Electrostatic immobilization of pectinase on negatively charged AOT-Fe3O4 nanoparticles

Enzyme immobilization on magnetic nanoparticles (MNPs) has been a field of intense studies in biotechnology during the past decade. The present study suggests MNPs negatively charged by docusate sodium salt (AOT) as a support for pectinase immobilization. AOT is a biocompatible anionic surfactant which can stabilize MNPs. Electrostatic adsorption can occur between enzyme with positive charge and oppositely charged surface of MNPs (ca. 100 nm). The effect of three factors, i.e. initial enzyme concentration, aqueous pH and AOT concentration in different levels was investigated on pectinase immobilization. Maximum specific activity (1.98 U/mg enzyme) of immobilized pectinase and maximum enzyme loading of 610.5 mg enzyme/g support was attained through the experiments. Initial enzyme concentration is significantly important on both loading and activity of immobilized enzyme, while pH and AOT concentration only affect the amount of immobilized enzyme. Immobilized enzyme on MNPs was recovered easily through magnetic separation. At near pH of immobilization, protein leakage in reusability of immobilized enzyme was low and activity loss was only 10–20% after six cycles. Since pH is associated with immobilization by electrostatic adsorption, the medium pH was changed to improve the release of protein from the support, as well. MNPs properties were investigated using Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, and Dynamic Light Scattering (DLS) analysis.     

A method for determination of xanthine in meat by amperometric biosensor based on silver nanoparticles/cysteine modified Au electrode

A method is described for construction of an amperometric xanthine biosensor based on covalent Immobilization of xanthine oxidase (XOD) onto citrate capped silver nanoparticles deposited on Au electrode surface through cysteine self assembled monolayers (SAM). The biosensor showed optimum response within 5 s at pH 7.0 and 35 °C, when polarized at 0.5 V vs. Ag/AgCl. The linear working range of biosensor for xanthine was from 2 to 16 μM, with a detection limit of 0.15 μM and sensitivity of 0.17 mA/μM/cm². The mean analytical recovery of exogenously added xanthine in fish meat extract (5 g/l and 10 g/l) was 96.2 ± 2.3% and 95.2 ± 3.4%, respectively. Within and between batches coefficients of variation were <2.6% and <3.4%, respectively. The biosensor measured xanthine in fish, chicken, pork, and beef meat. The enzyme electrode lost 20% of its initial activity after its regular 180 uses over a period of 60 days, when stored at 4 °C in dry state.     

Synthesis and characterization of cysteine functionalized silver nanoparticles for biomolecule immobilization

A facile method for the aqueous phase synthesis of cysteine-functionalized silver nanoparticles by potato extract has been reported in the present work. These functionalized nanoparticles were then used for the covalent immobilization of a biomolecule, alkaline phosphatase, on its surface through carbodiimide coupling. Different reaction parameters such as cysteine concentration, reducing agent concentration, temperature, pH and reaction time were varied during the nanoparticles’ formation, and their effects on plasmon resonance were studied using Ultraviolet–visible spectroscopy. Fourier transform infrared spectroscopy was used to confirm the surface modification of silver nanoparticles by cysteine and the particle size analysis was done using particle size analyzer, which showed the average nanoparticles’ size of 61 nm for bare silver nanoparticles and 201 nm for the enzyme-immobilized nanoparticles. The synthesized nanoparticles were found to be highly efficient for the covalent immobilization of alkaline phosphatase on its surface and retained 67 % of its initial enzyme activity (9.44 U/mg), with 75 % binding efficiency. The shelf life of the enzyme-nanoparticle bioconjugates was found to be 60 days, with a 12 % loss in the initial enzyme activity. With a simple synthesis strategy, high immobilization efficiency and enhanced stability, these enzyme-coated nanoparticles have the potential for further integration into the biosensor technology.     

Directed immobilization of CGTase: The effect of the enzyme orientation on the enzyme activity and its use in packed-bed reactor for continuous production of cyclodextrins

In this study, two different approaches were assessed in order to direct the immobilization of a cyclodextrin glycosyltransferase on functionalized silica support, one by amino groups using glutaraldehyde activation (Si-NH-G-CGTase) and other by disulfide bond through the Cys on the enzyme surface (Si-SH-CGTase). The efficiency of the immobilization of the enzyme by the Cys in Si-SH was four times higher than with the amino group linkage in Si-NH-G (2.86% and 11.91%, respectively). After immobilization, the optimum pH remained at 5.5 for the two derivatives and the optimum temperature was 70 °C for the free enzyme, 80 °C for Si-SH-CGTase and 90 °C for Si-NH-G-CGTase. Both preparations were used for continuous production of cyclodextrins, and Si-NH-G-CGTase presented higher total productivity, retaining 100% of its initial activity for at least 200 h, while the Si-SH-CGTase presented only 40% at the same time. The Si-SH-CGTase could be reloaded with new enzymes linked by disulfide bonds and was able to be used for more than 200 h.     

Green synthesis of silver nanoparticles with Dalbergia spinosa leaves and their applications in biological and catalytic activities

The phytosynthesis of silver nanoparticles (AgNPs) by Dalbergia spinosa leaves (DSL) in aqueous extract was investigated. AgNPs were characterized by UV–visible absorption spectroscopy (UV–vis), transmission electron microscopy (TEM) and Fourier transform infra red spectrophotometry (FTIR). The results showed that the increase in the initial extract concentration at room temperature increased the mean size and widened the size distribution of the AgNPs, leading to a red shift and broadening the surface plasmon resonance absorption (439 nm). The results showed that the reducing sugars and flavonoids were primarily responsible for the bioreduction of silver ions and that their reductive capability was promoted at 36 °C. TEM analysis showed that the AgNPs were nearly spherical in shape with an average size of 18 ± 4 nm. When evaluated for in vitro antioxidant activity by DPPH, NO, hydrogen peroxide radicals, reducing power and CUPRAC assay methods in addition to anti-inflammatory activity by HBRC method, the silver nanoparticles exhibited considerably enhanced antioxidant and anti-inflammatory activity at the test doses when compared with that of the standards and the plant extract. Finally, the antibacterial activity of the AgNPs against two Gram-positive bacteria and two Gram-negative bacteria showed moderate antibacterial activity when compared with the standard and the plant extract. The synthesized silver nanoparticles were also effective in the catalytic reduction of 4-nitrophenol (4-NP) into 4-aminophenol (4-AP).     

Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate

Objective of this study is to realize appropriate enzyme immobilization onto a suitable support material and to develop a model which enables reactions catalyzed with different enzymes arranged in order. Thence, this model was potential for developing a multi-enzyme system. The reactions need more than one enzyme can be realized using immobilized form of them and the enzymes will be in one support at wanted activities. In this study, sodium alginate was used as immobilization material and glycidyl methacrylate was grafted onto sodium alginate. Thus reactive epoxy groups were added to sodium alginate which also has carboxyl groups. Average molecular weight of sodium alginate was determined using Ubbelohde viscosimetri. The molecular mass of sodium alginate was calculated as 15,900 Da. Graft polymerization was made in two steps. Firstly, sodium alginate was activated with benzophenone using UV-light at 254 nm. Secondly, glycidyl methacrylate was grafted under UV-light at 365 nm onto activated sodium alginate. Grafted glycidyl methacrylate was determined gravimetric and titrimetric. Additional groups after grafting were showed with FT-IR spectrum. 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide was used for immobilization urease from carboxyl groups at pH 5.0. Suitable 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide/–COOH ratio was found 1/10 and immobilized product activity was 197 U/g support. Reaction medium pH was 8.0 for immobilization from epoxy group. Optimum immobilization reaction time was found as 2 h and immobilized product activity was 285 U/g support. Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate was made from –COOH and epoxy groups, respectively.     

Immobilization of apricot pectinesterase (Prunus armeniaca L.) on porous glass beads and its characterization

Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzyme. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. Optimum pH and temperature were determined to be 8.0 and 50 °C, respectively. The immobilized PE exhibited better thermostability than the free one. Kinetic parameters of the immobilized enzyme (K_m and V_max values) were also evaluated. The K_m was 0.71 mM and the V_max was 0.64 μmol min^?1 mg^?1. No drastic change was observed in the K_m and V_max values. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.     

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.     

Aqueous extract of gum olibanum (Boswellia serrata): A reductant and stabilizer for the biosynthesis of antibacterial silver nanoparticles

A simple and ecofriendly biosynthetic process has been developed for silver nanoparticles using the aqueous extract of gum olibanum (Boswellia serrata), a renewable natural plant biopolymer. The water soluble compounds in the gum serve as dual functional reducing and stabilizing agents. The effect of concentration of gum and silver nitrate; and reaction time on nanoparticle synthesis was studied. The UV–visible spectroscopy, transmission electron microscopy and X-ray diffraction techniques were used to characterize the synthesized nanoparticles. By tuning the reaction conditions, size controlled spherical nanoparticles of around 7.5 ± 3.8 nm was achieved. Using Fourier transform infrared spectroscopy and Raman spectroscopy, a probable mechanism involved in reduction and stabilization of nanoparticles has been explained. The produced silver nanoparticles exhibited substantial antibacterial activity on both the Gram classes of bacteria. By virtue of being biogenic and encapsulated with proteins, these surface functionalized nanoparticles can be easily integrated for various biological applications.     

Silver glyconanoparticles functionalized with sugars of sweet sorghum syrup as an antimicrobial agent

Bio-directed synthesis of metal nanoparticles is gaining importance due to their biocompatibility, low toxicity and eco-friendly nature. We used sweet sorghum syrup for a facile and cost-effective green synthesis of silver glyconanoparticles. Silver nanoparticles were formed due to reduction of silver ions when silver nitrate solution was treated with sorghum syrup solutions of different pH values. The nanoparticles were characterized by UV–vis, TEM (transmission electron microscopy), DLS (dynamic light scattering), EDAX (energy dispersive X-ray spectroscopy), FT-IR (fourier transform infrared spectroscopy) and XRD (X-ray diffraction spectroscopy). The silver glyconanoparticles exhibited a characteristic surface plasmon resonance around 385 nm. At pH 8.5, the nanoparticles were mono-dispersed and spherical in shape with average particle size of 11.2 nm. The XRD and SAED studies suggested that the nanoparticles were crystalline in nature. EDAX analysis showed the presence of elemental silver signal in the synthesized glyconanoparticles. FT-IR analysis revealed that glucose, fructose and sucrose present in sorghum syrup acted as capping ligands. Silver glyconanoparticles prepared at pH 8.5 had a zeta potential of ?28.9 mV and were anionic charged. They exhibited strong antimicrobial activity against Gram-positive, Gram-negative and different Candida species at MIC values ranging between 2 and 32 μg ml^?1. This is first report on sweet sorghum syrup sugars-derived silver glyconanoparticles with antimicrobial property.     

Immobilization of penicillin G acylase on paramagnetic aldehyde-functionalized mesostructured cellular foams

Paramagnetic aldehyde-functionalized mesostructured cellular foams (PAMCFs), synthesized by grafting 3-aminopropyltriethoxysilane modified Fe₃O₄ (NH₂-Fe₃O₄) nanoparticles with larger particle size than the window pore size of MCFs on the outer surface of aldehyde-functionalized mesostructured cellular foams (AMCFs), were investigated as efficient supports for immobilization of penicillin G acylase (PGA). The results show that NH₂-Fe₃O₄ nanoparticles were successfully grafted on the outer surface of AMCFs and PGA molecules were mainly immobilized covalently on the inner surface of PAMCFs, which was because amino groups of NH₂-Fe₃O₄ nanoparticles or PGA molecules reacted with aldehyde groups of AMCFs or PAMCFs to form imine bonds. PGA/PAMCFs-15 showed a rather high initial activity of 9563 U g⁻¹ and retained 89.1% of its initial activity after recycled for 10 times. PGA/PAMCFs are easily recycled by magnetic field in order to replace tedious separation of high-speed centrifugation for mesoporous materials.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to enhance catalytic activity and stability. Although stability of enzyme was accomplished with immobilization approaches, activity of the most of the enzymes was declined after immobilization. Herein, we synthesize the flower shaped-hybrid nanomaterials called hybrid nanoflower (HNF) consisting of urease enzyme and copper ions (Cu²⁺) and report a mechanistic elucidation of enhancement in both activity and stability of the HNF. We demonstrated how experimental factors influence morphology of the HNF. We proved that the HNF (synthesized from 0.02 mg mL⁻¹ urease in 10 mM PBS (pH 7.4) at +4 °C) exhibited the highest catalytic activity of ∼2000% and ∼4000% when stored at +4 °C and RT, respectively compared to free urease. The highest stability was also achieved by this HNF by maintaining 96.3% and 90.28% of its initial activity within storage of 30 days at +4 °C and RT, respectively. This dramatically enhanced activity is attributed to high surface area, nanoscale-entrapped urease and favorable urease conformation of the HNF. The exceptional catalytic activity and stability properties of HNF can be taken advantage of to use it in fields of biomedicine and chemistry.     

Immobilization of lipase on epoxy-activated Purolite® A109 and its post-immobilization stabilization

In this study, Purolite^® A109, polystyrenic macroporous resin, was used as immobilization support due to its good mechanical properties and high particle diameter (400 μm), which enables efficient application in enzyme reactors due to lower pressure drops. The surface of support had been modified with epichlorhydrine and was tested in lipase immobilization. Optimized procedure for support modification proved to be more efficient than conventional procedure for hydroxy groups (at 22 °C for 18 h), since duration of procedure was shortened to 40 min by performing modification at 52 °C resulting with almost doubled concentration of epoxy groups (563 μmol g⁻¹). Lipase immobilized on epoxy-modified support showed significantly improved thermal stability comparing to both, free form and commercial immobilized preparation (Novozym^® 435). The highest activity (47.5 IU g⁻¹) and thermal stability (2.5 times higher half-life than at low ionic strength) were obtained with lipase immobilized in high ionic strength. Thermal stability of immobilized lipase was further improved by blocking unreacted epoxy groups on supports surface with amino acids. The most efficient was treatment with phenylalanine, since in such a way blocked immobilized enzyme retained 65% of initial activity after 8 h incubation at 65 °C, while non-blocked derivative retained 12%.     

Enhanced stability of Kluyveromyces lactis β galactosidase immobilized on glutaraldehyde modified multiwalled carbon nanotubes

The present study demonstrates covalent immobilization of Kluyveromyces lactis β galactosidase on functionalized multi-walled carbon nanotubes (MWCNTs). Highly efficient surface modification of MWCNTs was achieved by glutaraldehyde for binding greater amount of enzyme. X-ray diffraction analysis and UV visible spectroscopy of MWCNTs showed them to be entirely dispersive in aqueous solution. Transmission electron microscopy showed that MWCNTs were of 20 nm size. Thermogravimetric analysis further revealed the stability of glutaraldehyde modified MWCNT as an ideal matrix for enzyme immobilization. The optimal pH for soluble and immobilized β galactosidase was observed at pH 7.0 while the optimal operating temperatures were observed at 40 °C and 50 °C, respectively. Moreover, our findings demonstrated that β galactosidase immobilized on surface functionalized MWCNTs retained greater biocatalytic activity at higher galactose concentration, and upon repeated uses as compared to enzyme in solution.     

Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum

Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an optimum temperature and pH of 50 °C and 9.0, respectively. Stability was well maintained, with high levels of activity at a pH of up to 11.0, including high stability at a temperature of up to 50 °C after 60 min of incubation. The inhibition test demonstrated strong inhibition by PMSF, a serine protease inhibitor, and Kinetic constants km and kcat for BAPNA were 0.517 mM and 5.0 S^?1, respectively. The purified enzyme was also as active as casein, as analyzed by zymography. Therefore, we consider trypsin a promising enzyme for industrial processes, owing to its stability in a wide range of pH and temperature and activity even under immobilization.     

Polyaniline grafted polyacylonitrile conductive composite fibers for reversible immobilization of enzymes: Stability and catalytic properties of invertase

Polyacylonitrile fibers (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The effect of aniline concentration on the grafting efficiency and on the electrical surface resistance of PAN/PANI composite fibers was investigated. The surface resistance of the conductive composite fibers in this work was found to be between 8.0 and 0.5 kΩ/cm. As the amount of grafted PANI increased on the PAN fibers the electrical resistance of composite fibers decreased. The PAN/PANI composite fibers were characterized by SEM and FTIR studies. Composite PAN/PANI fibers were used for reversible immobilization of invertase. The immobilization efficiency and the activity of the immobilized invertase (from 1.0 mg/mL invertase solution at pH 5.5) were increased with increasing PANI contents of the composite fibers. The maximum amount of immobilized enzyme onto composite fibers containing 2.0% PANI was about 76.6 mg/g. The optimum pH for the free enzyme was observed at 5.0. On the other hand, immobilized invertase yielded a broad optimum pH profile between pH 5.0 and 7.0. Immobilized invertase exhibited 83% of its original activity even after two months storage at 4 °C while the free enzyme showed only 7% of its initial activity.     

Development of a transparent,non-cytotoxic,silver ion-exchanged glass with antimicrobial activity and low ion elution

We investigated the antimicrobial, cytotoxicity, skin irritation, and ion elution behaviors of glass doped with silver ions with respect to its application to electronic equipment such as phones and tablet screens. The microbes tested were Escherichia coli, Staphylococcus aureus, and Penicillium funiculosum. AgNO₃ powder was spread on both sides of aluminosilicate glass, and it was heated to 250–280 °C for 10 min. Under optimized heating conditions (260 °C, 10 min), the antimicrobial activity of ion-exchanged glass against bacteria and fungi was over 99.9% after 24 weeks. The glass failed to irritate the skin of experimental animals and was considered non-cytotoxic. The maximum amount of Ag ions that were eluted from the ion-exchanged glass into drinking water was measured at 0.037 ± 0.003 μg L⁻¹, an amount which is several orders of magnitude below the standard limit of 0.1 mg L⁻¹ in drinking water. Ag ion-exchanged glass had characteristics suitable for use as a display screen, such as a light transmittance of 90% and a surface roughness of 0.704 nm. Our findings suggest that glass doped with silver ions is more hygienic than non-doped glass is, and should be applied to display screens and glassware.     

Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g^?1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.     

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.