Analysis of Hydrolysates by HPLC

Chitooligomers are analyzed by two HPLC sets. The first set comprises a Tosoh TSKgel Amide-80 column (4.6 x 250 mm; flow rate, 1.0 mL/min; eluent, 65:35 MeCN-water). The second set comprises an Asahipak NH2P-50 column (4.6 x 250 mm; flow rate, 1.0 mL/min; eluent, 70:30 MeCN-water). Quantitative analysis is done using an authentic mixture of oligomers, and a UV detector at 210 nm.

N-Acetylation of the hydrolysates doubled the amount of total (GlcNAc) and (GlcNAc)3, and increased the amount of (GlcNAc)4 ninefold. The amount of (GlcNAc)n after hydrolysis and N-acetylation increased with the increase of degree of acetylation of partially N-acetylated chitosans (PNACs). The optimum degree of acetylation for the formation of oligomers is ca. 70%. The yields of (GlcNAc)n (n = 2 - 5) after gel filtration chromatography are 16.3, 10.3, 18.0, and 1.7 mg, respectively, from the hydrolysates of 72% N-acetylated chitosan (100 mg). This procedure is useful for the production of (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4.

Figure 3 shows the effect of the concentration of acetic acid and methanol on degree of acetylation. The chitosan concentration is 0.3% and the molar ratio of acetic anhydride to amino groups is 0.60 (17).

Partially reacetylated chitosans are suitable for hydrolysis with lysozyme. The recommended concentrations for acetylation are: chitosan 1%, acetic acid 0.4%, methanol 50%.

The highest digestibility of chitosans is due to the blocks of NAcGlc sequences. The hexamers containing three or more acetylated units contribute mostly to the initial degradation velocity. This information is based on the Michaelis-Menten analysis of the degradation data for various hexamers under the action of lysozyme (18). The substrate specificities of human and hen egg white lysozymes with respect to partially N-acetylated chitosans are undistinguishable. Tailor-made chitosans with a predetermined degradation rate in the human body can be made by simply controlling their degree of acetylation (19).

Lysozyme does not seem to lead straightforwardly to the production of oligomers: partially N-acylated chitosans (N-acetyl-N-hexanoyl) subjected to the

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Effect of the concentration of methanol and acetic acid on the degree of acetylation: acetic acid, 0.4% (), 1% (•). and 3% (A); molar ratio of acetic anhydride to amino group, 0.605; chitosan, 0.3%. From Aiba (17).

action of lysozyme show final average molecular weights of 1-10*104 over a contact period of 24 h (20).

Immoblized Papain

Depolymerization is carried out in 1% lactic acid at 20°C in the presence of immobilized papain: at fixed times, aliquots are taken, filtered on filter paper to remove the immobilized enzyme and diluted (10-20 fold) with aqueous solutions of acetic acid and sodium acetate, in such a way as to prepare solutions for n GPC, and LS measurements of the following composition: 0.5 M acetic acid, 0.2 M sodium acetate, and 1.0 g/L lactic acid.

The elemental analysis and the molecular parameters for the control chitosan (0.23) and for the same contacted with immobilized papain for 7 d and isolated as the control sample are found to be the following: Chitosan (0.23): N 7.42, C 40.77, H 6.91, N/C 0.182; n 11.77 dl/g, Mw 780,000, 4 2.10-3, Rg 1190 A°. Papain-treated chitosan (0.23) (7 d): N 7.31, C 40.11, H 6.69, N/C 0.182, n 3.66, Mw 183,000, 4 3.10-3, RG 700 A°.

The reaction is followed using a viscometer (Haake Rotovisco RV-20, M5) driven by a personal computer with Haake Rotation software. The doublewalled NV rotor is housed in an 11-mL cup filled with the solution under study (10 mL), to which 1 mL of enzyme solution is added with the aid of a syringe after reaching the desired temperature and just before starting measurements. The measurements are done at a shear rate value (200 s-1) which does not gen-

20 40 60 BO 100

Concentration of rnelhanol ( Vt J

20 40 60 BO 100

Concentration of rnelhanol ( Vt J

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erate excessive mechanical degradation. Such shear rate is reached in 0.1 min and protracted for 9.8 min with 0.1 min deceleration to 0. Three hundred eightyfour readings are automatically recorded in the 10 min period. The initial 0.1 min during which the rotor accelerated and mixed the solutions is not taken into consideration when calculating the initial velocity. Initial velocities (mPa.s.min-1) are read graphically from the points plotted in the subsequent 0.3-min period. The overall error involved is within 5%. Measurements are also done on controls, where the lipase solution is replaced with water. In general, the final enzyme concentration is 0.45 mg/mL, unless otherwise stated. The viscosity decrease percent (VDP) is calculated as VDP = (V0 - Vt) / V0, where V0 is the initial viscosity and Vt is the viscosity after 10 min of incubation with lipase preparation in the viscometer. The slight viscosity decrease recorded for the control is taken into account. The initial velocity for the enzymatic reaction is expressed in terms of mPa.s.min-1, and is the slope of the VDP vs time curve in the time interval 0.1-0.4 min. Other measurements (intrinsic viscosity and gel permeation chromatography) are performed as described by Terbojevich et al. (21).

The plot of viscosity vs time for the chitosan lactate salt solution (10 g/L) containing wheat germ lipase preparation at pH 5.8 and 25°C indicates that the hydrolytic reaction is fast and still in progress after the 10 min measurement time period. Three curves are presented in Fig. 4 for the following lipase concentrations: 4.5, 45, and 450 mg/L. Measurements taken in this range showed a logarithmic dependence of Vi on the enzyme concentration. When the data are replotted in the double logarithmic presentation for three temperatures, curves slightly bent downward are obtained, indicative of deviation from usual kinetic models, and confirming the progress of the reaction beyond the time selected for the experiment.

The effect observed after 10 min treatment in terms of VDP is in the range of 50-60% for lipase concentration 0.45 g/L. Therefore the lipase concentration 0.45 g/L is preferred.

When the recombinant lipase is used, the unspecific activity is present and leads to measurable viscosity reduction, but the depolymerization of chitosan is sluggish compared to the wheat-germ lipase. In fact, the viscosity of the chitosan solutions is reduced to one half only after one d rather than within minutes, as it is the case for wheat germ lipase. The recombinant lipase can be activated by CaCl2 when the substrate is chitosan; it is reversibly inhibited by high acetate concentrations, and keeps its activity for days at 50°C.

The gel permeation chromatography curves indicate that the recombinant lipase preferentially acts on chains with high degree of polymerization, because the corresponding fractions are progressively disappearing with time (left hand side part of the plot in Fig. 5), while the average molecular weight values

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Variation Awn Presence Wheat

Variation of the intrinsic viscosity vs time, for chitosan lactate in the presence of wheat germ lipase at three concentrations: 4.5, 45, and 450 mg/mL. Chitosan lactate concentration 10 g/L; pH 5.4. From Muzzarelli et al. (11).

Variation of the intrinsic viscosity vs time, for chitosan lactate in the presence of wheat germ lipase at three concentrations: 4.5, 45, and 450 mg/mL. Chitosan lactate concentration 10 g/L; pH 5.4. From Muzzarelli et al. (11).

Gel Permeation Chromatography Oils

Gel permeation chromatography curves for the depolymerization of Katakura CTA-3 chitosan under the action of recombinant lipase, after various contact times: 0, 6, 24, 48 h and 3, 4, 5 d. From Muzzarelli et al. (11).

Gel permeation chromatography curves for the depolymerization of Katakura CTA-3 chitosan under the action of recombinant lipase, after various contact times: 0, 6, 24, 48 h and 3, 4, 5 d. From Muzzarelli et al. (11).

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move towards lower values and, the Mw/Mn ratio decreases from 4.03 to 2.54 in the 5-d period. These enzymatic reactions do not obey a simple kinetic model and, of course, the Lineweaver-Burk plot is impossible to draw.

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