Kingma, Jacob

van Beilen, J.B.

Witholt, Bernard

We have studied the hydroxylation of a wide range of linear, branched and cyclic alkanes and alkylbenzenes by the alkane hydroxylase system of Pseudomonas oleovorans GPo1 in vivo and in vitro. In vivo hydroxylation was determined with whole cells of the recombinant PpS8141; P. putida PpS81 carrying plasmid pGEc41. In vitro hydroxylation was determined with a reconstituted hydroxylase system consisting of AlkB (the membrane-bound catalytic monooxygenase component), AlkG (rubredoxin), and spinach ferredoxin reductase. The introduction of one or two methyl substituents in linear alkanes hampers, but does not block the conversion of alkanes. However, substrates were not hydroxylated when a tertiary carbon was present. Substituted cyclic alkanes were oxidized with high enantiomeric excess at ring positions trans-4 relative to the substituents, bur not at tire methyl- or ethyl-substituents themselves. trans-1,4-dimethylcyclohexane and t-butylcyclohexane were not hydroxylated at all. Several alkylbenzenes, such as ethylbenzene and its 3- and 4-substituted derivatives, were hydroxylated at rates close to or superior to the rate for n-nonane. Isopropylbenzene and n-butylbenzene were oxidized at intermediate rates, while toluene and n-propylbenzene were converted at relatively low rates, only in vivo, and only when present in high concentrations. t-Butylbenzene was nor oxidized at all. These results indicate that the P. oleovorans alkane hydroxylase system can be employed in the production of a considerable range of aliphatic and aromatic alcohols. The data also provide information on the dimensions of the substrate-binding site.</p

SUBSTRATE-SPECIFICITY OF THE ALKANE HYDROXYLASE SYSTEM OF PSEUDOMONAS-OLEOVORANS GPO1

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