Immobilization of Candida antarctica lipase B (CALB) on surface-modified rice husk ashes (RHA) via physical adsorption and cross-linking methods

In the present study, the recovery of activity of Candida antarctica lipase B (CALB) immobilized onto surface-modified rice husk ash (RHA) was 90% for both cross-linking and adsorption methods. Both cross-linked and adsorbed immobilized preparations were very stable, retaining more than 48% of their activity over the range of temperatures studied. The optimum temperature and optimum pH values were 37?°C and 7.0, respectively for both immobilized preparations, while the relative activities after storage at 4.0?°C for 60 days were 55% and 65% using cross-linking and adsorption methods, respectively. Also, the activity of the immobilized lipase began to decrease after 10 cycles, more than 58% of the initial activities were still retained after 10 cycles for both immobilization methods. These results indicated that lipase immobilized by cross-linking and adsorption not only effected activity recovery, but also remarkably effected stability, reusability and application adaptability. It can be concluded that, surface-modified RHA can be used as alternative supports for immobilization of CALB for polymerization reactions.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Immobilization and characterisation of a lipase from a new source, Bacillus sp. ITP-001

A new source of lipase from Bacillus sp. ITP-001 was immobilized by physical adsorption on the polymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. The support and immobilized lipase were characterised, compared to the lyophilised lipase, with regard to the specific surface area, adsorption–desorption isotherms, pore volume (Vp) and size (dp) by nitrogen adsorption, differential scanning calorimetry, thermogravimetric analysis, chemical composition analysis, Fourier transform infrared spectroscopy and biochemical properties. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0, whereas the optimum pH for the free enzyme was at pH 7.0; the optimum temperature of activity was 80 and 37 °C for the free and immobilized enzyme, respectively. The inactivation rate constant for the immobilized enzyme at 37 °C was 0.0038 h^?1 and the half-life was 182.41 h. The kinetic parameters obtained for the immobilized enzyme gave a Michaelis–Menten constant (K _m) of 49.10 mM and a maximum reaction velocity (V _max) of 205.03 U/g. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that it could be reused for 10 successive cycles, duration of each cycle (1 h), maintaining 33 % of the initial activity.     

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Immobilized CALB Catalyzed Transesterification of Soybean Oil and Phytosterol

Candida antarctica lipase B (CALB) was immobilized on Fe₃O₄/SiO_x-g-P(GMA) polymer carrier to catalyzed the transesterification of soybean oil and phytosterol. The enzyme loading of the obtained particles was 98.7 mg/g supports and the enzyme activity was 1226.5 U/g. The average particle size was 100.5?±?1.30 nm and the magnetization was 15.80 emu/g. The immobilized enzyme showed higher activities at a wider range of pH and temperatures. Its optimum reaction temperature was up to 50 °C; increased by 5 °C compared to the free enzyme. The obtained magnetic immobilized Fe₃O₄/SiO_x-g-P(GMA) lipase was nanoscale. First-grade soybean oils were used as a substrate. System pH was adjusted to 7.0. The optimal reaction temperature was 50 °C and the reaction time was 3 h. The phytosterol concentration of 5% and immobilized CALB of 2% were obtained. The conversion rate of transesterification reaction between soybean oil and phytosterol was 86.2%. The use of magnets can quickly separate the immobilized enzymes from the substrates. The relative activity of the immobilized enzymes was 83.0% when reused seven times. The prepared immobilized CALB can improve efficiently enzyme activity and reutilization.     

Activity and adsorption of lipase from Nigella sativa seeds on Celite at different pH values

Lipase from Nigella sativa seeds was immobilized by adsorption on Celite 535 from phosphate buffer solutions varying pH values of 5.0–8.0 at 25?°C. Langmuir isotherms described the adsorption equilibria well for lipase adsorption at all pH range. The saturation capacity for adsorption of lipase increased from 14.5 to 24.3 mg g^?1 Celite as the adsorption pH was reduced from 8 to 5, but the adsorption equilibrium constant remained constant and was determined to be 1.92 × 10⁵ M^?1. The adsorbed enzymes showed different activity values depending on the pH of the adsorption medium. The immobilized enzymes prepared at pH 6 displayed the highest activity values.     

Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Abstract

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of conditions showed that the maximal loading of lipase on PA-M reached 230.2 mg g^{? 1} at pH 9.0 and 35°C. An increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed. Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on PA-M was thermodynamically favorable. Covalent crosslinking of the lipase adsorbed extended the pH range and increased the optimal temperature of the lipase activity. The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase) retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase, covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.     

Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the K_m values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.     

Investigation of deactivation thermodynamics of lipase immobilized on polymeric carrier

In the present work, we have investigated biochemical thermo-kinetic stability of lipases immobilized on a biocompatible polymeric material. Immobilization of lipase Candida rugosa (CRL) was carried out on biocompatible blend of poly vinyl alcohol (PVA) and chitosan (CHY) support via entrapment and glutardehyde (Glu) cross-linking method to produce PVA:CHY:CRL and PVA:CHY:Glu:CRL as robust biocatalyst. These immobilized lipases were characterized by various physico-biochemical characterization techniques. Later on, thermal and solvent stability of polymer immobilized lipase was determined in term of half-life time (t _0.5), D values, enthalpy (ΔH°), entropy (ΔS°), and free energy (ΔG°) of deactivation at different temperatures and in various solvents. The thermodynamic deactivation stability trend was found as: cross-linked lipase CRL > entrapped lipase CRL > free lipase CRL. Moreover, kinetic parameters, such as K _m, V _max, and catalytic efficiency, were also determined to understand the kinetic features. The polymer immobilized enzyme was reused to investigate the economic viability of the developed biocatalyst.

     

Stability improvement of immobilized lactoperoxidase using polyaniline polymer

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55?°C, which has been increased about 10?°C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60?days whereas the native enzyme lost 80?% of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K_m and K_m.app were calculated to be 0.6 and 0.4; also V_max and V_max.app were 1.3 and 0.9 respectively.     

Investigation on the immobilization of Pseudomonas isoamylase onto polysaccharide matrices

This study examined Pseudomonas isoamylase immobilized onto polysaccharide matrices, among which included agarose, cellulose, and raw corn starch. For chemical binding of polysaccharides activated with tosyl chloride, a high specific activity of 23144?U/g-starch was obtained as compared with matrices of cellulose and agarose with 3229?U/g-cellulose and 84?U/g-agarose, respectively. For raw corn starch, isoamylase desorption occurred when the immobilized enzyme by physical adsorption was subjected to 0.05?M acetate buffer with pH?5.2 at 40?°C; this is despite the considerable affinity between the enzyme and the matrix. In contrast, no detectable activity leached from the matrix for chemical binding, regardless of whether maltose, i.e. an affinity species to isoamylase, was added. For immobilized starch-isoamylase, its optimal activity performance was obtained in broader pH?ranges of 3.5–5.5 and 5?°C higher than those of the free enzymes. More specifically, the free enzyme's activity markedly decreased within five hours while the immobilized starch-isoamylase exhibited a fairly stable behavior over a three day incubation period at 40?°C. After 175 days of storage at 4?°C, the residues of relative activity of 75% and 45% were obtained with respect to immobilized and free isoamylases, respectively.     

Immobilized copper-ion affinity adsorbent based on a cross-linked β-cyclodextrin polymer for adsorption of Candida rugosa lipase

Lipase from Candida rugosa was immobilized on a β-cyclodextrin-based polymer by adsorption and subsequent cross-linking with epichlorohydrin (EP-CD). The ligand iminodiacetic acid (IDA) was then bonded with the cross-linked β-cyclodextrin (EP-CD-IDA). This affinity adsorbent was further chelated with Cu^{2 +} for the purpose of binding affinity and stability. The properties of the immobilized lipase were assayed and compared with those of the free enzyme. Results showed that 266 µg protein with an activity of 17.85 U was bound per gram of matrix, giving 188% of the specific activity of the free enzyme and a total recovered activity of 79.7% under the optimum conditions. The pH and thermal stabilities of lipase were improved after immobilization on the β-cyclodextrin-based polymer (EP-CD-IDA-Cu^{2 +}). In addition, experimental results indicated that the residual activity of the immobilized lipase was 50% after eight cycles of reuse.     

Preparation and application of poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan hydrogels for immobilization of lipase

In the present of this study, two novel polymeric matrixes that are poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan was synthesized and applied for immobilization of lipase. For the immobilization of enzyme, two different immobilization procedures have been carried out via covalently binding and entrapment methods. On the free and immobilized enzymes activities, optimum pH, temperature, storage and thermal stability was investigated. The optimum temperature for free, covalently immobilized and entrapped enzymes was found to be 30, 35 and 30 degrees C, respectively. Optimum pH for both free and immobilized enzymes was also observed at pH 8. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for free and immobilized lipases. Furthermore, the reuse numbers of immobilized enzymes also studied. It was observed that after 40th use in 5 days, the retained activities for covalently immobilized and entrapped lipases were found as 39% and 22%, respectively. Storage and thermal stability of enzyme was also increased by as a result of immobilization procedures.     

Immobilization of thermostable exo-inulinase from mutant thermophilic Aspergillus tamarii-U4 using kaolin clay and its application in inulin hydrolysis

In this study, attempts were made to immobilize purified exo-inulinase from mutant thermophic Aspergillus tamarii-U4 onto Kaolinite clay by covalent bonding cross-linked with glutaraldehyde with an immobilization yield of 66% achieved. The free and immobilized inulinases were then characterized and characterization of the enzymes revealed that temperature and pH optima for the activity of the free and immobilized enzymes were both 65?°C and pH 4.5 respectively. The free inulinase completely lost its activity after incubation at 65?°C for 6 h while the immobilized inulinase retained 16.4% of its activity under the same condition of temperature and incubation time. The estimated kinetic parameters K_m and V_max for the free inulinase as estimated from Lineweaver-Burk plots were 0.39?mM and 4.21?µmol/min for the free inulinase and 0.37?mM and 4.01?µmol/min for the immobilized inulinase respectively. Inulin at 2.5% (w/v) and a flow rate of 0.1?mL was completely hydrolysed for 10?days at 60?°C in a continuous packed bed column and the operational stability of the system revealed that the half-life of the immobilized inulinase was 51?days. These properties make the immobilized exo-inulinase from Aspergillus tamarii-U4 a potential candidate for the production of fructose from inulin hydrolysis.     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

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.     

Immobilization of goat brain aminopeptidase B in calcium alginate beads

Aminopeptidase B, an arginyl aminopeptidase, was purified from goat brain with a purification factor of ～280 and a yield of 2.7%. It was entrapped in calcium alginate together with bovine serum albumin. The optimal conditions for immobilization for maximum activity yield were 1% CaCl₂ and 2.5% alginate. The immobilized enzyme retained ～62% of its initial activity and could be used for five successive batch reactions with retention of 30% of the initial activity. The pH and temperature optima of the free and immobilized enzyme were pH 7.4, 45°C and pH 7.8, 50°C respectively, while the pH and thermal stability as well as the stability of the enzyme in organic solvents were improved significantly after entrapment. The K_m value for the immobilized enzyme was about twofold higher than that of the soluble enzyme. Because of this increased stability, the immobilized enzyme may be useful in the meat processing industry.