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.     

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.     

Improved yield and stability of amylase by multipoint covalent binding on polyglutaraldehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions

In this study raw starch digesting amylase (RSDA) from Aspergillus carbonarius (Bainier) Thom IMI 366159 was stabilized by covalent binding on polyglutaraldehyde (PG), glutaraldehyde (G) activated chitosan beads or post immobilization cross linking of enzyme adsorbed on chitosan. Presence of Ca²⁺ ions (0.5–1.5 mM) activated the PG and G derivatives but repressed the crosslinked enzyme. Optimum pH for cross linked derivative increased by 2 units but was unaltered for PG and G derivatives. Immobilized amylase exhibited improved thermal and storage stability. Immobilized derivatives had no loss of activity after 1 month storage and retained above 90% activity after 10 batch reactions of 60 min each. Immobilization successfully stabilized RSDA and immobilized enzyme from A. carbonarius can be applied in numerous industries for cheap, cost effective and environmentally friendly starch hydrolytic processes to simple sugars.     

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.     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24 h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor

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

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 catalase onto Eupergit C and its characterization

Bovine liver catalase was covalently immobilized onto Eupergit C. Optimum conditions of immobilization: pH, buffer concentration, temperature, coupling time and initial catalase amount per gram of carrier were determined as 7.5, 1.0 M, 25 °C, 24 h and 4.0 mg/g, respectively. V_max and K_m were determined as 1.4(±0.2) × 10⁵ U/mg protein and 28.6 ± 3.6 mM, respectively, for free catalase, and as 3.7(±0.4) × 10³ U/mg protein and 95.9 ± 0.6 mM, respectively, for immobilized catalase. The thermal stability of the immobilized catalase in terms of half-life time (29.1 h) was comparably higher than that of the free catalase (9.0 h) at 40 °C. Comparison of storage stabilities showed that the free catalase completely lost its activity at the end of 11 days both at room temperature and 5 °C. However, immobilized catalase retained 68% of its initial activity when stored at room temperature and 79% of its initial activity when stored at 5 °C at the end of 28 days. The highest reuse number of immobilized catalase was 22 cycles of batch operation when 40 mg of immobilized catalase loaded into the reactor retaining about 50% of its original activity. In the plug flow type reactor, the longest operation time was found as 82 min at a substrate flow rate of 2.3 mL/min when the remaining activity of 40 mg immobilized catalase was about 50% of its original activity. The resulting immobilized catalase onto Eupergit C has good reusability, thermal stability and long-term storage stability.     

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.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

Biodiesel production from sunflower,soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer

This study aims at carrying out lipase-catalyzed synthesis of fatty acid methyl esters (biodiesel) from various vegetable oils using lipase immobilized onto a novel microporous polymeric matrix (MPPM) as a low-cost biocatalyst. The research is focused on three aspects of the process: (a) MPPM synthesis (monolithic, bead, and powder forms), (b) microporous polymeric biocatalyst (MPPB) preparation by immobilization of lipase onto MPPM, and (c) biodiesel production by MPPB. Experimental planning of each step of the study was separately carried out in accordance with design of experiment (DoE) based on Taguchi methodology.Microporous polymeric matrix (MPPM) containing aldehyde functional group was synthesized by polyHIPE technique using styrene, divinylbenzene, and polyglutaraldehyde. Thermomyces lanuginosus lipase was covalently attached onto MPPM with 80%, 85%, and 89% immobilization efficiencies using bead, powder, and monolithic forms, respectively. Immobilized enzymes were successfully used for the production of biodiesel using sunflower, soybean, and waste cooking oils. It was shown that immobilized enzymes retain their activities during 10 repeated batch reactions at 25 °C, each lasting 24 h. Since the developed novel method is simple yet effective, it could have a potential to be used industrially for the production of chemicals requiring immobilized lipases.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

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

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

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

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

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

Immobilization of 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.     

Enzymatic transformation of 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) by immobilized α-cyclodextrin glucanotransferase from recombinant Escherichia coli

This work aims to produce 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin with immobilized α-cyclodextrin glucanotransferase (α-CGTase) from recombinant Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on α-CGTase immobilization were examined, and optimal immobilization conditions were determined as the following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl₂ 3 g/L, sodium alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free α-CGTase, immobilized α-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 °C). The continuous production of AA-2G from ascorbic acid and β-cyclodextrin in the presence of immobilized α-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free α-CGTase. The immobilization procedure developed here was efficient for α-CGTase immobilization, which was proved to be a prospective approach for the enzymatic production of AA-2G.     

Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane

Poly(itaconic acid) grafted and/or Fe(III) ions incorporated chitosan membranes were used for reversible immobilization of catalase (from bovine liver) via adsorption. The influences of pH and initial catalase concentration on the immobilization capacities of the CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membranes have been investigated in a batch system. Maximum catalase adsorption onto CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membrane were found to be 6.3 and 37.8 mg/g polymer at pH 5.0 and 6.5, respectively. The CH-g-poly(IA)-Fe(III) membrane with high catalase adsorption capacity was used in the rest of the study. The K_m value for immobilized catalase on CH-g-poly(IA)-Fe(III) (25.8 mM) was higher about 1.6-fold than that of free enzyme (13.5 mM). Optimum operational temperature was observed at 40 °C, a 5 °C higher than that of the free enzyme and was significantly broader. The optimum operational pH was same for both free and immobilized catalase (pH 7.0). Thermal stability was found to increase with immobilization. Free catalase lost all its activity within 20 days whereas immobilized catalase lost 23% of its activity during the same incubation period. It was observed that the same support enzyme can be repeatedly used for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity. In addition, the CH-g-poly(IA)-Fe(III) membrane prepared in this work showed promising potential for various biotechnological applications.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

Surface functionalized mesoporous activated carbon for the immobilization of acidic lipase and their application to hydrolysis of waste cooked oil: Isotherm and kinetic studies

This study deals with the surface functionalization of mesoporous activated carbon, using ethylenediamine and glutaraldehyde to facilitate the strong immobilization of acidic lipase (AL) onto MAC. The AL was produced from Pseudomonas gessardii by using slaughterhouse lipid waste as the substrate. The AL immobilized on functionalized mesoporous activated carbon (ALFMAC) was applied for the hydrolysis of waste cooked oil (WCO). The optimum conditions for the immobilization of AL onto functionalized mesoporous activated carbon (FMAC) were 90 min; pH 3.5; and 35 °C; which resulted at the maximum immobilization of 5440 U/g of FMAC (3.693 mg of AL/g of FMAC or the yield 2.7% or the expressed activity 103.7% or the activity per unit area of FMAC 1.08 mg of AL/m²). The ALFMAC showed better thermal and storage stabilities than the free AL. The ALFMAC retained a 98% and a 92% initial activity at 40 °C and 50 °C, respectively, while the AL showed the thermal stability (residual activities) 65% and 38%, respectively. The storage stability of ALFMAC at 4 °C showed 100% initial activity up to 15 days from the initial day of the storage, whereas AL showed only 88% initial activity up to 15 days. The FMAC and ALFMAC were characterized by using scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD) analysis. The K_m values of the ALFMAC and AL were 0.112 mM and 0.411 mM, respectively. The v_max values of the ALFMAC and AL were 1.26 mM/min and 0.53 mM/min, respectively. Immobilization of AL onto FMAC obeyed the Freundlich and Redlich–Peterson isotherm models. The non-linear models of pseudo first, and second order, intra-particle diffusion, Bangham, and Boyd plot were also performed to understand the dynamic mechanism of immobilization. ALFMAC showed a 100% hydrolysis of WCO up to 21 cycles of reuse, and 60% up to 45 cycles. The hydrolysis of WCO was confirmed by using FT-IR spectra.     

Synthesis of geranyl acetate in non-aqueous media using immobilized Pseudomonas cepacia lipase on biodegradable polymer film: Kinetic modelling and chain length effect study

Pseudomonas cepacia lipase (PCL) was immobilized on ternary blend biodegradable polymer made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alcohol (PVA). Immobilized biocatalyst was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), % water content, protein and lipase activity assay. The lipase activity assay showed enhanced activity of immobilized lipase than crude lipase. Higher half life time (t_1/2) and lower deactivation rate constant (K_d) was found for the n-hexane among various tested solvent. Influence of various reaction parameters on enzyme activity were studied in detail. When geraniol (1 mmol) and vinyl acetate (4 mmol) in toluene (3 mL) were reacted with 50 mg immobilized lipase at 55 °C; then 99% geraniol was converted to geranyl acetate after 3 h. Various kinetic parameters such as r_max, K_i(A), K_m(A), K_m(B) were determined using non-linear regression analysis for ternary-complex and Bi–Bi ping-pong mechanism. The kinetic study showed that reaction followed ternary-complex mechanism with inhibition by geraniol. Activation energy (Ea) was found to be lower for immobilized lipase (13.76 kCal/mol) than crude lipase (19.9 kCal/mol) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 4 fold increased catalytic activity than crude lipase and recycled five times.