ARTICLE PHARMACEUTICAL BIOTECHNOLOGY

DOI: 10.5504/50YRTIMB.2011.0029

PREPARATION OF CHITOSAN BEADS FOR TRYPSIN IMMOBILIZATION

M. Kamburov, I. Lalov University of Chemical Technology and Metallurgy, Biotechnology Department, Sofia, Bulgaria Correspondence to: Michail Kamburov E-mail: m_kamburov@yahoo.com

ABSTRACT
The present study deals with the potential application of chitosan macrobeads for immobilization of trypsin. Macrobeads were prepared by precipitation and their mechanical properties were improved by covalent crosslinking using glutaraldehyde. Scanning electron microscopy studies showed that the beads size varied between 2.5 mm and 3.5 mm depending on the chitosan content and molar excess of glutaraldehyde. Trypsin (EC.3.4.21.4) was chosen as model enzyme for immobilization on the chitosan gel beads pre-activated with glutaraldehyde. Catalytic properties and kinetic parameters of free and immobilized trypsin were determined and the impact of various technological parameters on enzyme activity was investigated. The obtained results showed that the optimal pH and temperature for the hydrolytic reaction catalyzed by immobilized trypsin for low molecular weight substrates were pH 8.0 and 50oC, respectively. The effect of substrate concentration was also studied and the values of the constants thus determined were: Km=1,54 mM/L and Vmax 3.3 M/min. Keywords: chitosan beads, trypsin, trypsin immobilization reactive functional groups, high hydrophilicity, mechanical stability and rigidity. The properties of the immobilized enzymes depend on its molecular characteristics, nature of the support material and the immobilization method. Furthermore, as a part of a two-phase system, the immobilized enzyme suffers from inevitable mass transfer limitations, which leads to unfavorable effects on its catalytic performance. Of all the carrier materials applied for enzyme immobilization, chitosan has many advantages. It is a natural polyaminosaccharide, and is also one of the worlds most plentiful and renewable organic resources. Chemically, chitin is composed of (1-4) linked 2-acetamido-2-deoxy--D-glucose residues, which form a long linear polymer (Fig. 5A). Chitosan is obtained from chitin by partial Ndeacetylation in a strong alkali medium. The properties of the chitosan thus produced depend on the degree of deacetyletion (Fig. 5B) (9). Chitosan is insoluble in water but it is soluble in acidic solutions of pH lower than 6.5. Chitosan possesses distinctive chemical and biological properties. Its polyglucoseamine chains contain reactive amino and hydroxyl groups, amenable to chemical modifications. Chitosans unique biological properties include biocompatibility, biodegradability, nontoxicity, affinity to proteins and physiological inertness. As enzyme immobilization matrices, chitosanbased materials are used in the form of powders (23), flakes and gels of different geometrical configurations (37, 40). Preparation of chitosan gels is promoted by the fact that chitosan dissolves in dilute solutions of organic acids (formic, acetic and citric acids) to form viscous solutions that precipitate in high pH solution and form waterinsoluble complexes. Thus, chitosan gels in the form of beads, membranes, fibers and sponges can

Introduction
Solid phase biochemistry is an established branch of biochemistry used in areas where the liquid phase biochemistry is inapplicable or difficult to apply (9). The enzymes immobilized on solid supports have many advantages in comparison with those in solution, however, their high cost, instability and low regeneration rate have limited their appliance in research. Due to the poor recovery and regeneration of enzyme in aqueous solutions, immobilization has drawn a lot of attention during the last few decades. Immobilization is usually achieved through attaching the enzyme onto or within the solid matrix. As a result, heterogeneous enzyme systems are obtained, whereas in the living cell the enzymes are attached to the cellular membranes. Thus the latter systems are much more stable, reliable and favorable for the enzyme activity. The enzymes attached to the solid supports are more robust and resistant to environmental changes. Additionally, immobilized enzymes have the advantage of being applicable in both batch and continuous processes and are easily removed from the reactor. Although immobilized enzymes usually show lower catalytic activity than the free ones, they are more stable, can be reused and therefore they are cheaper and more effective in large scale applications. Enzyme immobilization has drawn the attention of many researchers (15, 22, 24, 26). In principle, the enzymes can be immobilized by physical (weak interactions between the matrix and the enzyme) and chemical (covalent bonds) methods. Various matrices can be used for immobilization. A suitable matrix should possess high affinity to proteins,

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ARTICLE PHARMACEUTICAL BIOTECHNOLOGY be manufactured. The methods of chitosan gel preparation can be loosely divided into four groups: solvent evaporation method, neutralization method, cross-linking method and ionotropic gelation method (1, 8, 10, 16, 17, 20, 33, 36). Neutralization method is mainly used for the preparation of spherical beads of different size and porosity, obtained by dropwise addition of chitosan solution to a solution of NaOH, followed by crosslinking. Enzymes may be immobilized on such beads either by adsorption or covalent binding to the matrix. Glutaraldehyde is usually used as a cross-linking and activating agent (41), due to its bifunctionality, reliability and easy use. The model proteolytic enzyme chosen for the immobilization in this study was trypsin because of its high efficiency, specificity and wide use in biotechnology and medicine (2, 12, 14, 21, 39, 42).

DOI: 10.5504/50YRTIMB.2011.0029 production of chitosan beads is shown in Fig. 1. Chitosan solution was added dropwise through a flat-tip needle with an internal diameter of 0.413 mm (flow rate=0.4 ml/min) to an aqueous solution of sodium hydroxide (10% w/v) and ethanol in a volume ratio of 4:1 under discontinuous magnetic stirring. Since chitosan is not soluble at high pH, the drops solidified due to polymer precipitation. The obtained beads were filtered and washed with distilled water to remove residual sodium hydroxide and were stored in water. In order to form a covalent network and be activated, the beads were treated with glutaraldehyde. To this end 1.5 g swollen beads was suspended in 25 ml aqueous solutions under mild magnetic stirring. Being a dialdehyde, glutaraldehyde (OHC(CH2)3CHO) reacts with the primary amine groups of chitosan thus forming Schiff bases Fig. 5C. The concentration of glutaraldehyde in the solution is given as a molar ratio of glutaraldehyde molecules: amine groups. Two different molar ratios (1:1 and 1:10) were studied exposing the beads to the glutaraldehyde for 4 h at 20C under mild stirring. The chitosan beads were then washed with distilled water to remove residual glutaraldehyde and were stored in water.

Material and Methods

Reagents Chitosan from shrimp shells - low-viscousity (ChL) and Chitosan from crab shells - highly-viscous (Ch-H) were purchased from Fluka and trypsin powder from bovine pancreas (EC 3.4.21.4) and N-benzoyl-DL-arginine-p-nitroanilide.HCl were bought from Sigma. Glutaraldehyde (25%) was purchased from Merck Co. All other chemicals were of analytical grade. Determination of the degree of deacetylation of chitosan First derivative UV-VIS spectrophotometric analysis of N-acetylglucosamine standard solutions in 0.01 M acetic acid was carried out by Tan et al. (39) on a Varian-Cary 100 UV spectrophotometer. The vertical distance to each N-acetylglucosamine solution spectrum, H was measured at 199 nm. A linear calibration curve was obtained by plotting the H values against the corresponding Nacetylglucosamine concentration. Chitosan samples (0.01 g) were dissolved in 100 ml 0.01 M acetic acid and the first derivative spectrum was measured. The vertical distance, H values was determined and the contribution due to each N-acetylglucosamine was obtained from the calibration curve. The DD% of the unknown chitosan samples was determined using the following equation: DD=100-[A/(W-204A)/161+A]X100 [1]

Fig. 1. Experimental design for production of chitosan macrobeads

Scanning electron microscope studies The chitosan microparticles were freeze-dried and analyzed with scanning electron microscopy. Samples were coated with a thin gold layer, using a fine coater JEOl JFC-1200 and monitored microscopically (JEOL JSM-5510, Japan). Immobilization of Trypsin Trypsin was immobilized by adding 1.25 g of activated macrobeads to 9 ml 0.1 M phosphate buffer (pH 7.0) containing 20 mg trypsin. The reaction was run at 4C under mild stirring for 16 h. The beads were then filtered and washed with 0.1 M phosphate buffer (pH 7.0) until no soluble

where A is the amount of N-acetylglucosamine determined , W is the mass of chitosan used (18). Preparation of Macrobeads Chitosan-H (0.2 g) and Chitosan-L (0.6 g) were dissolved in 10 ml 2 % (w/v) acetic acid under overnight mild stirring. The system used for

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ARTICLE PHARMACEUTICAL BIOTECHNOLOGY protein was detected and stored at 4C. The yield of immobilized trypsin was estimated according to Eqs. 2: W)].C.V Immobilized protein=[(A0A)/(A0. [2]

DOI: 10.5504/50YRTIMB.2011.0029 Determination of kinetic parameters Km and Vmax. Constant amounts of free and immobilized trypsin were mixed with BAPNA solutions of increasing concentration. The initial rate of reaction was measured for each sample using a discontinuous assay following the Lineweaver-Burk plot (4).
1

where A0 is the absorbance at 280 nm of the free trypsin solutions, A is the absorbance at 280 nm of the trypsin solutions after immobilization, C is the concentration of the free trypsin solution (mg/ml), V is the volume of free trypsin solution (ml), and W is the weight of the dried sample (mg). Relative activity was determined according to equation 3: Relative activity=A/Acalculated [3]

V is the initial reaction rate, Vm is the maximum reaction rate, [S] is substrate concentration, and Km is the Michaelis-Menten constant.

. +

[4]

Results and Discussion

where A is the measured activity of the sample, Acalculated is the activity of trypsin in solution. Measurement of Trypsin Activity Determination of amidase activity Amidase activity of free and immobilized trypsin was determined by measuring the amount of pnitroaniline produced by the trypsin-catalyzed hydrolysis of N-benzoyl-DL-arginine-pnitroanilide.HCl using a discontinuous assay as described by Schleuning and Fritz (35) with slight modifications. Assay solution contained 4.4 ml of 0.1 M triethanolamine buffer (pH 8.0), 0.2 ml of 2.3 mM BAPNA, and 0.4 ml of sample. Absorbance was measured at 405 nm and a molar absorbency coefficient () of 9950 M-1.cm2 was used in the calculations. Specific activity of free and immobilized enzymes was expressed in mol pnitroaniline/min.mg trypsin. Determination of proteolytic activity Proteolytic activity of free and immobilized trypsin was assayed by the Anson's method (3) and hydrolysis rate of denatured hemoglobin was measured. One unit of enzyme activity is defined as the absorbance of 0.001 at 280 nm of trichloracetic acid soluble hydrolysis products per minute under standard conditions. The specific activity was calculated in units of enzyme activity per mg of protein. Determination of physicochemical properties of free and immobilized trypsin Effect of pH on enzyme activity The dependence of enzyme activity on pH was investigated for free and immobilized trypsin in buffers with pH values from 5.0 to 10.0 at 37C. Effect of temperature on enzyme activity The effect of temperature on enzyme activity was studied for the interval 20-80C for free and immobilized trypsin at pH 7.0. Chitosan is commercially available in various molecular weights and degrees of deacetylation (11). The degree of deacetylation is one of the main parameters pre-determining the areas of application of chitosan (4, 30). Since the degree of deacetylation depends on the manufacturing method, it is essential to measure the degree of chitosan deacetylation, prior to its use as a matrix for enzyme immobilization. There are various approaches for determining the degree of chitosan deacetylation, such as: nuclear magnetic resonance spectroscopy (13); hydrogen bromide titrimetry (7); infrared spectroscopy (34) and first derivative UVspectrophotometry (31, 39). The DD values of chitosan can be associated with the analytical methods employed. The concept of derivatizing spectral data was first introduced in 1950s and proved to have a lot of advantages. The development of computer technology made the method easily applicable. The zero-order spectrum obeys Beers law (A=bc), which is a linear relationship between the substance concentration and absorbance. This also holds true for the first order derivative [5], (32). UV spectra of two types of chitosan - Chitosan low .

viscousity (Ch-L) and Chitosan high-viscousity (Ch-H) are shown in Fig. 2B and UV spectra of various standard N-acetilglucosamine solutions are presented in Fig. 2A. The first derivative spectra of all UV spectra are shown on Fig. 3A and Fig. 3B. The amplitudes of the analyzed chitosan were measured and the effect of GlcNAc was calculated using the calibration curve. DD was estimated for both Ch-L (90%) and Ch-H (78%). Khan et al. (18) calculated the DD values using different methods for chitosan samples and estimated that they depended on the type of method employed. The DD values were noted to be highest (90%) if measured by FDUV-spectrophotometry.

[5]

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DOI: 10.5504/50YRTIMB.2011.0029

Absorbance

A
Wavelenght (nm) 1 2

Fig. 2. UV spectra of standard solutions of N-acetylglucosamine (A) (1=40.7 g/ml, 2=30.5 g/ml, 3=24.4 g/ml, 4=20.3 g/ml, 5=17.4 g/ml, 6=12.2 g/ml) and UV spectra of solutions of chitosan (B) (Ch-H=100 g/ml, Ch-L=118 g/ml)

dA/d

Wavelenght (nm) 1 2

Fig. 3. FDUV of standard solutions of N-acetylglucosamine (A) and FDUV of solutions of chitosan (B)

dA/d

Wavelenght (nm) Ch

Although both macro- and micro- chitosan beads were suitable for enzymes immobilization, we preferred to use in this study chitosan macrobeads prepared by the precipitation method (38). According to the latter, the formation of macrospheres is a result of a surface phenomenon consisting in interaction between the chitosan solution and the coagulant (NaOH). It induces

phase separation and formation of precipitated beads. The chitosan drops coagulates into spherical beads of diameter 3.10.3 mm. When drying at 50oC until constant weight, they shrunk to 1.00.2 mm. The wet beads were stored frozen at -60oC and consequently freeze-dried for 16 h.

A.

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B.

C.

Fig. 4. Scanning electron micrographs of a chitosan macrobead (A), microporous surface of beads processed from Ch-L (B) and Ch-H (C)

SEM images of freeze-dried chitosan beads are shown in Fig. 4A, Fig. 4B and Fig. 4C. As seen from the figure, the macroparticles were not completely spherical in shape. Their surface was porous and rough. Fig. 4B and Fig. 4C show the morphology of chitosan macroparticles of chitosan with different degree of polymerization (Ch-L and Ch-H). As seen from the figure, the MW of chitosan affects significantly the morphology of the beads. The average size of freeze-dried chitosan macrobeads was arround 1.5 mm. Glutaraldehyde has been used for many years for enzyme immobilization (27). It reacts with several functional protein groups, such as amine, thiol, phenol, and imidazole, since nucleophilic amino acid residues are the most reactive ones. Glutaraldehyde reacts reversibly with amino groups over a wide pH range (pH 3.0), excluding the interval between pH 7.0 and 9.0. The most preferred functional group in proteins for crosslinking is the -amino group of lysyl residues. Besides its high pKa constant (>9.5), it is presumed that the small percentage of amines present at low pH is sufficient for the reaction with glutaraldehyde. Most proteins contain many lysine residues, usually located on the surface of their molecules. Furthermore, lysine residues are occasionally found in the catalytic centers, which is a predisposition for sustaining enzymatic activity after immobilization of the enzymes to solid supports. The presence of reactive amino groups in chitosan makes it suitable for an enzyme carrier (5, 18, 31). Glutaraldehyde forms covalent imine bonds with the amino groups of chitosan, as a result of rearrangement of the primarily formed Schiff base (Fig. 5C). Monsan (29) has also discussed the possibility of glutaraldehyde reaction via ,unsaturated double bonds, formed as a result of its polymerization (aldolic condensation). Such products with amines produce Michael-type adducts, which are stable even when subjected to acid hydrolysis.

Fig. 5. Structure of chitin (A) and chitosan (B). Crosslinking and activation of chitosan with glutaraldehyde (C). Schematic representation of immobilization of trypsin on chitosan matrix (D)

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ARTICLE PHARMACEUTICAL BIOTECHNOLOGY The proposed method of immobilization of trypsin on chitosan beads includes the reactions represented on Fig. 5D. The pH dependency of the cross-linking of chitosan by GA is presupposed by a decrease in the reactivity of protonated amino groups in the reaction with carbonyl groups. The reaction proceeds with a high rate (42) in acidic media (pH 4.0) where the degree of protonation of the NH2 groups is 0.9 and therefore only 10% of them are active in reactions of nucleophilic addition. The cross-linking reaction proceeds with a very high rate at pH>6.5, i.e. when the chitosan NH2 groups are deprotonated. Parameters, characterizing trypsin immobilization on chitosan beads are presented in Table 1. As seen from the table, protein content in the dry beads was higher in the case when chitosan was treated with 10 molar excess of GA. The yield of enzyme immobilization however, depended on the

DOI: 10.5504/50YRTIMB.2011.0029 chitosan content in wet beads. The specific proteolytic activity of trypsin decreased with increasing of glutaraldehyde molar ratio (GA:NH2=1:10). The results from studying the external mass transfer limitations for BAPNA substrate showed that at a speed of stirring of 300 rpm/min the relative enzyme activity approached 95%. This indicates that the diffusion of low molecular substrate BAPNA is a rate limitation factor for the mass transfer processes. Hydrodynamic measurements are important for studying enzyme reactions with high molecular weight substrates (hemoglobin, casein). Such reactions are limited by the substrate diffusion in the particles. Respectively the external diffusion resistance to mass transfer should be reduced. Our experiments proved that the relative activity towards hemoglobin dropped down to 60%.

Parameters/Samples Chitosan content in wet beads [%] Diameter of wet beads [mm] Treatment with GA at molar ratio GA:NH2 groups Protein content [mg/g] dry beads Specific activity to BAPNA * [U/mg protein] Relative activity to BAPNA * [%] Proteolytic activity to hemoglobin * [U/mg protein] Relative activity to hemoglobin * [%] Specific activity to BAPNA ** [U/mg protein] Relative activity to BAPNA ** [%]

TABLE 1 Characterization of immobilized trypsin on chitosan beads Ch L Ch L Ch H Ch H 1:1 1:10 1:1 1:10 7.1 3.2 1:1 62 24.0 21.5 105.4 13.8 78.3 70.2 329.3 43.1 7.7 2.8 1:10 180 12.8 11.5 61.9 8.1 70.8 63.5 236.8 31.0 3.5 3.4 1:1 45 34.2 30.8 162.7 21.3 95.0 85.2 458.4 60.2 3.9 3.1 1:10 120 17.2 15.4 91.7 12.0 82.5 74.0 372.1 48.7

Proteolytic activity to hemoglobin ** [U/mg protein] Relative activity to hemoglobin ** [%]

Specific activity to BAPNA of free trypsin=111.5 U/mg Proteolytic activity to hemoglobin of free trypsin=764 U/mg *Stirrer speed - 60 rpm/min, ** Stirrer speed - 300 rpm/min

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ARTICLE PHARMACEUTICAL BIOTECHNOLOGY The interdependence of the relative activity of free and immobilized trypsin (sample Ch-H 1:10) and pH is shown on Fig. 6. The optimal pH for enzyme reaction remained unchanged up to pH 8.0. When immobilized, the pH- activity profile of the bound trypsin was broader, which means that the enzyme was protected at a wider range of pH. This phenomenon was observed only when the enzyme was covalently bound to the matrixes. The temperature-activity profiles are shown at Fig. 7. The optimum temperature of free trypsin under experimental conditions was 40C, whereas the optimal temperature of immobilized trypsin shifted to 50C. This indicates that the effects of temperature on both free and immobilized trypsin were different. Normally, enzymes are denatured when the temperature exceeds 60C, whereas the covalently coupled enzymes were heat resistant. The kinetic results for free and immobilized trypsin are shown on Fig. 8, as Lineweaver-Burk plots are obtained at various concentration of substrate (BAPNA). The linear nature of the Lineweaver-Burk plots proves that in both cases enzyme reactions followed the Michaelis-Menten kinetics. As the Km is a characteristic constant for enzyme activity, it was calculated for both immobilized and free enzymes. The Km for free trypsin was found to be 1.18 mM/L, whereas for the immobilized trypsin it was slightly higher (Km=1.54 mM/L). Since the Km values were of the same magnitude, this means that the catalytic function of the enzyme was not significantly impaired by the coupling process. The increase in Km might be a result of the lower accessibility of the substrate to the active site of the immobilized enzyme, which could be due to several factors, such as conformation changes in the protein molecule induced by the attachment to the matrix, steric, and diffusion effects.

DOI: 10.5504/50YRTIMB.2011.0029

Fig. 7. Effect of temperature on catalytic activity of free and immobilized trypsin

Fig. 8. Lineweaver-Burk plots of free and immobilized trypsin for substrate BAPNA

Conclusions
Chitosan beads in the millimeter range (macrobeads) were prepared to be used for immobilization of enzymes. Their morphology, diameter and other characteristics were studied by physicochemical methods and scanning electron microscopy. The beads were activated at different molar ratios of glutaraldehyde and were used for immobilization of trypsin as a model. The obtained results showed that the higher ratios of GA resulted in a higher yield of immobilized protein for botyh Ch-L and Ch-H samples (120-180 mg protein/g dry beads). The immobilization did not affect neither pH no the temperature optimum of the enzyme. Higher specific and proteolytic activity was registered with the Ch-H sample. The excellent kinetic properties of the immobilized enzyme demonstrate that the trypsin-chitosan system is suitable for practical applications. The relative

Fig. 6. Effect of pH on catalytic activity free and immobilized trypsin

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ARTICLE PHARMACEUTICAL BIOTECHNOLOGY activity of immobilized trypsin measured with the substrate BAPNA was 10-30%. It increased up to 85% when the stirring speed was elevated to 300 rpm/min. Identically, the relative proteolytic activity approached 60% at high stirring speed activity when hemoglobin was used as substrate. The latter could be explained by steric and diffusion limitations.

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