Candida Carbon

Respiratory, or oxidative, metaboHsm produces more energy than fermentation. Complete oxidation of one mol of glucose to carbon dioxide and water may produce up to 36 mol ATP in the tricarboxyHc acid (TCA) cycle or related oxidative pathways. More substrates can be respired than fermented, including pentoses (eg, by Candida species), ethanol (eg, by Saccharomjces), methanol (eg, by Hansenu/a species), and alkanes (eg, by Saccharomjces lipoljticd). 

Both alkanes and gas oil can be used as carbon and energy sources. Commercially, Candida tropicalis and Candida lipolytica have been used (35,36). The fermentation contains two immiscible Hquid phases (the alkane and the water) the semisoHd yeast and the gaseous air phase. In contrast to yeasts grown on carbohydrates, where maximum yields are 50%, yeasts grown on alkanes generally give yields of 95—105% based on the weight of the alkane. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

Candida boidinii was cultured at pH 3.51, 5.49 and 7.01, respectively. Czapek s Dox medium with citrus pectin (GENU Pectin, Denmark), sodium pectate or citrus pectin with 20% of D-galactopyranuronic acid (Fluka, Switzerland) as a carbon source were used. The growth curves were performed by measuring the optical density (OD) at 660 nm. 

Shyadehi AZ, DC Lamb, SL Kelly, DE Kelly, W-H Schunck, JN Wright, D Corina, M Akhtar (1996) The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14a-demethyl-ase of Candida albicans (other names are lanosterol 14a-demethylase, P-450]4p, and CYP51). J Biol Chem 271 12445-12450. 

Dalmau, E., Montesinos, J.L., Lotti, M. and Casas, C., Effect of different carbon sources on lipase production by Candida rugosa. Enzyme Microb. Technol., 2000, 26, 657-663. 

A novel continuous-flow SCCO2 process for the kinetic resolution of 1-phenyethanol enantiomers (Figure 30) using Novozym 435 immobilized enzyme from Candida antarctica was described by Matsuda et al. [51], The lipase enzyme, selectively acetylated the R)-alcohol component. A mixture of starting material and vinyl acetate was passed through the enzyme with supercritical carbon-dioxide (Figure 31). The reaction zone was pressurized and heated, so the reaction could be performed imder supercritical conditions, synthesizing the desired (i )-acetate with 99.7% ee. and 47% yield. 

DNB was biodegraded under aerobic conditions to carbon dioxide by a microbial strain, Candida pulcherrima, isolated from soil contaminated with 1,3-DNB manufacture wastes (Dey and Godbole 1986). Some biotransformation products detected in the metabolic pathway of biodegradation of... 

Scientists at Huddersfield University in collaboration with Avecia have developed a DKR process involving the combination of immobilized Candida rugosa lipase and an iridium-based racemization catalyst (Scheme 2.30). By using carbonate 62 as the acyl donor, the racemic secondary amine 61 was converted to the corresponding carbamate (R)-63 in high yield and enantiomeric excess [32]. 

The activity of three ester spHtting enzymes, Candida antarctica lipase B (CALB), Mucor miehei lipase (MML) and esterase, towards the carbonate surfactant was studied. While CALB and esterase were found to catalyze the hydrolysis of the carbonate bond, MML showed no activity. 

DKR of esters bearing an electron-withdrawing group at the a-carbon can be performed easily under mild reaction conditions because of the low pKa of the a-proton. Tsai et al. have reported an efficient DKR of rac-2,2,2-trifluoroethyl a-chorophenyl acetate in water-saturated isooctane [15]. They used lipase MY from Candida rugosa for the KR and trioctylamine as the base for racemization. (R)-a-chlorophenylacetic acid was obtained in 93% yield and 89.5% ee (Scheme 5.2). 

The integration of a catalyzed kinetic enantiomer resolution and concurrent racemization is known as a dynamic kinetic resolution (DKR). This asymmetric transformation can provide a theoretical 100% yield without any requirement for enantiomer separation. Enzymes have been used most commonly as the resolving catalysts and precious metals as the racemizing catalysts. Most examples involve racemic secondary alcohols, but an increasing number of chiral amine enzyme DKRs are being reported. Reetz, in 1996, first reported the DKR of rac-2-methylbenzylamine using Candida antarctica lipase B and vinyl acetate with palladium on carbon as the racemization catalyst [20]. The reaction was carried out at 50°C over 8 days to give the (S)-amide in 99% ee and 64% yield. Rather surpris-... 

Enantioselective enzymatic transesterifications have been used as a complementary method to enantioselective enzymatic ester hydrolyses. The first example of this particular type of biotransformation is the synthesis of the optically active 2-acetoxy-l-silacyclohexane (5 )-78 (Scheme 19). This compound was obtained by an enantioselective transesterification of the racemic l-silacyclohexan-2-ol rac-43 with triacetin (acetate source) in isooctane, catalyzed by a crude lipase preparation from Candida cylindracea (CCL, E.C. 3.1.1.3)62. After terminating the reaction at 52% conversion (relative to total amount of substrate rac-43), the product (S)-78 was separated from the nonreacted substrate by column chromatography on silica gel and isolated in 92% yield (relative to total amount of converted rac-43) with an enantiomeric purity of 95% ee. The remaining l-silacyclohexan-2-ol (/ )-43 was obtained in 76% yield (relative to total amount of nonconverted rac-43) with an enantiomeric purity of 96% ee. Repeated recrystallization of (R)-43 led to an improvement of enantiomeric purity by up to >98% ee. Compound (R)-43 has already earlier been prepared by an enantioselective microbial reduction of the l-silacyclohexan-2-one 42 (see Scheme 8)53. The l-silacyclohexan-2-ol (R)-43 is the antipode of compound (.S j-43 which was obtained by a kinetic enzymatic resolution of the racemic 2-acetoxy-l-silacyclohexane rac-78 (see Scheme 15)62. For further enantioselective enzymatic transesterifications of racemic organosilicon substrates, with a carbon atom as the center of chirality, see References 64 and 70-72. 

Primary alcohols have been successfully used as substrates for lipases. Monterde et. Al60 reported the resolution of the chiral auxiliary 2-methoxy-2-phenylethanol 1 via Candida antarctica lipase B (CAL-B)-catalyzed acylation using either vinyl acetate (R=H) or isopropenyl acetate (R= CH3) as acyl donor (cf. fig. 8) and the alkoxycarbonylation using diallyl carbonate as the alkoxycarbonylation agent in THF at 30 °C (cf. fig. 9). 

---