Fatty alcohol oxidases involved in alkane-degradation by yeasts

[摘要] English: Fatty alcoholoxidases (EC 3.1.1.13) (FAOD) are enzymes induced by growth of yeast onlong chain alkanes. These enzymes catalyse the oxidation of long chain (fatty) alcohols tofatty aldehydes. Many products of the alkane-assimilation pathway such as dicarboxylicacids, long chain alcohols and aldehydes are of industrial importance in the production ofdetergents, lubricants, surfactants and cosmetics. Currently, production of these productsinvolve extraction from natural sources or synthesis from petrochemicals, but neithermethod is satisfactory. The potential of synthesizing such value-added products usingalkane degrading yeasts is thus being investigated. Knowledge of the genes coding forenzymes responsible for production of these products by yeasts would facilitate geneticmanipulation of the yeasts, so that it becomes possible to accumulate products of thealkane-assimilation pathway.Isolation of fatty alcohol oxidase from C. tropicalis OC3 was carried out by firstharvesting and disrupting the cells to release the enzyme. The cell-free crude extract wassubjected to differential centrifugation to obtain the FAOD-containing peroxisomalfraction. The peroxisomal fraction was solubilized with a detergent, CHAPS, to releasethe enzyme from the membranes. Isolation of the FAOD enzyme was achieved usingammonium sulphate fractionation followed by hydrophobic interaction chromatographyon a Hexyl agarose 4XL column. Other chromatographic columns which were tried andfound to be unsuitable for purification of this enzyme include the anion-exchangers QAESephadexand DEAE-Toyopearl 650M, as well as affinity chromatography MIMETIC™dye ligands, Blue 1 and Yellow 2. The MIMETIC�?Blue 2 column seems to be the idealcolumn for purification of the FAOD enzyme, however very unfortunately, the hunt forbetter columns went on for too long and in the end there was no time to try the MimeticBlue 2 column again.The final purification protocol for FAOD from C. tropicalis OC3 resulted in a 73-foldpurification, a specific activity of 1.17U/mg and a final yield of about 48%. One majorband with an approximate molecular mass of 75 000 to 80 000 was obtained after SDSPAGE.The purified enzyme had an optimum activity at pH 9.5 and 35°C. The pHstability of the enzyme was found to be in the range pH 7.5 to 10 although the enzymeretained only about 60% activity at pH 7.0. The enzyme was not stable at temperaturesabove 20°C, exhibiting an approximate half-life of 4 hours at 20°C and only 30 minutesat 30°C. Substrate specificity studies showed that this FAOD prefers primary andsecondary alcohols in the range C9 to Cl2. Even though it has been reported (Dickinsonand Wadforth, 1992) that long-chain alkane-diols and ω-hydroxy acids are substrates forthis group of enzymes we found that 1,2-hexdecanediol, 16-hydroxydodecanoic acid andI2-hydroxydodecanoic acid were poorly oxidized by this FAOD. An anomally was thatthe cells from which the enzyme was isolated were grown on hexadecane but the enzymeshowed very low activity for hexadecan-l-ol. We found that in addition to FAOD theyeast cells also produced a fatty alcohol dehydrogenase (FADH) enzyme. This enzymemight enable the yeast to grow on a variety of hydrocarbon sources even when its FAODcellular levels are low.Even though SDS-PAGE results showed that the FAOD protein was not homogeneous,we concluded from the nature of the elution profiles and the specific activity values thatthe isolated FAOD enzyme is probably pure enough to submit for amino acid sequencing.However, Vanhanen et al. (2000) recently published three gene sequences of what theycall long-chain fatty acid alcoholoxidases from C. cloacae and C. tropicalis. It wouldnow probably be easier using this information to locate the FAOD gene(s) of our C.tropicalis OC3 strain.

[发布机构] University of the Free State