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A method for purification of cis-9,trans-11 isomer of conjugated linoleic acid (CLA) is described. Lipase from Candida cylindracea (CCL) was highly selective towards cis-9,trans-11 isomer and was chosen to catalyze esterification of a mixture of two CLA isomers. The effect of various parameters on the esterification process of an almost equimolar mixture of cis-9,trans-11 (44.0%), trans-10,cis-12 (42.5%) and other CLA isomers (13.5%) was studied. The influence of alcohol substrate, CLA/alcohol molar ratio, lipase amount, reaction temperature, pH and time of the reaction on the total esterification and cis-9,trans-11 isomer purity in ester fraction were examined. The highest isomeric excess of cis-9,trans-11 isomer on trans-10,cis-12 CLA and good esterification degree was observed when the esterification condition were as follows: CLA/2-propanol molar ratio, 1:5; water, 200% wt of CLA; CCL, 24 μg of CCL/mg CLA; temperature 30 C; time of reaction 6 h and when mixture enriched in cis-9,trans-11 CLA was used as a substrate for esterification. A preparative scale purification combining crystallization and one step enzymatic process were made to afford purified cis-9,trans-11 (94.2%) with total recovery of 31%.

The operational stability of the lipase enzyme from Candida rugosa (formerly known as Candida cylindracea) has been investigated. The activity was found to be dependent on shear rate, temperature and emulsifier concentration. The enzyme remained active (the activity was about 80% of the original) up to a shear rate of 122 s−1 for 2 hours at 40dC. Concentrated enzyme was more resistant to shear rate than the diluted enzyme. It was also found that the presence of emulsifier increased the enzyme inactivation. The enzyme lost almost all its activity in 45 minutes at an emulsifier concentration of 0.5%.

This work deals with the resolution of DL-menthol with propionic acid by Candida cylindracea lipase (Ccl) in organic solvent reaction systems and a reverse micelles system of sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (AOT). The activity and stability as well as enantioselectivity of the lipase in two systems were studied. The results indicate that the lipase showed higher stability in reverse micelles than in organic solvent, which proved that the reverse micelles system has potential application for maintaining the activity of the enzyme for a long time. This is because lipase molecules can be entrapped in water-containing micro-drops of reverse micelles, avoiding direct-contract with unfavorable organic medium. The enantioselectivity (E > 30, eep = 92.5) in the two systems is relatively high, although the conversion is moderate. The influence of the characteristic parameters of the two systems, such as pH, temperature, w0 (molar ratio of water to AOT in reverse micelles systems) and water content (organic solvent) on the conversion of DL-menthol was also investigated. Copyright © 2005 Society of Chemical Industry

Extensive screenings using commercially available enzymes were performed in relation to the asymmetric hydrolysis reactions of the (±)- cis- ( 1 , 3 and 5 ) and (±)- trans -1,2,3,4,6,7,8,8a-octahydro-8a-methyl-6- oxo -naphthyl acetates ( 7 , 9 and 11 ). The combinations of enzymes from plants and cis -equatorial compounds as well as the combinations of lipases from microorganisms and trans -axial-5-methoxycarbonyl acetate showed high enantiomeric ratio in asymmetric hydrolysis. However, the enantiomeric ratio of the combinations of lipases from microorganisms and trans -axial isomers except trans -axial-5-methoxycarbonyl derivative was less satisfactory. High E values (>200) were observed when β-amylase from wheat and lipase from Candida cylindracea were used for the (±)- cis -acetate 1 and the (±)- trans -isomer 7 , respectively, to obtain the corresponding chiral products with high enantiomeric ratios (>99:1).

Enzymatic kinetic resolution of racemic aryl β - hydroxy ketones in organic media catalyzed by Candida cylindracea lipase is reported.

The enzymatic kinetic resolution of (±)-4-acetoxy[2.2]paracyclophane by Candida cylindracea lipase was investigated in water and in a two-phase aqueous organic system. The (+)-(R)-4-hydroxy- and (+)-(S)-4-acetoxy[2.2]-paracyclophanes were isolated in excellent yields and high enantiomeric excesses. The resolution was carried out on multi-gram scale in hexane-water at 40° C.

Lipase unit is defined as that amount of enzyme needed to produce 1 μmol non-esterified fatty acid in one minute under specified conditions. Non-esterified fatty acid is traditionally measured by acid-base titration. This was done in the present study in place of High-performance liquid chromatography (HPLC) method. HPLC with a light scattering detector was conducted to measure non-esterified fatty acids to determine the activity of lipase from Candida cylindracea. HPLC result was essentially the same as that obtained by titration, the former method being more sensitive.

Asymmetric enzymatic hydrolysis of pro-chiral 3,3-disubstituted 2,4-diacetoxycyclohexa-1,4-dienes 2 affords in high yields optically pure 2,2-disubstituted 3-acetoxycyclohex-3-enones 1 (>98% ee). Under mild conditions Candida cylindracea lipase (enzyme/substrate ratio = 2%) hydrolyses specifically the pro-S enol ester function of the pro-chiral starting material 2.

A series of acetoxy derivatives of androstane was deacetylated in organic solvents by several lipases. The most satisfactory results were obtained with lipase from Candida cylindracea (CCL) and Candida antarctica (CAL). In some derivatives, CCL and CAL showed an overwhelming regioselectivity towards the removal of the 3β- or the 17β-acetyl group (see Table 2). Three new steroid derivatives were obtained through this approach. A hypothetical rationale for the behaviour of these enzymes is given.

Lipase from Candida cylindracea and Candida antarctica catalyzes the removal of acetyl groups from 3β-acetoxypregn-5-en-20-one and 3β-acetoxy-20-(S)-hydroxycholest-5-en-23-one through a transesterification reaction in organic solvents.

Aziridine carboxylates alcoholysis by lipases depends of the N-substituent. N-alkyl and N-aryl compounds have been resolved with medium to good enantiomeric purity by enzymatic alcoholysis catalyzed by pig pancreatic (PPL) or Candida cylindracea lipase (CCL).

Enantiomerically pure alkyl (1S, 4R)- and (1R, 4S)-4-acetamido-cyclopent-2-ene-carboxylates are obtained from their corresponding racemates by hydrolysis with PLE or the lipase from Candida cylindracea.

The activity and enantioselectivity of Candida cylindracea lipase enzyme are affected by a number of solvents. There is a good correlation between the activity and enantioselectivity of the lipase and the log P values of the solvents. Replacement of water with water-mimicking ethylene glycol increased the activity and enantioselectivity of the enzyme. The results show that the dominant factor is the enzyme—water interaction.

Lipases from Candida cylindracea and Candida antarctica catalyze the aminolysis of activated and nonactivated esters respectively. The degree of enantioselectivity depends on the amine.

Methyl 6-O-butyryl-α-D- and -L-galactopyranoside and methyl 6-O-butyryl-α-D- and -L-mannopyranoside, which present three contiguous secondary hydroxy groups in different orientations, have been acylated using three hydrolytic enzymes. Porcine pancreatic, Candida cylindracea, and Pseudomonas fluorescens lipases in organic solvents. Some generalization of the obtained results is discussed. Methyl 6-O-butyryl-α-D- and -L-galactopyranoside and methyl 6-O-butyryl-α-D- and -L-mannopyranoside, which present three contiguous secondary hydroxy groups in different orientations, have been acylated using three hydrolytic enzymes. Porcine pancreatic, Candida cylindracea, and Pseudomonas fluorescens lipases in organic solvents. Some generalization of the obtained results is discussed.

Lipases from Candida cylindracea and from Pseudomonas fluorescens efficiently catalyse the benzyl to alkyl transesterification in organic solvents under mild conditions in nearly quantitative yields.

Lipase from Candida cylindracea and lipase from Chromobacterium viscosum allowed the synthesis of silyl-chiral optically active compounds by mono-transesterification of 2-sila-1,3-propanediols.

Desymmetrization of meso -hydrobenzoin by irreversible acyl transfer using vinyl acetate as the acyl donor in the presence of Rhizopusjavanicus lipase or Candida cylindracea lipase enables the preparation of ( R )-1-acetoxy-( S )-2-hydroxy-1,2-diphenylethane and ( S )-1-acetoxy-( R )-2-hydroxy-1,2-diphenylethane respectively in excellent optical purity.

Lipase from Candida cylindracea and Candida antarctica catalyzes the removal of acetyl groups from 3β-acetoxypregn-5-en-20-one and 3β-acetoxy-20-(S)-hydroxycholest-5-en-23-one through a transesterification reaction in organic solvents.

The synthesis of some acyloxy-methoxy-cinnamic acid derivatives, azidohydroxy butanoates, and azidohydroxy butanedioates in enantiomerically pure form is presented. Racemic diastereomerically pure educts were prepared in few steps. These racemates are resolved with lipases from Candida cylindracea (CC) and Pseudomonas fluorescens (P).

Lipases from Candida cylindracea and from Pseudomonas fluorescens efficiently catalyse the benzyl to alkyl transesterification in organic solvents under mild conditions in nearly quantitative yields.

A preference for (S)-enantiomers has been observed in the course of the esterification of racemic 2-methylalkanoic acids catalyzed by lipase from Candida cylindracea in heptane.

The enzyme-catalysed condensation polymerization of 11-hydroxyundecanoic acid in hexane with the lipase from Candida cylindracea is reported. Molecular weights up to 35 000 are attained. A time course study of the polymerization process reveals that oligomers are formed relatively rapidly and that these oligomers then condense to generate higher molecular weight polyesters.

Enzymatic kinetic resolution of racemic aryl β - hydroxy ketones in organic media catalyzed by Candida cylindracea lipase is reported.

Publisher Summary This chapter provides a comparative study of thermal inactivation of enzymes in supercritical carbon dioxide. For study, esterase EP10 from Pseudomonas marginata was obtained. Following assays were used for enzyme activity measurements before and after treatment with SC-CO2. The linear increase of absorbance after 5 minutes of incubation at h 420nm is used for determination of enzyme activity. For lipase increase of absorbance at λ 572nm is used to measure activities. All absorbance measurements were performed at 25°C with a spectrophotometer equipped with a temperature controller. In the study, esterase EP10 shows a rapid decrease of enzyme activity at temperatures above 75°C. Even after incubation for only 1 hour at 75°C enzyme activity of esterase EP10 decreased, whereby a small increase is observed in case of lipase from Candida cylindracea. The activities of the untreated enzymes were set to 100% and is shown on the ordinate. Within the first hours of treatment of esterase EP10 with SC-CO2 at 75°C there is a rapid loss of enzyme activity but lipase from Candida cylindracea is stable for 24 hours. The residual activities of the enzymes after 24 hours at 75°C and 150 bar are shown. Enzymes show no significant loss of activity after incubation for 1 hour at 150 bar in SCCO2 at temperatures below 65°C.

From meso -conduritol D tetraacetate four homochiral partial derivatives, namely (+)-(1 R ,2 R ,3 S ,4 S )-1-hydroxy-2,3,4-triacetoxy-5-cyclohexene, (−)-(1 R ,2 R ,3 S ,4 S )-2-hydroxy-1,3,4-triacetoxy-5-cyclohexene, (−)-(1 R ,2 R ,3 S ,4 S )-1-benzoyloxy-3,4-diacetoxy-2-hydroxy-5-cyclohexene and (+)-(1 R ,2 R ,3 S ,4 S )-3,4-diacetoxy-1,2-dihydroxy-5-cyclohexene, have been prepared through enzymatic reactions catalysed by one of the following lipases: from porcine pancreas, from Mucor miehei and from Candida cylindracea . These compounds are of potential utility in the synthesis of cyclitols and aminocyclitols. As an example, the preparation of the previously unreported (+)-conduramine C-4 is also reported.

Esterification of five- and six-membered-ring-containing alcohols catalysed by three different lipases (those from porcine pancreas, Candida cylindracea and Geotrichum candidum) was studied. Some conversions gave high stereochemical purity, but all gave low yields.

Aziridine carboxylates alcoholysis by lipases depends of the N-substituent. N-alkyl and N-aryl compounds have been resolved with medium to good enantiomeric purity by enzymatic alcoholysis catalyzed by pig pancreatic (PPL) or Candida cylindracea lipase (CCL).

The Candida cylindracea lipase catalyzed enantioselective esterification of 2-chloropropionic acid was studied in different solvents with constant initial water content in the reaction mixture. The effect of solvents and solvent mixtures with different hydrophobicity (e.g. log P value) on the activity and enantioselectivity of the enzyme was investigated. The log P values showed a linear correlation with the initial reaction rate as well as with the enantioselectivity. The rate increased while the enantioselectivity slightly decreased with increasing log P values. In solvent mixtures the activity of the enzyme was generally determined by the solvent with the lowest hydrophobicity while the correlation between the average log P value and the enantioselectivity was linear.

The selectivity of Candida cylindracea lipase can be enhanced five-fold via covalent immobilization in acyl-transfer reactions using vinyl acetate as acyl-donor. When acid anhydrides are employed instead, the selectivity can be improved by one order of magnitude by controlling the micro-pH.

Methods for the preparation of chiral nonracemic 2-methylalkanols are reviewed with emphasis on microbial processes. Special attention is devoted to two reactions: 1. (S)-2-methylalkanols were obtained from the bakers’ yeast reduction of 2-methyl-3-(2-thienyl)propenals to the corresponding (S)-2-methylthienylpropanols followed by Raney nickel reduction and 2. Racemic 2-methylalkanoic acids were resolved via esterification using lipase from Candida cylindracea.

In lipase catalysed resolution of alcohols the choice of the acid moiety is important. Likewise the choice of the alcohol moiety may determine the resolution of acids. In Candida cylindracea catalysed esterification of 2-chloropropionic acid the choice of the alcohol determine which enantiomer of the acid that will react. In this paper reaction kinetics in relation to the role of acids and alcohols in enantioselectivity will be discussed.

Cholesterol esterase (EC 3.1.1.13) from the microorganism Candida cylindracea has been crystallized in two forms. Crystals, typically 0.30 × 0.15 × 0.10 mm in size, diffract rotating anode generated x-ray to beyond 3 A are suitable for data collection for an x-ray crystallographic investigation. A monoclinic crystal form in the space group P21 was found to have cell dimensions of a = 122.9 A , b = 101.0 A , c = 95.2 A and β = 108.3° . The asymmetric unit of cell contains two dimers of 129 kDa each. A second crystal form, in th triclinic space group P1, has cell dimensions of a = A , b = 88.7 A , c = 58.6 A , α = 93.3° , β = 113.8° and γ = 96.0°, and has one dimer per asymmetric unit.

A series of geometric isomeric mixtures of steroidal 3-(O-Carboxymethyl) oxime methyl esters 1–4 were hydrolyzed with high diastereoselectivity for the anti isomer with crude lipase from Candida cylindracea.

Lipase from Candida cylindracea and lipase from Chromobacterium viscosum allowed the synthesis of silyl-chiral optically active compounds by mono-transesterification of 2-sila-1,3-propanediols.

N-Substituted aziridine-2-carboxylates and -2,3-dicarboxylates have been resolved with good to excellent stereochemical purity by enzymatic hydrolysis catalysed by lipase from Candida cylindracea.

Enzymatic hydrolysis of racemic 2-(mono-,di- and trichlorophenoxy)-propionic acids methyl esters by α-chymotrypsin, pig liver esterase and porcine pancreatic or Candida cylindracea lipases was found to be poorly to moderately enantioselective; in most cases, the R-ester was preferentially hydrolyzed.

(1 R , 4a R , 10a S )-(−)-1-Methoxycarbonyl-1, 4a-dimethyl-1,2,3,4,4a,9,10, 10a-octahydrophenanthrene (deoxypodocarpic acid methyl ester 1), a useful intermediate for the synthesis of various diterpenes, was synthesized from ( R )-(+)-6-ethoxycarbonyl-2, 6-dimethyl-1-cyclohexenyl acetate 4. The chiral starting material was prepared by the enantioselective hydrolysis of the corresponding racemate using lipase OF from Candida cylindracea .

(+)- S -2-(6-Methoxy-2-naphthyl)propionic acid ( 1 ) has been prepared via enzymatic enantlospecific hydrolysis of(±)-chloroethyl-2-(6-methoxy-2-naphthyl) propionate ( 3 ), catalyzed by the lipase of Candida cylindracea.

Addition of lipase from Candida cylindracea to butter caused concentrations of free fatty acids to increase with increasing storage temperatures (−28 to 6°C) and times (0 to 60 days). The free fatty acids concentrations in butters also increased with greater numbers of freeze-thaw cycles (one to four), especially at the higher thawing temperature of 21°C. Evidence was obtained for two lipases in a sample of rancid commercial butter. These lipases showed different positional specificity for free fatty acids, and the activity of each enzyme was either dependent upon or independent of storage temperature.

Enzymatic hydrolysis of differently substituted carbohydrates with lipase from porcine pancreas and Candida cylindracea has been investigated. Selective removal of a n-butyryl group in the presence of an acetyl ester (e.g. 16 → 17) and a t-butylcarbonyl ester (e.g. 18 → 19) was feasible with lipase from Candida cylindracea .

The amino groups of Candida cylindracea lipase were modified with N-succinimidyl carboxylates at pH 7.5. Some of the modified enzymes had higher activities than that of the native enzyme possibly because the hydrophobicity enhanced contact between the enzyme and substrate.

Glycerol poly (oxyethylene) ether (GPEE) was found to effectively stabilize Candida cylindracea lipase in an aqueous solution (100U/ml) at 50°C. That is, this lipase became thermally stable in the presence of GPEE. Polyoxyethyleneglycol (PEG) and polypropyleneglycol (PPG) were also tested as stabilizing agents of this lipase in an aqueous solution and found to be effective.The relationship between the amount of additives in the lipase solution and their stabilizing extent was complicated. GPEE stabilized the lipase in proportion to its concentration and PEG brought about maximum stabilization at a concentration of 2.5mmol/20ml of solution. But stabilization became progressively less with increasing amount of PEG.Consequently, each additive is considered to have indivisual mechanism for imparting stability to a lipase solution.

Enzymatic resolution of endo-7-oxabicyclo [2.2.1] hept-2-yl butyrates 3 and 5 using lipase from Candida cylindracea led to optically pure bicyclic alcohols and esters being important intermediates for the synthesis of biologically active compounds.

A lipase (Candida cylindracea) catalyzed the reaction between ethyl propiolate and aromatic amines to afford propargylamides.