Immobilization of urease using glycidyl methacrylate grafted nylon-6-membranes

The graft copolymerization of glycidyl methacrylate (GMA) onto nylon-6-membrane using benzophenone (BP) as an initiator was carried out in an alcoholic aqueous solution. The acrylic double bond of GMA participated in the grafting onto the nylon-6-membrane backbone with the epoxy groups remaining unaffected. At the end of the grafting reaction, urease was immobilized onto the modified membrane. BP concentration, GMA concentration and organic solvent seperation were studied by determining the grafting percentage. The influence of urease concentration on the immobilization efficiency was also studied. With keeping other conditions constant, the optimum conditions were shown as following [BP]: 5 × 10⁻² mM; [GMA]: 10 M; [urease]: 10 mg/ml, organic solvent: methanol.     

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Immobilization of maltogenase onto polyurethane microparticles from poly(vinyl alcohol) and hexamethylene diisocyanate

A series of porous polyurethane (PU) microparticles from poly(vinyl alcohol) (PVA) and hexamethylene diisocyanate (HMDI) using different ratios of components were obtained by one step method. Molar compositions of PU microparticles were estimated by determination of nitrogen, isocyanate and hydroxyl groups. PU carriers which were synthesized using optimal initial molar ratios of PVA and HMDI were applied for immobilization of maltogenase (MG) from Bacillus stearothermophilus. Immobilized enzyme exhibited higher catalytic activity and enhanced temperature stability in comparison with the native MG. Maximal loading 7.78 mg/g wet carrier was reached when PU microparticles with initial molar ratio of PVA and HMDI = 1:3 was used as a carrier for immobilization. The high efficiency of immobilization (EI) was obtained using PU microparticles when initial molar ratio of HMDI and PVA was 1:1–1:10. High stability of MG immobilized onto PU microparticles during storage was demonstrated. Immobilized starch hydrolyzing enzyme was successfully tested in batch and column type reactors for hydrolysis of potato starch. MG immobilized onto PU enables easy separation from the reaction medium and reuse of the immobilized preparation over seven reaction cycles in bath operation and at least three cycles in column type reactor.     

Pseudomonas fluorescens lipase immobilization on polysiloxane–polyvinyl alcohol composite chemically modified with epichlorohydrin

The technique based on sol–gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane–polyvinyl alcohol (POS–PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS–PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS–PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS–PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis–Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS–PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis–Menten constant value (K_m) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities.     

Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained ⿼50% of the free enzyme activity at 6.4 mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.     

Synthesis and characterization of cyclodextrin-based polymers as a support for immobilization of Candida rugosa lipase

The objective of this study was to prepare cross-linked β-cyclodextrin polymers for immobilization of Candida rugosa lipase. The structures of synthesized macrocyclic compounds were characterized by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. Properties of the immobilized systems were assessed and their performance on hydrolytic reaction were evaluated and compared with the free enzyme. The influence of activation agents (glutaraldehyde (GA) and hexamethylene diisocyanate (HMDI)) and thermal and pH stabilities of the biocatalyst was evaluated. After the optimization of immobilization process, the physical and chemical characterization of immobilized lipase was performed. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme. It can be observed that the free lipase loses its initial activity within around 80 min at 60 °C, while the immobilized lipases retain their initial activities of about 56% by HMDI and 82% by GA after 120 min of heat treatment at 60 °C.Results showed that the specific activity of the immobilized lipase with glutaraldehyde was 62.75 U/mg protein, which is 28.13 times higher than that of the immobilized lipase with HMDI.     

Immobilization of lipase in organic solvent in the presence of fatty acid additives

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min⁻¹ mg⁻¹ as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min⁻¹ g⁻¹ immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.     

An integrated approach to produce biodiesel and monoglycerides by enzymatic interestification of babassu oil (Orbinya sp)

Two screenings of commercial lipases were performed to find a lipase with superior performance for the integrated production of biodiesel and monoglycerides. The first screening was carried out under alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding ethyl esters (biodiesel). The second screening was performed under glycerolysis conditions to yield monoglycerides (MG). All lipases were immobilized on silica–PVA composite by covalent immobilization. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface Methodology (RSM) was used to determine the optima operating conditions for each biotransformation. For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction at 39 °C using an oil-to-ethanol molar ratio of 1:7. For MG production, optima conditions corresponded to oil-to-glycerol molar ratio of 1:15 at 55 °C, yielding 25 wt.% MG in 6 h reaction. These results show the potential of B. cepacia lipase to catalyze both reactions and the feasibility to consider an integrated approach for biodiesel and MG production.     

Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles

Mucor javanicus lipase was effectively immobilized on silica nanoparticles which were prepared by Stöber method. Glycidyl methacrylate (GMA), which bears a reactive epoxide group, was incorporated onto the surface of the nanoparticles and the epoxide groups were directly used for multipoint coupling of the enzyme. We also introduced amine residues by coupling ethylene diamine (EDA) to the epoxide group of GMA. M. javanicus lipase was covalently immobilized onto the amine-activated silica nanoparticles by using glutaraldehyde (GA) or 1,4 phenylene diisothiocyanate (NCS) as a coupling agent. The lipase loading capacities of the EDA-GA and EDA-NCS nanoparticles (81.3 and 60.9 mg g⁻¹, respectively) were much higher than that of the unmodified GMA nanoparticles (18.9 mg g⁻¹). The relative hydrolytic activities in an aqueous medium of the lipases immobilized on EDA-GA and EDA-NCS attached silica nanoparticles (115% and 107%, respectively) were significantly high and almost in the same range with the free enzyme. This may be due to the improvement of the enzyme–substrate interaction by avoiding the potential aggregation of free lipase molecules. The immobilized lipases were also more resistant to temperature inactivation than the free form. This work demonstrates that the size-controlled silica nanoparticles can be efficiently employed as host materials for enzyme immobilization leading to high activity and stability of the immobilized enzymes.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Immobilized lipase on macroporous polystyrene modified by PAMAM-dendrimer and their enzymatic hydrolysis

The novel enzyme carrier, polyamidoamine (PAMAM) dendrimers modified macroporous polystyrene, has been synthesized by Michael addition and firstly used in the immobilization of porcine pancreas lipase (PPL) effectively by covalent attachment. The resulting carrier was characterized with the Fourier transform infrared spectra (FT-IR), scanning electron microscopy (SEM), elemental analysis and thermogravimetric (TG) analysis. Meanwhile, the amount of immobilized lipase was up to 100 mg g⁻¹ support and the factors related with the enzyme activity were investigated. The immobilization of the PPL improved their performance in wider ranges of pH and temperature. Thermal stability of the immobilized lipase also increased dramatically in comparison with the free ones and the immobilized lipase exhibited a favorable denaturant tolerance. As a biocatalyst, the immobilized lipase for batch hydrolysis of olive oil emulsion retained 85% activity after 10 times of recycling. This well-reusability of immobilized lipase was very valuable and meaningful in enzyme technology.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

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.     

Amino silicones finished fabrics for lipase immobilization: Fabrics finishing and catalytic performance of immobilized lipase

Finishing of silk fabric was achieved by using amino-functional polydimethylsiloxane (PDMS) and lipase from Candida sp. 99-125 was immobilized on the treated silk fabrics. Hydrophobic fabrics were obtained by dipping the native fabric in 0.125–0.25% (w/v) PDMS solution and dried at 70 °C. The direct adsorption on PDMS-treated fabric was verified to be a better strategy for lipase immobilization than that by covalent binding. Compared to unfinished fabrics, the hydrolytic activity of immobilized enzyme on the finished fabric was improved by 1.6 times. Moreover, the activity of immobilized enzymes on hydrophobic fabrics was significantly improved in different concentrations of strong polar solvents such as methanol and ethanol, and in common organic solvents with different octanol–water partition coefficients (Log P). Enzymatic activity and stability in 15% water content system (added water accounted for the total reaction mixtures, v/v) showed more than 30% improvement in each batch. The amino–silicone finished fabric surface was investigated by scanning electron microscopy and X-ray photoelectron spectroscopy. The hydrophobic fabric immobilized enzyme could be recycled for more than 80 times with no significant decrease in esterification activity. PDMS-treated woven silk fabrics could be a potential support for lipase immobilization in catalytic esterification processes.     

Enhancing performance of lipase immobilized on methyl-modified silica aerogels at the adsorption and catalysis processes: Effect of cosolvents

Hydrophobic silica aerogels modified with methyl group were applied as support to immobilize Candida rugosa lipase (CRL). At the adsorption process, different alcohols were used to intensify the immobilization of CRL. The results showed that n-butanol wetting the hydrophobic support prior to contacting with enzyme solution could promote lipase activity, but the adsorption quantity onto the support decreased. Based on this, a novel immobilization method was proposed: the support contacted with enzyme solution without any alcohols, and then the immobilized enzymes were activated by 90% (V) n-butanol solution. The experimental results showed that this method could keep high adsorption quantity (413.0 mg protein/g support) and increase the lipase specific activity by more than 50%. To improve the stability of immobilized lipase, the support after adsorption was contacted with n-octane to form an oil layer covering the immobilized lipases, thus the leakage can be decreased from over 30–4% within 24 h. By utilizing proper cosolvents, a high enzyme activity and loading capacity as well as little loss of lipase was achieved without covalent linkage between the lipase and the support. This is known to be an excellent result for immobilization achieved by physical adsorption only.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016     

Immobilization of pancreatic lipase on polyvinyl alcohol by cyanuric chloride

Porcine pancreatic lipase (EC 3.1.1.3) was covalently immobilized onto 2,4,6-trichloro-s-triazine (cyanuric chloride) activated polyvinyl alcohol (PVA). The influence of activating agent and enzyme concentration on the immobilization process were evaluated.Hydrolytic activities of free and immobilized enzyme were determined and the immobilization yield was estimated by measuring the quantity of protein, both in free enzyme solution and in washing solutions after immobilization. After the optimization of immobilization process, the physical and chemical characterization of immobilized enzyme was performed. Additionally, the thermal, pH, storage, and operational stability of the immobilized and free enzymes were tested. Obtained data showed that the immobilized enzyme seemed better and offered some advantages in comparison with free enzyme.     

Immobilization and activity of Rhizomucor miehei lipase. Effect of the matrix properties prepared from nonionic fluorinated surfactants

We report the immobilization of Rhizomucor miehei lipase (RmL) onto mesoporous silica materials, in particular the investigations concerning the effects of the level of silica condensation and of the pore size on the enzyme activity. The efficiency of the immobilization was revealed by FTIR spectroscopy. Infrared was also used to determine the quantity of adsorbed enzyme. Immobilization efficiency increased when the RmL concentration in the buffer solution was changed from 2 to 10 mg/mL. Nevertheless, while upon enzyme immobilization the mesopore ordering was sustained for the support recovered after hydrothermal treatment at 100 °C, a structure collapse occurred for the one prepared at 80 °C. The difference in behavior is attributed to the lower hydrothermal stability of this material, which reflects the lower level of silica condensation. The enzyme-containing mesostructured silica was effectively used to catalyze the model esterification reaction of lauric acid with 1-propanol, as the immobilized lipase retained its catalytic activity. A linear relationship was observed between the reaction rate and the amount of catalyst. RmL immobilized on mesoporous materials presented a satisfactory reusability, while the remaining activity of RmL after 4 months of storage was 47% of the initial one.     

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA_app) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g⁻¹ of gel for GLU, 7.76 mg g⁻¹ of gel for GLY, and 7.65 mg g⁻¹ of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g⁻¹ of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.     

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.