Purification, Partial Characterization and Substrate Specificity of a Squalene Cyclase fromBacillus acidocaldarius

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Abstract
From the thermophilic Bacillus acidocaldarius, a membrane bound cyclase catalyzing the formation of 22(29)-hopene (diploptene) and 22-hopanol (diplopterol) from squalene was enriched 670-fold to a purity of 95% as judged by gel chromatography. The specific activity of the purified enzyme in the presence of Triton X-100 is 0.22 .mu.mol product formation per min and mg protein at 54.degree.C. The molecular mass is 150 kDa and that of the subunits 80 kDa as determined by FPLC gel chromatography and sodium dodecyl sulfate gel electrophoresis, respectively. Not only squalene but also E,E-homofarnesol, homogeraniol and homofarnesyl (1,5,9-trimethyl-4,8-decadienyl)ether are substrates. The ether bond of the latter substrate is split by the enzyme. The products of the aforementioned three additional substrates are ambroxan (dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]-furan), octahydro-4,4,7a-trimethylbenzofuran and ambroxan together with 1,5,9-trimethyl-4,8-decadienol. The cyclization rates of these substrates compared to squalene are 3%, 0.05% and 0.03%, respectively. The half-life of the enzyme activity is 80 h at 45.degree.C and 18 h at 54.degree.C.