Bioresource Technology 89 (2003) 3539

Chitosan from Syncephalastrum racemosum used as a lm support for lipase immobilization

R.V.S. Amorim
a

a,b,*

, E.S. Melo a, M.G. Carneiro-da-Cunha a, W.M. Ledingham a, G.M. Campos-Takaki c

Laboratrio de Imunopatologia Keizo AsamiLIKA and Departamento de Bioqumica, Universidade Federal de Pernambuco-UFPE, o Av. Professor Moraes Rego s/n, Cidade Universitria, 50670-901 Recife, PE, Brazil a b Departamento de Biologia Molecular, Universidade Federal da ParabaUFPB, Campus ICidade Universitria, a 58051-900, Jo~o Pessoa, PB, Brazil a Ncleo de Pesquisas em Ci^ncias AmbientaisNPCIAMB and Departamento de Qumica, Universidade Catlica de PernambucoUNICAP, u e o Recife, PE, Brazil Received 2 April 2001; received in revised form 12 September 2002; accepted 11 January 2003

Abstract Chitosan from a native Mucoralean strain, Syncephalastrum racemosum, isolated from herbivorous dung (Northeast-Brazil), was used as a lm support for lipase immobilization. S. racemosum showed highest chitosan yield (152 mg g dry mycelia weight1 ; 15.2% of dry mycelia weight) among the nine strains screened, which presented 89% D -glucosamine. A chitosan lm was used for lipase (EC 3.1.1.3) immobilization using glutaraldehyde as a bifunctional agent. The immobilized lipase retained 47% (12.6 lmol s1 m2 ) 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 lm made from commercially available crustacean chitosan. 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Chitosan; Mucoralean; Film; Immobilization; Lipase

1. Introduction Chitosan, a cationic biopolymer consisting of (1,4)linked 2-amino-deoxy-b-D -glucan, is a deacetylated derivative from chitin, the second most abundant polysaccharide in nature. It has been described as occurring in the cell wall of some fungi, particularly in the Zygomycetes (Miyoshi et al., 1992; Tan et al., 1996). Commercially available chitosan is obtained from crustacea and has been used in a wide variety of applications. Its membrane has several uses including food processing, protein purication, and skin replacement technology (Muzzarelli, 1983). It has also been used as a support for enzyme immobilization, since it oers con-

* Corresponding author. Address: Laboratrio de Imunopatologia o Keizo AsamiLIKA, Universidade Federal de Pernambuco-UFPE, Av. Professor Moraes Rego s/n, Cidade Universitria, 50670-901 a Recife, PE, Brazil. Fax: +55-81-3271-8485. E-mail address: ramorim@dbm.ufpb.br (R.V.S. Amorim).

siderable advantages such as form versatility (powder, gel beads, ocks, bres, capsules and membranes), low biodegradability and cost, high anity towards proteins and absence of toxicity (No and Meyers, 1995). En zymes such as catalase (Cetinus and Oztop, 2000), tyro sinase (Carvalho et al., 2000), dextranase (Abdel-Naby et al., 1999), and beta-galactosidase (Shin et al., 1998) have been immobilized on commercial crustacean chitosan. Commercial lipases are expensive and methods to extend their active life have been intensively investigated and developed. Numerous methods of lipase immobilization are available, but adsorption is the most frequently reported methodology in the literature (Gunnlaugsdottir et al., 1998). However, adsorption is stabilised by weak forces and surfactants can often solubilise most of the lipolytic activity. The aim of this work was to screen the production of chitosan from native and culture collection Mucoralean strains looking for its utilization as a support for lipase covalent immobilization.

0960-8524/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-8524(03)00035-X

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2. Methods 2.1. Materials Lipase (EC 3.1.1.3) from Candida cylindraceae (type VII), arabic gum, Triton X-100, TrisHCl buer, crustacean chitosan (C-0792) and p-nitrophenyl-palmitate (pNPP) were obtained from Sigma Chemical Co. (St., Louis, Mo., USA). Glutaraldehyde (25% v/v) and isopropanol were purchased from Merck & Co., Inc. (Germany). All other chemicals used were of analytical grade. Mucoralean strains: Mucor circinelloides, Syncephalastrum racemosum and Circinella muscae were isolated from herbivorous dung (Northeast-Brazil). Cunninghamella bertholletiae (IFM 46.114), Cunninghamella echinulata (URM 2136), Cunninghamella ramosa (URM 1918), Cunninghamella blakesleeana (URM 168), Cunninghamella elegans (IFM 30.505) and Mucor rouxii (ATCC 24.905) were obtained from: URMDepartment of Mycology-UFPE-Brazil; ATCCAmerican Type Culture Collection and IFMInstitute for Food Microbiology, Chiba University, Japan. 2.2. Chitosan production, extraction and analysis The strains were maintained on potato dextrose agar (PDA) slants at 4 C. Cultures were sub-cultured on PDA plates, incubated at 28 C for 5 days and the spore suspension was used to inoculate 200 ml of nutrient broth (YPD medium; Bartinicki-Garcia and Nickerson, 1962) to a nal concentration of 104 spores ml1 . The fungi strains were grown at 28 C for 120 h on an orbital shaker at 150 rpm. Mycelia from Mucoralean strains were harvested by vacuum ltration, washed with distilled water and freeze dried. Chitosan extraction was carried out as described by Synowiecki and Al-Khateeb (1997). The IR spectra of chitosan was carried out using the KBr method and the degree of acetylation (DA) determined according to the method of Roberts (1992) using the relative absorbance at wavenumbers of 1655 3450 cm1 . Colorimetric measurement of chitosan was based on hydrolysis with 4 M hydrochloric acid for 12 h at 105 C and the liberated D -glucosamine estimated using the ElsonMorgan reaction as described by Blix (1968). Thermogravimetric analyses were carried out using a SHIMADZU TGA-50 analyser, under nitrogen ux and at 10 C/min temperature gradient. 2.3. Lipase immobilization on chitosan lm Chitosan lms were prepared as described by Carneiro-da-Cunha et al. (1999) and then thoroughly washed with l M NaOH and distilled water. The lms (%4 cm2 ) were activated with 5% glutaraldehyde solution (v/v) for 3 h. After that they were thoroughly washed

with distilled water and immersed in 10 ml of lipase solution (20 mg ml1 ) in 50 mM TrisHCl buer pH 8.0 overnight at 4 C. The lms were subsequently washed three times with 1 M NaCl, afterwards with 0.1% of Triton X-100 in TrisHCl 50 mM buer and nally with TrisHCl 50 mM buer. The lipolytic activity was determined according to the method of Winkler and Stuckmann (1979) and consisted of the hydrolysis of pNPP at 37 C. One unit of activity was dened as the amount of lipase that yields 1 lmol of free fatty acid per minute under the assay conditions. Operational stability of the immobilized biocatalyst was established by conducting a series of successive assays using the same enzyme sample. After each reaction, the biocatalyst was washed with 1 M NaCl and 50 mM TrisHCl buer pH 8.0. 2.4. Statistical analysis Analysis of variance and Tukeys studentized range test (Snedecor and Cochran, 1980) were used to determined dierences in mean values of the date from 3 to 6 replicates. The variance analyses were carried out with the software Statistic (Statsoft, Inc., Tulsa, USA, 1997). A two-factor experiment (Mucoralean strains and cultivation period) in randomised complete block design was used and results are expressed as average standard deviation (X sd). The treatments averages with the same letters do not dier signicantly (p 6 0:05).

3. Results and discussion 3.1. Screening of fungal strains for chitosan production Extractable chitosan content was examined from nine Mucoralean strains including three wild fungi isolated from herbivorous dung in Northeast Brazil. The potential ability of these fungi to produce chitosan was compared with the collection strains. Data on the chitosan yields of dierent Zygomycetes strains after 2 and 5 days of culture is shown in Fig. 1. The variance analyses between the chitosan production and growth time revealed signicant dierences (p 6 0:05) among Mucoralean strains. The chitosan contents in dry mycelia varied widely among the strains of Mucoralean studied, even between species of the same genus. This variation is clearly observed in Cunninghamella genus where the extractable chitosan ranged from 2.3% to 12.9%. The Tukey test (p 6 0:05) indicated that C. bertholletiae and C. echinulata declined in chitosan content over time and presented the highest chitosan content in 2 days of culture. The other species revealed dierent behaviours, with C. ramosa exhibiting the highest chitosan yield of 12.3% with 5 days of culture.

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Fig. 1. Chitosan percentage from dry mycelia weight from dierent Mucoralean strains (C. bertholetiaeCB; C. echinulataCE; C. ramoseCR; C. blakesleeanaCBL; C. elegansCEL; M. rouxiiMR; M. circinelloidesMC; S. racemosumSR and Circinella muscae CM), grown in YPD medium. Mean values from three replicates. Averages with dierent letters dier statistically by Tukey test (p 6 0:05).

Among all Mucoralean strains, S. racemosum, a native strain, was the best strain for chitosan production, maintaining highest amount of extractable chitosan in 2 and 5 days of culture, namely, 13.4% and 15.2%, respectively (Fig. 1). The results on chitosan production obtained in the current work are better than the responses observed by Miyoshi et al. (1992). In their study, other Zygomycetes strains including Cunninghamella species had a chitosan yield ranging from 1.2% to 10.4% of dry mycelia with 2 days of growth, with Absidia coerulea showing the highest yield for chitosan. Screening dierent Zygomycetes strains, Tan et al. (1996) observed that C. echinulata exhibited the highest chitosan yield with 7% of dry mycelia obtained with 4 days of culture using dierent culture conditions and chitosan extraction method. In the current work, using the same fungus, chitosan yields of 12.9% and 5.1% for 2 and 5 days of culture, respectively, were found, indicating the inuence of growth time on the chitosan contents found in the fungi. The fungus M. rouxii has been intensively investigated in the literature in relation to chitosan production. In 1979, White et al. reported a chitosan content of 6 9% of dry mycelia weight. In the current work, the chitosan yield from M. rouxii ATCC 24 905 was 3.3% and 5.0% for 2 and 5 days of culture, respectively, using dierent culture conditions than White et al. (1979). Synowiecki and Al-Khateeb (1997) studied the inuence of growth time of M. rouxii on the contents of the chitosan as well as the yield of chitosan during the isolation process. They demonstrated that the content of extractable chitosan reached a maximum (7.3% of the dry mycelia) after 2 days of culture and prolonged growth up to 5 days did not inuence the yield of ex-

tractable chitosan. S. racemosum studied in current work exhibited similar behaviour regarding chitosan production during the course of growth time. The dry weight of mycelia (biomass) and extractable chitosan increased over a period of time until 2 days and prolonged growth did not inuence the yield of extractable chitosan. The biomass reached a density of 8 g l1 at approximately 2 days of cultivation and the high chitosan yield 157 mg (g dry mycelia weight)1 (15.7% of dry mycelia) was observed in 3 days of cultivation. However, low variation was observed after 2 days, indicating that later exponential growth phase of the fungus is the best period of growth for optimal chitosan production. During growth of S. racemosum, the pH of the culture remained stable until 36 h, after that the pH reached 7.5 coinciding with the stationary growth phase. Chitin deacetylase is an enzyme that catalyses the conversion of chitin to chitosan by the deacetylation of N -acetyl-glucosamine residues in the fungal cell wall. The highest chitosan yields from the initial growth stage suggest that the chitosan formed by chitin deacetylase was prevalent in this stage, conrmed by previous results with this fungus where highest chitin deacetylase activity of 0.05 U mg protein1 was found in 2 days of culture. Davis and Bartinicki-Garcia (1984) reported similar observations indicating that during initial growth chitin is less crystalline and thus more susceptible to this enzyme. 3.2. Chitosan characterization Analysis of the D -glucosamine residue composition of chitosan from fungi revealed that S. racemosum has the highest D -glucosamine content 88.9% (Fig. 2). However, signicant variation (p 6 0:05) occurred only in

Fig. 2. D -glucosamine (% of chitosan) from dierent Mucoralean strains (C. bertholetiaeCB; C. echinulataCE; C. ramoseCR; C. blakesleeanaCBL; C. elegansCEL; M. rouxiiMR; M. circinelloidesMC; S. racemosumSR and Circinella muscaeCM) grown in YPD medium. Mean values from six replicates. Average with dierent letters dier statistically by Tukey test (p 6 0:05).

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comparison with C. ramosa, M. circinelloides and C. muscae. No statistically signicant eect was observed when the culture was grown during 2 or 5 days. High D -glucosamine content is considered an important property since amino groups present in chitosan are the reactive functional groups used for direct reactions with glutaraldehyde and enzymes in the immobilization process (Krajewska, 1991). To support the fact that acetic acid-extracted materials from fungal cells were chitosan, the infrared spectrum prole of the fraction extracted from S. racemosum was conducted. The spectrum was similar to those shown by crustacean chitosan with the characteristic bands of chitosan such as the hydroxyl band at 3450 cm1 , the amide band at 16551550 cm1 , the amine band at 16301550 cm1 and the CH band at 3250 cm1 . In addition, the degree of N -acetylation can be measured from IR spectra by the method of Roberts (1992). The estimate is determined from the ratio of the absorbance of the amide II band at %1655 cm1 to that of the CH band at 3450 cm1 and was 28% in chitosan from S. racemosum and 20% from crustacean (Sigma). Amorim et al. (2001) found the degree of N -acetylation to be 20% in chitosan from C. elegans with 2 days of cultivation. However, Miyoshi et al. (1992) found the degree of N -acetylation to be 59% and 35% from chitosan of M. rouxii and C. blakesleeana, respectively. Moreover, the large positive charge density due to the low degree of N -acetylation of chitosans is an important chemical property for their utilization as support for enzyme immobilization. The thermogravimetric analysis showed that the chitosan preparations obtained from the cells of S. racemosum contain about 4% of inorganic impurities, while the commercial crustacean chitosan contains 2%. The dierence can be attributed to the fact that the fungal chitosan has been submitted only to a pre-purication process with water, ethanol and acetone, during the nal step of chitosan extraction. McGahren et al. (1984) found that the impurities present in the hyphal material are likely to be of three types, proteinaceous material due to inadequate alkaline treatment, triolein and related glycerides, and sodium salts of oleic and related acids. 3.3. Immobilization of lipase onto chitosan lm and stability studies C. cylindraceae lipase was immobilized on a lm of chitosan obtained from mycelia of S. racemosum and from a crustacean source (Sigma) using glutaraldehyde as a bifunctional agent. The operational stability of the immobilized systems was evaluated by reusing them four times and assaying their activities. The lipase activity dramatically dropped to about 45% of the initial activity

for the second use and remained stable until the fourth use in both preparations. The lipase activities for fourth use on chitosan lm from S. racemosum and from crustacean source were 47% and 42% of their initial activities, respectively, dropping from 26.8 to 12.6 lmol s1 m2 and from 30.0 to 12.6 lmol s1 m2 . A similar result for residual activity of immobilized lipase on chitosan from a crustacean source was also reported by Carneiro-da-Cunha et al. (1999), where the lipase activity was 12.0 lmol s1 m2 after the fourth use, which represented 5% of the initial activity (245.0 lmol s1 m2 ). The highest residual activity of 42%, found in the current study on the same support, is probably due to the utilization of Triton X-100 for support pre-wash, before the rst lipolytic activity determination. Many physically adsorbed enzymes were probably washed out before the rst activity determination and this accounts for the dierence in the initial activity. Carneiro-da-Cunha et al. (1999) had previously determined that successive solubilization of the weakly absorbed immobilized enzyme was observed when Triton X-100 was present in the emulsion during the enzymatic assays. The Triton X-100 washing was introduced in the present work because of this observation. A decrease in activity after the rst washing procedure and further stabilization of the immobilized enzymatic preparation has been described in the literature for covalent binding immobilization of other enzymes on chitosan. Abdel-Naby et al. (1999) reported a dextranase from Penicillium funicolosum 258 with residual activity of 63% and Cetinus and Oztop (2000) a catalase with residual activity of about 50%. Other enzymatic preparations have been reported using chitosan as support. Spagna et al. (2001) found residual activity with a-L -rhamnopyranosidase from Aspergillus niger immobilized on chitosan and on diethylaminoethyl chitosan (DE-chitosan), both also activated with glutaraldehyde, showing activities of 21 and 22 U g1 of support, respectively. Most of the early studies reported in the literature on the immobilization of lipases refer to just the rst use of the biocatalyst, such as 12.0 lmol s1 m2 reported by Pronk et al. (1988) for an immobilized lipase on cellulose support. Currently, it is possible to nd reports of immobilization with more than one use, with dierent enzymes, supports and processes. Oliveira et al. (2000), in successive esterication activity assays, after 12 cycles, retained 77% (from 210.0 to 161.7 mmol g1 min1 ) of an immobilized lipase activity by adsorption on styrene divinylbenzene copolymer (STYDVB). Murray et al. (1997), studying C. rugosa lipase immobilization on a lipophilic particulate support (porous Accurel EP400 power, a low density polyethylene powder, from Akzo Nobel, Germany) observed a residual activity of 65% after ve hydrolysis cycles. Therefore, comparing results

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found in the literature with those present in the current study becomes dicult sometimes since it is often unclear whether the enzyme is just adsorbed or covalenty bound to the support and information on washings of support is often omitted. In conclusion, the results show that S. racemosum is a good producer of alternative source chitosan with ability for application as immobilization support.

Acknowledgements The authors acknowledge the nancial support of CAPES, UNICAP, PRONEX and CNPq. We thanked the reviewers for their valuable contributions. We are also grateful to Dra. Maria Helena Alves from Universidade do Vale do Acara for kindly supplying the u Mucoralean wild strains and to Dr. Cosme Rafael Martinez for statistical analysis assistance. References
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