Biomaterials 23 (2002) 36373644

Molecular weight and degree of deacetylation effects on lipase-loaded chitosan bead characteristics
Ibrahim A. Alsarraa, Seema S. Betigeria, Hua Zhanga, Barry A. Evansb, Steven H. Neaua,*
a

School of Pharmacy, Division of Pharmaceutical Sciences, University of Missouri-Kansas City, 108E Katz Pharmacy Building, 5100 Rockhill Road, Kansas City, MO 64110, USA b GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27790, USA Received 5 June 2001; accepted 12 March 2002

Abstract The effects of the molecular weight (MW) and degree of deacetylation (DD) of chitosan on chitosan hydrogel beads were characterized, and the entrapment efciency, release of entrapped lipase, and activity of immobilized Candida rugosa lipase were investigated. Fresh and freeze-dried beads were characterized. A solution of lipase was prepared in a 1.5% (w/v) chitosan and 1% (v/v) acetic acid medium, and then dropped into a tripolyphosphate solution to prepare the beads. The release studies were performed over 36 h. The enzyme activity was assayed using the Sigma lipase activity method. Chitosan with high MW and DD resulted in a higher loading. A lower activity was observed for beads produced with high DD chitosan. MW did not have a marked effect on the activity. The release study revealed that enzyme release increased to a maximum when the bead was manufactured with a low MW and a moderate to high DD chitosan sample. Freeze drying did not affect the release or the activity of the lipase. Chitosan with a high MW and DD can thus improve loading and reduce the release of lipase in these beads. The choice of chitosan can affect the activity normalized for lipase loading, and beads with desirable qualities can be produced. r 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Chitosan; Lipase; Hydrogel beads; Candida rugosa; Hierarchical regression analysis

1. Introduction Chitin (poly N-acetyl-d-glucosamine) is an abundant polymer that can be derived from the outer shell of crustaceans (e.g. shrimp and crab). It is also naturally present in the cell walls of some microorganisms and fungi. Chitosan is usually produced by alkaline hydrolysis of chitin, a process which results in N-deacetylation and depolymerization [1]. Chitosan has numerous applications in pharmaceutical systems and medicine. It has been used as a lm coating material [2], as an excipient in direct compression tablets, as a tablet disintegrant for improvement of drug dissolution, and for controlling drug release in oral formulations [3]. Chitosan is being evaluated in a number of medical applications, including its use in wound dressings, as a hemostatic agent, and in drug delivery systems. Chit*Corresponding author. Tel.: +1-816-235-2425; fax: +1-816-2355190. E-mail address: neaus@umkc.edu (S.H. Neau).

osans key properties for its medical applications are: (1) biocompatibility, (2) non-toxicity, (3) its ability to absorb liquids, and (4) its ability to form protective lms and coatings [4]. Recently, it has been found that chitosans with medium (400,000800,000) molecular weight were able to enhance the transnasal absorption of peptides and other polar drugs such as insulin, calcitonin, and morphine metabolites [5]. One difculty with the use of chitosan is that it is commercially available with a wide range of molecular weight (MW) and degree of deacetylation (DD) [6]. With the increasing availability of commercial chitosan products such as powders, solutions, gels, lms, and beads, and the diversity of chitosans sources, each of which may have an effect on chitosan properties, basic and applied research on chitosan will inevitably continue. The use of entrapped enzymes can increase the productivity of the biocatalytic process by concentrating the catalyst in the reaction media, as well as improving its catalytic and stability properties. A previous study in our laboratory has revealed that chitosan is a good

0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 0 9 6 - 0

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candidate for the immobilization of lipase when compared to other polysaccharides, namely, alginate and agarose [7]. Chitosan exhibits higher entrapment efciency, higher loading, and higher activity. In addition, the beads prepared using chitosan were rugged enough for the release study. The technique that has been employed in the previous study was immobilization by entrapment, a process that restricts the movement of the enzyme, to microencapsulate the lipase. It should be noted that only one type of chitosan, Sea Cure 242 (Protan Inc., Redmond, WA), was used in that study [7]. Since chitosan is commercially available with different DD and MW, it is important to take into consideration the effects of these parameters. In this study, chitosan with different MW and DD was used to microencapsulate lipase, a unique enzyme that has numerous pharmaceutical and industrial applications [8]. Lipases occupy a prominent place among biocatalysts and have a wide spectrum of biotechnological applications [9]. Lipases have been successfully used to catalyze the hydrolysis of triglycerides for the production of fatty acids, a reaction which we used in this study. The aim of the present study was to investigate the effects of MW and DD of chitosan on the lipase entrapment efciency of the chitosan beads, the release of lipase from the beads, the activity of lipase when loaded in the beads, the effect of freeze drying on lipase and chitosan bead properties, and the characteristics of the beads before and after freeze drying.

were used to prepare lipase-loaded hydrogel beads. The MW was assessed using a viscometric technique [10] and the DD was measured using a literature circular dichroism method [11]. Each chitosan solution was prepared by dissolving 150 mg in 8 ml of 1% (v/v) acetic acid. After 10.25 mg of C. rugosa lipase (EC 3.1.1.3) was dissolved in 2 ml of 1% (v/v) acetic acid, the two solutions were mixed. The beads were formed by dropping the 10 ml of bubble-free mixture through a disposable plastic syringe with a 22-gauge needle, using a KD Scientic (Boston, MA) Model 100 pushpull syringe pump at a speed of 70 ml/h, into 20 ml of a gently agitated 0.136 m solution of the gelling counterion, sodium tripolyphosphate (TPP), prepared in pH 7.2, 0.05 m TrisHCl buffer. The beads were cured in the TPP solution for 75 min and then washed twice using 3 ml of the TrisHCl buffer. The decanted solutions and washes were collected for further study. The decanted gelling medium for each batch consistently gave a neutral pH (7.070.03). The resulting beads were either used as such or freeze dried for further studies. Similar procedures were used to prepare placebo beads, which have no entrapped lipase. 2.3. Freeze drying procedure Freeze drying was accomplished using a Virtis Model 10MR-SA (Gardiner, NY). Fresh chitosan beads were rapidly frozen at 801C, held there for 8 h, then placed on a precooled shelf of the freeze dryer for 14 h. Primary drying was begun at 351C to 401C by application of a vacuum (100 mmHg). Secondary drying was completed at room temperature under the same vacuum. The drying process was completed over 22 h. 2.4. Entrapment study The decanted 20 ml of TPP solution and the two 3 ml washings were analyzed by using the bicinchoninic acid (BCA) protein assay [12]. Briey, a known volume of each sample was drawn and diluted with an appropriate volume of TrisHCl buffer. A combination of reagent A (sodium carbonate, sodium bicarbonate, and sodium tartrate in 0.2 n sodium hydroxide), reagent B (bicinchoninic acid solution), and reagent C (4% cupric sulfate pentahydrate) was added to the sample and the mixture was incubated for 1 h at 601C. The decanted solutions of the placebo beads were used as a blank. Different concentrations of 0.010.04 mg/ml of C. rugosa lipase were used as standards. The enzyme lost in these solutions was quantied by measuring the absorbance at 562 nm using a Beckman DU 7400 UV-Vis spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). The entrapment efciency percentage was obtained by calculating the percentage of the lipase entrapped

2. Experimental methods 2.1. Materials Candida rugosa lipase (EC 3.1.1.3), the pentasodium salt of tripolyphosphate (TPP), trizma buffer, Sigma lipase substrate, and thymolphthalein indicator solution were purchased from Sigma Chemical Company (St. Louis. MO). Micro-BCA protein solution was obtained from Pierce Company (Rockford, IL). Highly deacetylated ground chitosan, GC-92 (92% DD), and ground chitosan with a 75% degree of deactylation, GC75, were purchased from DCV Chitin Technologies (Wilmington, DE). Other chitosans (Sea Cure 242, 342, 442, and 443) from Protan Inc. (Redmond, WA) were gifts from G. D. Searle (Skokie, IL). The 0.05 m, pH 7.2 TrisHCl buffer was prepared by adjusting the pH of a solution of trizma base with 1 n HCl. 2.2. Encapsulation procedure Different samples of chitosan, with viscosity-average % molecular weights (Mv ) of 4.65, 6.25, 8.16, 8.28, 10.6, and 13.4 l05 and degrees of deacetylation of 92.0%, 77.8%, 76.0%, 85.6%, 77.0%, and 74.7%, respectively,

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based on the initial 10.25 mg mass of lipase present in each chitosan solution. 2.5. Release study The chitosan beads were suspended in screw-capped vials containing 10 ml of Tris buffer. At specic times, 0.5 ml samples were taken and replaced each time by 0.5 ml of Tris buffer to maintain sink conditions throughout the experiment. The release medium was kept at 251C with gentle shaking using a Magniwhirls constant temperature shaker bath. The samples and the blank, subjected to a similar process, were analyzed for their protein content using the micro-BCA protein assay. The reason that the release study was performed at 251C instead of 371C is due to the fact that one of our goals was to investigate the potential use of the entrapped enzyme in biotechnological applications, in which room temperature is commonly used [13]. 2.6. Determination of lipase enzymatic activity The enzymatic activity of lipase-loaded chitosan beads was measured using the Sigma lipase activity method [14]. Accurately weighed 8.0 mg samples of fresh and freeze-dried beads were separately added to the reaction media, which consists of 1 ml of trizma buffer, 3 ml of Sigma lipase substrate, and 3.5 ml of deionized water. Negative and positive controls were also studied. The positive control was the reaction mixture in which blank beads with free lipase were added, whereas the negative control was the reaction mixture with blank beads, and the free lipase was added just before the titration with NaOH. The reaction mixtures were agitated and incubated for 30 min in the water bath at 371C. The Sigma procedure involves hydrolysis of triglycerides from olive oil into fatty acids, diglycerides, and, to a negligible extent, monoglycerides and glycerol. The amount of fatty acids formed in 30 min, under the specic conditions of the test, is a measure of lipase activity in the sample. The fatty acids formed are quantied by titration with sodium hydroxide. The activity of free and encapsulated lipase was calculated in Units/ml. The value for the average activity of the placebo version of the respective bead was subtracted from the resulting values for each measurement for fresh and freeze-dried beads. 2.7. Characterization of the chitosan beads 2.7.1. Friability Friability studies were conducted in triplicate using a Vanderkamp Model 10805 friabilator (Vankel Industries Inc., Edison, NJ). The beads were passed through a US Standard Sieve series (Newark Wire Cloth Co., Newark, NJ). The beads retained on sieve number 16

(aperture 1.19 mm) were weighed and placed in the plastic chamber of the friabilator with 25 3 mm glass beads. The beads were subjected to 100 revolutions at 25 rpm. The fragments were separated by resieving the beads and the beads were weighed again. The friability was determined as the percent loss in weight of the beads. 2.7.2. Bead size Bead diameters were measured using an optical microscope (magnication 10 ). To determine the average bead size, measurements were carried out for 50 beads out of the beads produced. 2.7.3. Yield (number of beads) The number of beads were counted for each batch of fresh beads made from each type of chitosan.

3. Statistical analyses Two separate statistical analyses were conducted on the data. First, data were expressed as the mean of three experiments7the standard deviation (s.d.) and were analyzed using one-way analysis of variance (ANOVA), followed by Tukeys post-test. Statistical differences yielding po 0.05 were considered signicant. Second, the effects of the degree of deacetylation and molecular weight on loading, activity, bead size, number of beads produced, and maximum release were studied using an empirical mathematical model. A quadratic model is a common choice for experimenters because it is general enough to accommodate linear, quadratic, and interactive predictors [15]. The following quadratic equation was used in this study: R aDD bMW cDD2 dMW2 eMWDD f ; where R is the response variable, a; b; c; d; e; and f are the estimated regression coefcients, and MWDD is the product of molecular weight and degree of deacetylation. The procedure used to estimate the statistical signicance of a particular model is known as hierarchical regression analysis (HRA). HRA is a commonly reported multiple regression technique which can be dened as a series of multiple regression analyses in which a new predictor is added to or dropped from those used in the previous analysis. The decision concerning which variable to add or remove at each point in the series is made by the investigator [16] or can be automated by many software packages. In this study, the backward elimination procedure (a specic form of HRA) was used to reduce the number of terms in the model using the software package Design-Expert [17]. After starting with the full quadratic regression model, regression coefcients were removed one by one from the model until the terms remaining in the model were

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Load (mg) 13.40

Molecular weight (10^5)

all statistically signicant (a 0:10). For example, for the loading response, the quadratic terms were not statistically signicant and were dropped one at a time to arrive at the nal model presented in Table 1. In order to fully understand the nal empirical models, contour plots were used as a graphical aid. See Fig. 1 for an example. The lines represent the predicted response from the statistical model at different values of the predictor variables on the x and yaxis. The dots represent the actual data points. The contour plots can be used to evaluate trends in the response variable and can also help the experimenter dene optimal operating ranges for the predictor variables.

7.5 6.5 11.21 6 5.5 5 9.03 4 4.5 7

4. Results and discussion The model parameters, standard errors, and R2 values are shown in Table 1. Each of the models adequately ts

6.84

3.5

Table 1 Fitted mathematical models parameters Response Regression coefcients Coefcient standard errors 0.244 0.322 0.346 0.317 R2 of the tted model

4.65 74.70 79.13 83.55 87.98 92.40

Degree of deacetylation (%)

Fig. 1. Contour plot of the effects of DD and MW on the entrapment (loading) efciency of fresh chitosan beads.

Loading 22.65 0.257DD 3.255MW 0.043MWDD Leaching 44.28 0.975DD 1.382MW 0.005DD2 0.018MWDD Activity/load 797.7 10.29DD 375.2MW 5.853MW2 3.529MWDD Number of beads 3551 847.5DD 26.87MW 4.923DD2 9.932MW2 2.833MWDD % Weight loss 251.0 5.359DD 8.942MW 0.028DD2 0.056MW2 0.102MWDD 0.311 0.533 0.607 0.772 0.874 0.003 0.015 0.014 0.021 0.020

0.943

the respective experimental data, as supported by the R2 values. Readers should avoid extrapolating conclusions to factor ranges not explored in this study. 4.1. Entrapment efciency

0.999

0.999

The purpose of the entrapment efciency study was to examine the ability of the polymer to retain the enzyme during the various steps of the manufacturing processes. Entrapment efciency was calculated by determining the loss of the lipase during the entire manufacturing processes, including the loss during the gelation time in the TPP solution, the loss during the curing time, and the loss during the washing of the beads. The entrapment efciency results of different chitosan samples are listed in Table 2. It can be seen that the entrapment efciency of 242, 342, 442, and GC-75, chitosan samples with a similar DD are not statistically different. Results for beads containing Sea Cure 443 indicate that a higher degree of deacetylation resulted in a higher load (po0:05). As shown in Fig. 1, the results indicate that a higher DD and MW should provide a higher loading. Chitosan samples with a higher DD would have more positively charged amine groups upon dissolving them in the acetic acid solution, and therefore more positive charges would be available to interact with the negatively charged counterion leading to the formation

I.A. Alsarra et al. / Biomaterials 23 (2002) 36373644 Table 2 Loading/entrapment efciency and leaching/release studies of chitosan beadsa Chitosan DD7s.d. MW (105) Loading7s.d. (mg) (%)
b

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Leaching7s.d. Fresh beads (mg) (%)

c

Freeze-dried beads (mg) 0.5270.011 0.2970.120 0.4170.012 0.7170.033 0.5670.011 0.7870.011 (%)c 15.95 7.00 11.08 21.19 12.31 31.84

242 342 442 GC-75 443 GC-92

a b

77.870.97 76.071.46 77.071.72 74.771.00 85.671.25 92.470.90

6.25 8.16 10.6 13.4 8.28 4.65

3.2670.096 3.8070.075 3.7070.057 3.3570.158 4.5570.036 2.4570.280

31.8 37.0 36.0 32.6 44.3 23.9

0.4070.013 0.2970.007 0.3270.011 0.4170.012 0.2770.023 0.7170.029

12.27 7.77 8.64 12.24 5.93 28.98

The values are given as the mean7s.d. of three experiments. Load expressed as the percent of lipase mass in the chitosan solution that was entrapped in the beads. c Leaching expressed as the percentage of the loaded lipase that appeared in the release medium.

0.9 0.8 0.7 0.6 Protein Amount 0.5 0.4 0.3 0.2 0.1 0 0

Sea Cure 242 Ground Chitosan-75

Sea Cure 342 Ground Chitosan-92

Sea Cure 442 Sea Cure 443

10

15

20 Time (hour)

25

30

35

40

Fig. 2. Release proles for fresh chitosan beads.

of more ionic bonds and a more intricate network than in products manufactured with chitosan samples with lower degrees of deacetylation. It was observed in the entrapment study that the major loss of lipase was during the actual gelation time when the drop is not yet fully gelled to form the bead. In this gelling time, the porosity of the bead would be high, allowing more lipase to escape easily to the gelling medium. 4.2. Release studies The aim of this study was to consider the potential for reuse of the beads. The release of lipase into the pH 7.2, 0.05 m TrisHCl buffer was performed over a 36-h period to evaluate the loss of lipase from the beads after the manufacturing process was complete. Release studies were conducted in TrisHCl buffer because this buffer was also used during the manufacturing process. In addition, lipase was found to be stable in this buffer

[18]. The maximum release of lipase from fresh and freeze-dried beads is shown in Table 2. There was no signicant difference in lipase release between the freezedried and fresh beads for any given chitosan sample. The release proles for both fresh (Fig. 2) and freezedried beads (data not shown) show that there was an immediate release within the rst 4 h, and the remaining lipase was essentially retained. The percentage that was released was typically low (less than or equal to 12%) with the exception of high deacetylated ground chitosan (GC-92) where the results were statistically different from that for other types of chitosans (po0:05). This result could be attributed to both MW and DD effects since it is the chitosan sample with the highest DD and the lowest MW. From our observations, the release studies could not be conducted for more than 36 h because of the susceptibility of the release medium to microbial contamination, which was observed as an increase in the turbidity of the release medium as time

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progressed beyond 36 h. The effects of DD and MW on maximum release over the 36 h study can be described well by a contour plot (Fig. 3). The plot demonstrates that the release increased to a maximum at low values of MW and moderate to high value of DD. 4.3. Activity study This study was performed using a simple reaction for the conversion of triglycerides essentially to diglycerides

Maximum release (mg) 13.40

11.21 Molecular weight (10^5)

0 0.25

9.03

and fatty acids. The fatty acids were titrated using NaOH, and thus quantication was simple. The results of the activity studies for both fresh and freeze-dried chitosan beads are shown in Table 3. It can be observed that the average activities of beads were reduced signicantly when the bead was freeze dried (po0:05). In order to estimate a relative lipase activity, a plot of the relationship between enzyme activity and the amount of free lipase was constructed. It was found that the observed activities of lipase entrapped in both fresh and freeze-dried beads were about 50% of the activities predicted from the lipase loading, as presented in Table 3. The higher degree of retained activity exceeds that typically reported in the literature (ca. 2030%) for immobilized lipase in biotechnological applications [19,20]. The lowered observed activities in beads containing 242, 342, 442, 443, GC-75, and GC-92, compared to the predicted activities, might be due to a polymeric interaction with the enzyme, either physical or chemical in nature. It could also be related to other reasons for loss of activity, including a limited ability of the substrate to readily diffuse through the beads, or for the product to diffuse out. Activity normalized for lipase loading (activity/load) decreased as the DD increased, on the average. However, the MW did not have a marked effect on the normalized activity in the region of exploration (the contour lines are roughly vertical), as illustrated in Fig. 4. 4.4. Bead characteristics

6.84

0.25

0.5

0.75 4.65 74.70 79.13 83.55 87.98 92.40

Degree of deacetylation (%)

Fig. 3. Contour plot of the effects of DD and MW on the mass of lipase released from fresh chitosan beads.

With the exception of beads containing GC-92, the freeze-dried bead friabilities were not statistically signicantly different from each other (po0:05) (see Table 4). The friability results indicate that the beads are rugged enough for use in different applications. It can be seen from the contour plot (Fig. 5) that the percentage weight loss increased to a maximum at low to moderate

Table 3 Comparison of observed lipase activities of fresh and freeze-dried chitosan beads with the predicted activitya Chitosan Average predicted activity b7s.d. (Units/ml) Average observed activityc7s.d. (Units/ml) Fresh beads 242 342 442 GC-75 443 GC-92
a b

Freeze-dried beads 1086750.0 1446795.0e 1206781.0e 1060736.0e 1010720.0e 550750.0e

2253746.7 2523770.0d 2509750.0d 2295778.0d 2783790.0d 17097163d

1316776.0 1780761.0 1606781.0 1393751.0 13107111 626725.0

The values are given as the mean7s.d. of three experiments. The predicted activity was based on the actual amount of the lipase that was entrapped. c The value for the average activity of the placebo version of the respective bead was subtracted from the resulting values for each fresh and freezedried bead (n 3). d Statistical differences from corresponding values of the average observed activities (po0:05) by one-way ANOVA followed by Tukeys post-test. e Statistical differences from corresponding values of the fresh beads (po0:05) by one-way ANOVA followed by Tukeys post-test.

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Weight loss (%)

Activity/load (Units/ml/mg) 13.40

13.40

0 50 100 150 200 450 400 9.03 350 300 250

11.21 Molecular weight (10^5)

11.21 Molecular weight (10^5)

0.5 0 1 9.03 1.5

2 1.5 6.84 1 0.5 0 3

6.84

2.5

4.65 74.70 79.13 83.55 87.98 92.40

4.65 74.70 79.13 83.55 87.98 92.40

Degree of deacetylation (%)

Fig. 4. Contour plot of the effects of DD and MW on the activity/load of fresh chitosan beads.

Degree of deacetylation (%)

Fig. 5. Contour plot of the effects of DD and MW on friability.

Table 4 Bead characteristics Chitosan 242 342 442 GC-75 443 GC-92 Mean number of beads7s.d. 20273.78 41974.16 36071.15 37272.52 15072.08 12071.00 % Weight loss7s.d. 1.2970.503 1.3270.201 1.7770.671 1.7270.321 1.4270.260 2.5770.445

13.40

Yield

11.21 500 Molecular weight (10^5) 400 400 300 9.03 200 200 300

values of MW and moderate to high values of DD. The yield is presented in Table 4. Fig. 6 indicates that, at low DD, no MW effect on yield is evident since the response lines are essentially vertical. The beads produced were approximately 23 mm in diameter, as assessed by optical microscopy. For each particular batch of beads, however, a narrow size distribution was achieved. The relative standard deviation associated with the mean bead size never exceeded 7.0%. Beads prepared using tripolyphosphate are spheres, as was observed and described in the literature [2,21]. Since there were not enough degrees of freedom to estimate error (and hence calculate p-values) to develop the mathematical models for each response, the initial term to be dropped from the models was based on the magnitude of the coded regression coefcient. The rst term to be dropped was the term with the smallest coded

100 6.84 0

4.65 74.70 79.13 83.55 87.98 92.40 Degree of deacetylation (%)

Fig. 6. Contour plot of the effects of DD and MW on yield.

regression coefcient. Since the coded regression coefcients for the friability and for the yield were each of roughly the same magnitude, the most conservative

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I.A. Alsarra et al. / Biomaterials 23 (2002) 36373644 [4] Sandford P. Chitosan: commercial uses and potential applications. In: Skj( k-Br! k G, Anthonsen T, Sandford P, editors. Chitin a c and chitosan sources, chemistry, biochemistry, physical properties and applications. London: Elsevier Applied Science, 1988. p. 5169. [5] Aspden T, Illum L, Skaugrud . Chitosan as a nasal delivery system: evaluation of insulin absorption enhancement and effect on nasal membrane integrity using rat models. Eur J Pharm Sci 1996;4:2331. [6] Domard A. pH and c.d. measurements on a fully deacetyated chitosan: application to CuIIpolymer interactions. Int J Biol Macromol 1987;9:98104. [7] Seema S Betigeri. Immobilization of lipase using hydrophilic polymers in the form of beads. M.S. thesis, Faculty of the University of Missouri-Kansas City, 1999. [8] Desnuelle P. Lipases. In: Desnuelle P, editor. Molecular and cellular basis of digestion. Amsterdam: Elsevier, 1986. p. 27596. [9] Bickerstaff GF. Impact of genetic technology on enzyme technology. Genet Eng Biotech 1995;15:1330. [10] Chen R, Hwa H. Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane. Carbohydr Polym 1996;29:25360. [11] Domard A. Determination of N-acetyl content in chitosan samples by c.d. measurements. Int J Biol Macromol 1987;9:3538. [12] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:7685. [13] Kadir S. Production of biotech compounds. In: Crommelin D, Sindelar R, editors. Pharmaceutical biotechnology: an introduction for pharmacists and pharmaceutical scientists. Amsterdam, Netherlands: Harwood Acdemic Publishers, 1997. p. 5370. [14] Tietz N, Fiereck E. A specic method for serum lipase determination. Clin Chim Acta 1966;13:3527. [15] Hunter W. Response surface methods. In: Box G, Hunter W, Hunter J, editors. Statistics for experimenters: an introduction to design, data analysis, and model building. Somerset, NJ: Wiley, 1978. p. 51037. [16] Licht M. Multiple regression and correlation. In: Grims L, Yarnold P, editors. Reading and understanding multivariate statistics. Washington, DC: American Psychological Association, 1995. p. 1964. [17] Design Expert, v 5.0.5, Stat-Ease Inc, Minneapolis MN. [18] Hertzberg S, Kvittingen L, Anthonsen T, Skj( k-Braek G. a Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions. Enzyme Microbiol Technol 1992;14:427. [19] Kondo T, Muramatsu N. Enzyme inactivation in microencapsulation. In: Nixon JR, editor. Microencapsulation. New York, NY: Marcel Dekker, 1976. p. 6775. [20] Wood DA, Whateley TL. A study of enzyme and protein microencapsulationsome factors affecting the low apparent enzymatic activity yields. J Pharm Pharmacol 1982;34:5527. [21] Bodmeier R, Oh KH, Pramar Y. Preparation and evaluation of drug containing chitosan beads. Drug Dev Ind Pharm 1989;15:147594.

modeling approach is to assume that all the terms are important and t the models accordingly. It is not possible to calculate prediction standard errors when this is the case, but the predictions themselves are valid. Therefore, the full quadratic equations for both models were used to describe the effects of DD and MW on friability and the yield.

5. Conclusions Lipase can be immobilized successfully by entrapment in chitosan hydrogel beads. Chitosan with high MW and DD can improve lipase loading, and lessen the release of the entrapped lipase. It can also be concluded that the freeze drying process caused a reduction in lipase activity. On the basis of an amount of free enzyme equivalent to the actual lipase entrapped, about 50% retention of activity was seen with lipase entrapped in these chitosan beads. The friability results indicate that the beads are rugged enough for use in different applications. The hierarchical regression analysis proved to be worthwhile. The equations so developed can be predictive of the MW and DD effects on the selected responses. The choice of a chitosan sample with suitable MW and DD could produce hydrogel beads characterized by a good lipase entrapment efciency and with other desirable characteristics.

Acknowledgements The authors would like to thank G. D. Searle (Skokie, IL) for the gift of chitosan samples. This work was supported in part by the NIH (GM1073-01A1).

References
[1] Hirano S, Ohe Y, One H. Selective N-acetylation of chitosan. Carbohydr Res 1976;47:31520. [2] Miyazaki S, Yamaguchi H, Takada M, Hou W, Takeichi Y, Yasubuchi H. Pharmaceutical application of biomedical polymers. XXIX. Preliminary study on lm dosage form prepared from chitosan for oral drug delivery. Acta Pharm Nord 1990;2:4016. [3] Kristl J, Smid-Korbar J, Struc E, Schara M, Rupprecht H. Hydrocolloids and gels of chitosan as drug carriers. Int J Pharm 1993;99:139.