Isolation and Characterization of a Cyclohexane-Metabolizing Xanthobacter sp

Abstract

An unusual Xanthobacter sp., capable of independent growth on cyclohexane as the sole source of carbon and energy, has been isolated from soil by using classical enrichment techniques. The mean generation time for growth on cyclohexane was 6 h. The microorganism showed a limited ability to utilize hydrocarbons, with only alicyclic hydrocarbons closely related to cyclohexane supporting growth. Ultrastructural studies indicated the presence of electron-transparent vesicles in the cyclohexane-grown Xanthobacter sp., but the presence of complex intracytoplasmic membranes could not be identified. A soluble inducible enzyme capable of oxidizing cyclohexane was identified in cell extracts. This enzyme had a pH optimum of 6.5, an absolute specificity for NADPH, and a stoichiometric requirement for molecular 02 which was consistent with the formation of cyclohexanol. The enzyme showed no activity towards straight chain alkanes and only a limited activity towards unsaturated ring compounds. Enzymatic studies with cell extracts have indicated the main route of metabolism of cyclohexane by this Xanthobacter sp. to proceed via cyclohexane-* cyclohexanol-* cyclohexanone-* 1-oxa-2-oxocycloheptane (r-caprolactone)-* 6-hydroxyhexanoate (6-hydroxycaproate)-*-* adipic acid. Alternative routes involving initial double hydroxylation of the cyclohexane ring may operate fortuituously but are unlikely to represent major pathways for the dissimilation of cyclohexane by this microorganism.

Continuous cyclohexane oxidation to cyclohexanol using a novel cytochrome P450 monooxygenase from Acidovorax sp. CHX100 in recombinant P. taiwanensis VLB120 biofilms

“…(Anderson et al, 1980), 10 U g CDW À1 for Xanthobacter sp. (Trower et al, 1985), 19 U g CDW À1 for Nocardia sp. (Stirling et al, 1977), and 64 U g CDW À1 for Acidovorax sp.…”

“…Nature is an infinite source to screen and isolate novel biocatalysts because of selective pressures continuously opening up new trajectories in the evolution and development of microorganisms. Several microorganisms are reported to grow with cyclohexane as a sole carbon and energy source (Anderson et al, 1980;Trower et al, 1985). These microorganisms are often an excellent source for novel biocatalysts because growth performance is directly linked to enzymatic efficiencies.…”

“…The rate limiting step in cyclohexane mineralization is the initial oxyfunctionalization reaction of the alkane. Depending on the respective studies, this activity was reported either as specific oxygen consumption rate (mmole O 2 mg CDW À1 h À1 ) (Anderson et al, 1980;Trower et al, 1985) or growth rate (h À1 ) (Anderson et al, 1980;Trower et al, 1985). Based on the biomass yield (g CDW g cyclohexane À1 ), initial activities can be calculated to approximately 4 U g CDW À1 for Rhodococcus sp.…”

Proposed involvement of a soluble methane monooxygenase homologue in the cyclohexane‐dependent growth of a new Brachymonas species

“…We have been looking for a bioconversion route to the production of adipic acid and other long‐chain α,ω‐dicarboxylic acids, starting from relatively cheap cyclic alkanes. The proposed route to adipic acid starts with the hydroxylation of cyclohexane into cyclohexanol (Stirling et al ., 1977; Trower et al ., 1985a) followed by the oxidation of cyclohexanol into adipic acid through cyclohexanone, caprolactone, 6‐hydroxycaproate and 6‐oxo‐caproate (Fig. 1).…”

“…Bacteria growing on cyclic alcohols and ketones are easy to isolate, and the genes responsible for the degradation of these molecules have now been characterized in many organisms (Cho et al ., 1991; Iwaki et al ., 1999; 2002; Brzostowicz et al ., 2000; 2002; 2003; Cheng et al ., 2000; Kostichka et al ., 2001). In contrast, bacteria capable of growing on cyclic alkanes are difficult to isolate (De Klerk and Van der Linden, 1974; Walker and Colwell, 1976; Perry, 1984; Solano‐Serena et al ., 2000a,b), and only a few bacteria that use cyclohexane as a carbon source have been described (Stirling et al ., 1977; Anderson et al ., 1980; Trower et al ., 1985a; Kästner et al ., 1994; Lang, 1996; van der Werf et al ., 2001). Until now, nothing was known about their relevant metabolic genes.…”