Kinetics study of palm oil hydrolysis using immobilized lipase Candida rugosa in packed bed reactor

Continuous hydrolysis of palm oil triglyceride in organic solvent using immobilized Candida rugosa on the Amberlite MB-1 as a source of immobilized lipase was studied in packed bed reactor. The enzymatic kinetics of hydrolysis reaction was studied by changing the substrate concentration, reaction temperature and residence time(tau) in the reactor. At 55 degrees C, the optimum water concentration was found to be 15 % weight per volume of solution (%w/v). The Michaelis-Menten kinetic model was used to obtain the reaction parameters, Km(app) and V max(app). The activation energies were found to be quite low indicating that the lipase-catalyzed process is controlled by diffusion of substrates. The Michaelis-Menten kinetic model was found to be suitable at low water concentration 10-15 %w/v of solution. At higher water concentration, substrate inhibition model was used for data analysis. Reactor operation was found to play an important role in the palm oil hydrolysis kinetic.     

Covalent immobilization of Candida rugosa lipase on aldehyde functionalized hydrophobic support and the application for synthesis of oleic acid ester

This study focuses on Candida rugosa lipase (CRL) immobilization by covalent attachment on poly(ethylene terephthalate)-grafted glycidyl methacrylate (PET-g-GMA) fiber. The immobilization yielded a protein loading of 2.38?mg?g^-1 of PET-g-GMA fiber. The performances of the immobilized and free CRLs were evaluated with regard to hydrolysis of olive oil and esterification of oleic acid. The optimum activity pH of the CRL was changed by immobilization to neutral range. The maximum activity of the free and immobilized CRLs occurred at 40 and 45?°C respectively. The immobilized lipase retained 65% of its original activity at 50?°C for 2?h. It was found that the immobilized lipase stored at 4?°C retained 90% of its original activity after 35?days, whereas the free lipase stored at 4?°C retained 69% of its original activity after the same period. In the esterification experiments, the immobilized CRL could maintain a high activity at a water content range from 1.5 to 6% (v/v), while the activity of free CRL showed a clear dependence on water content and decreased rapidly at above 3% (v/v) water content. In addition, after five reuses, the esterification percent yield of the immobilized CRL slightly decreased from 29 to 27%.     

Conversion of sunflower oil to biodiesel by alcoholysis using immobilized lipase

Transesterification reaction was performed using sunflower oil and short-chain alcohol by immobilized lipases in organic solvents. The fatty acid ester, which is the product of this reaction, can be used as a diesel fuel that does not produce sulfur oxide and minimize the soot particulate. Immobilized porcine pancreatic lipase (PPL) and Candida rugosa lipase (CRL) showed the satisfactory activity in these reactions. Immobilization of lipases was carried out using inorganic absorbance Celit 545 particle as a carrier. Organic solvent like hexane in reactions was required when methanol and ethanol were used as alcoholic substrate. The reaction could be performed in absence of solvent when 1-propanol and 1-butanol were used as short-chain alcohol. The activities of immobilized lipases were highly increased in comparison with free lipases because its activity sites became more effective. Immobilized enzyme could be repeatedly used without difficult method of separation and the decrease in its activity was not largely observed.     

Cholate-dependent killing of Giardia lamblia by human milk.

We showed previously that nonimmune human milk (NHM) kills Giardia lamblia trophozoites in vitro and presented evidence that killing requires the bile salt-stimulated lipase of milk. Since this enzyme is activated by bile salts, killing should be dependent on the presence of bile salts. We now show that killing by fresh NHM or NHM stored at -70 degrees C is totally dependent on sodium cholate (a bile salt). With less than 0.4 mM cholate, no parasites were killed, whereas with 1 mM cholate, greater than 99.7% were killed by 5% NHM in 30 min. Moreover, killing activity was completely heat labile. The G. lamblia-killing activity of human milk was greatly altered by storage at -10 or -20 degrees C. In less than 23 days, the 50% lethal dose decreased, cholate dependence was lost, and killing activity became heat stable. In contrast, the activity of milk stored at -70 degrees C remained unchanged. Milk lipase activity, like killing activity, became cholate independent during storage at -10 or -20 degrees C. On the basis of these results, we hypothesize that killing of G. lamblia by fresh NHM or NHM stored at -70 degrees C depends on bile salt-stimulated lipase, which must be activated by bile salts. In contrast, NHM stored at -20 degrees C accumulated free fatty acids which kill G. lamblia. In support of this thesis, milk stored at -10 degrees C had a concentration of 18.7 mM free fatty acids compared with only 1.1 mM in an identical sample stored at -70 degrees C.     

Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups

A method for immobilization of Candida rugosa lipase to two types of chitosan beads by activating the hydroxyl groups of chitosan using carbodiimide coupling agent has been successfully developed. The ability of carbodiimide to activate the hydroxyl groups of chitosan was confirmed using the electron spectroscopy for chemical analysis (ESCA) technique. The properties of lipase immobilized using dry and wet chitosan beads were also investigated and compared. Immobilization enhanced the enzyme stability against changes of pH and temperature. High storage stability of 30 days and an increased enzyme activity of 2,110% were observed in wet immobilized lipase. Immobilized lipase using dry and wet chitosan beads retained 78% and 85% of its initial activity after 10 batch hydrolytic cycles. The kinetic parameters Km and Vmax were determined for the free and immobilized lipase. The activation energy (Ea) was found to decrease for immobilization of lipase on chitosan beads.     

Chitosan-tethered poly(acrylonitrile-co-maleic acid) hollow fiber membrane for lipase immobilization

A protocol was used to prepare a dual-layer biomimetic membrane as support for enzyme immobilization by tethering chitosan on the surface of poly(acrylonitrile-co-maleic acid) (PANCMA) ultrafiltration hollow fiber membrane in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxylsuccin-imide (NHS). The chemical change of the chitosan-modified PANCMA membrane surface was confirmed with Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Lipase from Candida rugosa was immobilized on this dual-layer biomimetic membrane using glutaraldehyde (GA), and on the nascent PANCMA membrane using EDC/NHS as coupling agent. The properties of the immobilized enzymes were assayed and compared with those of the free one. It was found that both the activity retention of the immobilized lipase and the amount of bound protein on the dual-layer biomimetic membrane (44.5% and 66.5 mg/m2) were higher than those on the nascent PANCMA membrane (33.9% and 53.7 mg/m2). The kinetic parameters of the free and immobilized lipases, Km and Vmax, were also assayed. The Km values were similar for the immobilized lipases, while the Vmax value of the immobilized lipase on the dual-layer biomimetic membrane was higher than that on the nascent PANCMA membrane. Results indicated that the pH and thermal stabilities of lipase increased upon immobilization. The residual activity of the immobilized lipase after 10 uses was 53% on the dual-layer biomimetic membrane and 62% on the nascent PANCMA membrane.     

Lysosomal hydrolysis of lipids in a cell culture model of smooth muscle foam cells

Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique. These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase. Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes. After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes. To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine. Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not. Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid. However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not. We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.     

Preparation and properties of lipases immobilized on different supports

Porcine pancreatic lipase and Candida cylindracea lipase were immobilized on Celite and Amberlite IRA-938. Activities and stabilities of immobilized lipases were investigated. The immobilized lipase derivatives on Celite exhibited grater residual activity and more resistance to thermal inactivation than their immobilized counterpart on Amberlite IRA-938. The apparent optimum temperatures of the immobilized lipases were 7-10 degrees C higher than that of the free enzymes. The native lipase and lipases immobilized on Celite showed same behaviors of pH dependence. But the pH optimum values for lipases immobilized on Amberlite IRA-938 were shifted to the acidic region relative to that of free enzymes. The stabilities of free and immobilized lipases were also investigated.     

Induction of an extracellular esterase from Candida albicans and some of its properties.

An extracellular esterase from Candida albicans A-714 was found to be induced in a medium containing 0.7% yeast nitrogen base and 2.5% Tween 80 (polyoxyethylenesorbitan compounds). Enzyme activity, which exists predominantly in the extracellular space, was measured by a colorimetric method using alpha-naphthyl palmitate as a substrate. The induction level of the esterase activity was found to be well correlated with fungal growth and was dependent on the Tween 80 concentration. Such esterase activity was observed only in medium containing Tween 80 or other Tweens as the sole carbon source and therefore was not observed in either peptone-glucose medium or peptone-glucose medium supplemented with Tween 80. The induced esterase was heat labile and had maximum activity at pH 5.5. Enzyme activity was stimulated by the addition of sodium taurocholate, an activator of lipase. Thin-layer chromatography revealed that this enzyme does not hydrolyze triolein and L-alpha-lecithin, suggesting that it is a monoester hydrolase (not a lipase in the strict sense of the word). Esterase activity was examined in 85 clinical isolates of Candida species; C. albicans, C. tropicalis, and C. parapsilosis tended to have higher enzyme activities than C. kefyr, C. krusei, C. glabrata, and C. guilliermondii. Although the physiological properties of this esterase are not clear at present, it was found to be crucial for fungal growth under specific conditions.     

Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization

Nanofibrous membranes with an average diameter of 100 and 180 nm were fabricated from poly(acrylonitrile-co-maleic acid) (PANCMA) by the electrospinning process. These nanofibrous membranes contain reactive groups which can be used to covalently immobilize biomacromolecules. Two natural macromolecules, chitosan and gelatin, were tethered on these nanofibrous membranes to fabricate dual-layer biomimetic supports for enzyme immobilization in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS). Lipase from Candida rugosa was then immobilized on these dual-layer biomimetic supports using glutaraldehyde (GA), and on the nascent PANCMA fibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized lipases were assayed. It was found that there is an increase of the activity retention of the immobilized lipase on the chitosan-modified nanofibrous membrane (45.6+/-1.8%) and on the gelatin-modified one (49.7+/-1.8%), compared to that on the nascent one (37.6+/-1.8%). The kinetic parameters of the free and immobilized lipases, K(m) and V(max), were also assayed. In comparison with the immobilized lipase on the nascent nanofibrous membrane, there is an increase of the V(max) value for the immobilized lipases on the chitosan- and gelatin-modified nanofibrous membranes. Results also indicate that the pH and thermal stabilities of lipases increase upon immobilization. The residual activities of the immobilized lipases are 55% on the chitosan-modified nanofibrous membrane and 60% on the gelatin-modified one, after 10 uses.     

Immobilization of uricase upon polypyrrole-ferrocenium film

In this study, uricase was immobilized by a glutaraldehyde/gelatine crosslinking procedure onto polypyrrole film. The K(m) value for immobilized enzyme 0.44 mM was much higher than that of the free enzyme 0.39 mM. V(max) values were 8.4 x 10(-2) mM/dak and 7.1 x 10(-2) mM/dak for free and immobilized enzyme, respectively. The optimal pH values for free and immobilized enzymes were 8.5 and 8.0, respectively. The optimum temperature for both free and immobilized uricase was 35 degrees C and 55 degrees C. The enzyme activity after storage for 7 weeks was found to be 42% and 49% of the initial activity values for free and immobilized enzymes, respectively. The amperometric current obtained after 30 measurements at a constant uric acid concentration of 5.0 x 10(-5) M was found to be 77.7% of initial activity.     

Properties of a thermostable extracellular lipase from Bacillus megaterium AKG-1

An extracellular lipase isolated from Bacillus megaterium AKG-1 had an optimum activity at 55 degrees C/pH 7.0. It retained 100% activity at 50 degrees C for 30 min with a half life of 30 min at 70 degrees C. A 20-70% increase in lipase activity was observed in presence of acetone (20% v/v), DMSO (20% v/v) and isopropanol (10% v/v). The enzyme activity was 92, 98 and 107% after 24 h, on treatment with 10% (v/v) acetone, benzene and isopropanol respectively. Deoxycholic acid, sodium deoxycholate, lithocholic acid, rhamnolipid, Brij 52 and cholic acid stimulated the lipase activity by 76, 36, 24, 24, 23.6 and 13%, respectively. Addition of reducing agents like sodium sulphite, sodium metabisulphite and L-cysteine-HCl, at 10 mM concentration stimulated lipase activity by 127, 146 and 150% respectively. The lipase appeared to show enantioselectivity in hydrolyzing racemic 3-acetoxy-beta-lactam as it hydrolyzed only the (+) enantiomer.     

Molecular weight and degree of deacetylation effects on lipase-loaded chitosan bead characteristics

The effects of the molecular weight (MW) and degree of deacetylation (DD) of chitosan on chitosan hydrogel beads were characterized, and the entrapment efficiency, release of entrapped lipase, and activity of immobilized Candida rugosa lipase were investigated. Fresh and freeze-dried beads were characterized. A solution of lipase was prepared in a 1.5% (w/v) chitosan and 1% (v/v) acetic acid medium, and then dropped into a tripolyphosphate solution to prepare the beads. The release studies were performed over 36 h. The enzyme activity was assayed using the Sigma lipase activity method. Chitosan with high MW and DD resulted in a higher loading. A lower activity was observed for beads produced with high DD chitosan. MW did not have a marked effect on the activity. The release study revealed that enzyme release increased to a maximum when the bead was manufactured with a low MW and a moderate to high DD chitosan sample. Freeze drying did not affect the release or the activity of the lipase. Chitosan with a high MW and DD can thus improve loading and reduce the release of lipase in these beads. The choice of chitosan can affect the activity normalized for lipase loading, and beads with desirable qualities can be produced.     

Biochemical properties of free and immobilized Candida rugosa lipase onto Al2O3: a comparative study

Lipase from Candida rugosa (CRL) was immobilized by physical adsorption onto Al(2)O(3). The immobilization yield was more than 95% during 30 min. The properties of free and immobilized enzyme were also searched and compared. The optimum pH was shifted from 7 to 8.5 by immobilization of the enzyme. The maximum activity of the free and the immobilized enzymes occured at 35 and 40°C, respectively.     

Syntheses of polyamides containing uracil and adenine

2-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)methylsuccinic acid ( 2a ) and 2-(6-aminopurin-9-yl)methylsuccinic acid ( 2b ) were synthesized through the addition reaction of uracil and adenine, respectively, to dimethyl methylenesuccinate, followed by hydrolysis of the resulting dimethyl esters. Dicarboxylic acid 2a was further converted to the corresponding di-p-nitrophenyl ester 3a and dicarboxylic acid 2b was converted to the di(cyanomethyl) ester 3b . The active esters 3a and 3b were polycondensed with diamines such as 1,2-diaminoethane, 1,6-diaminohexane, 4-aminomethylbenzylamine, and piperazine in solution, giving polyamides containing uracil and adenine as the side group. The polyamides were found to have molecular weights in the range of about 1000–5000. The polyamides derived from 1,2-diaminoethane and piperazine were soluble in water.     

Separation and properties of the haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii

Desalted ammonium-sulphate (0-65%) precipitates from the cell-free supernates of 16-24-h cultures of Listeria monocytogenes Boldy and L. ivanovii (previously L. monocytogenes) Type 5 were eluted through Sephadex G-200. The enzyme activities gave rise to two main peaks. The first peak (approximate mol. wt of protein 150,000) contained only phosphatase activity (assayed by hydrolysis of 4-nitrophenylphosphate at pH 5.0 and 7.0). The second peak (approximate mol. wts of proteins 40,000-60,000) contained the haemolysin activity and the following hydrolytic activities (assay substrates are given in parentheses): phospholipase C (phosphatidyl choline and 4-nitrophenyl-phosphoryl-choline); phosphodiesterase (bis-4-nitrophenyl-phosphate); acid phosphatase (4-nitrophenylphosphatase); and esterases and lipases (4-nitrophenyl acetate, naphthyl-acetate and -oleate, triacetin and triolein). DEAE-Sephadex chromatography of appropriate fractions from the Sephadex G-200 purification step separated the first peak into two phosphatases and resolved the second peak into its constituent activities. Polyacrylamide gel electrophoresis showed that the individual fractions from the DEAE-Sephadex step consisted of mixtures of protein. The effects of pH and potential activators and inhibitors on the active proteins purified by DEAE-Sephadex chromatography were examined.     

Molecular identification and antifungal susceptibility testing of clinical isolates of the Candida rugosa species complex and proposal of the new species Candida neorugosa

Candida rugosa is a poorly known fungal species occasionally involved in human infections. A molecular analysis of the sequences of the D1/D2 domains and the internal transcribed spacer (ITS) region of the ribosomal genes of 24 clinical isolates phenotypically identified as C. rugosa demonstrated that only 10 (41.6%) isolates belonged to that species. The other isolates were identified as Candida pararugosa (41.6%) and Candida pseudorugosa (8.3%). The remaining two isolates, from human and equine infections, respectively, were clearly different from the others and represent a new species proposed here as Candida neorugosa. The closest species by D1/D2 sequences was the type strain of C. rugosa, with only 92.3% similarity. C. neorugosa can also be differentiated from all other species of the C. rugosa complex by phenotypic features. The eight antifungal drugs tested showed high in vitro activity against the 24 isolates included in the study.     

A continuous-flow technique for analysis of stoichiometry and transport kinetics of gastric H,K-ATPase

A continuous-flow method was developed for determining the stoichiometry of the gastric proton pump H,K-ATPase (EC 3.6.1.36) in its hydrolysis of ATP and translocation of H+ and the K+ congener 86Rb+. H,K-ATPase-containing vesicles which had been isolated from pig gastric mucosa were incubated at 37 degrees C for 2 h in 150 mM 86RbCl, 0.5 mM ethylenebis(oxyethylenenitrilo)tetra-acetic acid and 3 mM 2-(N-morpholino)ethane sulphonic acid (Mes) adjusted to pH 6.1 with Tris, and then applied onto a 0.45 micron pore size cellulose acetate filter. The immobilized vesicles were superfused with 0.15 mM Mes/Tris buffer, pH 6.1, containing 150 mM choline chloride and 0.2 mM MgCl2. After changing to a medium containing 0.1 mM ATP, the amounts and rates of H+ uptake, 86Rb+ efflux and ATP hydrolysis were measured. The initial ratio of Rb+ transported to ATP hydrolysed gave values of 0.96 +/- 0.26 (mean +/- SD, n = 28). The initial ratio of ATP-dependent Rb+ efflux to H+ uptake gave values of 0.92 +/- 0.28 (mean +/- SD, n = 28). The Mg-ATPase activity was measured in vesicles which had been incubated with choline chloride instead of RbCl. This activity was 15.8 +/- 8.7% (mean +/- SD) of the total ATPase activity in the initial fractions used for calculation of the stoichiometry. It is argued that this Mg-ATPase may be an intrinsic activity of the H,K-ATPase and that the relation between these activities is dependent on the amount of K+ (or Rb+) present in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reutilization of fatty acid carbons for lung lipid synthesis

The utilization of glucose, palmitate, and oleate for the synthesis of lung lipids was studied in isolated rat lungs. Lungs were ventilated with 5% CO2 in air and perfused for 100 min with a Krebs-Ringer bicarbonate buffer (pH 7.4), containing 3% fatty acid-free albumin and either 5.6 mM [U-14C]glucose or 0.25 mM [1-14C]palmitate, or 0.25 mM [1-14C]-oleate. At the end of 100 min of perfusion with these precursors, between 73 and 85% of total lipid 14C was recovered as phospholipid. Glucose carbon was mainly incorporated into triglyceride fatty acids (TG-FA) and phosphatidylcholine fatty acids (PC-FA) of 16- and 18-carbon chain length. After perfusion with [14C]palmitate and [14C]oleate, only 65 and 20% of 14C was recovered as PC 16-carbon and 18-carbon FA, respectively. The remaining 14C was mainly recovered as FA shorter than the 14C precursors. Schmidt degradation analysis of lipid FA demonstrated considerable labeling of alkyl carbons on perfusion with the carboxyl-labeled precursors, indicating that FA degradation products were used for synthesis of lipid FA. This process was enhanced on addition of glucose to the perfusate.     

Syntheses of polyamides containing theophylline and thymine

2-(1,3-Dimethyl-2,6-dioxo-2,6-dihydropurin-7-yl)methylsuccinic acid ( 2a ) and 2-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)methylsuccinic acid ( 2b ) were synthesized via the addition reaction of theophylline and thymine, respectively, to dimethyl methylenesuccinate, followed by hydrolysis of the resulting ester. The dicarboxylic acid derivatives 2a and 2b were further converted to their di-p-nitrophenyl esters 3a and 3b , which were allowed to polycondense with diamines such as 1,6-diaminohexane, 1,2-diaminoethane, 3-aminomethylbenzylamine, and piperazine in solutions. The resulting polyamides are white powders with molecular weights in the range of about 2000–6000. The DP of the polyamides varies with the kind of diamines and solvent used. All polyamides are soluble in DMSO and formic acid, the polyamides deriving from esters 3a and 3b and 1,2-diaminoethane and piperazine are also soluble in water.