Immobilization of lipase onto micron-size magnetic beads

A novel and economical magnetic poly(methacrylate-divinylbenzene) microsphere (less than 8 microm in diameter) was synthesized by the modified suspension polymerization of methacrylate and cross-linker divinylbenzene in the presence of magnetic fluid. Then, surface aminolysis was employed to obtain a high content of surface amino groups (0.40-0.55 mmolg(-1) supports). The morphology and properties of these magnetic supports were characterized with scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and a vibrating sample magnetometer. These magnetic supports exhibited superparamagnetism with a high specific saturation magnetization (sigma(s)) of 14.6 emicrog(-1). Candida cylindracea lipase was covalently immobilized on the amino-functionalized magnetic supports with the activity recovery up to 72.4% and enzyme loading of 34.0 mgg(-1) support, remarkably higher than the previous studies. The factors involved in the activity recovery and enzymatic properties of the immobilized lipase prepared were studied in comparison with free lipase, for which olive oil was chosen as the substrate. The results show that the immobilized lipase has good stability and reusability after recovery by magnetic separation within 20s.     

Immobilization of a protease on modified chitosan beads for the depolymerization of chitosan

Neutral protease was immobilized on chitosan (CS), carboxymethyl chitosan (CMCS), and N-succinyl chitosan (NSCS) hydrogel beads. And the biocatalysts obtained were used to prepare low molecular weight chitosan (LMWC) and chitooligomers. Weight-average molecular weight of LMWC produced by neutral protease immobilized on CS, CMCS and NSCS hydrogel beads were 3.4 kDa, 3.2 kDa and 1.9 kDa, respectively. The effects of immobilization support and substrate on enzymatic reaction were analyzed by measuring classical Michaelis-Menten kinetic parameters. The FT-IR, XRD and potentiometric determination results indicated decrease of molecular weight led to transformation of crystal structure, but the degree of N-deacetylation and chemical structures of residues were not changed compared to initial chitosan. The degree of polymerization of chitooligomers was mainly from 2 to 7. We observed a strong dependence of the immobilized enzyme properties on the chemical nature of the supports, which leads to different microenvironment of neutral protease and changes the hydrolyzing process.     

Immobilization of catalase into chemically crosslinked chitosan beads

Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pH–activity curve, the temperature–activity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 °C. The K_m value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in V_max value from 32,000 to 122 μmol (min mg protein)⁻¹. It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization.     

Immobilization of pancreatic lipase on chitin and chitosan

In this study, porcine pancreatic lipase (EC 3.1.1.3) was immobilized on chitin and chitosan by adsorption and subsequent crosslinking with glutaraldehyde, which was added before (conjugation) or after (crosslinking) washing unbound proteins. Conjugation proved to be the better method for both supports. The properties of free and immobilized enzymes were also investigated and compared. The results showed that the pH optimum was shifted from 8.5 to 9.0 for both the immobilized enzymes. Also, the optimum temperature was shifted from 30 to 40 degrees C for chitin-enzyme and to 45 degrees C for chitosan-enzyme conjugates. The immobilization efficiency is low, but the immobilized enzymes have good reusability and stability (storage and operational). Besides these properties, the immobilized lipases were also suitable for catalyzing esterification reactions of fatty acids and fatty alcohols, both with a medium chain length. According to our results, esterification activities of immobilized lipases were two- and four-fold higher for chitosan- and chitin-enzyme, than for the free enzyme, respectively. The immobilization procedure shows a great potential for commercial applications of the immobilized lipase, a relatively low cost commercial enzyme.     

Immobilization of "Disguised" yeast in chemically crosslinked chitosan beads

A new simple method for the preparation of chemically crosslinked chitosan beads is presented. It consists of the dropwise addition of 2-3% (w/v) low molecular weight chitosan solution containing 2% (w/v) glyoxal in 1% (w/v) tetrasodiumdiphosphate, pH 8.0. Immobilized viable baker's yeast (Saccharomyces cerevisiae) could be obtained via gel entrapment within the new beads when means preventing their direct contact with soluble chitosan were provided, "disguising" the cells until gelation and crosslinking were completed. Such means included cell suspension in castor oil or mixing with carboxymethyl-cellulose powder. Application of these means was shown to be necessary, as cells exposed to soluble chitosan immediately lost their viability and glycolytic activity. Yeast disguised in castor oil was also protected from bead reinforcement by glutaraldehyde treatment, significantly strengthening bead stability while operating under acidic conditions. This capability was demonstrated by continuous ethanol production by chitosan entrapped yeast. (c) 1994 John Wiley & Sons, Inc.     

Enantioselective synthesis of (S)‐naproxen using immobilized lipase on chitosan beads

S‐naproxen by enantioselective hydrolysis of racemic naproxen methyl ester was produced using immobilized lipase. The lipase enzyme was immobilized on chitosan beads, activated chitosan beads by glutaraldehyde, and Amberlite XAD7. In order to find an appropriate support for the hydrolysis reaction of racemic naproxen methyl ester, the conversion and enantioselectivity for all carriers were compared. In addition, effects of the volumetric ratio of two phases in different organic solvents, addition of cosolvent and surfactant, optimum pH and temperature, reusability, and inhibitory effect of methanol were investigated. The optimum volumetric ratio of two phases was defined as 3:2 of aqueous phase to organic phase. Various water miscible and water immiscible solvents were examined. Finally, isooctane was chosen as an organic solvent, while 2‐ethoxyethanol was added as a cosolvent in the organic phase of the reaction mixture. The optimum reaction conditions were determined to be 35 °C, pH 7, and 24 h. Addition of Tween‐80 in the organic phase increased the accessibility of immobilized enzyme to the reactant. The optimum organic phase compositions using a volumetric ratio of 2‐ethoxyethanol, isooctane and Tween‐80 were 3:7 and 0.1% (v /v/v), respectively. The best conversion and enantioselectivity of immobilized enzyme using chitosan beads activated by glutaraldehyde were 0.45 and 185, respectively.     

Preparation and characterization of a thermostable and biodegradable biopolymers using natural cross-linker

The present study describes preparation and characterization of a thermally stable and biodegradable biopolymer using collagen and a natural polymer, alginic acid (AA). Required concentration of alginic acid and collagen was optimized and the resulting biopolymer was characterized for, degree of cross-linking, mechanical strength, thermal stability, biocompatibility (toxicity) and biodegradability. Results reveal, the degree of cross-linking of alginic acid (at 1.5% concentration) with collagen was calculated as 75%, whereas it was 83% with standard cross-linking agent, glutaraldehyde (at 1.5% concentration). The AA cross-linked biopolymer was stable up to 245°C and Exhibits 5-6-fold increase in mechanical (tensile) strength compared to plain collagen (native) materials. However, glutaraldehyde cross-linked material exhibits comparatively less thermal stability and brittle in nature (low tensile strength). With regard to cell toxicity, no cytotoxicity was observed for AA cross-linked material when tested with mesenchymal cells and found degradable when treated with collagenase enzyme. The nature of bonding pattern and the reason for thermal stability of AA cross-linked collagen biopolymer was discussed in detail with the help of bioinformatics. A supplementary file on efficacy of AACC as a wound dressing material is demonstrated in detail with animal model studies.     

Immobilization of lipase from Pseudomonas fluorescens on glyoxyl-octyl-agarose beads: Improved stability and reusability

The lipase from Pseudomonas fluorescens (PFL) has been immobilized on glyoxyl-octyl agarose and compared to the enzyme immobilized on octyl-agarose. Thus, PFL was immobilized at pH 7 on glyoxyl-octyl support via lipase interfacial activation and later incubated at pH 10.5 for 20 h before reduction to get some enzyme-support covalent bonds. This permitted for 70% of the enzyme molecules to become covalently attached to the support. This biocatalyst was slightly more stable than the octyl-PFL at pH 5, 7 and 9, or in the presence of some organic solvents (stabilization factor no higher than 2). The presence of phosphate anions produced enzyme destabilization, partially prevented by the immobilization on glyoxyl-octyl (stabilization factor became 4). In contrast, the presence of calcium cations promoted a great PFLstabilization, higher in the case of the glyoxyl-octyl preparation (that remained 100% active when the octyl-PFL preparations had lost 20% of the activity). However, it is in the operational stability where the new biocatalyst showed the advantages: in the hydrolysis of 1 M triacetin in 60% 1.4 dioxane, the octyl biocatalyst released >60% of the enzyme in the first cycle, while the covalently attached enzyme retained its full activity after 5 reaction cycles.     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.     

Immobilization of anaerobic sludge using chitosan crosslinked with lignosulfonate

A new method for immobilization of anaerobic sludge in chitosan is described. It is based on the reaction of basic NH₂ groups of chitosan with the acidic sulfonic-groups of lignosulfonate to form sulfonilamide linkages. The new procedure features simplicity, low-cost and mild immobilization conditions. Batch tests of acetate consumption along with a continuous reactor operation confirmed the effectiveness of the immobilization technique for maintenance of long-term stability of the polymer. Received 27 March 1997/ Accepted in revised form 01 October 1997     

Modification of collagen with a natural cross-linker, procyanidin

We have investigated the modification of collagen with a natural plant polyphenol, procyanidin under acidic conditions. Fourier transform infrared spectroscopy (FTIR) and Atomic force microscopy (AFM) studies demonstrate that the hydrogen bond interactions between collagen and procyanidin does not destroy the triple helix conformation of collagen, and the fibril aggregation occurs because of the cross-linking with procyanidin. The water contact angle (WCA) tests indicate that the hydrophobicity of the procyanidin modified collagen films can be improved. Whereas, the water vapor permeability (WVP) of the films decrease with the increasing procyanidin content due to the formation of denser structure. Moreover, differential scanning calorimetry (DSC) and thermogravimetric (TG) measurements reveal that the collagen/procyanidin films have improved thermal stability in comparison with pure collagen. The present study reveals that procyanidin stabilizes collagen as a cross-linker and preserves its triple helical structure.     

Immobilization of proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents.

alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.     

Immobilization of hydrogenase on glass beads

Hydrogenase from $\text{Clostridium pasteurianum}$ was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Immobilization of lipase on cotton cloth using the layer-by-layer self-assembly technique

Lipase from Thermomyces lanuginosus was assembled into multiple layers on polyethylenimine treated cotton flannel cloth, utilising the enzymes property of forming bimolecular aggregates via layer-by-layer (LBL) immobilization technique. An increase in lipase activity with increasing enzyme layers confirmed lipase aggregation. A study to compare the activity of enzyme bound by classical LBL technique, containing alternate layers of polyethylenimine and lipase and the modified approach indicated above, showed that more enzyme was bound to cloth in the modified approach. A total of 13 U/cm² of enzyme were bound to cloth till the fifth layer whereas only 10.2 U/cm² were bound till the fifth bilayer in the classical approach. The successful assembly of lipase molecules has shown that this modified technique is a promising approach to immobilize enzymes that aggregate through hydrophobic interactions as nano-films on cloth.     

Immobilization of ovomucoid on chitosan

The inhibitory activity of ovomucoid from duck egg white, immobilized on chitosan with the use of glutaraldehyde or carbodiimide as cross-linking agents, was studied. Glutaraldehyde proved to be a more preferable cross-linking agent than carbodiimide. When chitosan is used as a protein carrier, the possibility of shifting the pH optimum of these compounds should be taken into account.     

Immobilization of ovomucoid on chitosan

The inhibitory activity of ovomucoid from duck egg white, immobilized on chitosan with the use of glutaraldehyde or carbodiimide as cross-linking agents, was studied. Glutaraldehyde proved to be a more preferable cross-linking agent than carbodiimide. When chitosan is used as a protein carrier, the possibility of shifting the pH optimum of these compounds should be taken into account.