Enhancement of growth and chitosan production by Rhizopus oryzae in whey medium by plant growth hormones

Page 1

A

biaschp©

K

1

gccwtif[cctttmi

f

0d

International Journal of Biological Macromolecules 42 (2008) 120–126

Enhancement of growth and chitosan production by Rhizopus oryzae inwhey medium by plant growth hormones

Sudipta Chatterjee, Sandipan Chatterjee, Bishnu P. Chatterjee, Arun K. Guha ∗Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadvpur, Kolkata 700032, India

Received 16 April 2007; received in revised form 1 October 2007; accepted 4 October 2007Available online 12 October 2007

bstract

The effect of some plant growth hormones, viz., gibberellic acid, indole-3-acetic acid, indole-3-butyric acid, and kinetin on chitosan productiony Rhizopus oryzae in deproteinized whey was studied. Hormones, at different concentrations, increase the mycelial growth by 19–32%. However,ncrease in chitosan content of the mycelia was relatively small (1.7–14.3%) over the control. Maximum enhancement was observed with gibberelliccid. Fifty percent more chitosan could be obtained from 1 L of whey containing 0.1 mg/L gibberellic acid. Hormones, at higher dose, instead oftimulation inhibited both growth and mycelial chitosan content. This study showed that hormones have no influence on degree of deacetylation of

hitosan but increase the quality of the chitosan by increasing weight average molecular weight and decreasing polydispersity. All the hormonesad been found to enhance chitin deacetylase activity of R. oryzae by 1.067–1.267-fold and may be one of the reasons for increased chitosanroduction.

2007 Elsevier B.V. All rights reserved.

eacety

[z1itRtoKpc

org

eywords: Chitosan; Rhizopus oryzae; Whey; Plant growth hormone; Chitin d

. Introduction

Chitosan, a linear hydrophilic polysaccharide of �-1, 4-lucosamine, is obtained by thermo-chemical deacetylation ofhitin, which is found in the exoskeleton of crab, shrimp, lobster,rawfish and insects. Chitosan can also be isolated from the cellall of certain groups of fungi particularly zygomycetes. Chi-

osan finds numerous applications in food and pharmaceuticalndustries and also in environmental biotechnology, particularlyor the removal of toxic metal ions and dyes from wastewater1–5]. Chitosan isolated from fungi is of uniform physico-hemical properties than obtained by deacetylation of crustaceanhitin [6,7]. Recent research is therefore focused on the produc-ion of chitosan by fermentation of fungus especially belongingo zygomycetes group. Another advantage of fungal chitosan is

hat the production as well as physico-chemical properties, e.g.,

olecular weight of this chitosan can be manipulated by chang-ng the parameters of the fermentation. For example, Goksungur

∗ Corresponding author. Tel.: +91 33 2473 5904/4971x502;ax: +91 33 2473 2805.

E-mail address: [email protected] (A.K. Guha).

arsmacos

141-8130/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2007.10.006

lase

8] showed that production of chitosan by fermentation of Rhi-opus oryzae in molasses medium can be increased from 961 to109.32 mg/L by optimizing aeration rate, agitation speed andnitial sugar concentration. Yoshihara et al. [9] have shown thathe production of chitinous substances, especially chitosan by. oryzae can be enhanced by adding d-psicose at a concentra-

ion of 5–12 g/L to a synthetic medium containing low amountf glucose. Of the culture parameters studied, Arcidiacono andaplan [7] showed that length of incubation and medium com-osition affected biomass production and molecular weight ofhitosan in case of Mucor rouxii.

It is known that plant hormones are involved in several stagesf plant growth and development, but only a few conflictingeports are available regarding their effect on growth of microor-anisms. Makarem and Aldridge [10] found that gibberellic acidt an optimum concentration of 10 mg/L increased cell divisionate of different strains of Hansenula wingei; but this hormonehowed no effect on the growth of a number of bacteria andoulds [11]. Indole-3-butyric acid and 2,4-dichloro phenoxy

cetic acid caused an increase in the fresh weight and dry matterontent of Claviceps purpurea mycelia in static culture; on thether hand, indole-3-acetic acid enhanced the fresh weight butimultaneously inhibited dry matter production [12]. Abscisic

Page 2

f Biol

aBwskaetbdf

aomti

2

2

faoaa(ow(w

2

yatfiasstiwew

2

mol

hAr9wSoe

2

2w

dtwpwttoia

2

m

2

a7fcsPis[

me

3

bkctwG

S. Chatterjee et al. / International Journal o

cid stimulated growth of Fusarium culmorum [13]. Guha andanerjee [14] reported enhancement of biomass production asell as protein content of Agaricus campestris grown under

ubmerged condition in presence of indole-3-acetic acid andinetin. Similar effect of indole-3-acetic acid, gibberellic acidnd kinetin was reported by Paul et al. [15] and Mukhopadhyayt al. [16], respectively, for Kluyveromyces fragilis and Pleuro-us sajor-caju both grown in whey medium. Therefore, it haseen a subject of great interest to study whether chitosan pro-uction can be enhanced by adding plant growth hormones toermentation medium.

In this work, we investigated the influence of gibberellic acid,uxins and kinetin on growth and production of chitosan by R.ryzae in whey medium. The study is also important because itay help in utilizing whey, the largest by-product of dairy indus-

ry causing environmental pollution, to produce a commerciallymportant substance chitosan.

. Materials and methods

.1. Materials

R. oryzae (MTCC 262) used in this study was obtainedrom the Institute of Microbial Technology, Chandigarh, Indiand maintained on potato-dextrose agar slants. Whey wasbtained from local sweetmeat manufacturers and deproteinizeds described by Paul et al. [15]. Lactose content of whey wasdjusted to 4.5%. Gibberellic acid (GA3), indole-3-acetic acidIAA), indole-3-butyric acid (IBA) and kinetin (KIN) werebtained from Sigma Chemical Co., USA. Other chemicalsere purchased from E. Merck, Germany. Hormone solution

0.1%) was prepared in absolute alcohol except kinetin, whichas dissolved in 0.1 M NaOH.

.2. Fermentation medium and inoculation

R. oryzae was grown in deproteinized whey containing 0.2%east extract, 0.8% diammonium hydrogen phosphate and pHdjusted to 5.4. The medium was dispensed in 50 ml quan-ities in 250-ml-Erlenmeyer flasks and autoclaved at 121 ◦Cor 15 min. Hormones were added aseptically to the cold ster-le medium in concentrations described below in results, andmount of alcohol in all media including control was theame (2%). The flasks were inoculated with a spore suspen-ion (3.2 × 106 spores/flask), which was prepared by suspendinghe spores of the fungus in 0.9% saline after its full growthn potato-dextrose agar plates at 30 ◦C for 4 days. The flasksere incubated at 30 ◦C with agitation (120 rpm) for 72 h. At the

nd of fermentation, mycelia were separated by centrifugation,ashed with water, dried by lyophilization, and weighed.

.3. Chitosan and crude chitin extraction

Chitosan and crude chitin were isolated from the lyophilizedycelia (3–10 g) following the procedure described by Syn-

wiecki and Al-Khateeb [17] with little modification. In brief,yophilized mycelia were autoclaved at 121 ◦C for 15 min after

0btm

ogical Macromolecules 42 (2008) 120–126 121

omogenizing in a waring blender with 1N NaOH (1:40, w/v).lkali insoluble mass was thoroughly washed with water and

efluxed with 100 volumes of 2% acetic acid (v/v) for 24 h at5 ◦C. This was centrifuged, and acid insoluble residue wasashed with water, ethanol and acetone to obtain crude chitin.upernatant was adjusted to pH ∼9.0 with NaOH to precipitateut chitosan. This was washed with chilled water, followed bythanol and acetone and air-dried.

.4. Analyses

.4.1. Degree of deacetylation, weight average moleculareight and polydispersity index

Degree of deacetylation of chitosan was measured by firsterivative UV spectroscopic method [18] using Shimadzu spec-rophotometer. Weight average molecular weight of chitosanas determined by Haake Rheometer as described in our earlierublication [19]. Molecular size distribution pattern of chitosanas studied by dynamic light scattering experiment [1]. Chi-

osan solution (0.1%) in 2% acetic acid was prepared and filteredhrough 0.22 �m membrane (Millipore). Light scattering studyf the degassed solution was performed in Nano ZS Malvernnstrument, UK using He–Ne laser at 632.8 nm at 25 ◦C and atn angle 90◦.

.4.2. Lactose and proteinLactose and protein were measured by phenol sulfuric acid

ethod [20] and Bradford method [21], respectively.

.4.3. Chitin deacetylaseMycelial extract of R. oryzae was prepared by sonicating

suspension of the biomass in 50 mM Tris–HCl buffer (pH.2) containing 100 mM NaCl and 0.2 mM phenyl methyl sul-onyl fluoride. This was centrifuged at 10,000 rpm in a Sorvallentrifuge for 30 min at 4 ◦C. Requisite amount of ammoniumulfate was added to the supernatant to achieve 80% saturation.recipitated proteins were collected by centrifugation, dissolved

n minimum volume of Tris buffer as above and dialyzed exten-ively against the same buffer to remove ammonium sulfate22].

Glycol chitin, the substrate, was prepared according to theethod of Araki and Ito [23]. Chitin deacetylase activity of the

xtract was measured according to Kauss and Bausch [24].

. Result

The effect of supplementation of whey medium with gib-erellic acid, indole-3-acetic acid, indole-3-butyric acid andinetin on mycelial growth of R. oryzae and mycelial chitosanontent is presented in Figs. 1–4, respectively. It appears fromhe figures that all the hormones enhanced mycelial growth asell as its chitosan content at an optimum concentration. ThusA3 when added to the whey medium at a concentration of

.1 mg/L increased both mycelial growth and chitosan contenty 32 and 14.3%, respectively (Fig. 1). Both IAA and IBA athe optimum concentration of 3 mg/L (Figs. 2 and 3), enhanced

ycelial growth as well as chitosan content but to a different

Page 3

122 S. Chatterjee et al. / International Journal of Biological Macromolecules 42 (2008) 120–126

FoDe

ethctmtofto0

ao

FoDe

ctio

nby

R.o

ryza

e

itosa

nco

nten

tin

g/g

ofce

liaIn

crea

sein

chito

san

cont

ent

ofm

ycel

ia(%

)C

hito

san

prod

uctio

n(g

/Lof

med

ium

)In

crea

sein

chito

san

prod

uctio

n(%

)

19±

0.00

1–

0.74

9±

0.08

4–

36±

0.00

1214

.29

±0.

821.

131

±0.

1051

.0±

1.7

25±

0.00

145.

04±

0.65

1.01

±0.

0934

.8±

1.4

21±

0.00

131.

68±

0.58

0.90

8±

0.10

421

.2±

1.2

23±

0.00

123.

36±

0.62

0.94

7±

0.10

26.4

±0.

9

ediu

mat

thei

rop

timum

conc

entr

atio

n(m

g/L

).

ig. 1. Effect of GA3 on mycelial growth and chitosan content of Rhizopusryzae in whey. Represents (—�—) mycelial growth and (—�—) chitosan.ata represent an average of five independent experiments ±S.D. shown by

rror bar.

xtent. Enhancement of both mycelial growth and chitosan con-ent caused by supplementation of whey medium with IAA wasigher than that by IBA. IAA increased mycelial growth andhitosan content by 28.2 and 5.04%, respectively, over the con-rol as against 19.2 and 1.68% by IBA. KIN also increased both

ycelial growth and chitosan content by 22.2 and 3.36%, respec-ively, at the concentration of 2 mg/L (Fig. 4). Thus by additionf plant growth hormones at a very low concentration to theermentation medium, substantial increase in chitosan produc-ion can be achieved. For example, 50% more chitosan can bebtained from one litre whey medium by supplementing it with

.1 mg GA3 (Table 1).

Table 2 describes the physico-chemical properties of chitosans altered by the addition of hormone in the medium. Degreef deacetylation of chitosan isolated from R. oryzae grown in

ig. 2. Effect of IAA on mycelial growth and chitosan content of Rhizopusryzae in whey. Represents (—�—) mycelial growth and (—�—) chitosan.ata represent an average of five independent experiments ±S.D. shown by

rror bar. Tabl

e1

Eff

ecto

fad

ditio

nof

plan

tgro

wth

horm

ones

tow

hey

med

ium

ongr

owth

and

chito

san

prod

u

Hor

mon

esad

ded

toth

em

ediu

mM

ycel

ialg

row

th(d

ryw

eigh

t)in

g/L

ofm

ediu

mIn

crea

sein

myc

elia

lgr

owth

(%)

Ch

my

Con

trol

a6.

30±

0.30

–0.

1G

A3

(0.1

)8.

32±

0.44

32.0

6±

0.71

0.1

IAA

(3.0

)8.

08±

0.31

28.2

5±

0.66

0.1

IBA

(3.0

)7.

51±

0.25

19.2

1±

0.62

0.1

KIN

(2.0

)7.

70±

0.23

22.2

2±

0.58

0.1

Res

ults

show

nar

eav

erag

eof

five

repl

icat

eex

peri

men

ts(±

).H

orm

ones

wer

ead

ded

toth

em

aC

ontr

olfla

sks

rece

ived

noho

rmon

e.

Page 4

S. Chatterjee et al. / International Journal of Biol

Fig. 3. Effect of IBA on mycelial growth and chitosan content of Rhizopusoryzae in whey. Represents (—�—) mycelial growth and (—�—) chitosan.Data represent an average of five independent experiments ±S.D. shown byerror bar.

Fig. 4. Effect of KIN on mycelial growth and chitosan content of RhizopusoDe

pdowwa

ds

tscwtiameMG

4

gireipcmwasbgboosnimmcso

TC

H

CGIIK

Rfl

ryzae in whey. Represents (—�—) mycelial growth and (—�—) chitosan.ata represent an average of five independent experiments ±S.D. shown by

rror bar.

resence of hormone was more or less the same in all cases. Poly-ispersity index (PDI), which is the measure of the distribution

f molecular weights in a given polymer, was greatly reducedhen hormone, especially IAA, GA3 or KIN, was added to thehey medium. Weight average molecular weight increased in

ll cases by about 2-fold from 120 kDa to maximum 271 kDa

diir

able 2hanges in physico-chemical properties of chitosan due to hormone supplementation

ormone added to the medium Degree of deacetylation Weight average mo

ontrol 87.2 ± 0.58 120 ± 1.5A3 (0.1) 87.4 ± 0.62 224 ± 2.16

AA (3.0) 87.3 ± 0.52 248 ± 1.85BA (3.0) 87.2 ± 0.49 271 ± 1.95IN (2.0) 87.5 ± 0.57 262 ± 1.82

esults shown are average of five replicate experiments (±). Figures in parenthesesasks received no hormone.

ogical Macromolecules 42 (2008) 120–126 123

ue to hormone addition and these were reflected in molecularize distribution.

Table 3 shows that the hormones at their optimum concentra-ion decreased crude chitin content of the fungal mycelia withimultaneous increase in chitosan content. Thus chitosan/crudehitin ratio increased considerably due to addition of hormone inhey medium. Chitin deacetylase plays an important role in chi-

osan biosynthesis in fungi. It hydrolyzes N-acetamido groupsn the chitin chains to form chitosan [22]. Chitin deacetylasectivity of R. oryzae grown in whey medium in presence of hor-ones was measured. Result shows (Table 4) that activity of this

nzyme increased due to addition of hormone in the medium.aximum increase in activity of the enzyme was noted withA3.

. Discussion

It has been reported from different laboratories that plantrowth hormones show both stimulatory [10,15] and alsonhibitory [11,25] effects on microbial growth. It has also beeneported from our laboratory that the plant growth hormones cannhance protein content of fungal or yeast biomass. The presentnvestigation was therefore undertaken to monitor whether theselant growth hormones have any beneficial effect on growth andhitosan production by R. oryzae in whey medium under sub-erged condition. Whey, a by-product of the dairy industry,as used as medium for the growth of R. oryzae since it is

n inexpensive carbon source. This may also help to alleviateome environmental pollution caused by whey due to its highiochemical oxygen demand (BOD). R. oryzae was allowed torow under submerged condition in whey medium up to 72 hecause optimum production of biomass as well as chitosanccurred within this period (data not shown). , the results of onlyne time point have been presented in the figures. Maximumtimulation in growth of R. oryzae and its chitosan content wasoted with GA3 at a concentration of 0.1 mg/L (Fig. 1), whichs far below the optimum concentration required by other hor-

ones (Table 1). About 14.3% increase in chitosan content/g ofycelia was obtained with this hormone. Our findings although

ontradicted the previous report of Evans [26] that GA3 had noignificant effect on the growth of microorganisms but corrob-rated other findings in this regard, showing better growth and

evelopment of fungi in presence of GA3 [27,28]. The observedncrease in mycelial growth and chitosan content of R. oryzaen whey medium by both IAA (Fig. 2) and IBA (Fig. 3) cor-oborated the earlier reports on growth stimulation by different

of whey medium with plant growth hormone

lecular weight in kDa Polydispersity index Average diameter in nm

0.22 ± 0.025 163.2 ± 1.820.051 ± 0.02 416.6 ± 2.050.101 ± 0.0161 392.8 ± 1.720.286 ± 0.029 331.2 ± 1.750.115 ± 0.018 387.2 ± 1.89

indicate the amount of hormone added to the whey medium in mg/L. Control

Page 5

124 S. Chatterjee et al. / International Journal of Biological Macromolecules 42 (2008) 120–126

Table 3Influence of hormones on crude chitin and chitosan content of R. oryzae mycelia

Hormone Amount of chitosan in g/g of mycelia Amount of crude chitin in g/g of mycelia Chitosan:crude chitin in mycelia

Control 0.119 ± 0.001 0.241 ± 0.0014 0.489 ± 0.018GA3 (0.1) 0.136 ± 0.0012 0.189 ± 0.0012 0.720 ± 0.016IAA (3.0) 0.125 ± 0.0014 0.201 ± 0.0015 0.622 ± 0.014IBA (3.0) 0.121 ± 0.0013 0.208 ± 0.0012 0.582 ± 0.02K 04 ±C

actcwfbIftnsoodpatotw4m

owataum

TI

H

CGIIK

Raa

ccawtoDtsmiadhlsmwo

cloNN[mg

IN (2.0) 0.123 ± 0.0012 0.2

ontrol and figures in the parentheses are same as indicated in Table 2.

uxins [29,30,14]. It may be presumed that the auxins mightontrol fungal cell elongation [31] and differentiation [27] ashey do in case of higher plants. However the present findingsontradicted in this respect of an earlier report of Gruen [32]ho concluded that auxins had no growth-promoting role on

ungi. The observed increase in growth and chitosan productiony KIN (Fig. 4) was more or less same to that obtained withBA. KIN has also been reported to increase the growth of dif-erent fungi and yeast [13–15]. In all cases hormones increasedhe growth and chitosan production in a dose dependant man-er. At a concentration above the optimum, hormones instead oftimulation decreased the growth of fungus (Figs. 1–4). Similarbservations regarding the influence of hormone on the growthf microorganisms were also reported [10,12,27,29]. It is evi-ent from Table 1 that more than 50% increase in chitosanroduction can be achieved by adding GA3 to whey mediumt a concentration of 0.1 mg/L. Increase in chitosan produc-ion was not only due to enhanced mycelial growth in presencef hormone but also due to increase in chitosan content ofhe mycelia. Better growth of mycelia in presence of hormoneas reflected in more lactose utilization, which increased from8.9% to 54.9–61.6% depending on the hormone added to theedium.Table 2 strongly suggests that hormones have no influence

n the degree of deacetylation of chitosan but increased theeight average molecular weight by 2-fold. Thus the weight

verage molecular weight of chitosan increased from 120 kDao maximum 271 kDa due to influence of IBA. Arcidiacono

nd Kaplan [7] reported earlier that the weight average molec-lar weight of chitosan depends on the composition of theedium. High molecular weight chitosan is preferred in many

able 4nfluence of hormones on chitin deacetylase production by R. oryzae

ormones Specific activity of chitindeacetylase (units/mg ofcrude protein)

Increase in specificactivity of chitindeacetylase (%)

ontrola 0.0375 ± 0.006 –A3 (0.1) 0.0475 ± 0.005 26.7 ± 0. 70

AA (3.0) 0.0452 ± 0.006 20.5 ± 0.65BA (3.0) 0.0400 ± 0.005 6.67 ± 0.56IN (2.0) 0.0413 ± 0.009 10.13 ± 0.6

esults shown are average of five replicate experiments (±). One unit of enzymectivity is defined as the amount of the enzyme required to produce 1 �mol ofcetate/min under the experimental conditions described.a Control and figures in the parentheses are same as indicated in Table 2.

ao

fimtti

esitoeha

0.0016 0.603 ± 0.019

ases; for example, film prepared with high molecular weighthitosan has higher tensile strength and elongation propertys well as moisture absorption capacity [33]. High moleculareight chitosan can improve lipase loading and also reduce

he release of entrapped lipase [34]. Further, chitosan vectorsf lower molecular weight are less efficient in retaining theNA upon dilution [35]. Dynamic light scattering with chi-

osan isolated from R. oryzae grown in presence of hormonehows the molecular distribution pattern (Fig. 5). All the hor-ones, except IBA, reduced the polydispersity index of chitosan

ndicating improvement of the quality of the chitosan. It isssumed that such difference in polydispersity may arise fromifference in chitosanase activity of the fungus in presence oformone. Above findings were also confirmed from molecu-ar size distribution results (Table 2). Variation of molecularize of chitosan obtained from R. oryzae in presence of hor-one gives an insight to a weight average molecular weight,hich is in good agreement with the experimental resultsbtained.

The biosynthesis of chitosan in fungi is carried out byoordinated action of chitin synthase and chitin deacety-ase. The first enzyme synthesizes chitin by polymerizationf N-acetylglucosaminyl residue from uridine 5′-diphospho--acetylglucosamine. Chitin deacetylase then hydrolyzes the-acetamido groups in the chitin chains to convert it to chitosan

22]. Since this enzyme plays an important role in chitosan for-ation, its activity was measured. All the hormones increased

rowth and chitosan content of the mycelia. Therefore, the rel-tive amount of crude chitin and chitosan in the mycelia of R.ryzae grown in presence of hormones was determined.

Amount of crude chitin present in the mycelia was alwaysound to be higher to that of chitosan (Table 3). Hormonesncreased chitosan/crude chitin ratio of the mycelia and maxi-

um increase was noted with GA3. Increase in this ratio was dueo the decrease in crude chitin content of the mycelia with simul-aneous increase in chitosan. As expected, maximum increasen chitosan and decrease in chitin content was noted with GA3.

Since chitosan is formed by deacetylation of chitin by thenzyme chitin deacetylase, its activity in the mycelia was mea-ured. Specific activity of chitin deacetylase increased due tonfluence of hormone from 6.7 to 26.7% (Table 4). Thus allhese hormones especially GA3 increased the chitosan content

f the mycelia by increasing deacetylation of chitin throughnhanced chitin deacetylase activity. How these plant growthormones enhance the activity of this enzyme is yet to benswered.

Page 6

S. Chatterjee et al. / International Journal of Biological Macromolecules 42 (2008) 120–126 125

F wheyo

5

1

2

3

4

ig. 5. Dynamic light scattering pattern of chitosan from hormone supplementedf five independent experiments ±S.D. shown by error bar.

. Conclusions

The main conclusions that can be drawn from this study are:

. Plant growth hormones enhanced both mycelial growth andchitosan content of R. oryzae in a dose dependent man-ner. Maximum stimulation in this respect was noted with

gibberellic acid, which enhanced mycelial growth and itschitosan content by ∼32 and ∼14%, respectively, at aconcentration of 0.1 mg/L. Thus it is possible to increase chi-tosan production by more than 50% by supplementing wheymedium with gibberellic acid.

5

. (a) Control, (b) GA3, (c) IAA, (d) IBA and (e) KIN. Data represent an average

. At a dose above the optimum, all the hormones instead ofstimulation decreased both growth and chitosan content ofR. oryzae.

. Hormones did not change the degree of deacetylation butincreased the quality of chitosan by way of increasing itsmolecular weight and decreasing polydispersity.

. Hormones increased the chitosan/crude chitin ratio of R.oryzae mycelia.

. All the hormones especially gibberellic acid, enhanced chitin

deacetylase activity which hydrolyzes N-acetamido groupsin the chitin chains to form chitosan, by ∼26%. Thismay be one of the reasons for increase in the chitosanproduction.

Page 7

1 f Biol

A

ol

R

[[

[[[[

[

[[

[

[

[[

[[[[

[

[

[[

[[[

26 S. Chatterjee et al. / International Journal o

cknowledgement

Authors are thankful to Prof. Anjan Das Gupta of Departmentf Biochemistry, Calcutta University for his help in dynamicight scattering study.

eferences

[1] S. Chatterjee, M. Adhya, A.K. Guha, B.P. Chatterjee, Process Biochem. 40(1) (2005) 395–400.

[2] S.A. White, P.R. Farina, I. Fulton, Appl. Environ. Microbiol. 38 (1979)323–328.

[3] S. Chatterjee, A.K. Guha, B.P. Chatterjee, Ind. J. Chem. Technol. 10 (2003)350–354.

[4] S. Chatterjee, S. Chatterjee, B.P. Chatterjee, A.K. Guha, Process Biochem.39 (2004) 2229–2232.

[5] S. Chatterjee, S. Chatterjee, B.P. Chatterjee, A.R. Das, A.K. Guha, J. Col-loid Interface Sci. 288 (1) (2005) 30–35.

[6] C. Crestini, B. Kovac, G. Giovaannozzi-Sermanni, Biotechnol. Bioeng. 39(1996) 281–286.

[7] S. Arcidiacono, D.L. Kaplan, Biotechnol. Bioeng. 39 (1992) 281–286.[8] Y. Goksungur, J. Chem. Technol. Biotechnol. 79 (9) (2004) 974–981.[9] K. Yoshihara, Y. Shinohara, T. Hirotsu, K. Izumori, J. Biosci. Bioeng. 95

(3) (2003) 293–297.10] E.H. Makarem, N. Alldridge, Can. J. Microbiol. 15 (1969) 1225–1231.11] P.W. Brain, G.W. Elson, H.G. Hemming, M.J. Radley, J. Sci Food Agric.

5 (1954) 602–612.12] J. Rerabek, Folia Microbiol. 15 (1970) 309–313.13] M. Michniewicz, Biologigia Plantarum (Praha) 29 (1987) 273–278.14] A.K. Guha, A.B. Banerjee, Acta Microbiol. Pol. 6 (1974) 133–134.15] D. Paul, A.K. Guha, B.P. Chatterjee, Biochem. Arch. 10 (1994) 277–283.

[

[

ogical Macromolecules 42 (2008) 120–126

16] R. Mukhopadhyay, S. Chatterjee, B.P. Chatterjee, A.K. Guha, ProcessBiochem. 40 (2005) 1241–1244.

17] J. Synowiecki, N.A.A.Q. Al-Khateeb, Food Chem. 60 (4) (1997) 605–610.18] R.A.A. Muzzarelli, R. Rochetti, V. Stanic, M. Weckx, Chitin Handbook,

European Chitin Society (1997) 109.19] S. Chatterjee, B.P. Chatterjee, A.K. Guha, Res. J. Microbiol. 1 (2006)

90–94.20] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem.

28 (1956) 350–356.21] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.22] D. Kafetzopoulos, A. Martinou, V. Bouriotis, Proc. Natl. Acad. Sci. 90

(1993) 2564–2568.23] Y. Araki, E. Ito, Eur. J. Biochem. 55 (1975) 71–78.24] H. Kauss, B. Bausch, Methods Enzymol. 161 (1988) 518–523.25] A.A. Oravec, N.C. Strovilas, J.L. Beal, A. Tye, Nature 184 (1959) 1405.26] M.L. Evans, Encyclopedia of Plant Physiology N.S. 10th, Springer-Verlag,

Berlin, Germany, 1984.27] K. Tomita, T. Murayama, T. Nakamura, Plant Cell Physiol. 25 (1984)

355–358.28] M. Michniewicz, B. Rozej, Acta Physiologia Plantarum 10 (1988) 227–

236.29] M. Michniewicz, B. Rozej, Acta Physiologia Plantarum 9 (1987) 219–227.30] T. Nakamura, Y. Kawanabe, E. Takiyama, N. Takahashi, T. Murayama,

Plant Cell Physiol. 19 (1978) 705–709.31] N. Yanagishima, Plant Cell Physiol. 4 (1963) 257–264.32] H.E. Gruen, Ann. Rev. Plant Physiol. 8 (1959) 405–440.33] J. Nunthanid, S. Puttipipatkhachorn, K. Yamamoto, G.E. Peck, Drug Dev.

Ind. Pharm. 27 (2) (2001) 143–157.34] I.A. Alsarra, S.S. Betigeri, H. Zhang, B.A. Evans, S.H. Neau, Biomaterials

23 (2002) 3637–3644.35] M. Huang, C.W. Fong, E. Khor, L.Y. Lim, J. Contr. Rel. 106 (2005)

391–406.