Enzyme replacement therapy for Morquio A: an active recombinant <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine-6-sulfate sulfatase produced in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2–4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.     

Purification and characterization of an azoreductase from Escherichia coli CD-2 possessing quinone reductase activity

An oxygen-insensitive intracellular enzyme that is responsible for the decolorization of azo dyes was purified from Escherichia coli CD-2. The molecular weight of the purified enzyme was estimated as 27,000 ± 500 Da. Protein identification indicated that the enzyme had high sequence homology with E. coli K12 quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. With methyl red as substrate, the optimal pH value and temperature were 6.5 and 37 °C, respectively. The enzyme was stable under different physiochemical conditions. The azoreductase activity was restrained by SDS and was almost completely inhibited by Co²⁺ and Hg²⁺. K_m and V_max values were 0.18 mM and 8.12 U mg^?1 of protein for NADH and 0.05 mM and 6.46 U mg^?1 of protein for methyl red, respectively. The purified enzyme could efficiently decolorize methyl red with both NADH and NADPH as electron donors.     

Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation

The pectinolytic enzyme obtained from Penicillium viridicatum RFC by solid-state fermentation was purified to homogeneity by pretreatment with kaolin (40 mg mL⁻¹) and ultrafiltration, followed by chromatography on a Sephadex G50 column. The apparent molecular weight of the enzyme was 24 kDa. Maximal activity occurred at pH 6.0 and at 60 °C. The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of highly esterified pectin. The presence of 10 mM Ba²⁺ increased the enzyme activity by 96% and its thermal stability by 30%, besides increasing its stability at acid pH. The apparent K_m with apple pectin as substrate was 1.82 mg mL⁻¹ and the V_max was 81 μmol min⁻¹ mg⁻¹.     

Purification and partial characterisation of a thermostable xylanase from salt-tolerant Thermobifida halotolerans YIM 90462T

ThxynA, an extracellular xylanase of T. halotolerans YIM 90462^T, was purified to homogeneity from a fermentation broth by ultra-filtration, ammonium sulphate precipitation, hydrophobic chromatography and ion exchange chromatography. The purified xylanase has a molecular mass of 24 kDa and is optimally active at 80 °C and pH 6.0. The enzyme is stable over a broad pH range (pH 6.0–10.0) and shows good thermal stability when incubated at 70 °C for 1 h. The K_m and V_max values of the enzyme are 11.6 mg/mL and 434 μmol mg^?1 min^?1, respectively, using oat spelt xylan as a substrate. Moreover, the enzyme seemingly has both xylanase activity and cellulase activity. These unique properties suggest that it may be useful for industrial applications.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Purification and characterization of a new laccase from Shiraia sp.SUPER-H168

A new laccase from Shiraia sp.SUPER-H168 was purified by ion exchange column chromatography and gel permeation chromatography and the apparent molecular mass of this enzyme was 70.78 kDa, as determined by MALDI/TOF-MS. The optimum pH value of the purified laccase was 4, 6, 5.5 and 3 with 2,6-dimethoxyphenol (DMP), syringaldazine, guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as substrates, respectively. The optimum temperature of the purified laccase was 50 °C using DMP, syringaldazine and guaiacol as substrates, but 60 °C for ABTS. Inhibitors and metal ions of SDS, NaN₃, Ag⁺ and Fe³⁺ showed inhibition on enzyme activity of 10.22%, 7.86%, 8.13% and 67.50%, respectively. Fe²⁺ completely inhibited the purified laccase. The K_cat/K_m values of the purified laccase toward DMP, ABTS guaiacol and syringaldazine were 3.99 × 10⁶, 3.74 × 10⁷, 8.01 × 10⁴ and 2.35 × 10⁷ mol^?1 L S^?1, respectively. The N-terminal amino acid sequence of the purified laccase showed 36.4% similarity to Pleurotus ostrestus. Approximately 66% of the Acid Blue 129 (100 mg L^?1) was decolorized by 2.5 U of the purified laccase after a 120 min incubation at 50 °C. Acid Red 1 (20 mg L^?1) and Reactive Black 5 (50 mg L^?1) were decolorized by the purified laccase after the addition of Acid Blue 129 (100 mg L^?1).     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.     

Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli,purification, characterization and use in continuous cyanide degradation

A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile. V_max and K_m for HCN were ca. 6.8 mmol min⁻¹ mg⁻¹ protein and 109 mM, respectively. V_max for fumaronitrile and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3 μmol min⁻¹ mg⁻¹, respectively) but K_m was also lower (ca. 14.7 and 3.7 mM, respectively). Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18–34 C-terminal aa residues. The enzyme exhibited the highest activity at 45 °C and pH 8–9; it was unstable at over 35 °C and at below pH 5.5. The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6 h at 28 °C. A reactor loaded with an excess of the catalyst was used to degrade 25 mM KCN. A conversion rate of over 80% was maintained for 3 days.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

A thermoalkaliphilic halotolerant esterase from Rhodococcus sp. LKE-028 (MTCC 5562): Enzyme purification and characterization

A newly isolated Rhodococcus sp. LKE-028 (MTCC 5562) from soil samples of Gangotri region of Uttarakhand Himalayan produced a thermostable esterase. The enzyme was purified to homogeneity with purification fold 62.8 and specific activity 861.2 U mg^?1 proteins along with 26.7% recovery. Molecular mass of the purified enzyme was 38 kDa and values of K_m and V_max were 525 nM and 1666.7 U mg^?1 proteins, respectively. The esterase was active over a broad range of temperature (40–100 °C) and pH (7.0–12.0). The esterase was most active at pH 11.0. The optimum temperature of enzyme activity was 70 °C and the enzyme was completely stable after 3 h pre-incubation at 60 °C. Metal ions like Ca²⁺, Mg²⁺ and Co²⁺ stimulated enzyme activities. Purified esterase remarkably retained its activity with 10 M NaCl. Enzyme activity was slightly increased in presence of non-polar detergents (Tween 20, Tween 80 and Triton X 100), and compatible with oxidizing agents (H₂O₂) and reducing agents (β-mercaptoethanol). Activities of the enzyme was stimulated in presence of organic solvents like DMSO, benzene, toluene, methanol, ethyl alcohol, acetone, isoamyl alcohol after 10 days long incubation. The enzyme retained over 75% activity in presence of proteinase K. Besides hyperthermostability and halotolerancy the novelty of this enzyme is its resistance against protease.     

Production of an anti-MUC1 C595 dbFv antibody fragment in recombinant Escherichia coli

The production of C595 diabody fragment (dbFv) in Escherichia coli (E. coli) HB2151 clone has been explored. The comparison of fermentation processes mode demonstrated that a higher biomass inoculum operation enhanced C595 dbFv production. It was demonstrated that a concentration of 12.1 mg l⁻¹ broth of dbFv and a cell concentration of 23.6 g l⁻¹ broth were achieved at the end of 75 l fermentation.     

Toward the production of flavone-7-O-β-d-glucopyranosides using Arabidopsis glycosyltransferase in Escherichia coli

Flavonoid glycosides are highly attractive targets due to their dominant roles in clinical, cosmetic production and in the food industry. In this research, an Escherichia coli strain bearing the reconstructed uridine-diphosphate glucose (UDP-glucose) pathway cassette and a putative glycosyltransferase from Arabidopsis thaliana, was developed as a host for the production of apigenin-7-O-β-d-glucoside (APG) and baicalein-7-O-β-d-glucoside (BCG) from exogenously supplied flavone aglycones (apigenin and baicalein, respectively). In order to improve the yield, genetic engineering of E. coli strains for optimization of intracellular UDP-glucose generation, as well as media optimization were carried out. The production was scaled up using a fed batch fermentation, and the maximal yield of products reached 90.88 μM (39.28 mg L^?1) and 76.82 μM (33.19 mg L^?1) of APG and BCG, respectively. And, the maximum bioconversion rate corresponded to 90.88% and 76.82% of apigenin and baicalein, respectively.     

Enzyme Replacement in a Human Model of Mucopolysaccharidosis IVA In Vitro and Its Biodistribution in the Cartilage of Wild Type Mice

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K_uptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.     

A novel low-temperature-active exo-inulinase identified based on Molecular-Activity strategy from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis

A novel glycosyl hydrolase family 32 exo-inulinase (InuAGN25) gene was cloned from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis. InuAGN25 showed the highest identity of 54.3% with a putative levanase recorded in GenBank. Molecular-Activity strategy was proposed to predict InuAGN25 to be a low-temperature-active exo-inulinase before experiments performance. Molecular analyses included progressive sequential, phylogenetic and structural analyses. InuAGN25 was effectively expressed in Escherichia coli. The purified recombinant InuAGN25 showed characteristics of low-temperature-active enzymes: (1) the enzyme retained 55.8% of the maximum activity at 20 °C, 35.8% at 10 °C, and even 8.2% at 0 °C; (2) the enzyme exhibited 75.8, 30.5 and 10.8% of the initial activity after preincubation for 60 min at 45, 50 and 55 °C, respectively; (3) K_m values of the enzyme toward inulin were 2.8, 3.0, 3.2 and 5.8 mg ml⁻¹ at 0, 10, 20 and 40 °C, respectively. Fructose was the main product of inulin and Jerusalem artichoke tubers hydrolyzed by the purified recombinant InuAGN25 at room temperature, 10 °C and 0 °C. These results suggested the Molecular-Activity strategy worked efficiently and made InuAGN25 promising for the production of fructose at low temperatures.     

Molecular cloning and high-level expression of a β-galactosidase gene from Paecilomyces aerugineus in Pichia pastoris

A β-galactosidase gene (designated PaGalA) was cloned for the first time from Paecilomyces aerugineus and expressed in Pichia pastoris under the control of the AOX1 promoter. The coding region of 3036 bp encoded a protein of 1011 amino acids with a deduced molecular mass of 108.7 kDa. The PaGalA without the signal peptide was cloned into a vector pPIC9K and was expressed successfully in P. pastoris as active extracellular β-galactosidase. The recombinant β-galactosidase (PaGalA) was secreted into the medium at an extremely high levels of 22 mg ml⁻¹ having an activity of 9500 U ml⁻¹ from high density fermentation culture, which is by far the highest yield obtained for a β-galactosidase. The purified enzyme with a high specific activity of 820 U mg⁻¹ had a molecular mass of 120 kDa on SDS-PAGE. PaGalA was optimally active at pH 4.5 and a temperature of 60 °C. The recombinant β-galactosidase was able to hydrolyze lactose efficiently at pH 5.0 and 50 °C. It also possessed transglycosylation activities at high concentrations of lactose. PaGalA exhibited better lactose hydrolysis efficiency in whey than two other widely used commercial lactases. The extremely high expression levels coupled with favorable biochemical properties make this enzyme highly suitable for commercial purposes in the hydrolysis of lactose in milk or whey.     

Characterization of a spore surface lipase from the biocontrol agent Metarhizium anisopliae

A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ～66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of K_m and V_max for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min^?1 mg^?1 and 0.712 mM and 5.696 mMol min^?1 mg^?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca²⁺, Mg²⁺ and Co²⁺ and inhibited by Mn²⁺. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 °C, respectively and under such conditions remained stable with no decrease in activity after 5 h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1 U mg^?1 after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85 kDa and K_m and V_max values of 272.3 mM and 166.7 μmol min^?1 ml^?1. The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600 mg ml^?1. Sucrose concentration (400 mg ml^?1) temperature (60 °C), and pH (5.6) favoured the synthesis of high levels of GF₃ and GF₄. Incubation time had a critical effect on the yield of FOS as the major products were GF₂ after 4 h and GF₄ after 8 h. A prolonged incubation of 16 h resulted in the conversion of GF₄ into GF₂ as a result of self hydrolase activity.     

Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae

The piceid-β-d-glucosidase that hydrolyzes the β-d-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-β-d-glucosidase was 60 °C, and the optimum pH was 5.0. The piceid-β-d-glucosidase was stable at less than 60 °C, and pH 4.0–5.0. Ca²⁺, Mg²⁺ and Zn²⁺ ions have no significant effect on enzyme activity, but Cu²⁺ ion inhibits enzyme activity strongly. The K_m value was 0.74 mM and the V_max value was 323 nkat mg⁻¹ for piceid.     

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.