Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Stabilization of ficin extract by immobilization on glyoxyl agarose. Preliminary characterization of the biocatalyst performance in hydrolysis of proteins

A protein extract containing ficin was immobilized on glyoxyl agarose at pH 10 and 25 °C. The free enzyme remained fully active after 24 h at pH 10. However the enzyme immobilized on the support retained only 30% of the activity after this time using a small substrate. After checking the stability of ficin preparations obtained after different enzyme-support multi-interaction times, it was found that it reached a maximum at 3 h (40-folds more stable than the free enzyme at pH 5). The immobilized enzyme was active in a wide range of pH (e.g., retained double activity at pH 10 than the free enzyme) and temperatures (e.g., at 80 °C retained three-folds more activity than the free enzyme). The activity versus casein almost matched the results using the small substrate (60%) at 55 °C. However, in the presence of 2 M of urea, it became three times more active than the free enzyme. The immobilized enzyme could be reused five cycles at 55 °C without losing activity.     

Improved production of 2-keto-3-deoxy-d-glycero-galactononulosonic acid (KDN) using FastPrep-CLEAs

FastPrep cross-linked enzyme aggregates of N-acetylneuraminate aldolase from Staphylococcus carnosus (ScNAL-FpCLEAs) were prepared in order to improve the synthesis of 2-keto-3-deoxy-d-glycero-galactononulosonic acid (KDN), an important building block for therapeutic glycolipids and a possible marker for human prostate cancer. ScNAL-FpCLEAs showed improved thermostability compared with the free enzyme, doubling its half-life at 60 °C. When the effect of substrate ratio (pyruvate:d-mannose) and temperature on the yield of KDN was studied at its optimum pH (pH 7.0), 90% conversion in only 8 h was reached in the presence of 0.6 M d-mannose and 1.2 M pyruvate at 37 °C. This is the highest conversion described to date for enzymatic KDN synthesis. In addition, ScNAL-FpCLEAs exhibited enhanced catalytic activity and stability and could be recycled 10 times with no loss of activity. These results suggest the biotechnological potential of using FastPrepCLEAs to obtain valuable biocatalysts.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Isolation and characterization of a novel organic solvent-tolerant and halotolerant esterase from a soil metagenomic library

Soil metagenome conceals a great variety of unexploited genes for industrially important enzymes. To identify novel genes conferring lipolytic activity, one metagenomic library comprising of 200,000 transformants were constructed. Among the 48,000 clones screened, 19 clones which exhibited lipolytic activity were obtained. After sequence analysis, 19 different lipolytic genes were identified. One of these genes, designated as estWSD, consisted of 1152 nucleotides, encoding a 383-amino-acid protein. Multiple sequence alignment and phylogenetic analysis indicated that EstWSD and its closest homologues may constitute a new family of bacterial lipolytic enzymes. The best substrate for the purified EstWSD among the ρ-nitrophenol esters examined was ρ-nitrophenol butyrate. Recombinant EstWSD displayed a pH optimum of 7.0 and a temperature optimum of 50 °С. This enzyme retained 52% of maximal activity after incubation at 50 °C for 3 h. Furthermore, EstWSD also exhibited salt tolerance with over 51% of its initial activity in the presence of up to 4.5 M NaCl for 1 h. In particular, this enzyme showed remarkable stability in 15% and 30% dimethylsulfoxide, ρ-xylene, hexane, heptane, and octane even after incubation for 72 h. To our knowledge, it is the first report to find a novel esterase belonging to a new lipolytic family and possessing such variety of excellent features. All these characteristics suggest that EstWSD may be a potential candidate for application in industrial processes.     

A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.     

Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding

An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activities of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 °C) than that of the free enzyme (55 °C). Kinetic parameters, Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max), were calculated as K_m = 13.375, 11.956, and 8.698 × 10^?4 mg/ml and V_max = 0.156, 0.163 and 0.17 × 10^?3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Characterization of lactase-conjugated magnetic nanoparticles

Conjugation of lactase to magnetic nanoparticles is of interest in biosensor and ingredient processing applications that require high enzyme concentration and catalyst separation from the reaction stream. However, little is known about the effects of these materials on the physicochemical attributes of conjugated lactase. Lactase (Aspergillus oryzae) was covalently attached by carbodiimide chemistry to carboxylic-acid functionalized magnetic particles having a hydrodynamic radius of 18 nm. The resulting enzyme–nanoparticle conjugates were characterized with regard to particle size, zeta potential, enzyme kinetics, temperature and pH stability, catalyst recovery, and secondary structure changes. Following attachment, the materials retained colloidal stability and individual particle characteristics with a zeta potential of ?33 mV compared to ?46 mV for the native particle. The conjugated enzyme showed no changes in secondary structure and exhibited significant catalytic activity with a catalytic efficiency of 2.8 × 10³ M^?1 s^?1 compared to 2.5 × 10³ M^?1 s^?1 for the native enzyme. Relative to the free enzyme, the conjugated enzyme was recovered for repeated use with 78% activity retained after five cycles. This work demonstrates that carboxylic-acid functionalized magnetic nanoparticles can be utilized as a means of producing a simple and effective conjugated-lactase system that achieves both particle and enzyme stability.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Immobilized β-glucosidase on magnetic chitosan microspheres for hydrolysis of straw cellulose

β-Glucosidase immobilized on magnetic chitosan microspheres for potential recycling usage in hydrolysis of cellulosic biomass was investigated. The immobilized enzyme had an activity of 6.4 U/g support under optimized condition when using cellobiose as substrate. Immobilization resulted in less increase of the apparent K_m, low drift of the optimal pH, as well as improved stability relative to the free enzyme. The immobilized β-glucosidase was applied to enzymatic hydrolysis of corn straw to produce 60.2 g/l reducing sugar with a conversion rate of 78.2% over the course of a 32-h reaction. This conversion rate was maintained above 76.5% after recycling the enzyme for use in eight batches (total 256 h), showing favorable operational stability of the immobilized enzyme.     

Effect of deglycosylation on the properties of thermophilic invertase purified from the yeast Candida guilliermondii MpIIIa

Invertase from Candida guilliermondii MpIIIa was purified and biochemically characterized. The purified enzyme (INV3a-N) is a glycoprotein with a carbohydrate composition comprising nearly 74% of its total molecular weight (MW) and specific activity of 82,027 U/mg of protein. The enzyme displayed optimal activity at pH 5.0 and 65 ˚C. The Km and V_max values for INV3a-N were 0.104 mM and 10.9 μmol/min/mg of protein, respectively, using sucrose as the substrate. The enzyme retained 50% and 20% of its maximal activity after 168 h and 30 days, respectively, at 50 ˚C. INV3a-N was fully active at sucrose concentrations of 400 mM and the activity of the enzyme dropped slowly at higher substrate concentration. Interestingly, the deglycosylated form of INV3a-N (INV3a-D) displayed 76–92% lower thermostability than that of INV3a-N at all temperatures assayed (50–70 ˚C), and was inhibited at sucrose concentrations of 200 mM. Findings here indicate glycosylation plays an important role, not only in the thermostability of INV3a-N, but also in the inhibition of the enzyme by sucrose. Since the enzyme is active at high sucrose concentrations, INV3a-N may be considered a suitable candidate for numerous industrial applications involving substrates with high sugar content or for improvement of ethanol production from cane molasses.     

Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor

A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 °C) when compared to PAMO in solution (no activity detected after 1 h at 60 °C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier.     

Functional and structural analyses of a 1,4-β-endoglucanase from Ganoderma lucidum

Ganoderma lucidum is a saprotrophic white-rot fungus which contains a rich set of cellulolytic enzymes. Here, we screened an array of potential 1,4-β-endoglucanases from G. lucidum based on the gene annotation library and found that one candidate gene, GlCel5A, exhibits CMC-hydrolyzing activity. The recombinant GlCel5A protein expressed in Pichia pastoris is able to hydrolyze CMC and β-glucan but not xylan and mannan. The enzyme exhibits optimal activity at 60 °C and pH 3–4, and retained 50% activity at 80 and 90 °C for at least 15 and 10 min. The crystal structure of GlCel5A and its complex with cellobiose, solved at 2.7 and 2.86 Å resolution, shows a classical (β/α)₈ TIM-barrel fold as seen in other members of glycoside hydrolase family 5. The complex structure contains a cellobiose molecule in the +1 and +2 subsites, and reveals the interactions with the positive sites of the enzyme. Collectively, the present work provides the first comprehensive characterization of an endoglucanase from G. lucidum that possesses properties for industrial applications, and strongly encourages further studying in the cellulolytic enzyme system of G. lucidum.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.     

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0–5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3 h. The addition of Mn²⁺, K⁺, Zn²⁺, Ca²⁺ and Al³⁺ inhibited the enzyme activity; it increased in the presence of Mg²⁺ and Cu²⁺ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K_m and V_max values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T₆₅₀ from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

High activity and selectivity immobilized lipase on Fe3O4 nanoparticles for banana flavour synthesis

Lipase (E.C.3.1.1.3) from Thermomyces lanuginosus (TL) was directly bonded, through multiple physical interactions, on citric acid functionalized monodispersed Fe₃O₄ nanoparticles (NPs) in presence of a small amount of hydrophobic functionalities. A very promising scalable synthetic approach ensuring high control and reproducibility of the results, and an easy and green immobilization procedure was chosen for NPs synthesis and lipase anchoring. The size and structure of magnetic nanoparticles were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The samples at different degree of functionalization were analysed through thermogravimetric measurements. Lipase immobilization was further confirmed by enzymatic assay and Fourier transform infrared (FT-IR) spectra. Immobilized lipase showed a very high activity recovery up to 144% at pH = 7 and 323% at pH = 7.5 (activity of the immobilized enzyme compared to that of its free form). The enzyme, anchored to the Fe₃O₄ nanoparticles, to be easy recovered and reused, resulted more stable than the native counterpart and useful to produce banana flavour. The immobilized lipase results less sensitive to the temperature and pH, with the optimum temperature higher of 5 °C and optimum pH up shifted to 7.5 (free lipase optimum pH = 7.0). After 120 days, free and immobilized lipases retained 64% and 51% of their initial activity, respectively. Ester yield at 40 °C for immobilized lipase reached 88% and 100% selectivity.