The Metabolism of Aromatic Ring Fission Products by Bacillus stearothermophilus Strain IC3

Abstract

BmifZus stearotkmphifus IC3 degraded the meta cleavage product uf catechol, 2-hydroxymuconic semialdehyde, to pyruvate and acetaldehyde via the rGOxalocrotcmate pathway. The pathway was identical to those previously delineated in several mesophilic organisms. However, all the enzymes showed activity at 55 "C and other properties (substrate specificities and effects of metal ions) also differed from those displayed by the mesophilic enzymes. AU e q m m of this meta cleavage pathway, except the 2-hydroxykohepta-2,4-dienoate hydrolase and 4-hydroxy-2-axovalerate aldolase activities, were induced by growth on phenol. INTRODUCTIONA number of thermophilic bacteria are able to utilize aromatic compounds as carboll and energy sources. A thermophilic Bacilhrs w a able to metabolize benzoate and phydroxybenmate, via gentisate, to pyruvate and fwarate (Buswell & Twomey, 1974;Buswell & Clark, 1976). Thenrwcs strains utilizing phenylacetate as a carbon source have dm been observed (Degryse et aZ., 1978). The metabolism of phenol by midentilied themophilei (Egorova, 1942(Egorova, , 1946) and Bacillus stearothemwphiltrs s.trains (Buswell, 1974 Buswell & Twomey, 1975; Golovacheva & Oreshkin, 1975) has also been widely described.An earlier report (Adams 8t Ribbons, 1988) established that a number of thennophilic bacilli were able to grow on aromatic compounds, including benzoate, phydmxybemte, phenylacetate, p-hydroxyphenylacetate, L-phenylalanine and phenol. One of these organisms, Bacilltrs stemottoennophilus IC3, metabolized phenol to 2-hydmxpuconic semialdehyde via catechol. In this paper we describe the further metabolism of 2-hydroxymwnic semialdehyde to pyruvate and acetaldehyde. MBTHODSI&tion, growth media and growth conditions of &rcilhrs stearvthemmphb IC3. These were as described by RIBBONSThe preparation of 2-oxopent-4-enoate was based upon the methods of Collinsworth et of. (1973). A 10 ml solution containing 250 pmol Tris buffer (pH 8.2), 0.5 mmol DL-allyldyCine, 25 units mamino acid oxidase and 1250 units catalase, was incubated at 25 "C for 30 min. The solution was then acidified to pH 1-5 with HCl, and the product extracted twice with 25 ml diethyl ether. The ether was removed and the precipitate resuspended in 5 ml dilute HCl (pH 1.5). The solutions were stored at -20 "C and used within 1 week.The following enzymes were obtained from Sigma: aldehyde dehydrogenase (EC 1.2.1.5) from bakers' yeast; *amino acid oxidase (EC 1.4.3.3) from porcine kidney; catalase (EC 1.11.1.6) from bovine liver; formate dehydrogenase (EC 1.2.1.2) from Pseudomonas oxulaticus; NADase (EC 3.2.2.5) from Neurospora crussu; and pyruvate dehydrogenase from bovine heart.Cell-fee extracts. These were prepared from 1 litre cultures that had been harvested in the mid-exponential growth phase. The cells were washed twice in 50 m-potassium phosphate buffer (PH 7.0) at mom temperature, and then resuspended in 5 ml fresh buffer containing 10% (v/v) acetone and 0.5 pmol phenylmethylsulphonyl fluoride. The resuspended cells were then sonicated ...

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