AmorimRVS03_Chitosan From Syncephalastrum Racemosum

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Chitosan from Syncephalastrum racemosum used as a film support

for lipase immobilization

R.V.S. Amorim a,b,*, E.S. Melo a, M.G. Carneiro-da-Cunha a, W.M. Ledingham a,G.M. Campos-Takaki c

a Laboratoorio de Imunopatologia Keizo Asami––LIKA and Departamento de Bioquıımica, Universidade Federal de Pernambuco-UFPE,

Av. Professor Moraes Rego s/n, Cidade Universitaaria, 50670-901 Recife, PE, Brazil b Departamento de Biologia Molecular, Universidade Federal da Paraııba––UFPB, Campus I––Cidade Universitaaria,

58051-900, Jo~aao Pessoa, PB, Brazil c N uucleo de Pesquisas em Ci ̂eencias Ambientais––NPCIAMB and Departamento de Quıımica, Universidade Catoolica de Pernambuco––UNICAP,

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 film support for lipase immobilization. S. racemosum showed highest chitosan yield (152 mg g dry mycelia weightÀ1; 15.2%

of dry mycelia weight) among the nine strains screened, which presented 89% DD-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 lmolsÀ1 mÀ2)

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.

Ó 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-DD-glucan, is a deacetylated

derivative from chitin, the second most abundant poly-

saccharide in nature. It has been described as occurring

in the cell wall of some fungi, particularly in the Zygo-

mycetes (Miyoshi et al., 1992; Tan et al., 1996).

Commercially available chitosan is obtained from

crustacea and has been used in a wide variety of appli-cations. Its membrane has several uses including food

processing, protein purification, and skin replacement

technology (Muzzarelli, 1983). It has also been used as a

support for enzyme immobilization, since it offers con-

siderable advantages such as form versatility (powder,

gel beads, flocks, fibres, capsules and membranes), low

biodegradability and cost, high affinity towards proteins

and absence of toxicity (No and Meyers, 1995). En-

zymes such as catalase (Cßetinus and €OOztop, 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 toextend their active life have been intensively investi-

gated and developed. Numerous methods of lipase im-

mobilization 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 sol-

ubilise 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.

* Corresponding author. Address: Laboratoorio de Imunopatologia

Keizo Asami––LIKA, Universidade Federal de Pernambuco-UFPE,

Av. Professor Moraes Rego s/n, Cidade Universitaaria, 50670-901

Recife, PE, Brazil. Fax: +55-81-3271-8485.

E-mail address: [email protected] (R.V.S. Amorim).

0960-8524/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0960-8524(03)00035-X

Bioresource Technology 89 (2003) 35–39

http://mail%20to:%[email protected]/

http://mail%20to:%[email protected]/



2. Methods

2.1. Materials

Lipase (EC 3.1.1.3) from Candida cylindraceae (type

VII), arabic gum, Triton X-100, Tris–HCl buffer, crus-

tacean chitosan (C-0792) and p -nitrophenyl-palmitate

(pNPP) were obtained from Sigma Chemical Co. (St.,

Louis, Mo., USA). Glutaraldehyde (25% v/v) and iso-

propanol were purchased from Merck & Co., Inc.

(Germany). All other chemicals used were of analytical

grade.

Mucoralean strains: Mucor circinelloides, Syncepha-

lastrum racemosum and Circinella muscae were isolated

from herbivorous dung (Northeast-Brazil). Cunning-

hamella bertholletiae (IFM 46.114), Cunninghamella

echinulata (URM 2136), Cunninghamella ramosa (URM

1918), Cunninghamella blakesleeana (URM 168), Cunn-

inghamella elegans (IFM 30.505) and Mucor rouxii

(ATCC 24.905) were obtained from: URM––Depart-ment of Mycology-UFPE-Brazil; ATCC––American

Type Culture Collection and IFM––Institute 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 final concentration of 104

spores mlÀ1

. Thefungi strains were grown at 28 °C for 120 h on an orbital

shaker at 150 rpm. Mycelia from Mucoralean strains

were harvested by vacuum filtration, washed with dis-

tilled 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 cmÀ1. Colorimetric measurement of chitosan was

based on hydrolysis with 4 M hydrochloric acid for 12 h

at 105 °C and the liberated DD-glucosamine estimated

using the Elson–Morgan reaction as described by Blix(1968). Thermogravimetric analyses were carried out

using a SHIMADZU TGA-50 analyser, under nitrogen

flux and at 10 °C/min temperature gradient.

2.3. Lipase immobilization on chitosan film

Chitosan films were prepared as described by Car-

neiro-da-Cunha et al. (1999) and then thoroughly wa-

shed with l M NaOH and distilled water. The films (%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 mlÀ1) in 50 mM Tris–HCl buffer pH 8.0

overnight at 4 °C. The films were subsequently washed

three times with 1 M NaCl, afterwards with 0.1% of

Triton X-100 in Tris–HCl 50 mM buffer and finally with

Tris–HCl 50 mM buffer. The lipolytic activity was de-

termined according to the method of Winkler and

Stuckmann (1979) and consisted of the hydrolysis of

pNPP at 37 °C. One unit of activity was defined 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 as-

says using the same enzyme sample. After each reaction,

the biocatalyst was washed with 1 M NaCl and 50 mM

Tris–HCl buffer pH 8.0.

2.4. Statistical analysis

Analysis of variance and TukeyÕs studentized rangetest (Snedecor and Cochran, 1980) were used to deter-

mined differences 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 cul-

tivation period) in randomised complete block design

was used and results are expressed as averageÆ standard

deviation ( X Æ sd). The treatmentÕs averages with the

same letters do not differ significantly ( 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 po-

tential ability of these fungi to produce chitosan was

compared with the collection strains. Data on the

chitosan yields of different 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 significant differences ( 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 ob-

served 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. echinu-

lata declined in chitosan content over time and pre-

sented the highest chitosan content in 2 days of culture.

The other species revealed different behaviours, with C.

ramosa exhibiting the highest chitosan yield of 12.3%

with 5 days of culture.

36 R.V.S. Amorim et al. / Bioresource Technology 89 (2003) 35–39



Among all Mucoralean strains, S. racemosum, a

native strain, was the best strain for chitosan produc-

tion, maintaining highest amount of extractable chito-

san 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 yieldfor chitosan.

Screening different 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 different 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, indi-

cating the influence of growth time on the chitosan

contents found in the fungi.

The fungus M. rouxii has been intensively investi-

gated 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

different culture conditions than White et al. (1979).

Synowiecki and Al-Khateeb (1997) studied the influ-

ence of growth time of M. rouxii on the contents of the

chitosan as well as the yield of chitosan during the iso-

lation 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 influence 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 pro-

longed growth did not influence the yield of extractable

chitosan. The biomass reached a density of 8 g lÀ1 at

approximately 2 days of cultivation and the high

chitosan yield 157 mg (g dry mycelia weight)À1 (15.7% of

dry mycelia) was observed in 3 days of cultivation.

However, low variation was observed after 2 days, in-

dicating 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 stagesuggest that the chitosan formed by chitin deacetylase

was prevalent in this stage, confirmed by previous re-

sults with this fungus where highest chitin deacetylase

activity of 0.05 U mg proteinÀ1 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 suscepti-

ble to this enzyme.

3.2. Chitosan characterization

Analysis of the DD-glucosamine residue compositionof chitosan from fungi revealed that S. racemosum has

the highest DD-glucosamine content 88.9% (Fig. 2). How-

ever, significant variation ( p 6 0:05) occurred only in

Fig. 1. Chitosan percentage from dry mycelia weight from different

Mucoralean strains (C. bertholetiae ––CB; C. echinulata ––CE; C. ra-

mose ––CR; C. blakesleeana ––CBL; C. elegans ––CEL; M. rouxii ––MR;

M. circinelloides ––MC; S. racemosum ––SR and Circinella muscae ––

CM), grown in YPD medium. Mean values from three replicates.

Averages with different letters differ statistically by Tukey test

( p 6 0:05).

Fig. 2. DD-glucosamine (% of chitosan) from different Mucoralean

strains (C. bertholetiae ––CB; C. echinulata ––CE; C. ramose ––CR; C.

blakesleeana ––CBL; C. elegans ––CEL; M. rouxii ––MR; M. circi-

nelloides ––MC; S. racemosum ––SR and Circinella muscae ––CM)

grown in YPD medium. Mean values from six replicates. Average with

different letters differ statistically by Tukey test ( p 6 0:05).

R.V.S. Amorim et al. / Bioresource Technology 89 (2003) 35–39 37



comparison with C. ramosa, M. circinelloides and C.

muscae. No statistically significant effect was observed

when the culture was grown during 2 or 5 days. High

DD-glucosamine content is considered an important prop-

erty since amino groups present in chitosan are the re-

active functional groups used for direct reactions with

glutaraldehyde and enzymes in the immobilization

process (Krajewska, 1991).

To support the fact that acetic acid-extracted mate-

rials from fungal cells were chitosan, the infrared spec-

trum profile 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

cmÀ1, the amide band at 1655–1550 cmÀ1, the amine

band at 1630–1550 cmÀ1 and the C–H band at 3250

cmÀ1.

In addition, the degree of N -acetylation can be mea-

sured from IR spectra by the method of Roberts (1992).

The estimate is determined from the ratio of the ab-sorbance of the amide II band at %1655 cmÀ1 to that of

the C–H band at 3450 cmÀ1 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 difference can be attributed to the fact that the

fungal chitosan has been submitted only to a pre-puri-

fication process with water, ethanol and acetone, during

the final 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 film and

stability studies

C. cylindraceae lipase was immobilized on a film 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 film from S. racemosum and from

crustacean source were 47% and 42% of their initial

activities, respectively, dropping from 26.8 to 12.6

lmolsÀ1 mÀ2 and from 30.0 to 12.6 lmolsÀ1 mÀ2.

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 sÀ1 mÀ2 after the fourth

use, which represented 5% of the initial activity (245.0

lmolsÀ1 mÀ2). 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 first lipolytic activity de-

termination. Many physically adsorbed enzymes were

probably washed out before the first activity determi-

nation and this accounts for the difference in the initial

activity. Carneiro-da-Cunha et al. (1999) had previ-

ously determined that successive solubilization of theweakly 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 obser-

vation.

A decrease in activity after the first washing proce-

dure and further stabilization of the immobilized enzy-

matic preparation has been described in the literature

for covalent binding immobilization of other enzymes

on chitosan. Abdel-Naby et al. (1999) reported a dex-

tranase from Penicillium funicolosum 258 with residual

activity of 63% and Cß

etinus and€

OOztop (2000) a catalasewith residual activity of about 50%.

Other enzymatic preparations have been reported

using chitosan as support. Spagna et al. (2001) found

residual activity with a-LL-rhamnopyranosidase from

Aspergillus niger immobilized on chitosan and on di-

ethylaminoethyl chitosan (DE-chitosan), both also ac-

tivated with glutaraldehyde, showing activities of 21 and

22 U gÀ1 of support, respectively.

Most of the early studies reported in the literature on

the immobilization of lipases refer to just the first use of

the biocatalyst, such as 12.0 lmolsÀ1 mÀ2 reported by

Pronk et al. (1988) for an immobilized lipase on cellulose

support. Currently, it is possible to find reports of im-mobilization with more than one use, with different

enzymes, supports and processes. Oliveira et al. (2000),

in successive esterification activity assays, after 12 cycles,

retained 77% (from 210.0 to 161.7 mmol gÀ1 minÀ1) 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 five hydrolysis cycles. Therefore, comparing results

38 R.V.S. Amorim et al. / Bioresource Technology 89 (2003) 35–39



found in the literature with those present in the current

study becomes difficult sometimes since it is often un-

clear 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 immobi-

lization support.

Acknowledgements

The authors acknowledge the financial 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 Uni-

versidade do Vale do Acarauu for kindly supplying the

Mucoralean wild strains and to Dr. Cosme Rafael

Martinez for statistical analysis assistance.

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AmorimRVS03_Chitosan From Syncephalastrum Racemosum

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