Candida Cylindracea Lipase Research Articles

ChemInform Abstract: Enzymatic Asymmetric Synthesis of cis-4-Cyclopentene-1,3-dimethanol Monoacetate.

Abstract Asymmetric synthesis of cis-4-cyclopentene-1,3-dimethanol monoacetate was performed via enzymatic hydrolysis of the diacetate or enzymatic acetylation of the diol precursor. Esterases and lipases were studied, and Candida cylindracea lipase was shown to afford the (−)- enantiomer with ee >97%.

ChemInform Abstract: Potential Applications of Enzyme-Mediated Transesterifications in the Synthesis of Bioactive Compounds

The yeast lipase Candida cylindracea (CCL) and porcine pancreatic lipase (PPL) have been used for regioselective deacylation of peracetylated benzopyrones, diphenylpropenones and acetophenones for the first time. The deacylation study on different classes of polyphenols has revealed that the presence of carbonyl group attached to the aromatic ring is needed by the lipases to exhibit regioselectivity towards hydrolysis of acetoxyl groups. The acetoxyl groups at positions other than the one at ortho position to the carbonyl group get selectively hydrolysed by PPL in orgnnic solvents. The trnnsesterification reactions using triflouroethylbutyrate (TFEB), catalysed by PPL and CCL on some polyols in dry organic solvents were also performed. It was found that the primary hydroxyl is acylated. In D-panthenol, the oxidised dextrorotatory form of which is a major constituent of vitamin B-complex, the primary hydroxyl group at the far end of the asymmetric carbon atom gets exclusively acylated. This work should be of importance in the synthesis of building blocks of biologically active natural products which may provide structural leads to anti AIDS and anticancer agents.

Lipase-catalyzed synthesis and characterization of novel lipidyl-cyclodextrins in solvent free medium

The lipase-catalyzed amidation reaction between fatty acyl donors and mono-6-amino-permethylated β-cyclodextrin (MBCD-NH 2 ) was achieved with high yield in solvent free medium. The ability of Mucor miehie and Candida cylindracea lipases, free or immobilized, to catalyze amidation reaction was shown. Products were purified and characterized. To optimize the reaction, some reaction conditions were assessed like temperature, type of shaking and amount of enzyme, on a model amidation reaction between butyl caprylate and MBCD-NH 2 . Among the biocatalysts tested, Lipozyme was found to be the most active, requiring low loading, being robust at 70 °C even under magnetic agitation.

The solvent effect of ethanol and n-hexane on the hydrolysis of triolein and tributyrin by Candida cylindracea lipase

The solvent effect of ethanol and n-hexane on the hydrolysis of triolein and tributyrin by Candida cylindracea lipase

Optimization of Enzymatic Hydrolysis of Chicken Fat in Emulsion by Response Surface Methodology

AbstractChicken fat in an emulsion prepared with mechanical shearing and high pressure homogenization (HPH) was hydrolyzed using Candida cylindracea lipase. A homogenization pressure of 50 MPa, which can generate smaller droplets and higher hydrolysis efficiency than mechanical shearing, was fixed to prepare the emulsion for hydrolysis optimization. Response surface methodology (RSM) was applied to study the effect of temperature, enzyme loading, shaking rate and reaction time on the hydrolysis process. The results showed that all three‐second‐order polynomial models adequately fitted the experimental data. Additionally, hydrolysis parameters for the optimal yields of free oleic and linoleic acids were also obtained using the desirability function: a temperature of 38 °C, an enzyme loading of 0.48% (g/g fat basis), a shaking rate of 100 rpm and a reaction time of 80 min. Under the optimal conditions, the yields of free oleic and linoleic acids were predicted as being 0.470 and 0.118 g/g fat with recoveries of 94.6 and 93.7%, respectively. During the hydrolysis process, the particle size increased with concomitant boosting of the degree of hydrolysis and the stability of the emulsion system was gradually undermined by this reaction process.

Optimization of benzoin synthesis in supercritical carbon dioxide by response surface methodology (RSM)

α-Hydroxy ketones are important precursors for biologically active compounds, especially benzoins have important applications in chemistry and pharmacy. In this study enantioselective synthesis of benzoin was studied in supercritical carbon dioxide (SCCO 2). Response surface methodology (RSM) was used to investigate the effects of temperature ( T), pressure ( P) and pH on enantiomeric excess of chiral benzoin using Candida cylindracea lipase in SCCO 2. A 2 3 factorial design was performed to optimize the chiral benzoin synthesis. The optimum conditions were found as 40 °C, 79 bar, pH 6.4 and at these conditions enantiomeric excess (ee) was obtained as 62% ( R-benzoin). At the optimum conditions, bases such as DMAP (4-dimethylaminopyridine) and triethylamine were added to reaction medium to increase kinetic resolution. The addition of DMAP considerably enhanced the enantiomeric excess (80% S-benzoin), while triethylamine had negative effect on ee.

Effect of Temperature on n-Tetradecane Emulsion in the Presence of Phospholipid DPPC and Enzyme Lipase or Phospholipase A2

Zeta potentials and effective diameters of n-tetradecane emulsions in 1 M ethanol were investigated in the presence of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) (1 mg/100 mL), Candida cylindracea lipase (CCL), and phospholipase PLA2 (1 mg/100 mL) at 20, 37, and 45 degrees C. The enzyme was added at the beginning of mechanical emulsion homogenization or 1 min before the end of stirring for 10 min at 10,000 rpm. It was found that DPPC decreases the negative zeta potentials at all three temperatures. The decrease was largest at 20 degrees C and smallest at 45 degrees C. The influence of the enzymes on the zeta potentials depended on the enzyme kind, time of its injection, and temperature. More negative values of the zeta potentials relative to n-C14H30/DPPC droplets were obtained if the lipase was present. Generally, the effective diameters correlate with the zeta potentials, i.e., lower zeta potential corresponds with bigger effective diameter. Possible reasons for the observed changes of the measured parameters are discussed.

Enzymatic Synthesis of Astaxanthin n-Octanoic Acid Esters

We examined the enzymatic synthesis of astaxanthin n-octanoic acid esters. Carriers for the immobilized enzyme and reaction conditions such as water content, reaction temperature, and time were examined using Candida cylindracea lipase (Lipase OF). Lipase OF) immobilized by a hydrophobic anion exchange resin (10% w/w content of lipase) gave the best yield in the esterification reaction of astaxanthin. Two milligrams of astaxanthin per 750 microL tri-n-octanoin (ca. 0.3%) was optimum because of the low solubility of tri-n-octanoin. The esters were obtained in a yield of 36.4% under the optimal reaction conditions.

Altering the Substrate Specificity ofCandida rugosaLIP4 by Engineering the Substrate-Binding Sites

Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.

Production of low molecular weight esters using vegetable oils or butter oil as reaction media

Corn oil, peanut oil, sunflower oil, soybean oil, canola oil, olive oil and butter oil were tested as replacements for nonaqueous solvents in the lipase mediated biosynthesis of ethyl butyrate, butyl butyrate, isoamyl butyrate and isoamyl acetate. The most effective oils tested were butter oil, soybean oil, peanut oil and olive oil (yield = 50–60% after 16 h incubation at 25°C) while corn oil was least effective (yield = 38%). Higher yields were obtained, for all ester syntheses, at a reaction temperature of 50°C. Drying agents were shown to have no effect or to inhibit the synthesis. Of the four enzymes tested, Candida cylindracea lipase was the most effective enzyme for ethyl butyrate synthesis.

The influence of antioxidant and post-synthetic treatment on the properties of biodegradable poly(butylene succinate)s modified with poly(propylene oxide)

Novel poly(ester-ether)s based on poly(butylene succinate) (PBS) as the hard segments and 30 mass % of poly(propylene oxide) (PPO) as the soft segments were synthesized with varying amount of the antioxidant (N,N'-diphenyl-p-phenylenediamine, DPPD). The influences of the addition of DPPD and the impact of post-synthetic treatment by precipitation on the molecular structure, thermal and physical properties, as well as on the storage stability of the biodegradable aliphatic copolyesters, were investigated. The structure and composition of the copolymers were determined by means of 1H-NMR spectroscopy. The molecular weight and polydispersity of the poly(ester-ether)s were evaluated from solution viscosity and GPC measurements. The thermal properties and stability were evaluated, respecttively, by means of DSC and non-isothermal thermogravimetry in an inert nitrogen atmosphere. The biodegradability potential of the polymers was studied in hydrolytic and enzymatic degradation tests with Candida cylindracea lipase by monitoring the weight loss of polymer films after incubation. The weight losses of the samples increased with time and were in the range from 1 to 5 mass % after 4 weeks. GPC analysis confirmed that there were changes in the molecular weight of the copolyesters during both hydrolytic and enzymatic degradation tests, leading to the conclusion that the degradation mechanism of poly(butylene succinate)s modified with PPO occurred through surface erosion and bulk degradation.

Production of n − 3 polyunsaturated fatty acid concentrate from sardine oil by lipase-catalyzed hydrolysis

The production of n − 3 polyunsaturated fatty acids ( n − 3 PUFAs) concentrate from oil extracted from Pacific sardines ( Sardinops sagax) was studied using lipase-catalyzed hydrolysis. Commercially available microbial lipases, from Candida Rugosa (CR), Candida cylindracea (CC), Mucor javanicus (MJ), and Aspergillus niger (AN) were used for enzymatic hydrolyses with extracted sardine oil, run at 37 °C with constant stirring for 1.5, 3, 6, and 9 h. Fatty acid composition analysis by gas chromatography showed that the refined unhydrolyzed oil contained 26.86% of eicosapentaenoic acid (EPA) and 13.62% of docosahexaenoic acid (DHA) (wt/wt%). CR lipase was the most effective in concentrating n − 3 PUFA. Hydrolysis with 250 U CR lipase increased EPA concentration to a relatively constant level of 33.74% after 1.5 h. DHA levels were also significantly increased from 13.62% to 29.94% with 500 U after 9 h. Compared to CR and CC lipases, MJ and AN lipases resulted in low n − 3 PUFA concentration. Triacylglycerol levels decreased significantly as reaction time progressed.

Enzymatic synthesis of benzoin in supercritical carbon dioxide

Enantioselective enzymatic hydrolysis of benzoyl-benzoin catalyzed by Candida cylindracea (CCL) lipase was carried out in SCCO 2. It was found that CCL lipase enantioselectively hydrolyzed the ( R)-benzoin. The enantiomeric excess of product (ee p) was maximized at 35 °C near the critical region 90 bar and obtained 61.3% at a 50% conversion. CCL did not catalyze the reaction in an atmospherical condition.

Biotransformations of ent-18-acetoxy-6-ketomanoyl oxides epimers at C-13 with filamentous fungi

Two ent-18-acetoxy-6-oxomanoyl oxides, epimers at C-13, have been prepared from ent-6α,8α,18-trihydroxylabda-13(16),14-diene (andalusol), isolated from Sideritis foetens, by means of several chemical pathways and a regioselective acylation with Candida cylindracea lipase (CCL). Biotransformation of these 13-epimeric ent-manoyl oxides by Fusarium moniliforme and Neurospora crassa produced mainly ent-1β- or ent-11α-hydroxylations, as well as their deacetylated derivatives, in both epimers. In addition, with the 13- epi substrate N. crassa originated other minor hydroxylations by the ent-α face at C-1 or at C-12, whereas an ent-11β-hydroxyl group, probably originated by reduction of an 11-oxo derivative also isolated, was achieved with the 13- normal substrate.

The acetates of p-nitrophenyl α- l-arabinofuranoside—Regioselective preparation by action of lipases

All possible di- O-acetates and mono- O-acetates of p-nitrophenyl α- l-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di- O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per- O-acetylated p-nitrophenyl α- l-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-di- O-acetates were obtained by acetylation of p-nitrophenyl α- l-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in organic solvents. The 5- O-acetate was regioselectively synthesised in 95% yield by acetylation of p-nitrophenyl α- l-arabinofuranoside catalysed by porcine pancreas lipase. Finally, the 2- and 3- O-acetates of p-nitrophenyl α- l-arabinofuranoside were obtained in two steps. The enzymatic di- O-acetylation of p-nitrophenyl α- l-arabinofuranoside by LPS-30 was followed by enzymatic hydrolysis of the primary acetyl group by CCL or LAY.

Immobilization of lipase onto micron-size magnetic beads

A novel and economical magnetic poly(methacrylate-divinylbenzene) microsphere (less than 8 μm in diameter) was synthesized by the modified suspension polymerization of methacrylate and cross-linker divinylbenzene in the presence of magnetic fluid. Then, surface aminolysis was employed to obtain a high content of surface amino groups (0.40–0.55 mmol g −1 supports). The morphology and properties of these magnetic supports were characterized with scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and a vibrating sample magnetometer. These magnetic supports exhibited superparamagnetism with a high specific saturation magnetization ( σ s) of 14.6 emu g −1. Candida cylindracea lipase was covalently immobilized on the amino-functionalized magnetic supports with the activity recovery up to 72.4% and enzyme loading of 34.0 mg g −1 support, remarkably higher than the previous studies. The factors involved in the activity recovery and enzymatic properties of the immobilized lipase prepared were studied in comparison with free lipase, for which olive oil was chosen as the substrate. The results show that the immobilized lipase has good stability and reusability after recovery by magnetic separation within 20 s.

Biodegradable aliphatic polyesters. Part I. Properties and biodegradation of poly(butylene succinate- co-butylene adipate)

A series of aliphatic homopolyesters and copolyesters was prepared from 1,4 butanediol and dimethylesters of succinic and adipic acids through a two-step process of transesterification and polycondensation. The synthesized polyesters were characterized by means of nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), viscosity measurements, differential scanning calorimetry (DSC), X-ray diffraction (XRD), and mechanical property measurements. The homopolymer poly(butylene succinate) exhibited the highest tensile strength, which decreased with increasing adipate unit content, passed through a minimum at copolyester composition close to equimolarity and then increased towards the value of poly(butylene adipate). It is interesting to note that in contrast to tensile strength, the elongation at break increased for adipate unit content of 20–40 mol%. The biodegradation of the polymers was investigated by soil burial and enzymatic hydrolysis using three enzymes, Candida cylindracea lipase, Rhizopus delemar lipase, and Pseudomonas fluorescens cholesterol esterase. It appears that the key factor affecting material degradation was its crystallinity.

Enzymatic esterification of DL‐menthol with propionic acid by lipase from Candida cylindracea

AbstractThis 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

The catalytic properties and mechanism of cyclohexane/DBSA/water microemulsion system for esterification

A model esterification reaction of hexanol and hexanoic acid in the cyclohexane/dodecylbenzenesulfonic acid (DBSA)/water microemulsion system has been investigated. Compared with cyclohexane/AOT/water microemulsion system and organic medium system, esterification reaction in the cyclohexane/DBSA/water system can perform relatively rapidly whether catalyzed by Candida cylindracea lipase (Ccl) or not, this demonstrated that DBSA itself can act as a kind of acid catalyst. Comparison of conversion in several acid-catalyzed reaction systems were also performed and the results showed that the conversion in DBSA system was the most highest, which proved the key factor affecting the conversion was not the acid strength, but the attribute of DBSA as a kind of surfactant. Furthermore, we also perform transesterification reaction of butanol and ethyl butyrate (and methyl butyrate) in the DBSA reverse microemulsion system; however, it cannot get remarkably high conversion like esterification, this reason may be alcohol produced during the transesterification cannot easily enter water droplet like water produced by the esterification. Comparison of conversion in the DBSA system with conversion in the O/W emulsion system DBSA as an emulsion agent also indicated that the conversion in the DBSA reverse microemulsion was much higher. Finally, the mechanism of reaction was also explored. After realizing that DBSA is both acid catalyst and surfactant, we concluded that it was concurrent function of acid catalyst and surfactant that played an important role in improving reaction rate and conversion of esterification.

Immobilization of Lipases on Different Carriers and Their Use in Synthesis of Pentyl Isovalerates

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA‐938.