Fatty alcohol oxidases involved in alkane-degradation by yeasts

English: Fatty alcoholoxidases (EC 3.1.1.13) (FAOD) are enzymes induced by growth of yeast on long chain alkanes. These enzymes catalyse the oxidation of long chain (fatty) alcohols to fatty aldehydes. Many products of the alkane-assimilation pathway such as dicarboxylic acids, long chain alcohols and aldehydes are of industrial importance in the production of detergents, lubricants, surfactants and cosmetics. Currently, production of these products involve extraction from natural sources or synthesis from petrochemicals, but neither method is satisfactory. The potential of synthesizing such value-added products using alkane degrading yeasts is thus being investigated. Knowledge of the genes coding for enzymes responsible for production of these products by yeasts would facilitate genetic manipulation of the yeasts, so that it becomes possible to accumulate products of the alkane-assimilation pathway. Isolation of fatty alcohol oxidase from C. tropicalis OC3 was carried out by first harvesting and disrupting the cells to release the enzyme. The cell-free crude extract was subjected to differential centrifugation to obtain the FAOD-containing peroxisomal fraction. The peroxisomal fraction was solubilized with a detergent, CHAPS, to release the enzyme from the membranes. Isolation of the FAOD enzyme was achieved using ammonium sulphate fractionation followed by hydrophobic interaction chromatography on a Hexyl agarose 4XL column. Other chromatographic columns which were tried and found to be unsuitable for purification of this enzyme include the anion-exchangers QAESephadex and 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 ideal column for purification of the FAOD enzyme, however very unfortunately, the hunt for better columns went on for too long and in the end there was no time to try the Mimetic Blue 2 column again. The final purification protocol for FAOD from C. tropicalis OC3 resulted in a 73-fold purification, a specific activity of 1.17U/mg and a final yield of about 48%. One major band 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 pH stability of the enzyme was found to be in the range pH 7.5 to 10 although the enzyme retained only about 60% activity at pH 7.0. The enzyme was not stable at temperatures above 20°C, exhibiting an approximate half-life of 4 hours at 20°C and only 30 minutes at 30°C. Substrate specificity studies showed that this FAOD prefers primary and secondary alcohols in the range C9 to Cl2. Even though it has been reported (Dickinson and Wadforth, 1992) that long-chain alkane-diols and ω-hydroxy acids are substrates for this group of enzymes we found that 1,2-hexdecanediol, 16-hydroxydodecanoic acid and I2-hydroxydodecanoic acid were poorly oxidized by this FAOD. An anomally was that the cells from which the enzyme was isolated were grown on hexadecane but the enzyme showed very low activity for hexadecan-l-ol. We found that in addition to FAOD the yeast cells also produced a fatty alcohol dehydrogenase (FADH) enzyme. This enzyme might enable the yeast to grow on a variety of hydrocarbon sources even when its FAOD cellular 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 that the 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 they call long-chain fatty acid alcoholoxidases from C. cloacae and C. tropicalis. It would now probably be easier using this information to locate the FAOD gene(s) of our C. tropicalis OC3 strain.