distilled water at pH 6.5. The 1988). The enzyme presumably catalyses one of the steps in the pathway in which alkanes undergo monoterminal oxidation carbon source was hexadecane (1 %). The cultures were incubated on an orbital shaker for 50 h at 30 °C before harvesting. and are converted into long-chain fatty acids before f-oxidation. Our early work established that the enzyme is membrane-bound, Preparation of microsomal membranes but no attempt was made to purify it. Later work showed that The procedure was that described by Kemp et al. (1988) with the enzyme in crude preparations is light-sensitive: activity was only minor alterations. These were that the harvested cells were completely lost in a few minutes on exposure to a blue light washed in 50 mM-Hepes buffer, pH 7.5, and that the membrane source (Kemp et al., 1990). It was not, of course, clear whether fraction, obtained after cell breakage in the French pressure cell this lability was due to an intrinsic property of the enzyme or to the generation of harmful radicals in the membranes under and differential centrifugation, was washed with 50 mM-Hepes bright illumination. Other work established that freeze-dried buffer, pH 8.0, containing 0.15 M-KCI. The latter step removed membrane preparations could be suspended in organic solvents some weakly associated protein before the membranes were stored as a suspension in 50 mM-Hepes buffer, pH 8.0, in liquid (e.g. octane), with the enzyme still showing good activity (Kemp N2. et al., 1991). As oxygen is the electron acceptor in the oxidation reaction, this result suggested that the enzyme might have a Enzyme activity and protein assays commercial application in the synthesis of long-chain aldehydes. Alcohol were The results referred to above indicated that purification and oxidase-catalysed reactions routinely performed spectrophotometrically at 405 nm in coupled assays with ABTS further characterization of the enzyme was necessary. Certainly and peroxidase as described by Kemp et al. (1988), but modified questions about the mechanism of action of the enzyme, the by using 20 mM-glycine/NaOH buffer, pH 9.0. Occasionally prosthetic group involved in catalysis and the light-sensitivity of enzyme activity was measured directly by using an oxygen the enzyme could not be resolved by using crude enzyme electrode (Rank Brothers, Cambridge, U.K.). The conditions for preparations. We have now developed a reliable purification this assay were as for the spectrophotometric assay, but ABTS method, and this, and some observations on the properties of the and peroxidase were omitted. The spectrophotometric assay is purified preparations, are presented here. based on the methanol oxidase assay of Haywood & Large MATERIALS AND METHODS (1981). The absorption coefficient for the radical cation of ABTS was taken to be e = 18400 M-l cm-' (Werner et al., 1970) with Materials 2 mol of radical cation being formed per mol of substrate Long-chain alcohols, diols, aldehydes, wo-hydroxy-fatty acids oxidized. and cholic acid were obtained from Aldrich Chemical Co., Substrates were prepared as solutions in acetone or in 1,4- Gillingham, Dorset, U.K. NAD+ was from Boehringer Mann- dioxan. A 20 Isl portion of substrate solution was added per 3 ml heim UK, Lewes, Sussex, U.K. 2,2'-Azino-bis-(3-ethylbenz- assay volume via a calibrated glass micro-syringe. For assays thiazoline-6-sulphonic acid) (ABTS), catalase, peroxidase, glu- where the oxygen concentration was varied, the range of con- cose oxidase and CHAPS were from Sigma Chemical Co., Poole, centrations was obtained by mixing appropriate volumes of Dorset, U.K. Bincinchoninic acid reagent was from Pierce assay mixture previously bubbled with N2 or 02 for 10 min. The Europe B.V., BA Oud Beijerland, The Netherlands. bubbling was done in graduated glass syringes held vertically in a retort stand. After gassing, the syringes were capped either with Yeast growth conditions a rubber seal or by mounting the needle. Liquid could then be Candida tropicalis was grown up in 100 ml starter cultures on transferred from one syringe to another under positive pressure. an orbital shaker at 30 °C on a medium containing (g/l) yeast The final mixture was transferred to an N2-filled 3 ml cuvette extract 5.0, malt extract 20.0 and glucose 5.0. After 2 days, 10 ml through a narrow sleeve in the cap. The reaction was then Abbreviation used: ABTS, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid). Vol. 282 326 F. M. Dickinson and C. Wadforth initiated by adding enzyme and substrate through the same including detergent in the eluting buffer. However, in this case capillary from calibrated micro-syringes. the overall yield from the step was decreased to about 500 of Protein determinations were by the method of Smith et al. that when detergent was present. (1985), using bincinchoninic acid. Gel filtration Purity of substrates Experiments were conducted on a 30 cm x 1.5 cm column of The purity of long-chain alcohols and aldehydes was assessed Sephacryl S-300 SF (Pharmacia, Uppsala, Sweden) equilibrated by g.l.c. at 140 or 160°C by using a Pye-Unicam PU 4500 with 50 mM-sodium phosphate buffer, pH 7.6, containing 0.5 % chromatograph fitted with a 1 % diethyleneglycol succinate sodium cholate. Samples (0.2 ml) of proteins (approx. 1.5 mg/ml.) column. All samples, except dodecane- 1,1 2-diol (which was were applied to the column. The column was connected to an dissolved in ethanol), were dissolved in n-hexane. LKB h.p.l.c apparatus (LKB, Bromma, Sweden). The proteins used to calibrate the column were /8-amylase (Mr = 200000), Purification of the enzyme yeast alcohol dehydrogenase (Mr = 150000), BSA (Mr = 66000), All steps were carried out at 0-4 'C and, because of the light- ovalbumin (Mr = 45000) and cytochrome c (Mr = 12400). sensitivity of the enzyme (Kemp et al., 1990), extracts were protected from the light. Chromatography of flavin samples The microsomal membranes from 3 litres of culture medium T.l.c. Samples were run at room temperature on 20 cm x 20 cm were suspended in 100 ml of 50 mM-Hepes buffer, pH 8.0, con- 100,tm-thick layers of Whatman Gel 300 DEAE-cellulose plates. taining 1 % (w/v) sodium cholate. The suspension was then The moving phase was 4% (w/v)Na2HPO4. immediately supplemented by addition of 2 ml of 50 mm- H.p.l.c. Samples were run on a Spherisorb S 50 D52 column phenylmethanesulphonyl fluoride. After a few minutes (25 cm x 4.6 cm) with the solvent methanol/2M-ammonium (NH4)2SO4 (240 g/l) was added to the membrane suspension, acetate (7:3, v/v). and the precipitated material was removed by centrifugation and discarded. Extraction for a longer period did not yield signific- RESULTS AND DISCUSSION antly more solubilized activity, and exposed the enzyme to a greater risk of proteolysis. More (NH4)2SO4 (55 g/l) was added, Homogeneity, stability and Mr of the purified enzyme and the precipitated material was then collected by centrifugation The results of a typical purification procedure are shown in and dissolved in 10 mM-NaHCO3 buffer, pH 9.2, containing Table 1. Only 1-2 mg quantities of enzyme are obtained from the 0.5 % (w/v) CHAPS. Phenylmethanesulphonyl fluoride was standard 3-litre culture of yeast. The purified enzyme is essentially added to the solution to give a final concentration of 1 mm, and homogeneous as judged by SDS/PAGE (Fig. 1). It is noteworthy the fraction was then dialysed for 2 h against 10 mM-NaHCO3 that, although detergent is essential to solubilize the enzyme buffer, pH 9.2, containing 0.5 % CHAPS. from microsomal membranes, in the last stages of purification The dialysed fraction was supplemented with I mM-phenyl- the detergent can be removed and the enzyme remain in solution methanesulphonyl fluoride and applied to a column of QAE- and fully active at concentrations of approx. 0.5 mg/ml. This cellulose (2.2 cm x 24 cm) equilibrated with 10 mM-NaHCO3 suggests that in its natural environment the enzyme makes buffer, pH 9.2, containing 0.5 % CHAPS.

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