Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.     

Synthesis of enantiomerically pure glycidol via a fully enantioselective lipase-catalyzed resolution

The efficient enzymatic synthesis of enantiopure 2,3-epoxypropanol (glycidol) has been achieved. The racemic glycidyl butyrate was successfully resolved by enzymatic hydrolysis using a strategy that combines different immobilization protocols and different experimental reaction conditions. A new enzyme (25 kDa lipase)—which is a lipase-like enzyme purified from the pancreatic porcine lipase (PPL) extract—immobilized on DEAE–Sepharose was selected as the optimal biocatalyst. The optimal results were obtained at pH 7, 25 °C and 10% dioxane using this biocatalyst and a very high enantioselectivity for the enzyme was displayed, obtaining both (R)-(−)-glycidyl butyrate and (R)-(+)-glycidol with enantiomeric excesses >99% (E >100). The hydrolysis of (R)-(−)-glycidyl butyrate produced pure (S)-(−)-glycidol.     

Collagen-Immobilized Lipases Show Good Activity and Reusability for Butyl Butyrate Synthesis

Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.     

A Calcium-Ion-Stabilized Lipase from <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> ZS04 and its Application in Resolution of Chiral Aryl Alcohols

An extracellular organic solvent-tolerant lipase-producing bacterium was isolated from oil-contaminated soil samples and was identified taxonomically as Pseudomonas stutzeri, from which the lipase was purified and exhibited maximal activity at temperature of 50 °C and pH of 9.0. Meanwhile, the lipase was stable below or at 30 °C and over an alkaline pH range (7.5–11.0). Ca²⁺ could significantly improve the lipase thermal stability which prompts a promising application in biocatalysis through convenient medium engineering. The lipase demonstrated striking features such as distinct stability to the most tested hydrophilic and hydrophobic solvents (25 %, v/v), and DMSO could activate the lipase dramatically. In the enzyme-catalyzed resolution, lipase ZS04 manifested excellent enantioselective esterification toward the (R)-1-(4-methoxyphenyl)-ethanol (MOPE), a crucial chiral intermediate in pharmaceuticals as well as in other analogs with strict substrate specificity and theoretical highest conversion yield. This strong advantage over other related schemes made lipase ZS04 a promising biocatalyst in organic synthesis and pharmaceutical applications.     

Covalent immobilization of Candida antarctica lipase B on nanopolystyrene and its application to microwave-assisted esterification

Nanopolystyrene was used as a solid support for the covalent immobilization of Candida antarctica lipase B (CalB) using the photoreactive reagent 1-fluoro-2-nitro-4-azido benzene (FNAB) as a coupling reagent. The obtained derivative was then used as a biocatalyst in a microwave assisted esterification experiment. Factors such as contact time, pH, and enzyme concentration were investigated during immobilization. The hydrolytic activity, thermal, and operational stability of immobilized-CalB were determined. The maximum immobilized yield (218 μg/mg support) obtained at pH 6.8 exhibited optimum hydrolytic activity (4.42 × 10³ mU p-nitrophenol/min). The thermal stability of CalB improved significantly when it was immobilized at pH 10, however, the immobilized yield was very low (93.6 μg/mg support). The immobilized-CalB prepared at pH 6.8 and pH 10 retained 50% of its initial activity after incubation periods of 14 and 16 h, respectively, at 60 ℃. The operational stability was investigated for the microwave assisted esterification of oleic acid with methanol. Immobilized-CalB retained 50% of its initial activity after 15 batch cycles in the microwave-assisted esterification. The esterification time was notably reduced under microwave irradiation. The combined use of a biocatalyst and microwave heating is thus an alternative total green synthesis process.     

Production of Thermostable Lipase by Thermomyces lanuginosus on Solid-State Fermentation: Selective Hydrolysis of Sardine Oil

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Preparation and characterization of lipase immobilized on reversibly soluble-insoluble N-(2-carboxylbenzoyl) chitosan

N-(2-carboxylbenzoyl) chitosan (CBC), a reversibly soluble-insoluble polymer with pH change, was prepared by modifying chitosan backbone with phthalic anhydride and employed as carrier for lipase immobilization. The obtained CBC exhibited reversible solubility in aqueous solution; it was soluble at pH above 3.8 and precipitated at pH below 3.4. The porcine pancreatic lipase was covalently immobilized on CBC with glutaraldehyde as the crosslinking agent. Under the optimal immobilization condition, the retention activity of the immobilized lipase was found to be 69.8 %. The maximum activity of lipase immobilized on CBC was observed at 40 °C, pH 8.0, while the free lipase presented maximum activity at 37 °C, pH 7.5. The lipase immobilized on CBC exhibited improved thermal and storage stabilities and retained 58.7 % of its initial activity after 9 times of repeated use.     

Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g^?1 dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K _m) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.     

Covalent attachment of microbial lipase onto microporous styrene-divinylbenzene copolymer by means of polyglutaraldehyde

A novel method for immobilization of Thermomyces lanuginosus lipase onto polyglutaraldehyde-activated poly(styrene-divinylbenzene) (STY-DVB), which is a hydrophobic microporous support has been successfully developed. The copolymer was prepared by the polymerization of the continuous phase of a high internal phase emulsion (polyHIPE). The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. Lipase from T. lanuginosus was immobilized covalently with 85% yield on the internal surface of the hydrophobic microporous poly(styrene-divinylbenzene) copolymer and used as a biocatalyst for the transesterification reaction. The immobilized enzyme has been fully active 30 days in storage and retained the activity during the 15 repeated batch reactions. The properties of free and immobilized lipase were studied. The effects of protein concentration, pH, temperature, and time on the immobilization, activity, and stability of the immobilized lipase were also studied. The newly synthesized microporous poly(styrene-divinylbenzene) copolymer constitutes excellent support for lipase. It given rise to high immobilization yield, retains enzymatic activity for 30 days, stable in structure and allows for the immobilization of large amount of protein (11.4mg/g support). Since immobilization is simple yet effective, the newly immobilized lipase could be used in several application including oil hydrolysis, production of modified oils, biodiesel synthesis, and removal of fatty acids from oils.     

Immobilization of Candida antarctica lipase B by covalent attachment to green coconut fiber

The objective of this study was to covalently immobilize Candida antarctica type B lipase (CALB) onto silanized green coconut fibers. Variables known to control the number of bonds between enzyme and support were evaluated including contact time, pH, and final reduction with sodium borohydride. Optimal conditions for lipase immobilization were found to be 2 h incubation at both pH 7.0 and 10.0. Thermal stability studies at 60 degrees C showed that the immobilized lipase prepared at pH 10.0 (CALB-10) was 363-fold more stable than the soluble enzyme and 5.4-fold more stable than the biocatalyst prepared at pH 7.0 (CALB-7). CALB-7 was found to have higher specific activity and better stability when stored at 5 degrees C. When sodium borohydride was used as reducing agent on CALB-10 there were no improvement in storage stability and at 60 degrees C stability was reduced for both CALB-7 and CALB-10.     

Immobilization of Candida cylindracea Lipase by Covalent Attachment on Glu-Modified Bentonite

Alkaline Ca-bentonite, obtained upon acid activation and base load of natural bentonite, has a good anion exchange capability. Glu-modified alkaline Ca-bentonites were further prepared by covalent binding with glutamic acid for the immobilization of lipase OF from Candida cylindracea. The obtained immobilized lipase demonstrated a significantly higher catalytic activity than that of unmodified alkaline Ca-bentonite, giving a specific activity of 62.1 U mg⁻¹ protein, twice that of the unmodified carrier, and a total activity of 391.2 U g⁻¹ support, retaining ~ 82.3% of the activity after being reused five times for olive oil emulsion hydrolysis. X-ray diffraction and Fourier transform infrared spectroscopy assays demonstrated the successful immobilization of the lipase on the surface of the bentonite. Upon immobilization, the thermostability of the lipase improved remarkably. At 50 °C, free lipase retained only 6.0% of its initial activity at 6 h, in comparison with 15% for Ca-Bent-lipase and 50% for Glu-Ca-Bent-lipase after 8 h. The Glu-Ca-Bent-lipase is proved as an effective biocatalyst for the biodiesel preparation, improving the transesterification reaction conversion from 52.8% in the condition of free lipase to 99.9% and keeping at 56.2% after being reused five times, while the free lipase was inactive upon two reuses. The above results provide a new route in the use of inexpensive bentonite for the enzyme immobilization.

     

The Purification and Characterization of Lipases from <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis>, and Their Immobilization and Use for Biodiesel Production from Coconut Oil

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)—purified 25.41-fold, recovery of 47.1%—and lipase B (32,000 Da)—purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca²⁺, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5–10.0 and 20–80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.     

Polyesters from Macrolactones Using Commercial Lipase NS 88011 and Novozym 435 as Biocatalysts

The demand for environmentally friendly products allied with the depletion of natural resources has increased the search for sustainable materials in chemical and pharmaceutical industries. Polyesters are among the most widely used biodegradable polymers in biomedical applications. In this work, aliphatic polyesters (from globalide and ω-pentadecalactone) were synthesized using a new commercial biocatalyst, the low-cost immobilized NS 88011 lipase (lipase B from Candida antarctica immobilized on a hydrophobic support). Results were compared with those obtained under the same conditions using a traditional, but more expensive, commercial biocatalyst, Novozym 435 (lipase B from C. antarctica immobilized on Lewatit VP OC). When NS 88011 was used in the polymerization of globalide, longer reaction times (240 min)—when compared to Novozym 435—were required to obtain high yields (80–90 wt%). However, higher molecular weights were achieved. When poly(ω-pentadecalactone) was synthesized, high yields and molecular weights (130,000 g mol^?1) were obtained and the enzyme concentration showed strong influence on the polyester properties. This is the first report describing NS 88011 in polymer synthesis. The use of this cheaper enzymatic preparation can provide an alternative for polyester synthesis via enzymatic ring-opening polymerization.     

Preparation,characterization, and potential application of an immobilized glucose oxidase

A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50°C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50°C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.     

Development and Characterization of a Solid-Phase Biocatalyst Based on Cyclodextrin Glucantransferase Reversibly Immobilized onto Thiolsulfinate-Agarose

Reduction of disulfide bonds and introduction of ??de novo?? thiol groups in cyclodextrin glucantransferase from Thermoanaerobacter sp. were assessed in order to perform reversible covalent immobilization onto thiol-reactive supports (thiolsulfinate-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0?% for the native and reduced enzyme, up to nearly 90?% for the thiolated enzyme. The mild conditions of the immobilization process (pH 6.8?C7.0 and 22?°C) allowed the achievement of 100?% coupling efficiencies when low loads were applied. Ionic strength was a critical parameter for the immobilization process; for high activity recoveries, 50?mM phosphate buffer supplemented with 0.15?M NaCl was required. The kinetic parameters, pH and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. For ??-cyclization activity, optimal pH range and temperature were 4.0?C5.4 and 85?°C. The possibility of reusing the support was demonstrated by the reversibility of enzyme?Csupport binding.     

Immobilization of Candida antarctica Lipase B by Adsorption to Green Coconut Fiber

An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t _1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction.     

trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific immobilization and fluorescent labeling

Lipase B from Candida antarctica (CAL-B) has been site-directedly modified by the introduction of a trans,trans-hexadiene moiety onto lipase molecules, identified by MALDI-TOF. This modification on CAL-B permitted its immobilization on Q-Sepharose supports in excellent yields (>95%) when native lipase was not immobilized at pH 7 and 25 °C. After the entire modification procedure, the catalytic activity of the protein on the solid support was surprisingly increased 2-fold. A tailor-made maleimide-fluorophore derivative was specifically covalently linked to the protein in high yield via a selective Diels-Alder reaction in aqueous media. Furthermore, the NBD-labeled-CAL-B was also immobilized on the ionic support, retaining around 80% of the specific activity. The preparation of this labeled-CAL-B was also possible by a Diels-Alder reaction on solid phase in excellent yields.     

Immobilization of Thermomyces lanuginosus Lipase by Different Techniques on Immobead 150 Support: Characterization and Applications

Thermomyces lanuginosus lipase (TLL) was immobilized on native and modified Immobead 150, with epoxy groups removed by hydrolysis and oxidized to add aldehyde on its surface. Immobilizations on both supports were performed by adsorption, adsorption and cross-linking, covalent attachment, multipoint covalent attachment, and, for the modified support, multipoint covalent attachment using ethylenediamine. Biocatalysts were evaluated for thermal and solvent stabilities, and the best biocatalyst was also tested after incubation in ionic liquids and used in the synthesis of butyl butyrate and isoamyl butyrate. Multipoint covalent immobilized TLL on the native Immobead 150 (Emulti) showed a half-life of 5.32 h at 70 °C, being approximately 30 times more stable than its soluble form; it showed high stability in acetone, hexane, and isooctane. Its enzymatic activity was up to 40 % when incubated in ionic liquids. Ester synthesis produced yields of esterification above 60 % in 24 h. Of all immobilization protocols, the Emulti performed best concerning the thermal, solvent, and ionic liquids stabilities. Emulti was successfully applied to the synthesis of butyl butyrate and isoamyl butyrate, which are very important products for the food and beverage industries.     

Kinetic studies of lipase from Candida rugosa

The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (K _d=0.98 h⁻¹), whereas for the immobilized lipase it was 1.10 h (K _d=0.63 h⁻¹). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The K _m was 0.15 mM and the V _max was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.