Fatty alcohol oxidases involved in alkane-degradation by yeasts

Abstract

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.

Afrikaans: Langketting alkohol oksidases (EC 3.1.1.13) (FAOD) is ensieme wat geinduseer word tydens groei van giste op lang ketting alkane. Hierdie ensieme kataliseer die oksidasie van lang ketting alkohole na die ooreenkomstige aldehiede. Baie produkte van die alkaan assimileringsweg soos dikarboksielsure, lang ketting alkohole en aldehiede is van industriele belang vir die produksie van detergente, smeermiddels, surfaktante en kosmetiese preparate. Tans behels die produksie van hierdie verbindings ekstraksie uit natuurlike bronne of sintese vanaf petrochemiese substrate, maar nie een van bogenoemde metodes werk bevredigend nie. Die moontlikheid om alkaan benuttende giste te gebruik vir die produksie van hierdie waardevolle produkte word dus ondersoek .. Kennis van die gene wat kodeer vir die ensieme verantwoordelik vir hierdie reaksies in giste sal dit moontlik maak om die giste geneties te manipuleer, sodat hulle van die waardevolle tussenprodukte kan ophoop. Om die lang ketting alkohol oksidase van C. tropicalis OC3 te isoleer is die selle eers geoes en gebreek. Die selvrye ru-ekstrak is onderwerp aan differensiele sentrifugasie om die oksidase bevattende peroksisoom fraksie te verkry. Die peroksisoom fraksie is gesolubiliseer met In detergent, CHAPS, om die ensiem vry te stel uit die membrane. Die alkohol oksidase ensiem is geisoleer deur ammonium sulfaat fraksionering gevolg deur hidrofobiese interaksie chromatografie op 'n Heksiel agarose 4XL kolom. Ander chromatografiese kolomme wat probeer is, maar nie geskik was vir die suiwering van die ensiem nie sluit in die anioon-uitruilers QAE-Sephadex en DEAE- Toyopearl 650M, asook affiniteitsmatrikse met MIMETIC™ kleurstofligande, Blue 1, Blue 2 and Yellow 2. Met die finale suiweringsprotokol kon die langketting alkohol oksidase van C. tropicalis OC3 geisoleer word met In suiweringsfaktor van 73, 'n spesifieke aktiwiteit van l.17 U/mg en 'n finale opbrengs van 48%. Een hoof band met 'n molekulere massa tussen 70 000 en 75 000 is verkry na SDS-PAGE. Die gesuiwerde ensiem het optimale aktiwiteit gehad by pH 9.5 en 35°C. Die pH stabiliteit van die ensiem was in die gebied pH 7.5 tot 10 alhoewel die ensiem slegs 60% van optimale aktiwiteit behou het by pH 7.0. Die ensiem was nie stabiel by temperature bo 20°C nie met 'n halfleeftyd van ongeveer 4 ure by 20°C en slegs 30 minute by 30°C. Substraat spesifisiteit studies het gewys dat hierdie ensiem primere en sekondere alkohole met ketting lengtes C9 tot C12 verkies. Alhoewel dit geraporteer is (Dickinson and Wad forth, 1992) dat lang ketting alkaan diole en (0- hydroksiesure substrate is vir hierdie groep ensieme, het ons gevind dat 1,2-heksaandiol, 16-hidroksieheksadekanoësuur and 12-hidroksiedodekanoësuur baie swak deur hierdie ensiem geoksideer word. Veral vreemd is die waarneming dat die ensiem geisoleer is vanaf selle wat opgegroei was op heksadekaan as substraat, maar heksadekan-l-ol baie swak okdsideer. Ons het gevind dat die giste behalwe die alkohol oksidase ook 'n lang ketting alkohol dehidrogenase (FADH) ensiem produseer. Laasgenoemde ensiem mag die gis in staat stelom op 'n verskeidenheid koolwaterstowwe te groei selfs wanneer die alkohol oksidase aktiwiteit laag is. Alhoewel SDS-PAGE getoon het dat die oksidase protein nie homogeen was nie, het ons op grond van die elueringsprofiele en die finale spesifieke aktiwiteit besluit dat die geisoleerde oksidase ensiem waarskynlik skoon genoeg was om 'n aminosuurvolgordebepaling te laat doen. Vanhanen et al. (2000) het egter onlangs die basispaar volgordes gepubliseer van die gene wat kodeer vir drie sogenaamde langketting vetsuur alkohol oksidases in C. c/oacae en C. tropica/is. Dit sal nou waarskynlik makliker wees om hierdie inligting te gebruik om die geen wat kodeer vir die langketting alkohol oksidase in ons C. tropicalis OC3 stam op te spoor.