Comparison of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase Immobilization Yield of Entrapment,Adsorption, and Covalent Bond Techniques

The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic properties. Sodium alginate and chitosan were the polymers selected to immobilize lipase by inclusion. The beads of each polymer were dried by freeze drying and fluidization. The results show that chitosan was more adapted to the inclusion of lipase. Even though freeze dried, bead activity was low compared to that of fluidized beads. The freeze-drying process seems to produce suitable beads for storage at 4 and 20 degrees C. The immobilization by adsorption was carried out on both celite and silica gel. Maximum immobilization yield of 76% was obtained with celite followed by 43% in silica gel. The enzyme adsorbed on the two supports exhibited greater stability at a certain temperature (50 degrees C) and in no polar solvents (Isooctane, n-heptane, and n-hexane). In addition, the lipase immobilized by covalent bond retained residual activity equitable to 70%. It was demonstrated that the enzyme immobilized by covalent bond showed greater activity (80%) after 5 months of storage.     

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.     

Stability of hydrophobic lipase derivatives immobilized on organic polymer beads

Lipase fromCandida rugosa was immobilized by attaching various hydrophobic groups to the enzyme molecule and adsorbing these hydrophobic lipase derivatives on several organic polymer beads. The immobilized enzymes were more thermostable in organic solvents compared to the native and modified Upases. Thermostability was highest with ΧAD2 beads, followed by ΧAD7 and RCOOH. Initially modifying the enzyme with hydrophobic modifiers did not have any effect on the enzyme thermostability. The best conditions for storing these enzyme preparations were at very low temperature in the lyophilized form and in a solution containing the reaction substrate. Interestingly, PEG-lipase immobilized on ΧAD7 beads showed increased operational stability when used in a stirred-tank reactor. The operational stability was further increased by a mild glutaraldehyde treatment of the enzyme preparation.     

Adsorption and activity of lipase from Candida rugosa on the chitosan-modified poly(acrylonitrile-co-maleic acid) membrane surface

Efforts have recently been made to improve the biocompatibility of support surface for enzyme immobilization, which could create a specific microenvironment for the enzymes and thus benefit the enzyme activity. In this work, one natural macromolecule, chitosan, was tethered on the surface of poly(acrylonitrile-co-maleic acid) (PANCMA) membrane to prepare a dual-layer biomimetic support for enzyme immobilization. Lipase from Candida rugosa was immobilized on this dual-layer biomimetic support by adsorption. The properties of the immobilized enzyme were assayed and compared with those of the free one. It was found that the adsorption capacity of lipase on the chitosan-tethered PANCMA membrane increases with the decrease of ionic strength and there is an optimum pH value for the adsorption. The activity retention of the immobilized lipase on the chitosan-tethered membrane by adsorption (54.1%) is higher than that by chemical bonding (44.5%). In comparison with the immobilized lipase by chemical bonding, there is a decrease of the K(m) value and an increase of the V(max) value for the immobilized lipase by adsorption. Additionally, the experimental results of thermal stabilities indicate that the residual activity of the immobilized lipase at 50 degrees C is 38% by adsorption and 65% by chemical bonding.     

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.     

Physical immobilization of Rhizopus oryzae lipase onto cellulose substrate: activity and stability studies

Rhizopus oryzae lipase (ROL) was immobilized by adsorption onto oxidized cellulose fibers and regenerated films. The maximum adsorption level increases with the raise in the amount of carboxylic groups on cellulose surface confirming that adsorption is being governed mainly by electrostatic interaction between the enzyme and the substrate. This hypothesis was further confirmed by zeta-potential measurements showing a decrease in the zeta-potential of the fibers after enzyme adsorption. XPS analysis showed an intensification of the N 1s peak attesting the presence of the enzyme on the surface. The effect of temperature, pH and solvent polarity on the immobilized enzyme activity and stability was investigated. The catalytic esterification of oleic acid with n-butanol has been carried on using hexane as an organic solvent. A high conversion yield was obtained (about 80%) at 37 degrees C with a molar ratio of oleic acid to butanol 1:1 and 150IU immobilized lipase. The adsorption achieved two successive cycles with the same efficiency, and started to lose its activity during the third cycle.     

Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> Lipase onto Chitosan

Lipase from Rhizomucor miehei (RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v^?1 at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.     

Immobilized Lipase from Candida sp. 99–125 on Hydrophobic Silicate: Characterization and Applications

Lipase Candida sp. 99–125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99–125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.     

Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption

Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase.     

Synthesis of SBA-15 from low cost silica precursor obtained from sugarcane leaf ash and its application as a support matrix for lipase in biodiesel production

Ordered mesoporous silica material was synthesized from a low-cost precursor, sugarcane leaf ash, was used as a support matrix for lipase for the production of biodiesel. The mesoporous samples were characterized using Fourier transform infra red spectroscopy. The surface topography and morphology of the mesoporous materials were studied using scanning electron microscope. The pore diameter, pore volume, Brunauer Emmett and Teller surface area of the mesoporous material were determined by N₂ gas adsorption technique. Different pore size Santa Barbara Acid-15 (SBA-15) samples were synthesized and their lipase immobilization capacity and specific enzyme activity of immobilization lipase were determined and compared. Lipase from Candida Antarctica immobilized on SBA-15 (C) had shown maximum percentage immobilization and specific enzyme activity. The immobilized lipase mesoporous matrix was used for biodiesel production from crude non-edible Calophyllum inophyllum oil. The percentage yield of fatty acid methyl ester, 97.6 % was obtained under optimized conditions: 100 mg of lipase immobilized on SBA-15, 6:1 methanol to oil molar ratio, the reaction of 2 g C. inophyllum oil with methanol.     

Immobilization and Properties of Lipase from Candida rugosa on Electrospun Nanofibrous Membranes with Biomimetic Phospholipid Moities

Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly（acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine）s（PANCMPCs） were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile（PAN） nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%（molar fraction） in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.     

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.     

Immobilization of Candida antarctica lipase B on the surface of modified sol–gel matrix

The use of modified sol–gel matrix to immobilize the enzyme Candida antartica lipase B (CALB) was investigated. Free hydroxyl groups on the matrix surface were exploited to covalently immobilize the enzyme. Based from the results, incorporating hydrophobic sol–gel precursor (ethyltrimethoxysilane) enhanced enzyme activity. An enzyme activity of 192.02 U/g beads with 80.88 % attachment was obtained. At alkaline pH, immobilization yield of enzyme increased. The attachment of enzyme on the surface of the matrix was confirmed by scanning electron microscope images. Covalently immobilized CALB on sol–gel supports has higher thermal stability with 2.7 times higher half-life compared to soluble enzymes at 60 °C. This enzyme immobilization system retains the enzyme residual activity even for repetitive use. Hence, the immobilization approach developed recommends its further application.     

Adsorption and activity of Candida rugosa lipase on polypropylene hollow fiber membrane modified with phospholipid analogous polymers

Efforts have recently been made toward the study of interactions of phospholipid with various enzymes. It seems that phospholipids may be directly involved in regulating the enzyme activity. In this work, three phospholipid analogous polymers (PAPs), containing hydrophobic octyloxy, dodecyloxy, and octadecyloxy groups (abbreviated as 8-PAP, 12-PAP, and 18-PAP, respectively), were tethered on polypropylene hollow fiber microfiltration membrane (PPHFMM) to create a biocompatible interface for lipase immobilization. Lipase from Candida rugosa was immobilized on these PPHFMMs by adsorption. The adsorption capacity, activity, and thermal stability of enzyme on the PAP-modified PPHFMMs were compared with those of enzyme on the nascent ones. It was found that, as for the PAP-modified PPHFMMs, the adsorption capacities of lipase are lower than that of the nascent ones, while the activity retention of immobilized lipase increases from 57.5% to 74.1%, 77.5%, and 83.2% respectively for the 8-PAP-, 12-PAP-, and 18-PAP-modified PPHFMMs. In addition, the experimental results of thermal stability show that the residual activity of the immobilized lipase at 50 degrees C for 2 h is 62% for the 8-PAP-modified PPHFMM, 59% for the 12-PAP-modified PPHFMM, and 66% for the 18-PAP-modified PPHFMM, which are also higher than that of the nascent ones.     

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.     

微球载体固定化碱性磷酸酶的底物／产物吸附现象

微球载体固定化碱性磷酸酶的底物／产物吸附现象史国利，马建标，何炳林（南开大学高分子化学研究所，天津，３０００７１）关键词碱性磷酸酶，固定化，底物，产物，吸附在酶固定化研究中载体对底物和产物的吸附会造成载体内部和载体周围底物和产物的浓度不均一［１］，从...     

Preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization

Nanofibrous membrane with a fiber diameter of 80-150 nm was fabricated from mixed chitosan/poly(vinyl alcohol) (PVA) solution by an electrospinning process. Field emission scanning electron microscope and transmission electron microscope were used to characterize the morphology of the nanofibrous membrane. It was found that chitosan nanofibrous membrane with stabilized morphology could be prepared through removing most of PVA from the nascent one with 0.5 M NaOH aqueous solution. This treatment also resulted in an obvious decrease in fiber diameter. The stabilized chitosan nanofibrous membrane was explored as support for enzyme immobilization due to the characteristics of excellent biocompatibility, high surface/volume ratio, and large porosity. Lipase from Candida rugosa was immobilized on the nanofibrous membrane using glutaraldehyde (GA) as coupling reagent. The properties of the immobilized lipase were assayed and compared with the free one. Results showed that, the observed lipase loading on this nanofibrous membrane was up to 63.6 mg/g and the activity retention of the immobilized lipase was 49.8% under the optimum condition. The pH and thermal stabilities of lipase were improved after it was immobilized on the chitosan nanofibrous membrane. In addition, the experimental results of reusability and storage stability indicated that the residual activities of the immobilized lipase were 46% after 10 cycles and 56.2% after 30 days, which were obviously higher than that of the free one.     

聚ε-己内酯的微米硅球固定化猪胰脂肪酶促合成

本文采用微米硅球固定化猪胰脂肪酶为催化剂合成聚ε-己内酯, 以期获得具有较高分子量、 良好生物相容性和使用安全性的生物可降解医用高分子材料.     

Immobilization of prostaglandin synthetase by hydrophobic adsorption

In this article, the immobilization of prostaglandin synthetase onn-alkyl or aryl amino-agar beads by hydrophobic adsorption is reported. The effects of different hydrophobic groups in the agar beads, pH of buffer, concentration of salts on the adsorption of prostaglandin synthetase, and the properties of immobilized prostaglandin synthetase were also studied. The results showed that 20–35 mg of microsome containing PG synthetase (protein content 8–15 mg) could be adsorbed on each gram ofn-dodecylamino-agar beads after suction drying the gel in the buffer of pH 5.5 (containing 0.5 mol/L KC1), 0.1 mol/L citric-phosphate at 4‡C. The remaining immobilized enzyme activity was over 80%. The optimum pH of immobilized PG synthetase is 8.0, similar to that of the native enzymes. The thermostability of immobilized PG synthetase in the buffer containing 0.5 mol/L KC1 was increased. Immobilized PG synthetase was used as a catalyst of synthesis of prostaglandin E₁. The preservation of activity after 10 working cycles was 86.2%.     

Preparation and characterization of pancreatic lipase immobilized in Eudragit-matrix

Pancreatic lipase (EC 3.1.1.3) was immobilized by entrapping in a commercial preparation of acrylic/methacrylic acid ester-based copolymer (Eudragit E 30 D). The activity of the immobilized lipase beads with a diameter of 1.5-2.0 mm was found to be lower than that of the free lipase. The optimum pH was shifted to the alkaline region and the thermal stability increased, whereas the optimum temperature level remained unchanged. The most important reason for the decreased activity was diffusion limitations. The diffusion of the substrate and products became more pronounced, and lipolytic activity increased upon addition of n-hexane into the reaction medium. The storage and operational stabilities of the immobilized lipase were investigated, and both characteristics were found to be increased when compared to the free enzyme. Furthermore, mechanical or magnetic stirring during the operation were found to have no influence on the carrier-matrix as determined by nephelometric measurements.