Glycerol Candida

Glycerol kinase (from Candida mycoderma, E coli, rat or pigeon liver glycerokinase)... 

Figure 2.9 Schematic model of the (R)-enantiomer of a secondary glycerol-like substrate as its tetrahedral intermediate inside the active site cleft of hpase B from Candida antarctica (CALB). The large group (-CH2-0-Rj) and the acyl part is inserted hke a V-shape into the cleft while the small group (-CH2-R2) at the stereocentre is located in the stereospecificity pocket which is next to the site of reaction.

Lipases (triacyl glycerol acyl hydrolases, E.C 3.1.1.3) are a unique class of hydrolases for asymmetric synthesis86,87,S9,90e, They are available from fungi, bacteria and mammalians. The lipases most commonly used so far are the commercially supplied pig pancreas lipase (PPL)136, Pseudomonas cepacia lipase (PCL)89,137 and Candida cylindracea lipase (CCL). In most cases only the crude lipases, consisting of a mixture of proteins which may even be other hydrolases, are successfully applied1373. 

Fraenkel et al. (17), who isolated mutant strains which had lost the ability to grow on glycerol, succinate, or acetate but grew normally on hexoses or pentoses. These organisms were shown to be deficient in a specific FDPase, which could be distinguished from the nonspecific acid hexosephosphatase present in both mutant or wild-type strains by the fact that the latter was present in the periplasmic space (86) and did not require a divalent metal cation. The properties of the specific FDPase were confirmed with a partially purified preparation (87) the E. coli enzyme was shown to be highly specific for FDP and to be active with very low concentrations of this substance. The requirement for a divalent cation was satisfied by Mg2+, which was far more effective than Mn2+ other divalent cations were either inactive or inhibitory. The partially purified enzyme showed optimum activity at pH 7.8, with very little activity below pH 7 or above pH 9. The enzyme resembled mammalian and Candida FDPases in its sensitivity to low concentrations of AMP it was approximately 50% inhibited at an AMP concentration of 2.5 X 10-° M. 

Ciani, M. and Ferraro, L. (1996). Enhanced glycerol content in wines made with immobilized Candida stellata cells. Appl. Environ. Microbiol. 62,128-132. 

Irimescu, R., Furihata, K., Hata, K., Iwasaki, Y., and Yamane, T. 2001. Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of l,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol. J. Am. Oil Chem. Soc., 78, 285-289. 

The immobilized Candida antarctica lipase (CAL) displayed superb performance in terms of the regiocontrol and yields of the enzymatic step (Halldors-son et al., 2003 Haraldsson, 2007). This is based on a rapid, irreversible transesterification of glycerol using 1.25-fold stoichiometric amount of vinyl esters of the MCFA in dichloromethane or chloroform at 0-4 °C. Excellent yields (90-92%) were obtained as based on pure material after recrystallization. 

Immobilised lipase (Novozym 435) from Candida antarctica supported on a macroporous acrylic resin was purchased from Novo Nordisk A/S (Bagsvaerd, Denmark). The cod liver oil used was of commercial quality and obtained from Lysi h.f. Iceland. Dimodan LS is a commercial MG product made by Grindsted, Denmark, this product contains mainly monooleoyl- and monolinoleoyl glycerol. 

Hydrolysis has traditionally been used for the production of fatty acids and glycerols, which find widespread apphcation in soaps and detergents, cosmetics, pharmaceuticals, and food products (174). Hydrolysis of soybean (181), canola (147, 208, 209), sunflower (149, 181, 210), tuna (150), and blackcurrant oils (145), tri-palmitin (146), triolein (211), and ethyl stearate (202) in SCCO2 has been reported. These investigations employed a variety of lipases, including immobilized lipase from porcine pancreas (211), Novozyme 435 (146, 181), Lipozyme (147, 150, 208, 209), non-immobilized Candida rugosa (150, 181), Lipase OL (150), and Lipolase lOOT (149, 181, 210). The effects of water content, enzyme load, operating conditions (temperature and pressure), pH, enzyme/substrate ratio, oil/buffer ratio, and CO2 flow rate (for continuous reactions) on the hydrolysis reaction were reported. 

Acrolein Glycerol Glycerol dehydrogenase Bacillus amaracrylus Bacillus welchii Candida boidinii... 

So far, only very little attention has been focussed on the use of zeolites in biocatalysis, i.e., as supports for the immobilization of enzymes. Lie and Molin [116] studied the influence of hydrophobicity (dealuminated mordenite) and hydrophilicity (zeolite NaY) of the support on the adsorption of lipase from Candida cylindracea. The adsorption was achieved by precipitation of the enzyme with acetone. Hydrolysis of triacylglycerols and esterification of fatty acids with glycerol were the reactions studied. It was observed that the nature of the zeolite support has a significant influence on enzyme catalysis. Hydrolysis was blocked on the hydrophobic mordenite, but the esterification reaction was mediated. This reaction was, on the other hand, almost completely suppressed on the hydrophilic faujasite. The adsorption of enzymes on supports was also intensively examined with alkaline phosphatase on bentolite-L clay. The pH of the solution turned out to be very important both for the immobilization and for the activity of the enzyme [117]. Acid phosphatase from potato was immobilized onto zeolite NaX [118]. Also in this study, adsorption conditions were important in causing even multilayer formation of the enzyme on the zeolite. The influence of the cations in the zeolite support was scrutinized as well, and zeolite NaX turned out to be a better adsorbent than LiX orKX. 

Regioselective acylations of polyhydroxylated compounds such as carbohydrates, glycerols, steroids, or alkaloids have been carried out with lipases, esterases, and proteases [13, 20]. One example is the Candida antartica lipase (immobilized on acrylic resin) catalyzed monoacylation of the signalling steroid ectysone (1) giving selectively the 2-C)-acetate 2 (eq. (1)). Using vinyl acetate for this transesterification the reaction was irreversibly pushed to the product side, since the liberated enol instantaneously isomerizes to acetaldehyde [21]. The sometimes unfavorable aldehyde is avoided when 1-ethoxyvinyl acetates [22], trichloro- or -fluoroethyl esters [23 a, b], oxime esters [23 c] or thioesters [23 d] are employed for the quasi-irreversible reaction courses. 

To find a suitable enzyme to catalyze the transesterification reaction (methyl ester to glycerol ester) under mild conditions, we screened a number of lipases and esterases. We found two lipases (Candida cylindracea lipase and porcine pancreatic lipase) to exhibit catalytic activity for this reaction. The reaction conditions entailed elevated temperatures, with the use of glycerol as the solvent and an acyl acceptor. The enzyme catalyzed the esterification of the carboxyl group of the pectin to the Cl (primary) alcohol of the glycerol to form the monoester. No substantial glycerol... 

Long crystals from ether, mp 146-147. [ajj 4T (pyridine). Freely so] in methanol, pyridine, 2,3 -butanediol 1 2-propanediol. Sparingly sol in ethanol, butanol, acetone, lnsol in water glycerol, ethyl aceiate, ether, benzene, petr ether. Shows in vitro activity againsi Cryptococcus neoforma ns> Candida albicans, and some saprophytic fungi. Practically ineffective in rabbits and mice suffering from fungus diseases. 

Nakano, H., Kitahata, S., Tominaga, Y, and Takenishi, S. 1991. Esterification of glycosides with glycerol and trimethylolpropane moieties by Candida cylindracea lipase. Agric. Biol. Chem. 55 2083-2089. 

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