Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads

The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 °C higher (45 °C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium.     

Immobilization of Candida rugosa lipase on hexagonal mesoporous silicas and selective esterification in nonaqueous medium

Candida rugosa lipases (CRLs) immobilized by physical adsorption, cross-linking and covalent binding methods on a MSU-H type mesoporous silica previously modified organically by different strategies, respectively, were examined as biocatalysts for esterification of conjugated linoleic acid (CLA) and ethanol in nonaqueous medium. MSU-H silica was modified by nonionic surfactant of triblock copolymer Pluronic P123, amino-functionalization and glutaraldehyde-grafting and confirmed by FT-IR analysis. Interaction mechanisms of CRLs and supports involve covalent and non-covalent interactions including electrostatic repulsion and hydrophobic interaction at pH 7. The immobilized CRLs containing surfactant were prepared by cross-linking via entrapping CRL aggregates inside the pores of silicas. The surfactant located inside the silicas could interfacially activate the immobilized CRLs and favored catalytic esterification. The biocatalyst containing 38 wt.% of surfactant afforded 1111.1 U/mg of specific activity about eight times higher than soluble CRL, and maximal 56.7% of total CLA esterification with 96.5% of 9c, 11t-CLA isomer esterification degree. The immobilized-CRL with 64.5 mg/g of loading amount of protein exhibited maximal hydrolytic activity of 2945.3 U/g-support for grafting glutaraldehyde. This derivative showed a high level of esterification activity and operational stability and remained 43.2–46.9% of total esterification for 32 h consecutive four runs.     

A novel immobilization method for nuclease P1 on macroporous absorbent resin with glutaraldehyde cross-linking and determination of its properties

Microbial nuclease P₁ from Penicllium citrinum was immobilized on macroporous absorbent resins: strong polar poly (styrene-co-DVB) resin (SPPSD), polymethacrylic ester resin and poly (styrene-co-DVB)-Br resin. The results showed that SPPSD was the best carrier. Three methods of glutaraldehyde cross-linking were used and simultaneous immobilization and cross-linking (CIS) was demonstrated to be the best method. The functional properties of immobilized nuclease P₁ were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized nuclease P₁ were obtained in 0.2 M acetate buffer at pH 4.5 and a temperature of 70 °C. An increase in K_m (from 3.165 to 18.125 mg mL^?1) and a decrease in V_max (from 1667.18 to 443.95 U min^?1 mL^?1) were recorded after immobilization. SPPSD-glutaraldehyde-nuclease P₁ exhibited better thermal stability than the free enzyme. The apparent activation energy (E_a) of the free and immobilized nuclease P₁ was 137.04 kJ mol^?1 and 98.43 kJ mol^?1, respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit.     

Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium

Racemic _DL-tert-leucine (_DL-Tle) was resolved to obtain enantiopure _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative with immobilized penicillin G acylase (PGA). The effects of pH, reaction temperature, substrate concentration and reaction time on the reaction were investigated. The reaction was conveniently carried out at 0.4 M substrate concentration in water at pH 8.0 and 30 °C. Under the optimized reaction conditions, _L-Tle was obtained in an enantiopure form (>99% ee) with 45.8% substrate conversion after 4 h. The thermal stability and operational stability of immobilized PGA were examined. Furthermore, the preparation of _L-Tle was successfully performed in a recirculating packed bed reactor (RPBR) system and immobilized PGA exhibited a long-term stability for 51 days with a slight decrease of activity. The isolated _D-enantiomer was racemized at 160 °C for 15 min and reused as substrate. The results obtained clearly demonstrated a potential for industrial application of immobilized PGA in the preparation of _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative.     

A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase

The objective of this work was to compare the properties of free and immobilized β-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 °C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The K_m and V_m values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 g_glucose/L min mg_enzyme, respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 g_lactose/min m³_catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55 °C. Alternatively, the immobilized form was most active at pH 5.0 at 60 °C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

Preparation and application of cross-linked aggregates of chloroperoxidase with enhanced hydrogen peroxide tolerance

A cross-linked enzyme aggregate (CLEA^®) of chloroperoxidase (CPO) was created that exhibited greatly improved stability in the presence of hydrogen peroxide concentrations as high as 1.2 M. The CPO-CLEA was generated by oxidizing the protein with sodium periodate and precipitating and cross-linking with ammonium sulfate and sodium borohydride. CLEA^® production parameters, including the concentrations of these three reagents, were optimized to maximize the activity of the biocatalyst in oxidizing 7-azaindole to 7-azaoxindole. Additionally, the in situ production of the CLEA^® was demonstrated, resulting in a process for converting >90% of 5 g/l 7-azaindole in <1 h while requiring neither gradual peroxide addition nor immobilized enzyme isolation.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Efficient digestion of chitosan using chitosanase immobilized on silica-gel for the production of multisize chitooligosaccharides

Chitosanase-coated silica-gels were prepared via cross-linking of the chitosanase onto silica-gels for the efficient production of multisize chitooligosaccharides (MCOs) in a continuous process. The kinetic aspects of immobilized chitosanase (IMMCTase) were investigated based on the reaction time, production of MCOs, and MALDI-TOF mass analyses to achieve maximum bioconversion of high molecular weight chitosan (HMWC) to MCOs. IMMCTase revealed a negligible loss of chitosanase activity after multi uses in continuous digestion of HMWC. The optimal temperature of IMMCTase was 37 °C, and kinetic parameters toward HMWC were determined to be K_m = 1.45 mM and V_max = 360 μmole/μg/min, respectively. Under optimal conditions, the recovery of enzyme activity of IMMCTase was determined to be 82.3%, thus indicating that it can still be reused few more times. In conclusion, use of IMMCTase resulted in rapid and efficient digestions of HMWC with consistent results to produce MCOs.     

Immobilization of urease on vapour phase stain etched porous silicon

Porous silicon layers fabricated by the reaction-induced vapor phase stain etch method were coated with 5% polyethylenimine. Urease from Canavalia brasiliensis beans was immobilized on this support through covalent linking with 2.5% glutaraldehyde. The pH and temperature profile of the immobilized and free urease exhibited higher activity at pH 6.5 and 37 °C. After being stored for 30 days at 4 °C, the immobilized enzyme had 75% of the initial activity. The maximum apparent Michaelis constant for free urease (K_m) was 94.33 mM whereas for immobilized urease was 53.04 mM. The maximum reaction velocity (V_max) for free urease was 3.51 mmol/min and for immobilized urease was 1.57 mmol/min.     

The study on effective immobilization of lipase on functionalized bentonites and their properties

Three different functionalized bentonites including acid activated bentonite (B_a), organically modified bentonite with cetyltrimethyl ammonium bromide (B_CTMAB) and the composite by acid activation and organo-modification (B_a-CTMAB) were prepared, and used for immobilization of lipase from bovine pancreatic lipase by adsorption. The amount of lipase adsorbed on the functionalized bentonites was in the following sequence: B_a > B_CTMAB > B_a-CTMAB, showing the strongest affinity of B_a for lipase among the three supports. However, the immobilized lipase on B_a-CTMAB showed the highest activity in the hydrolysis of olive oil by 1.67 times of activity of free lipase due to the hydrophobically interfacial activation and enlarged catalytic interface. While, the activity of immobilized lipase on B_a was lower than 20% of free lipase’s activity due to the absence of hydrophobic activation and negative impact of excessive hydrogen ions on the surface. The K_m values for the immobilized lipase on B_a-CTMAB (0.054 g/mL) and B_CTMAB (0.074 g/mL) were both lower than that of free lipase (0.115 g/mL), and the V_max values were higher for the immobilized lipases, exhibiting a higher affinity of the immobilized lipase toward olive oil than free lipase. In comparison to free lipase, the better resistance to heating inactivation, storage stability and reusability of the immobilized lipases on B_a-CTMAB and B_CTMAB were also obtained. The results show that the efficient and stable biocatalysts for industrial application can be prepared by using the low-cost bentonite mineral as the supports.     

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.     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding

An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activities of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 °C) than that of the free enzyme (55 °C). Kinetic parameters, Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max), were calculated as K_m = 13.375, 11.956, and 8.698 × 10^?4 mg/ml and V_max = 0.156, 0.163 and 0.17 × 10^?3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

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.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: Optimization and characterization

Laccase from Trametes versicolor was immobilized on Amberlite IR-120 H beads. Maximum immobilization obtained was 78.7% at pH = 4.5 and temperature T = 45 °C. Kinetic parameters, K_m and V_max values, were determined respectively as 0.051 mM and 2.77 × 10^?2 mM/s for free and 4.70 mM and 5.27 × 10^?3 mM/s for immobilized laccase. The Amberlite–laccase system showed a 30% residual activity after 7 cycles. On the other hand, the loss of activity for free laccase after 7 days of storage at 4 °C was 18.5% in comparison to Amberlite–laccase system with a loss of 1.4%, during the same period. Improved operational, thermal and storage stabilities of the immobilized laccase were obtained compared to the free counterpart. Therefore, the use of low-cost matrices, like Amberlite for enzyme immobilization represents a promising product for enzymatic industrial applications.     

Graft copolymerization of acrylamide on chitosan-co-chitin and its application for immobilization of Aspergillus sp. RL2Ct cutinase

The synthesis of chitosan (Chs) and chitin (Chi) copolymer and grafting of acrylamide (AAm) onto the synthesized copolymer have been carried out by chemical methods. The grafted copolymer was characterized by FTIR, SEM and XRD. The extracellular cutinase of Aspergillus sp. RL2Ct (E.C. 3.1.1.3) was purified to 4.46 fold with 16.1% yield using acetone precipitation and DEAE sepharose ion exchange chromatography. It was immobilized by adsorption on the grafted copolymer. The immobilized enzyme was found to be more stable then the free enzyme and has a good binding efficiency (78.8%) with the grafted copolymer. The kinetic parameters K_M and V_max for free and immobilized cutinase were found to be 0.55 mM and 1410 μmol min⁻¹ mg⁻¹ protein, 2.99 mM and 996 μmol min⁻¹ mg⁻¹ protein, respectively. The immobilized cutinase was recycled 64 times without considerable loss of activity. The matrix (Chs-co-Chi-g-poly(AAm)) prepared and cutinase immobilized on the matrix have potential applications in enzyme immobilization and organic synthesis respectively.