Tài liệu Báo cáo khóa học: Non-specific depolymerization of chitosan by pronase and characterization of the resultant products pptx

Tharanathan1 1 Department of Biochemistry and Nutrition,2Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore, India Pronase type XXV ser

Non-specific depolymerization of chitosan by pronase

and characterization of the resultant products

Acharya B Vishu Kumar1, Lalitha R Gowda2and Rudrapatnam N Tharanathan1

1

Department of Biochemistry and Nutrition,2Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore, India

Pronase (type XXV serine protease from Streptomyces

griseus) efficiently depolymerizes chitosan, a linear

bfi1,4-linked polysaccharide of 2-amino-deoxyglucose and

2-amino-2-N-acetylamino-D-glucose, to low-molecular

weight chitosans (LMWC), chito-oligomers (degree of

poly-merization, 2–6) and monomer The maximum

depolymeri-zation occurred at pH 3.5 and 37C, and the reaction obeyed

Michaelis–Menten kinetics with a Kmof 5.21 mgÆmL)1and

Vmaxof 138.55 nmolesÆmin)1Æmg)1 The molecu lar mass of

the major product, LMWC, varied between 9.0 ± 0.5 kDa

depending on the reaction time Scanning electron

microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X-ray diffractometry and

13C-NMR revealed them to possess a lower degree of acety-lation, hydration and crystallinity compared to chitosan Chitosanolysis by pronase is an alternative and inexpensive methodtoproduceavarietyofchitosandegradationproducts that have wide and varied biofunctionalities

Keywords: chitosan; chito-oligomers; low-molecular weight chitosan; pronase; structure

Chitosan is the de-N-acetylated derivative of chitin, a linear

polysaccharide of b1fi4-linked 2-deoxy-2-acetamido-D

-glu-cose units [1], that constitutes the exoskeleton of

inverte-brates and is one of the components of the cell walls of

fungi It has wide and varied applications in medicine,

agriculture, pharmaceuticals and the food industry [1,2],

which is attributed to the biofunctionality of the amine

moiety that confers both cationic (polyelectrolyte) and

chelating properties Despite being biocompatible, nontoxic

and multifunctional, the use of chitosan in vivo is hampered

by its high-molecular mass and high viscosity even at low

concentrations [2] Therefore, a prerequisite for efficient

utilization of chitosan is its depolymerization to

low-molecular weight chitosans (LMWC), chito-oligomers and

monomer The depolymerized products find additional

applications as hypo-cholesterolemic, antitumorigenic,

antimicrobial, immuno-enhancing agents, and also in the

treatment of osteoarthritis, gastritis, etc [3–5] A 9 kDa

LMWC suppressed Escherichia coli activity whereas that of

LMWC 5 kDa showed antihyperlipemic and

hypocholes-terolemic effects [2] Chito-oligomers with a degree of

polymerization (DP) > 6 showed antitumor activity

towards Sarcoma-180 and Meth-A tumors, and caused

activation of defence responses in plants Chitotriose exhibited maximum inhibitory effect towards angiotensin converting enzyme (ACE) [4]

Chitosan can be depolymerized by acid or enzymatic hydrolysis The former is harsh, time consuming, modifies the products and forms a large quantity of monomers [6]

In contrast, enzymatic hydrolysis produces specific products as the reaction can be precisely controlled Chitosanase, the enzyme of choice due to its specificity, degrades chitosan to chito-oligomers, but is, however, very expensive and unavailable in bulk for commercial exploitation [7]

The earlier concept of a ratio of one enzyme to one substrate/group of related substrates is no more a reality in most of hydrolases as evidenced in recent literature b1fi4 Glucanase, although specific for b1fi4-linked glucans, can hydrolyze mannans and cellobiose [8] Chitosanase from MyxobacterA-1, Streptomyces griseus HUT 6037, Bacillus sp.7-M and Bacillus megaterium degrades carboxymethyl-cellulose A b1fi3/1fi4 glucanase from Bacillus circulans WL-12 depolymerizes chitosan to low-molecular mass products [9–12] Susceptibility of chitosan to various nonspecific enzymes like wheat germ lipase, lysozyme, papain, cellulase, hemicellulase, b-glucosidase, etc., has been reported earlier, although enzyme purity was in doubt and could be contaminated with chitosanase [13,14] Recently, we have shown that a homogeneous isozyme of Aspergillus niger pectinase could depolymerize chitosan quantitatively to yield LMWC and chito-oligomers [15] The objective of the present study was to demonstrate yet another example of enzyme nonspecificity in catalyzing the cleavage of completely unrelated substrates An electropho-retically pure pronase preparation was used to depolymerize chitosan to LMWC, chito-oligomers and monomer, and structural characterization of the depolymerized products is presented herein

Correspondence to R N Tharanathan, Department of Biochemistry

and Nutrition, Central Food Technological Research Institute,

Mysore ) 570 013, India.

Fax: + 91 821 2517233, Tel.: + 91 821 2514876,

E-mail: tharanathan@yahoo.co.uk

Abbreviations: DP, degree of polymerization; LMWC, low-molecular

weight chitosan.

Enzyme: Pronase (type XXV serine protease from Streptomyces

griseus; EC 3.4.24.31)

(Received 30 October 2003, revised 12 December 2003,

accepted 22 December 2003)

Materials and methods

Materials

Chitosans (molecular mass, 150–600 kDa) and

phenyl-methylsulfonyl fluoride were obtained from Fluka Chemika

(Buchs, Switzerland) Pronase (type XXV protease from

Streptomyces griseus, EC 3.4.24.31), Sephadex G15 and

Celite were obtained from Sigma Shrimp chitin was

obtained from the CFTRI Regional Center at Mangalore,

India Other chemicals used were of highest purity available

Preparation of chitosan

Shrimp chitin was subjected to heterogeneous

N-deacety-lation to obtain chitosan [16], which was further purified

by dissolving in 1% acetic acid, filtered through a plug of

glass-wool to remove suspended particles and finally

precipitated with 2% sodium carbonate The precipitate

was water-washed, freeze-dried and stored at ambient

temperature until further use

Criteria of enzyme homogeneity

To establish the purity, the stock pronase solution

(10 mgÆmL)1) was subjected to capillary electrophoresis

(Prince 550 system, Prince Technologies, Emmen, the

Netherlands) using fused silica capillary (80 cm

length· 75 lm i.d.) connected to an UV detector

(280 nm) and Tris/glycine buffer (pH 8.8) at 10 kV,

100 mbar, 26 ± 1C The data acquisition and control

were performed usingDAXsoftware The enzyme solution

after suitable dilution (30 lg) was subjected to SDS/

PAGE (12.5% T, 2.7% C using Tris/glycine buffer of

pH 8.8) according to the method of Laemmli followed by

Coomassie Brilliant Blue staining [17] For further assay,

the stock solution was suitably diluted

Enzyme assay

Proteolytic activity of pronase was evaluated using casein

(1%) as the substrate at optimum conditions (pH 7.5 and

37C) and estimating the trichloroacetic acid soluble

peptides released (specific activity, unit¼ absorbance at

280 nm/reaction time· mg protein in the reaction

mix-ture) For chitosanolytic activity, 1% chitosan in 1%

aqueous acetic acid was treated with pronase in the ratio

100 : 1 (w/w), at optimum conditions (pH 3.5 and 37C)

To terminate the reaction, the mixture was heated for

5 min followed by the addition of an equal volume

of 2MNaOH and centrifugation (1000 g) of the reaction

mixture The supernatant was analyzed for reducing

groups (specific activity, unit¼ lmoles of reducing

equi-valents released per minute per mg of protein at optimum

conditions) [18]

Energy of activation (Ea) was determined by the slope

(–Ea/2.3R, where R is the gas constant) of Arrhenius plots

obtained by plotting logarithm of maximum enzyme

velocity determined at different temperatures (20–37C)

under standard reaction conditions against the reciprocal of

absolute temperatures (T¼ 273 + A, where A is the

temperature inC) [19]

Kinetics of the chitosanolytic activity The pH optimum was determined using chitosan solution (1%) at a pH value between 1.5 and 6.0 (pH was adjusted using 0.1M HCl/NaOH and above pH 6.5, chitosan was insoluble) and 1% casein solution of

pH 4.0–11.0 The temperature optimum was determined

by carrying out the reaction between 20 and 60C pH and temperature stability measurements were carried out

by preincubating the enzyme at different pH values/ temperatures followed by determination of the residual chitosanolytic activity (%) as described above by taking the aliquots of the enzyme at regular intervals The Michaelis constants, Km and Vmax were evaluated from the double reciprocal plot of the initial velocity versus substrate concentrations

Isolation of the products Chitosan solution (1%, dissolved in 1% acetic acid with pH adjusted to 3.5) was treated with pronase in the ratio,

100 : 1 (w/w), incubated for different periods at optimum conditions followed by arresting the reaction using an equal volume of 2MNaOH The precipitate (LMWC) obtained after centrifugation at 1000 g, was dialyzed against water using a membrane with a molecular mass cut-off of 2 kDa (Sigma Chemicals Co.) and freeze-dried

The supernatant containing chito-oligomers and mono-mer as well as the heat denatured enzyme, was passed through a charcoal-Celite column Unadsorbed saccha-rides (GlcN, GlcN-rich oligomers), enzyme and excess alkali added while arresting the reaction were collected by eluting with distilled water (Fraction IA), and the adsorbed saccharides (GlcNAc, GlcNAc-rich oligomers) were recovered using 60% ethanol as the eluant (Fraction II) Fraction IA was re-N-acetylated and subjected to a second charcoal-Celite chromatography phase wherein the water-washing removed the enzyme and alkali, and elution with 60% ethanol resulted in the recovery of re-N-acetylated GlcN and GlcN-rich oligomers (Fraction I) The fractions were concentrated by flash treatment at ambient temperature Simultaneously, after neutralization, the supernatant was passed through Sephadex G15 column (62· 0.8 cm; bed volume, 78 mL) to remove salt and denatured enzyme, and the fraction containing reducing groups (chito-oligomers + monomer) were pooled, followed by its freeze-drying

Determination of the molecular mass of chitosan and the depolymerization products

The molecular mass was measured using three techniques: (a) viscometric measurements; (b) gel permeation chroma-tography (GPC) and HPLC

Viscometric measurements The viscosity of chitosan and LMWC dissolved in sodium acetate buffer (0.5M acetic acid + 0.2Msodium acetate, pH 4.5) was measured using

an Ostwald viscometer [20] The average molecular mass was deduced using the Mark–Houwink’s equation

g¼ K · (molecular mass)awhere, g is the intrinsic visco-sity, K¼ 3.5 · 104and a¼ 0.76

Gel permeation chromatography The molecular mass of

chitosan was determined by GPC on a Sepharose CL-4B

column (Sigma Chemicals Co; bed volume, 180 mL)

equilibrated in sodium acetate buffer (pH 4.5) [21] The

column was precalibrated with dextrans (of known

mole-cularmass)andchitosanmolecularmassstandards.Fractions

I and II obtained after charcoal-Celite column

chromatogra-phy were subjected to GPC using a Biogel P2 column

(100· 0.8 cm, Bio-Rad laboratories) equilibrated with water

and calibrated with chito-oligomers and GlcNAc [18]

High performance liquid chromatography The molecular

mass of LMWC was determined using E-linear and E-1000

columns in series The mobile phase used was acetate buffer

(0.5Macetic acid + 0.2Msodium acetate, pH 4.5) at a flow

rate of 0.8 mLÆmin)1 and the detection was performed

using an RI detector (Shimadzu Corp., Kyoto, Japan) The

columns were calibrated with dextran standards The use of

acetate buffer minimized the interaction between the

column material and -NH2groups

Fractions I and II were subjected to HPLC on an

aminopropyl column (3.9· 300 mm, Waters Associates,

Ireland) using acetonitrile:water (70 : 30, v/v) as the mobile

phase at a flow rate of 1.0 mLÆmin)1and detected using an

RI detector Chito-oligomers and GlcNAc were used as

standards

Further characterization of chitosan and the

depolymerization products

Further characterization of chitosan and the

depolymeriza-tion products was performed using a variety of techniques

as detailed below

Environmental scanning electron microscopy (ESEM)

Freeze dried samples were spread on a double sided

conducting adhesive tape pasted onto a metallic stub

Samples were observed under SEM (LEO 435 VP, LEO

Electron Microscopy Ltd, Cambridge, UK) at 20 kV and

variable pressure

Infrared (IR) spectroscopy IR spectral studies were

performed on a Perkin Elmer 2000 spectrometer under dry

air at room temperature using KBr pellets Chitosan,

LMWC and freeze-dried chito-oligomers + monomer

(4 mg samples) were mixed thoroughly with 200 mg KBr

and 40 mg of the mixture was pelletized The reproducibility

of the spectra was verified on two preparations and the

degree of acetylation (DA) was determined using the formula

(A1655Æcm)1/A3450Æcm)1)· 100 / 1.33, where A is the

absorb-ance at these wavelengths, calculated from the baseline [22]

Potentiometric titration Chitosan, LMWC and

chito-oligomers + monomer (200 mg) were dissolved in 25 mL

of 0.1MHCl and the volume was made up to 100 mL with

distilled water and the ionic strength was adjusted to 0.1M

using KCl 0.1MNaOH containing 0.1MKCl was used as

the titrant Initially, the solution pH was brought to pH 2.0

by adding the titrant, after which it was added in stepwise

increments (0.5 mL, each time) The titration was

termin-ated when the solution pH reached 6.0 For each sample,

five replicates were performed A graph of pH as a function

of the volume of titrant was plotted, which gave a titration curve having two inflexion points, whose difference along the abscissa corresponded to the amount of acid required for the protonation of the amine groups of samples The number of equivalents of acid groups was calculated using the formula: [NH3+] (mequ iÆkg)1)¼ 1000 · molarity (moles/L)· volume of titrant (mL)/mass of sample (g) and the level of deacetylation was calculated by comparison between the number of free amino groups (per unit weight

of the sample) and the equivalent weight of GlcN [23]

Circular Dichroism (CD) CD spectra for native chitosan and LMWC (5 mgÆmL)1 in 0.1M perchloric acid; path length, 1 cm) were recorded on a Jasco J-810 automatic recording spectropolarimeter, continuously purged with N2 before and during the experiment (Japan Spectroscopic, Tokyo, Japan) Slits were programmed to yield 10 A˚ band width at each wavelength so that the resolution was more or less constant The spectra were recorded between 200 and

240 nm (far UV region) and baseline was obtained using 0.1M perchloric acid After accumulation of scans, the spectra were standardized to mean residual ellipticity expressed as h in degree· cm2per residue using the mean residual weight of GlcNAc [24]

X-ray diffractometry X-ray diffraction studies were car-ried out on an EG-7 G solid state germanium, liquid nitrogen cooled detector Scintag XDS-2000 diffractometer equipped with a h–h goniometer, 30 kV + 25 mA with CuKa radiation at 1.5414 nm (Enraf Nonius Co., Bohemin, NY) The relative intensity was recorded in a scattering range (2h) of 0–45 The crystallinity index (CrI, %) was determined using the formula (I110– Iam)· 100/I110, where

I110is the maximum intensity at 20 and Iamis the intensity

of amorphous diffraction at 16 [25]

Solid-state CP-MAS13C-NMR spectroscopy 13C-NMR spectra were obtained with a Bruker dsx300spectrometer at

75 MHz The cross polarization pulse sequence was utilized for all samples, which were spun at the magic angle at 6.2 kHz for native chitosan and 7 kHz for LMWC and chito-oligomers + monomer A contact time of 1 ms and a pulse repetition time of 5 ms were used, and more than 2000 scans were accumulated for each spectrum Approximately

300 mg of freeze-dried samples were inserted into a 7 mm ceramic rotor The DA was calculated using the equation,

ICH3/(IC1+ IC2+ … IC6)/6, where I is the intensities of C1–C6 as well as methyl carbons [26]

Liquid-state 13C-NMR spectroscopy Chitosan, LMWC and freeze-dried chito-oligomers + monomer (50 mg each) were dissolved in 1 mL solvent mixture of D2O-DCl (0.98 + 0.02 mL, respectively) After ensuring complete dissolution of the samples, the spectra were recorded with a Bruker amx400spectrometer at 100 MHz and 27C

Results and Discussion

Characterization of chitosan The molecular mass of purified chitosan was 71 ± 2 kDa

as determined by both viscometry and GPC The solid-state

CP-MAS13C-NMR and IR spectra indicated chitosan to

have a b-conformation, as evidenced by the absence of

splitting of signals corresponding to C3-C5 carbons (Fig 1)

and appearance of a single broad peak around 3371 cm

(Fig 2), in contrast to the presence of two peaks in case of

a-conformation The degree of acetylation (DA) determined

by IR and13C-NMR was in good agreement with each

other (Table 1) The crystallinity index (CrI) determined by

X-ray diffraction pattern was 70% (Table 1) and indicated

chemical homogeneity, and this was in close correspondence

with chitosans of Euphausia superba (68.4%) [27]

Effect of different solvents, molecular mass

and DA on chitosanolysis by pronase

Preliminary studies indicated that chitosan could be

degra-ded nonspecifically by proteases such as pepsin, papain,

protease, etc It was observed that chitosanolysis by pronase

was dependent upon the solubility, molecular mass and DA

of chitosan Muzzarelli et al (1994) compared acetic acid

with lactic acid as a chitosan solvent during the nonspecific

chitosanolysis by papain and reported lactic acid to be a

better solvent owing to the hydrolyzing action of the former

[28] Contrary to this, the specific activity of pronase indicated that chitosan dissolved in aqueous acetic acid (1%) and was the best solvent when compared to formic and lactic acids In acetic acid, being a weak acid (see pKa values, Table 2), chitosan was much less decomposed, [29] thus, resulting in better enzymic depolymerization Increase

in the molecular mass of chitosan from 71fi600 kDa and

DA from 15fi26% resulted in increased specific activity (Table 2) Ibrahim et al (2002) reported the effect of molecular mass and DA on lipase loaded chitosan bead characteristics, in which increases in molecular mass and

DA resulted in efficient loading and minimization of the release of entrapped lipase [30] The observed increase in specific activity with increasing molecular mass suggests the preference of pronase towards higher molecular mass chitosans Improved chitosanolysis with higher DA indica-ted that the affinity of enzyme is more towards chitosan with higher DA

Homogeneity and specific activity of pronase Capillary zone electrophoresis and SDS/PAGE of pronase showed the presence of a single homogeneous protein (Fig 3 and the inset) of molecular mass  20 kDa as determined using protein markers The enzyme notably showed a much higher specific activity towards proteolysis

of casein (4.14 U) as compared to 1.15 U towards chitosan depolymerization Although the specificity of pronase was much lower ( 43-fold less) in comparison with that of chitosanase (from Streptomyces griseus, 50 UÆmg)1, Sigma Chemical Co.), the advantage of pronase catalyzed chitos-anolysis was the production of LMWC in higher yields (>70%), which was lacking in chitosanase as a result of its specificity towards the formation of mono- and oligomers (DP 2–3), rather than LMWC

Enzyme kinetics The pH optimum of pronase towards proteolysis and chitosanolysis were 7.5 and 3.5, respectively, whereas the temperature optimum towards chitosanolysis was 37C (Fig 4A,B) Investigation of the pH and temperature stabilities indicated enzyme activation in the initial hour, which could be due to prior attainment of an active conformation by the enzyme, remaining stable up to

240 min (4 h) after which there was a decline (Fig 4C,D), indicating enzyme stability in the pH range 2.0–5.0 and 45–60C in the initial hours The effect of varying chitosan concentration on the initial velocity showed that chitosano-lysis by pronase follows Michaelis–Menten kinetics The use

of substrate concentration greater than 20 mgÆmL)1 was limited owing to increased viscosity of the solutions, affecting the enzyme penetration The Kmand Vmaxvalues obtained from a double reciprocal plot were 5.21 mgÆmL)1 and 138.55 nmolesÆmin)1Æmg)1, respectively Activation energy calculated for pronase towards proteolysis and chitosanolysis were 15.5 and 5.12 kcalÆmol)1, respectively, further supporting the fact that pronase prefers proteins over chitosan as the substrate, which was in accordance with the specific activities of pronase The maximum chitosano-lysis was observed at 240 min (4 h) beyond which there was

no further appreciable increase

Fig 1 Solid-state CP-MAS 13 C-NMR spectra of chitosan, LMWC

and freeze-dried chito-oligomers + monomer.

Characterization of LMWC

Chitosan showed b-conformation as evidenced by both13

C-NMR spectra and IR (Figs 1 and 2, respectively) [27], and

accordingly was more susceptible to the hydrolytic

proces-ses, due to individual chains arranged in a parallel manner

[1], resulting in a weaker hydrogen-bonding network

between the chains and thus enabling enzyme penetration

The percentage yield of LMWC, chito-oligomers and

monomer (GlcNAc) was dependent on the reaction time

(Table 1) There was approximately eightfold decrease in

the molecular mass following depolymerization, which occurred in the initial hour beyond which the decrease was not appreciable (Table 1) Unless stated otherwise, further characterization was performed with the LMWC obtained after 1 h reaction time

Environmental scanning electron microscopy Although chitosan is a linear polymer, its freeze-drying results in the aggregation of the chains by inter- and intrachain hydrogen bonding network resulting in the formation of particle-like structure [1] ESEM of LMWC (obtained after 5 h

Fig 2 Infrared (IR) spectra of chitosan

and LMWC.

Table 1 Characteristics of chitosan and low-molecular weight chitosan (LMWC), and the percentage yield of the depolymerization products Percentage yield is dependent on the incubation time (1–5 h) and is approximate.

Molecular mass (kDa)

Degree of acetylation (DA; %)

Crystallinity index (CrI; %)

Yield (%) IR

13 C-NMR (solid state) Chitosan

Depolymerization products

a Dependent on the incubation time (1–5 h)

incubation) indicated decreased particle size compared to

chitosan (Fig 5) in accordance with the lower molecular

mass The continuous fibrous/flat ribbon type appearance

of chitosan disappeared upon depolymerization and

resul-ted in the formation of discontinuous smaller pieces

IR spectra In both chitosan and LMWC, a well defined

band around 2922 cm)1due to the -CH vibration mode

represented a good internal reference for comparison of

band absorbance Absence of a sharp and convoluted

spectral band around 3600–3000 cm)1in the spectra of both

chitosan and LMWC indicated the absence of free -OH

groups and the involvement of both -OH…3 and

-CH2OH…6 in intra- and intermolecular hydrogen bonding

(Fig 2) [31] Line width and frequency position of the band

above 3000 cm)1corresponds to the intermolecular crystal

lattice of a molecule, which depends on the hydrogen

bonding network, and results in the incorporation of water

molecule into molecular structure The latter promotes a perfect crystal structure and increases CrI In chitosan, this band appeared at 3371 cm)1and there was a slight shifting

of the same in LMWC (3368 cm)1), indicating a decrease in the ordered structure This is probably due to the decrease in the DA (Table 1) of LMWC, which results in lower degree

of hydration of the molecule affecting the crystallinity index and thus the orderliness This was further confirmed by a decrease in the peak height at 1320 cm)1 in LMWC compared to chitosan, which corresponds to the GlcNAc residues [32] The region between 1420 and 1435 cm)1, considered to be polysaccharide conformation sensitive, is assigned to -CH2 bending, which depends on the confor-mation of the primary -OH group (C6) in a favorable orientation [33] The shift of the same to 1458 cm)1in the LMWC would probably indicate a change in the environ-ment of primary -OH groups and thus the overall hydrogen-bonding network

Potentiometric titration Potentiometric titration is a well-known analytical tool for quantifying acidic functional groups and is independent of sample molecular mass [23] The DA (%) calculated by titration (graphs, not shown) were found to be 26.3, 18.8 and 32.3, respectively, which were in good agreement with that calculated using IR spectra

CD-Spectra Compared to chitosan, in LMWC, there was

a decrease in the peak height near 211 nm (due to gfip* transition) (Fig 6) corresponding to the acetyl content, which is independent of conformation, chain length, ionic strength and pH [24] This decrease in the peak height continued with increased time of enzyme digestion

X-ray diffractometry The X-ray diffraction (Fig 7) pat-tern of chitosan was that of a typical hydrated form with a peak in the range 9–11 and 020 reflection appearing at 10.32 (d-spacing, 8.571 A˚), characteristic of a tendon type polymorph [34] The LMWC showed a shift of the same to 9.94 (d-spacing, 8.898 A˚), which is probably due to larger d-spacing as a result of increase in the unit cell dimension that results in the incorporation of fewer water molecules Decrease in the intensity of 020 reflections in LMWC was also indicative of a decrease in the acetamido group [33,34] The observed decrease in CrI (Table 1) was in good agreement with the fact that decreased DA results in less hydrated crystals that affects the CrI

Solid-state CP-MAS 13C-NMR This technique is known

to be very sensitive to changes in the local order structure Chitosan showed higher crystalline nature as evidenced by narrow line width of the peaks (Fig 1) Peak broadening in LMWC indicates decrease in crystalline nature (Fig 7), in support of its low CrI The splitting of peaks corresponding

to C1, 3, 4 and 5 ring carbons supports a rather different hydrogen-bonding network and thus the existence of multiple conformations in LMWC Chemical shift values for ring and methyl carbon atoms are given in Table 3 The observed difference in chemical shift values of C1 and C4 indicated a conformational change of the glycosidic linkage

in LMWC Multiplicity of the peak corresponding to C4

is independent of DA and is associated with deacetylation

Table 2 Effect of acids, molecular mass and DA on the chitosanolytic

activity of pronase.

Activity (l M reducing sugar releasedÆmin)1Æmg)1) Effect of different acids (1%) as the solvent

Acetic acid (pK a , 4.76) 3.4 1.10

3.0 0.89 Formic acid (pK a , 3.74) 3.4 1.08

Lactic acid (pK a , 3.73) 3.0 0.59

Effect of chitosans of different molecular mass (DA,  23–26%)

71 (kDa) – 1.15

150 (kDa) – 1.60

400 (kDa) – 1.67

600 (kDa) – 1.76

Effect of DA (%) on chitosanolysis by pronase

 15 (83 kDa) – 0.70

 19 (75 kDa) – 0.86

 25 (71 kDa) – 1.15

Fig 3 Capillary electropherogram of pronase in Tris/glycine buffer.

pH 8.3 detected at 280 nm Inset: SDS/PAGE of protein markers

(lane A) and pronase (lane B).

temperature [27] Nevertheless, it could also be due to

increased mobility of the individual chains resulting from

decrease in chain length, as evidenced by decreased

molecular mass of LMWC The DA calculated using

13C-NMR spectra was in accordance with the one

calcula-ted by IR-spectra (Table 1) The peak corresponding to

-CH3 showed a slight decrease in the height in case of

LMWC further supporting a decrease in the DA [33]

(Table 3 and Fig 1) In LMWC, the -C¼O peak showed

splitting and considerable difference in chemical shift value

(3.467 p.p.m.) compared to native chitosan, further

con-firming the conformational inhomogeneity

Liquid-state 13C-NMR 13C-NMR in the liquid state

(Fig 8) was performed as the peaks corresponding to

-CH3 and -C¼O were not obvious in solid-state spectra

There was a decrease in the peak height corresponding to

-CH3 in LMWC compared to chitosan, whereas

chito-oligomers + monomer showed slight increase in the same

The DA (%) calculated using the spectra was 26.02 and

18.97%, respectively, for chitosan and LMWC [22], which

were in good agreement with those calculated before

(Table 1)

Solubility of LMWC The solubility study was performed

according to the method described by Qin et al (2003) [35]

Although the molecular mass of LMWC was <10 kDa, it

was not readily soluble in aqueous medium (water-solubility

of LMWC, 1 h sample )72%, 5 h )63%), instead it required a very dilute acidic medium for complete solubilization (LMWC, 100% solubility compared to 13% solubility of chitosan in 0.01% acetic acid, chitosan required 1% acetic acid for complete dissolution) There was no evidence for annealed polymorphism in LMWC, as indicated by the absence of a 15 reflection in the X-ray diffraction pattern [25] This observation was contrary to the earlier reports that decrease in the molecular mass of chitosan is associated with increased solubility, attributed to decreased intermolecular interactions, such as van der Walls forces and hydrogen bonds, provided that the DA did not change after degradation [35] However, chitosanolysis by pronase was associated with decreased DA as evidenced by the spectral data and hence the observed decreased solubility (in water) could probably be due to the exposure and thus conversion of -NH2 groups on LMWC to the sodiated form (Na+, added to terminate the reaction), which could result in a different molecular conformation This was obvious from the d-spacing of LMWC (8.898 A˚), which was neither typical of tendon nor of L-2 polymorph Compared to the 1 h sample, LMWC obtained after 5 h incubation showed much lower solubility as there was a further decrease in the DA (Table 1), exposing more -NH2 groups for sodiation Cheng et al (2000) made use of two volumes of acetonitrile [36], instead of an equal volume of

2 N NaOH as in the present study, to arrest the catalytic reaction Similar use of acetonitrile in the present study

Fig 4 Effect of (A) pH and (B) temperature on pronase activity and the stability studies (C & D).

resulted in LMWC that was readily soluble in water, thus,

supporting the formation of sodiated LMWC when alkali

was added The drawbacks of using acetonitrile were, its

high cost and flash treatment of the supernatant at elevated

temperature, which may result in Maillard reaction

products limiting one step production of LMWC and oligomers + monomer Although 0.01% acetic acid was necessary for LMWC solubilization, the pH value of resulting solution was near neutrality ( 6.8)

Nevertheless, the decrease in molecular mass, DA and CrI, and molecular inhomogeneity of LMWC did not hinder its biofunctionality LMWC exhibited potent anti-microbial activity towards Bacillus cereus [106 colony forming units (CFU)] and Escherichia coli (103CFU), with

a minimum inhibitory concentration of 0.01 and 0.03% (w/v), respectively The inhibitory activity of LMWC was much superior over that of chitosan, and resulted in a progressive lysis of the bacterial cells as evidenced by SEM (unpublished observations)

Characterization of the mono-oligomeric mixture Chito-oligomers + monomer, due to their affinity, bind to charcoal through a weak van der Wall’s force and their binding capacity is dependent on charge/mass ratio The affinity decreases with increase in the charge/mass ratio of the binding molecules The bound fractions, due to their varying solubility, are desorbed using organic solvent gradient [37,38] In the present study, use of 60% ethanol resulted in the elution of all the bound chito-oligomers + monomer Fraction I (obtained after re-N-acetylation), by both GPC (Biogel P2 column) and HPLC (aminopropyl column) (Figs 8 and 9, respectively) showed the existence of dimer and trimer in abundance along with higher oligosac-charides (tetra- to hexamer), whereas Fraction II showed mainly dimer and tetramer along with GlcNAc

Fig 5 Environmental scanning electron microscopy ( 3500) of (A)

chitosan and (B) LMWC.

Fig 6 Circular dichroic (CD) spectra of chitosan and LMWC.

Fig 7 X-ray diffractograms of chitosan and LMWC.

IR-spectrum of the freeze-dried chito-oligomers +

monomer showed a DA value of 31.2% (spectrum not

shown), which was in accordance with that calculated using

13

C-NMR ( 30.83%, Fig 1) GPC-HPLC profiles of

Fraction I and II are depicted in Figs 9 and 10, respectively

In the solid-state13C-NMR spectra (Fig 1), appearance of

peaks at 25.831 and 173 600 p.p.m corresponding to -CH3

and -C¼O groups, respectively, indicated the release of

GlcNAc/GlcNAc-rich oligomers; this was further

con-firmed by liquid-state 13C-NMR (Fig 8) The DA value

calculated using liquid-state spectrum was 31.33%, which

was in good agreement with that calculated by solid-state

spectrum (Table 1)

The monomeric residue sequence in chitosan is of four

types, -GlcN-GlcN-, -GlcN-GlcNAc-,

-GlcNAc-GlcN-and -GlcNAc-GlcNAc-, of which the first is the major

type and the last one results from the heterogeneous

de-N-acetylation Formation of chito-oligomers as one of the

products and a drastic decrease in molecular mass of

chitosan indicates an endo-type activity of pronase

Addition of hexosaminidase (specific for the release of

GlcNAc from the nonreducing end) to LMWC and

Fraction II did not result in the release of reducing groups indicating action of pronase on -GlcNAc-GlcN-linkage resulting in the products (LMWC and oligomers) with GlcNAc at reducing ends In addition, the presence of GlcNAc in Fraction II and the absence of GlcN in Fraction I demonstrates the exo-type action of pronase and this was further confirmed by using (GlcNAc)2 and (GlcNAc)3 as substrates, where the activities were found

to be 0.09 and 0.18 units, respectively From these data,

Table 3 Solid-state CP-MAS13C-NMR chemical shift values of ring and methyl carbons of native chitosan, LMWC and chito-oligomers.

Sample

Chemical shift values (p.p.m.)

Chitosan 26.843 60.906 78.703 84.703 108.103 176.524 LMWC (1 h) 25.521 62.942 77.878 87.332 107.025 173.057 Chito-oligomers + monomer 25.831 61.025 78.051 87.140 107.562 173.600

Fig 8 Liquid state13C-NMR spectra of chitosan, LMWC and freeze

dried chito-oligomers + monomer.

Fig 9 Chromatographic profile of the chito-oligomeric Fractions (I and II) on Biogel P2 column (Bed volume, 80 mL).

along with that of hexosaminidase, it could be concluded

that during exo-type of activity, pronase prefers GlcNAc

at the nonreducing ends, i.e -GlcNAc-GlcNAc-linkage,

the action on which results in the release of GlcNAc and

products with GlcNAc at the reducing ends

The observed decrease in DA value of LMWC is due to

the release of GlcNAc and GlcNAc-rich oligomers as some

of the products An examination of DA values indicates that

the total DA of the products is equal to that of native

chitosan (19% for LMWC and 31.2% for chito-oligomers

+ monomer, the average of these is 25.1%, in agreement

with DA of native chitosan, Table 1) From these data, it

was also evident that in the reaction catalyzed by pronase,

only glycosidic bonds were cleaved leaving the N-acetyl

groups intact

Evaluation of the ionization constants of pronase

towards chitosanolysis

The ionization constants of enzymes that obey Michaelis–

Menten kinetics can be further evaluated by analyzing the

curves of log (Vmax/Km) vs pH, which yields pKEvalues,

and often provides valuable clues as to the identities of the

amino acid residues essential for enzymatic activity [39] In

the present study, the calculated pKEwas 4.0, suggesting

the involvement of aspartic (Asp, pKa3.90)/glutamic (Glu,

pK 4.07) acid residues for the catalysis Moreover, the pH

optima of pronase towards proteolysis and chitosanolysis were at two extremes (Fig 2A), suggesting possible varia-tions in the protein conformation Use of 1 mM phenyl-methanesulfonyl fluoride in the reaction medium brought about 100% inhibition of proteolysis, whereas, the chitos-anolytic activity was inhibited by only 30%, which sugges-ted the involvement of a serine residue in this nonspecific catalysis As a serine protease, pronase bears a triad consisting Asp, His and Ser residues in the catalytic site [39,40] Watanabe et al [41], by site directed mutagenesis, identified the involvement of crucial Glu and Asp residues during the chitinolytic activity of chitinase A1 from Bacillus circulans W-12 Replacement of SerfiAla decreased the catalytic activity without affecting the Km, and the mutant retained only 10% of the wild-type activity It was concluded that Ser might have an important role in maintaining the structural features of the catalytic site On similar lines, it may be tempting to speculate that Asp/ Glu+ Ser are probably involved during the pronase-catalyzed chitosanolysis

While screening the proteases for chitosanolysis, highest activity was shown by pepsin, followed by papain, pronase and a protease from Aspergillus niger (4.98, 1.78, 1.16 and 0.058 units, respectively) Though, pepsin and papain were inexpensive, the former showed a decrease in the activity during the course of time due to its auto-catalytic property and the latter, due to its higher initial velocity, resulting in the formation of LMWC of  4–5 kDa and below As pronase overcomes these drawbacks, it could be considered

as a better chitosanolytic agent in comparison with other proteases screened

In conclusion, depolymerization of chitosan using pro-nase, though unusual and nonspecific, is a viable alternative way of catalysis to produce LMWC, chito-oligomers (DP 2–6) and monomer (GlcNAc), quantitatively and econom-ically with multiple applications Although chitosanase is

the enzyme for chitosanolysis, it is expensive ( 15 $ per unit), unavailable in bulk and results in more monomer and oligomers (DP 2–3), whereas pronase is relatively inexpen-sive ( 0.13 $ per unit), easily available and LMWC of any desired molecular mass can be custom made by manipula-ting the reaction conditions Use of pronase in place of chitosanase is still feasible even though pronase quantity has

to be increased by 40-fold to mimic the specific activity of chitosanase Thus, the results add value to otherwise commercially available raw materials, viz chitosan from the offal of marine food processing industry (and pronase), and show the biofunctionalities of these materials

Acknowledgements

Authors thank Dr S Subramanian (Indian Institute of Science, Bangalore) for X-ray analysis, Dr A.G AppuRao and Dr Sridevi Annapurna Singh (Department of Protein Chemistry and Technology, CFTRI, Mysore) for CD measurements A.B.V.K thanks CSIR (New Delhi) for the senior research fellowship.

References

1 Tharanathan, R.N & Kittur, F.S (2003) Chitin – The undisputed biomolecule of great potential Crit Rev Food Sci Nutr 43, 61–87.

Fig 10 HPLC profiles of chito-oligomeric Fractions I and II separated

on charcoal/Celite column (values in the parenthesis indicate retention

time of the individual peaks in minutes).