Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Field-evolved resistance to chitin synthesis inhibitor insecticides by soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae), in Brazil.

Soybean looper (SBL), Chrysodeixis includens (Walker), is an economically important soybean and cotton pest in Brazil. Here, we selected an SBL strain resistant to teflubenzuron using F2 screening, estimated the resistance allele frequency, characterized the inheritance of resistance, investigated fitness costs, evaluated patterns of cross-resistance, and determined the magnitude of resistance. The teflubenzuron-resistant strain (Teflu-R) was selected from field-collected populations with an estimated allele frequency of 0.1700. Estimated LC50 values were 0.010 and 363.61 µg a.i. cm-2 for the susceptible (Sus) and Teflu-R strains, respectively, representing a 36,361-fold resistance ratio (RR). The LC50 values of reciprocal crosses were 1.02 and 0.59 µg a.i. cm-2, suggesting that resistance is autosomally inherited. The low survival of reciprocal crosses (16 and 20%) on teflubenzuron-sprayed leaves indicates incomplete recessive resistance. The number of segregations influencing resistance was 2.72, suggesting a polygenic effect. The Teflu-R strain showed longer development periods as well as lower survival and population growth than the Sus strain, revealing fitness costs. The Teflu-R strain also showed high cross-resistancesto other chitin inhibitor insecticides, such as novaluron (RR = 6147-fold) and lufenuron (RR = 953-fold), but low cross-resistance to methoxyfenozide, flubendiamide, and indoxacarb (RR < 3.45-fold). On discriminatory concentrations of teflubenzuron and novaluron, populations of SBL showed survival rates from 15 to 52%, indicating field resistance to these insecticides. Our findings indicated that resistance to teflubenzuron in SBL is autosomal, recessive, polygenic, and associated with fitness cost. We also found a high cross-resistance to other benzoylphenylureas and a high frequency of resistance to this mode-of-action in SBL in Brazil.

Heterologous expression of an active chitin synthase from Rhizopus oryzae.

Chitin synthases are highly important enzymes in nature, where they synthesize structural components in species belonging to different eukaryotic kingdoms, including kingdom Fungi. Unfortunately, their structure and the molecular mechanism of synthesis of their microfibrilar product remain largely unknown, probably because no fungal active chitin synthases have been isolated, possibly due to their extreme hydrophobicity. In this study we have turned to the heterologous expression of the transcript from a small chitin synthase of Rhizopus oryzae (RO3G_00942, Chs1) in Escherichia coli. The enzyme was active, but accumulated mostly in inclusion bodies. High concentrations of arginine or urea solubilized the enzyme, but their dilution led to its denaturation and precipitation. Nevertheless, use of urea permitted the purification of small amounts of the enzyme. The properties of Chs1 (Km, optimum temperature and pH, effect of GlcNAc) were abnormal, probably because it lacks the hydrophobic transmembrane regions characteristic of chitin synthases. The product of the enzyme showed that, contrasting with chitin made by membrane-bound Chs's and chitosomes, was only partially in the form of short microfibrils of low crystallinity. This approach may lead to future developments to obtain active chitin synthases that permit understanding their molecular mechanism of activity, and microfibril assembly.

Genetic basis of Spodoptera frugiperda (Lepidoptera: Noctuidae) resistance to the chitin synthesis inhibitor lufenuron.

BACKGROUND: An understanding of the genetic basis of insect resistance to insecticides is important for the establishment of insect resistance management (IRM) strategies. In this study we evaluated the inheritance pattern of resistance to the chitin synthesis inhibitor lufenuron in Spodoptera frugiperda. RESULTS: The LC50 values (95% CI) were 0.23 µg lufenuron mL(-1) water (ppm) (0.18-0.28) for the susceptible strain (SUS) and 210.6 µg mL(-1) (175.90-258.10) for the lufenuron-resistant strain (LUF-R), based on diet-overlay bioassay. The resistance ratio was ≈ 915-fold. The LC50 values for reciprocal crosses were 4.89 µg mL(-1) (3.79-5.97) for female LUF-R and male SUS and 5.74 µg mL(-1) (4.70-6.91) for female SUS and male LUF-R, indicating that the inheritance of S. frugiperda resistance to lufenuron is an autosomal, incompletely recessive trait. Backcrosses of the progeny of reciprocal crosses with the parental LUF-R showed a polygenic effect. The estimated minimum number of independent segregations was in the 11.02 range, indicating that resistance to lufenuron is associated with multiple genes in S. frugiperda. CONCLUSIONS: Based on genetic crosses, the inheritance pattern of lufenuron resistance in S. frugiperda was autosomal, incompletely recessive and polygenic. Implications of this finding to IRM are discussed in this paper.

The Impact of Selection with Diflubenzuron, a Chitin Synthesis Inhibitor, on the Fitness of Two Brazilian Aedes aegypti Field Populations.

Several Aedes aegypti field populations are resistant to neurotoxic insecticides, mainly organophoshates and pyrethroids, which are extensively used as larvicides and adulticides, respectively. Diflubenzuron (DFB), a chitin synthesis inhibitor (CSI), was recently approved for use in drinking water, and is presently employed in Brazil for Ae. aegypti control, against populations resistant to the organophosphate temephos. However, tests of DFB efficacy against field Ae. aegypti populations are lacking. In addition, information regarding the dynamics of CSI resistance, and characterization of any potential fitness effects that may arise in conjunction with resistance are essential for new Ae. aegypti control strategies. Here, the efficacy of DFB was evaluated for two Brazilian Ae. aegypti populations known to be resistant to both temephos and the pyrethroid deltamethrin. Laboratory selection for DFB resistance was then performed over six or seven generations, using a fixed dose of insecticide that inhibited 80% of adult emergence in the first generation. The selection process was stopped when adult emergence in the diflubenzuron-treated groups was equivalent to that of the control groups, kept without insecticide. Diflubenzuron was effective against the two Ae. aegypti field populations evaluated, regardless of their resistance level to neurotoxic insecticides. However, only a few generations of DFB selection were sufficient to change the susceptible status of both populations to this compound. Several aspects of mosquito biology were affected in both selected populations, indicating that diflubenzuron resistance acquisition is associated with a fitness cost. We believe that these results can significantly contribute to the design of control strategies involving the use of insect growth regulators.

Characterization and functional analysis of a chitin synthase gene (HcCS1) identified from the freshwater pearlmussel Hyriopsis cumingii.

The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain.

Effect of corn steep liquor (CSL) and cassava wastewater (CW) on chitin and chitosan production by Cunninghamella elegans and their physicochemical characteristics and cytotoxicity.

Microbiological processes were used for chitin and chitosan production with Cunninghamella elegans UCP/WFCC 0542 grown in different concentrations of two agro-industrial wastes, corn steep liquor (CSL) and cassava wastewater (CW) established using a 2² full factorial design. The polysaccharides were extracted by alkali-acid treatment and characterized by infrared spectroscopy, viscosity, thermal analysis, elemental analysis, scanning electron microscopy and X-ray diffraction. The cytotoxicity of chitosan was evaluated for signs of vascular change on the chorioallantoic membrane of chicken eggs. The highest biomass (9.93 g/L) was obtained in trial 3 (5% CW, 8% CSL), the greatest chitin and chitosan yields were 89.39 mg/g and 57.82 mg/g, respectively, and both were obtained in trial 2 (10% CW, 4% CSL). Chitin and chitosan showed a degree of deacetylation of 40.98% and 88.24%, and a crystalline index of 35.80% and 23.82%, respectively, and chitosan showed low molecular weight (LMW 5.2 × 10³ Da). Chitin and chitosan can be considered non-irritating, due to the fact they do not promote vascular change. It was demonstrated that CSL and CW are effective renewable agroindustrial alternative substrates for the production of chitin and chitosan.

Ultrasonication and steam-explosion as chitin pretreatments for chitin oligosaccharide production by chitinases of Lecanicillium lecanii.

In this study, chitin oligosaccharides have been successfully produced using chitinases from submerged fermentation of Lecanicillium lecanii. The highest Hex, Chit and Prot production was 0.14, 0.26 and 2.05 U/mg of protein, respectively, which were attained varying pH from 5 to 8 after 96 h. Culture conditions conducted at constant pH of 6 resulted in significantly lower enzyme production. The crude enzyme was partially purified by salting out with (NH4)2SO4 followed by size exclusion chromatography to isolate the chitinase mixture for further chitin hydrolysis assays. In this regard, chitin substrates were pretreated with sonication and steam explosion prior to enzymatic reaction. Structural changes were observed with steam explosion with 11.28% reduction of the crystallinity index attained with the lowest chitin/water ratio (0.1g/mL). Pretreated chitins reached the highest production of reducing sugars (0.37 mg/mL) and GlcNAc (0.59 mg/mL) in 23.6% yield.

Gene expression modulation by paraquat-induced oxidative stress conditions in Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), the most common systemic mycosis in Latin America. The infection is initiated by inhalation of environmentally dispersed conidia produced by the saprophytic phase of the fungus. In the lungs, P. brasiliensis assumes the parasitic yeast form and must cope with the adverse conditions imposed by cells of the host immune system, which includes a harsh environment, highly concentrated in reactive oxygen species (ROS). In this work, we used the ROS-generating agent paraquat to experimentally simulate oxidative stress conditions in order to evaluate the stress-induced modulation of gene expression in cultured P. brasiliensis yeast cells, using a microarray hybridization approach. The large-scale evaluation inherent to microarray-based analyses identified 2070 genes differentially transcribed in response to paraquat exposure, allowing an integrated visualization of the major metabolic changes that constitute the systemic defense mechanism used by the fungus to overcome the deleterious effects of ROS. These include overexpression of detoxifying agents, as well as of molecular scavengers and genes involved in maintenance of the intracellular redox potential. Particularly noteworthy was to verify that the oxidative stress resistance mechanism of P. brasiliensis also involves coordinated overexpression of a series of genes responsible for chitin-biosynthesis, suggesting that this pathway may constitute a specific regulon. Further analyses aiming at confirming and understanding the mechanisms that control such regulon may provide interesting new targets for chemotherapeutic approaches against P. brasiliensis and other pathogenic fungi.

Biotechnological processes for chitin recovery out of crustacean waste: a mini-review

Background: Chitin is an important natural resource. The annual worldwide production is estimated in approximately 10(10)-10(12) ton. It is produced by arthropods (insects and crustaceans), molluscs and fungi. Its main biological function is structural. Crustacean shells are the most important chitin source for commercial use due to its high content and ready availability. Chitin and its derivatives have great economical value because of their numerous applications: food, cosmetics, pharmaceuticals, textile industries, waste water treatment and agriculture. In nature, chitin is closely associated with proteins, minerals, lipid and pigments, which have to be removed. Results: Several techniques to extract chitin from different sources have been reported. The most common method for recovery of chitin from crustacean shells is the chemical procedure. It involves two mayor steps: elimination of inorganic matter (demineralization) and extraction of protein matter (deproteination) using strong acids and bases. However, these processes may cause depolymerization affecting the polymer properties such as molecular weight, viscosity and degree of acetylation. In addition, the chemical purification of chitin is hazardous, energy consuming and threatening to the environment. As an alternative to the chemical process, different biological processes have been investigated: microbiological fermentation and methodologies using enzymatic crude extracts or isolated enzymes. Conclusions: The results reported are extremely variable; however, they offer new perspectives for the production of chitin with the concomitant reduction of the environmental impact.

Silencing of Entamoeba histolytica glucosamine 6-phosphate isomerase by RNA interference inhibits the formation of cyst-like structures.

Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs) and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.

Chitin and L(+)-lactic acid production from crab (Callinectes bellicosus) wastes by fermentation of Lactobacillus sp. B2 using sugar cane molasses as carbon source.

Crab wastes are employed for simultaneous production of chitin and L(+)-lactic acid by submerged fermentation of Lactobacillus sp. B2 using sugar cane molasses as carbon source. Response surface methodology was applied to design the culture media considering demineralization. Fermentations in stirred tank reactor (2L) using selected conditions produced 88% demineralization and 56% deproteinization with 34% yield of chitin and 19.5 gL(-1) of lactic acid (77% yield). The chitin purified from fermentation displayed 95% degree of acetylation and 0.81 and 1 ± 0.125% of residual ash and protein contents, respectively.

Expression of Paracoccidioides brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant demonstrates its functionality as a chitin synthase gene.

We report the isolation and sequencing of CHS3, a gene that encodes one of several chitin synthases in Paracoccidioides brasiliensis, a medically important fungus restricted geographically to Latin America. The gene contains a single open reading frame of 3817 bp with two introns (71 and 86 bp) and encodes a 1220 amino acid polypeptide with high similarity to other fungal chitin synthases. Northern analysis reveals a high expression of CHS3 in the pathogenic yeast-like phase of the fungus and at the end of the mycelium-yeast transition. Expression of P. brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant enhanced calcofluor white staining in parallel to an increase in total chitin synthase activity and chitin content in its cell wall.

Effect of the chitin synthesis inhibitor triflumuron on the development, viability and reproduction of Aedes aegypti.

The control of Aedes aegypti is impaired due to the development of resistance to chemical insecticides. Insect Growth Regulators (IGR) exhibit distinct mechanisms of action and are considered potential vector control alternatives. Studies regarding the effects of sublethal IGR doses on the viability of resulting adults will contribute to eval-uating their impact in the field. We analyzed several aspects of Ae. aegypti adults surviving exposure to a partially lethal dose of triflumuron, a chitin synthesis inhibitor. A highly significant difference in the proportion of males and females was noted in the triflumuron-exposed group (65.0% males) compared to the controls (50.2% males). Triflumuron affected adult longevity, particularly for females; after 16 days, only 29.2% of males and 13.8% of females were alive, in contrast with 94% survival of the control mosquitoes. The locomotor activity was reduced and the blood-feeding ability of the treated females was also affected (90.4% and 48.4% of the control and triflumuron-exposed females, respectively, successfully ingested blood). Triflumuron-surviving females ingested roughly 30% less blood and laid 25% fewer eggs than the control females. The treated males and females exhibited a diminished ability to copulate, resulting in less viable eggs.

Effect of the chitin synthesis inhibitor triflumuron on the development, viability and reproduction of Aedes aegypti

The control of Aedes aegypti is impaired due to the development of resistance to chemical insecticides. Insect Growth Regulators (IGR) exhibit distinct mechanisms of action and are considered potential vector control alternatives. Studies regarding the effects of sublethal IGR doses on the viability of resulting adults will contribute to eval-uating their impact in the field. We analyzed several aspects of Ae. aegypti adults surviving exposure to a partially lethal dose of triflumuron, a chitin synthesis inhibitor. A highly significant difference in the proportion of males and females was noted in the triflumuron-exposed group (65.0 percent males) compared to the controls (50.2 percent males). Triflumuron affected adult longevity, particularly for females; after 16 days, only 29.2 percent of males and 13.8 percent of females were alive, in contrast with 94 percent survival of the control mosquitoes. The locomotor activity was reduced and the blood-feeding ability of the treated females was also affected (90.4 percent and 48.4 percent of the control and triflumuron-exposed females, respectively, successfully ingested blood). Triflumuron-surviving females ingested roughly 30 percent less blood and laid 25 percent fewer eggs than the control females. The treated males and females exhibited a diminished ability to copulate, resulting in less viable eggs.

Chitin synthesis inhibitor effect on Aedes aegypti populations susceptible and resistant to organophosphate temephos.

BACKGROUND: In Brazil, dengue vector control is hampered by the resistance of Aedes aegypti L. populations to organophosphates (OPs). Insect growth regulators (IGRs) are a promising alternative, as their mechanisms of action are different from those of conventional insecticides. The authors analysed the effect of the IGR triflumuron, a chitin synthesis inhibitor, on the Ae. aegypti insecticide-susceptible strain Rockefeller, as well as on field populations both susceptible (TemS) and resistant (TemR) to the OP temephos. RESULTS: Triflumuron arrested development and inhibited adult emergence of the Rockefeller strain in a dose-dependent way (EI(50) and EI(90) of 0.8 and 1.8 microg L(-1) respectively). A direct relationship between triflumuron concentration and the precocity of its effects was evident. TemS and TemR temephos resistance ratios (RR(90)) were 4.5 and 13.8, triflumuron RR(90) being 1.0 and 1.3 respectively. CONCLUSION: The IGR triflumuron exhibited a dose-dependent effect against the reference Ae. aegypti Rockefeller strain. It was also effective against two field populations, regardless of their OP resistance status. The present results are discussed in the context of utilization of chitin synthesis inhibitors as potential alternatives in the control of Ae. aegypti in Brazil.

A chitin-like component in Aedes aegypti eggshells, eggs and ovaries.

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.

Chitosomes: past, present and future.

José Ruiz-Herrera's discovery that chitin microfibrils could be made by a fungal extract paved the way for elucidating the intracellular location of chitin synthetase. In collaboration with Charles Bracker, chitosomes were identified as the major reservoir of chitin synthetase in fungi. Unique in size, buoyant density, and membrane thickness, chitosomes were found in a wide range of fungi. Their reversible dissociation into 16S subunits is another unique property of chitosomes. These 16S subunits are the smallest molecular entities known to retain chitin synthetase activity. Further dissociation leads to complete loss of activity. From studies with secretory mutants, yeast researchers concluded that chitosomes were components of the endocytosis pathway. However, key structural and enzymatic characteristics argue in favor of the chitosome being poised for exocytotic delivery rather than endocytotic recycling. The chitosome represents the main vehicle for delivering chitin synthetase to the cell surface. An immediate challenge is to elucidate chitosome ontogeny and the role of proteins encoded by the reported chitin synthetase genes in the structure or function of chitosomes. The ultimate challenge would be to understand how the chitosome integrates with the cell surface to construct the organized microfibrillar skeleton of the fungal cell wall.

Analysis of phenomena involved in the apical growth of Phycomyces blakesleeanus.

The effects of the Ca(2+)/H(+) exchanger A23187 and the K(+)/H(+) exchanger nigericin, the electrogenic membrane-potential depleters valinomycin and CCCP, and the calcium channel blockers ruthenium red, nifedipine, and nitrendipine on the apical growth of Phycomyces blakesleeanus were analyzed. While all of the compounds inhibited the growth of germlings in liquid medium, the Ca(2+) channel blockers were the least effective. Chitin synthesis in vivo was also sensitive to the inhibitors; here again, the calcium channel blockers were less efficient, and their effect occurred after a lag phase, in contrast to the electroneutral ionophores whose effects were immediate. The ionophores rapidly inhibited protein secretion, and reduced the number of secretory vesicles and chitosomes in the hyphal apex of P. blakesleeanus. The results suggest that not only tip-to-base calcium gradients but also transmembrane ionic gradients and membrane potential have a role in the apical growth of P. blakesleeanus. They are probably involved in the formation, migration, and/or fusion with the plasmalemma of secretory vesicles and chitosomes.

Chitosan from Syncephalastrum racemosum used as a film support for lipase immobilization.

Chitosan from a native Mucoralean strain, Syncephalastrum racemosum, isolated from herbivorous dung (Northeast-Brazil), was used as a film support for lipase immobilization. S. racemosum showed highest chitosan yield (152 mg g dry mycelia weight(-1); 15.2% of dry mycelia weight) among the nine strains screened, which presented 89% D-glucosamine. A chitosan film was used for lipase (EC 3.1.1.3) immobilization using glutaraldehyde as a bifunctional agent. The immobilized lipase retained 47% (12.6 micromol s(-1) m(-2)) of its initial catalytic activity after four cycles of reaction. This result is comparable (same order of magnitude) to that of the enzyme immobilized on film made from commercially available crustacean chitosan.