Encapsulation of β-Glucuronidase in Biomimetic Alginate Capsules for

Mar 3, 2007 - Protamine-Templated Biomimetic Hybrid Capsules: Efficient and Stable Carrier for Enzyme Encapsulation. Yufei Zhang , Hong Wu , Jian Li ,...
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Ind. Eng. Chem. Res. 2007, 46, 1883-1890

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Encapsulation of β-Glucuronidase in Biomimetic Alginate Capsules for Bioconversion of Baicalin to Baicalein Zhongyi Jiang, Yufei Zhang, Jian Li, Wen Jiang, Dong Yang, and Hong Wu* Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, People’s Republic of China

Baicalin was converted into baicalein by β-glucuronidase (GUS) encapsulated in liquid-core alginate capsules, mimicking the natural enzyme existence in lysosome. Taking both the physical properties of the capsule and the activity of the encapsulated enzyme into account, optimal conditions (1.0% w/v sodium alginate; 0.10 mol/L CaCl2; 30 min gelation time) for GUS encapsulation were determined. The encapsulated GUS retained up to 88% of its free-form activity with an encapsulation efficiency of 77%. Conversion of baicalin by free and encapsulated GUS resulted in the baicalein productivities of 80% and 65%, respectively. The optimal temperature (60 °C) and pH value (pH 7.0) for bioconversion were not changed after encapsulation. In addition, the encapsulated GUS showed no appreciable loss in activity after four repeated cycles, and 90% of its initial activity remained after 26-day storage at 4 °C. 1. Introduction Baicalin (5,6,7-trihydroxyflavone-7-β-D-glucuronide) and its aglycone, baicalein (5,6,7-trihydroxyflavone), are the major effective flavone components in the dry root of Scutellaria baicalensis Georgi (Scutellariae Radix) which is an important herb existing in many Chinese traditional medicines, such as in the famous recipe “Xiao Chai Hu Tang” (“Sho-saiko-to” in Japanese) effectively used for the treatment of chronic hepatitis. The pharmacological efficacy and application potential of baicalin and baicalein are mainly attributed to their strong antioxidation, anti-inflammation, and specific inhibition toward HIV and RLV, together with their promising anticancer function.1-4 Pharmacological study has found that baicalein shows higher activity than baicalin in the above medical efficacies. 5 Furthermore, baicalein is absorbed more quickly and in greater amounts than baicalin and shows a better bioavailability.6-8 However, the content of baicalein in Scutellariae Radix (ca. 0.2%) is, unfortunately, much less, about 50-fold lower than that of baicalin (9-14%). This contradiction between the higher activity and lower content of baicalein leads to explorations of converting baicalin to baicalein either by chemical conversion or by bioconversion. Chemical conversion approach includes chemical synthesis starting from trihydroxyacetophenone9 and acid hydrolysis of baicalin.10-12 In comparison, bioconversion seems more promising due to its inherent advantages such as high selectivity, high efficiency, mild reaction conditions, and environmental friendliness.13-16 Catalyzed by β-glucuronidase (GUS), the β-1,4-glucuronide bond of baicalin can be cleaved to produce baicalein.17-19 So far, a majority of microbial or enzymatic conversions of herbal medicines including baicalin are catalyzed by free-form biocatalysts whereas immobilized biocatalysts are obviously more preferable for potential industrialization.20-23 It has been found that the most common host of GUS in nature is the organelle of eukaryotic cells called lysosome. The structure of lysosome is like a capsule surrounded by a biomembrane consisting of a monolayer of lipoprotein, acting as a protective * To whom correspondence should be addressed. Tel.: +86-2227892143. Fax: +86-22-27892143. E-mail: [email protected].

shell as well as a barrier preventing possible leakage of the inside ca. 60 kinds of acid hydrolases.24 To mimic the natural existence of GUS, a capsule structure with liquid core inside and membrane shell outside is supposed for efficient encapsulation of GUS in this study. Calcium alginate is one of the most commonly used materials for enzyme encapsulation due to its natural biocompatibility, ease of formation, and mild physiological gelation conditions.25,26 Alginate capsules can be prepared by the interfacial gelation process to form a membrane around the drops of enzyme solution. Sodium carboxymethyl cellulose (CMC), polyethylene glycol (PEG), and glycerol are among the most commonly used thickening agents for maintaining the spherical shape of the liquid droplets. With the outward diffusion of divalent metallic ions (Ca2+ or Ba2+) from the inner liquid core, alginate is cross-linked and thus solidified. A number of enzymes and cells have been successfully encapsulated in alginate capsules with high activity retention and enhanced stability.27-31 The aim of the present work was to implement the bioconversion of baicalin to baicalein by GUS encapsulated in alginate capsule, mimicking the structure of its natural host (lysosome). The optimal encapsulation conditions were determined based on both the physical properties of the capsules and the encapsulated enzyme activity. The storage and recycling stability were also examined. 2. Materials and Methods 2.1. Materials. β-Glucuronidase (GUS) (EC 3.2.1.31) from Escherichia coli (type IX-A, lyophilized powder, 10000005000000 units/g protein) was purchased from Sigma Chemical Co. Sodium alginate (SA) and sodium carboxymethyl cellulose (CMC) were obtained from Tianjin Reagent Chemicals Co. Ltd. Anhydrous calcium chloride (CaCl2, analytical reagent grade, Tianjin Kermel Chemical Reagent Co. Ltd.) was used to induce gelation. Baicalin and baicalein standards for analysis were obtained from the National Institute for the Control of Pharmaceutical and Biological Products of China. Baicalin (purity g98%) used as substrate was purchased from Sichuan Xieli Pharmaceutical Co. Ltd. All the other chemicals were of analytical reagent grade.

10.1021/ie0613218 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/03/2007

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2.2. GUS Encapsulation. GUS was encapsulated in alginate capsules via an extrusion process followed by interfacial gelation.27 Tris-HCl buffer (30 mmol/L, pH 7.0) was used as a universal solvent to dissolve all the chemicals. CMC, a thickening agent to maintain the spherical shape of the capsules, was dissolved in CaCl2 solution of different concentrations (0.05, 0.10, 0.20, 0.30, and 0.40 mol/L) to give a final content of 2% w/v. GUS was then added to the CMC-CaCl2 solution to obtain the cationic mixture with an enzyme content of 0.1 mg/mL. The mixture was stirred for 30 min at room temperature. Two milliliters of the above CMC-CaCl2-GUS solution was extracted into a syringe and extruded through an injection needle (inner diameter 0.45 mm) into 40 mL of SA solution (0.5% w/v or 1.0% w/v). The distance between the tip of the needle and the SA solution surface was fixed at 20 cm. The SA solution was stirred during the whole gelation process to facilitate mass transfer as well as to prevent the capsules from sticking together. Alginate membrane was immediately formed upon the dripping of the liquid drops into the SA solution. After a specific period of gelation time (5-120 min), the mixture was poured into 160 mL of deionized water and stirred gently for 5 s before filtering with a piece of gauze. Ten milliliter of Tris-HCl buffer was used to remove the unreacted SA from the surface of capsules. To reinforce the alginate membrane, the capsules were transferred into CaCl2 solution with the same concentration as that in the liquid drops and allowed to be further cross-linked for 10 min. Finally, the capsules were filtered, rinsed with TrisHCl buffer twice, and stored in a sealed plastic bag at 4 °C. 2.3. Characterization of GUS-Containing Capsules. 2.3.1. Diameter and Membrane Thickness. The external diameter of alginate capsule was determined by measuring the diameters of 10 replicates using a caliper. For membrane thickness measurement, 10 capsule samples were cut into half and for each capsule the thickness at three randomly selected locations was measured under a polarizing microscope (NIKON Eclipse E600POL). The membrane thickness was thus an average of 3 × 10 data and the maximum standard deviation was 0.020 mm. 2.3.2. Morphology. The micromorphology of the capsules was observed by a scanning electron microscope (SEM) (Philips XL30 ESEM). 2.3.3. Swelling Property. Fresh capsules were immersed into Tris-HCl buffer (30 mmol/L, pH 7.0) at 37 °C. The weight of the capsules was monitored until a constant value was reached (Ws). Then the capsules were taken out and lyophilized to weight constancy (Wd). The swelling degree (Sw) was calculated as follows:

Sw ) (Ws - Wd)/Wd × 100% 2.3.4. Mechanical Strength. The mechanical strength of the capsules was examined using an Electronic Universal Testing Machine (CSS-44001, Changchun Research Institute for Testing Machines, China) at room temperature with a compressing rate of 5 mm/min until the capsule ruptured. Five replicates were tested and averaged. 2.3.5. Encapsulation Efficiency. Capsules were disrupted by cutting to release the GUS inside28 and the encapsulation efficiency was determined by the following equation,

encapsulation efficiency ) [GUS]capsule/[GUS]droplet × 100% where [GUS]capsule and [GUS]droplet are the concentrations of GUS in the final capsule and in the original CMC-CaCl2GUS liquid droplet, respectively. The GUS concentration was

determined by the micro-Bradford method.32,33 Two milliliters of Bradford reagent was added into 2 mL of GUS-containing solution. After being incubated for 5 min, the absorbance of the above solution at 595 nm was measured using a UV spectrophotometer (Hitachi U-2800). 2.4. Bioconversion Reaction and Enzyme Activity. Bioconversion of bailcalin to baicalein catalyzed by either free or encapsulated GUS was carried out and compared. The reaction kinetics and enzyme activities were studied by measuring the amounts of baicalein produced. GUS-containing alginate capsules were introduced into a beaker containing 20 mL of 0.09 mmol/L baicalin and 0.1% w/v Na2SO3, both dissolved in Tris-HCl buffer (30 mmol/L, pH 7.0). Na2SO3 was used here as an antioxidant. The beaker was sealed and the reaction was performed under stirring at 37 °C. At different time intervals, 100 µL of reacting solution was sampled and analyzed by HPLC (HP1100, Agilent) equipped with Agilent ZORBAX SB-C18 column. A mixture of methanol: H2O:H3PO4 (60:40:0.2) was employed as the mobile phase at a flow rate of 1 mL/min. The detection wavelength was set at 274 nm. For the experiments with free GUS, 300 µL of methanol was added to the 100 µL of sample to stop the reaction and make the free enzymes precipitate. After centrifugation at 10000 rpm, the supernatant was sampled and analyzed by HPLC. The enzyme activity unit was defined as the amount of GUS needed to produce 1.0 µmol of baicalein/h at 37 °C, pH 7.0. The relative activity of encapsulated GUS was represented by a ratio of the encapsulated enzyme activity to its free-form activity under the identical reaction conditions. The productivity was defined as the mole ratio of the amount of baicalein produced to the original amount of baicalin in the feed. The Michaelis constant (Km) and the maximum reaction rate (Vmax) were determined by varying the substrate concentration from 0.06 to 0.90 mmol/L. 2.5. Storage Stability. Free and encapsulated GUS were stored at 4 °C for a certain period of time. The storage stability was compared by storage efficiency defined as the ratio of free or encapsulated enzyme activity after storage to their initial activity.

storage efficiency (%) )

enzyme activity after storage × intial enzyme activity 100

2.6. Recycling Stability. The encapsulated GUS was filtered after each reaction batch, rinsed with Tris-HCl buffer and then added to the next reaction cycle. The recycling stabilities of encapsulated GUS were explored by measuring the enzyme activity in each successive reaction cycle and expressed by recycling efficiency.

recycling efficiency (% ) ) enzyme activity in the nth cycle × 100 enzyme activity in the 1st cycle 3. Results and Discussion 3.1. Formation and Characterization of GUS-Containing Alginate Capsules. 3.1.1. Morphology. Figure 1a is a typical picture of the capsules prepared in this study. Spherical capsules with an external diameter of ca. 3 mm were obtained. The membrane thickness was less than one-tenth of the capsule diameter. The perfect spherical shape was mainly owed to the suitable viscosity of the thickening agent. Previous experimental

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Figure 2. SEM photographs of the alginate membrane.

Figure 1. GUS-containing alginate capsules.

results (data not shown) indicated that the sphericity and uniformity of the capsules were most satisfactory when the CMC concentration was in the range of 1.5-2.0% w/v. In this study, CMC concentration was fixed at 2.0% w/v thereby. The micromorphology of the alginate membrane of the capsules observed by SEM is shown in Figure 2. Both the external and the internal surface of the alginate membrane were relatively smooth with a network structure. The internal surface had a denser cross-linked structure than the external surface (Figure 2a,b). Gelation started just at the moment when the cationic CMC-CaCl2-GUS droplets were added into the

anionic SA solution. The contact of Ca2+ ions with alginate at the spherical interface initiated an instantaneous ionic crosslinking, thus inhibiting further diffusion of alginate into the droplet. With the outward diffusion of Ca2+ ions from the inside core, the cross-linked membrane around the droplet grew. Along the diffusion direction, Ca2+ ions were consumed and the concentration reduced, resulting in an inhomogeneous denseto-loose structure. A typical spongelike porous alginate membrane was obtained as shown in Figure 2c. The capsule with a CMC liquid core and alginate membrane shell might be regarded as a simplified and enlarged form of lysosome for GUS encapsulation in nature. The enzyme molecules were physically confined in the liquid core by the membrane formed around the drops of enzyme solution. The liquid core provided the enzymes inside sufficient space to move and rotate as freely as in their free form, resulting in a much

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Figure 3. Membrane thickness growth with gelation time and CaCl2 concentration (1.0% w/v SA).

easier accessibility and contact between enzyme and substrate than that in any other solid encapsulation matrixes. The pore size of the semipermeable capsule membrane should satisfy two requirements: big enough for the substrates and products to move in and out freely but small enough to confine the enzyme inside. A number of studies in the literature were devoted to the characterization of the pore size of Ca-alginate membrane. Enzymes with smaller molecular weight (MW) than GUS (MW 290 kDa), such as glucose oxidase (MW 152 kDa), were encapsulated in alginate capsules and used as biocatalysts without significant leakage.28,29 It was also found that PEG molecule with a MW of 4 kDa could not penetrate through the alginate membrane prepared under certain conditions.30 Dembczynski and Jankowski reported that Ca-alginate membrane and capsules had an approximate exclusion limit of 21-25 kDa for dextran and 78-103 kDa for protein by means of inverse size exclusion chromatography.34 Based on these early results, it was assumed that GUS with a molecular weight as large as 290 kDa could be efficiently confined in the alginate capsule without significant leakage. The existence of GUS in the capsule was imagined to be like “fishes in a bag”. Direct study on encapsulation efficiency, instead of complicated pore size or weight cutoff measuring, was carried out for further assurance (see section 3.1.3). 3.1.2. Gelation Conditions. Figure 3 shows the growth of membrane thickness with gelation time and SA concentration at different CaCl2 concentrations. The membrane grew rapidly during the first 20 min and then leveled off. Higher CaCl2 concentration resulted in thicker membrane after the same gelation time. When CaCl2 concentration increased from 0.05 to 0.40 mol/L, the final membrane thickness increased from 0.040 ( 0.007 to 0.400 ( 0.020 mm. The influence of SA concentration on the membrane thickness at different CaCl2 concentrations was also studied (Figure 4). The higher the concentration of SA solution, the more cross-linkable sites for Ca2+ ions and thus the thicker the resulting membrane. This effect was more notable in the case of higher CaCl2 concentration (>0.30 mol/L). 3.1.3. Encapsulation Efficiency. Encapsulation efficiency reflects the possible leakage during the gelation period as shown in Figure 5. Alginate capsules formed at higher SA concentration retained more enzymes because of the strengthened blocking effect of the denser and thicker membrane. The highest encapsulation efficiency was obtained with a CaCl2 concentration between 0.20 and 0.30 mol/L. The capsules were prepared by two successive gelling stages: capsule formation stage and reinforcement stage. In the first stage, the viscous property of the droplet and the fast gelation reaction were both beneficial factors for confining the enzymes within the liquid core as much

Figure 4. Effects of SA concentration on membrane thickness at various CaCl2 concentrations (gelation time 10 min).

Figure 5. Effect of CaCl2 and SA concentrations on the encapsulation efficiency (gelation time 30 min).

as possible before they diffused out. Moreover, under the neutral (pH 7.0) gelling environment, GUS, whose pI is 4.8, carries a negative charge. The electrostatic repulsion interaction between the negatively charged enzyme and the negatively charged alginate made an additional favorable contribution for the confinement of enzymes within the liquid core. The loss of encapsulation efficiency might mainly occur during the reinforcement stage. The reinforcement proceeding from the outer surface of the membrane by further cross-linking between the free guluronic acid blocks (G-blocks) in alginate via Ca2+ ions caused a sharp shrinkage of the membrane as well as the inner size of the capsule.35 A certain amount of water was consequently extruded out of the core, carrying more or less enzyme molecules together leaking out of the capsule. When the SA concentration was fixed at 0.5% w/v or 1.0% w/v, this membrane and core shrinkage was not significantly severe at CaCl2 concentration lower than 0.20 mol/L. Therefore, the encapsulation efficiency was improved by increasing CaCl2 concentration from 0.05 to 0.20-0.30 mol/L due to the denser cross-linking membrane structure. With the further increase of CaCl2 concentration higher than 0.20-0.30 mol/L, not only free G-blocks but also junctions of G-blocks were further crosslinked, four or more G-blocks being linked side by side quickly.36 In this case, the capsule shrinkage during the reinforcement stage gradually prevailed, resulting in a decrease in encapsulation efficiency thereafter. The highest encapsulation efficiency obtained in this study was 93% at 1.0% w/v SA and 0.30 mol/L CaCl2. 3.1.4. Swelling Property. All the capsules reached swelling equilibrium in Tris-HCl buffer (30 mmol/L, pH 7.0) at 37 °C within 6 h and no breakage was found during the testing period (25 h). As shown in Figure 6, the swelling degree of alginate capsule was directly related to the cross-linking density which

Ind. Eng. Chem. Res., Vol. 46, No. 7, 2007 1887 Table 1. Compression Intensity of Capsules Prepared under Different Conditions CaCl2 concentration (mol/L)

SA concentration (% w/v)

gelation time (min)

compression intensity (N)

0.10 0.20 0.20 0.20 0.20 0.40 0.40 0.40

1.0 0.5 1.0 1.0 1.0 0.5 1.0 1.0

30 10 10 30 120 10 10 120

17.3 11.1 46.0 64.5 72.8 43.8 93.2 117.0

Table 2. Encapsulated GUS Activity against CaCl2 Concentration (1.0% w/v SA) CaCl2 concentration (mol/L) optimal gelation time (min) relative activity of encapsulated GUS (%)

Figure 6. Swelling degree of capsules at various CaCl2 concentrations: (a) effect of gelation time (1.0% w/v SA); (b) effect of SA concentration (gelation time 10 min).

Figure 7. Effect of CaCl2 concentration on free GUS activity.

was determined by gel composition and gelation time. The swelling was inhibited by using higher CaCl2 and SA concentration. Gelation time had less influence on the swelling degree when the CaCl2 concentration was sufficiently low (e0.10 mol/ L) or high (g0.40 mol/L) (Figure 6a). In these two extreme concentration ranges, the content of Ca2+ ions being either too insufficient or too excessive compared with the amount of SA, a rather loose or fairly dense structure would be formed which was less affected by gelation time. When the CaCl2 concentration was moderate (0.10-0.40 mol/L), gelation time, before the equilibrium of the cross-linking reaction was reached (∼20 min deduced from Figure 3), made a larger difference to swelling degree. After 30 min when the reaction was already finished, the effect of longer gelation time on the swelling property became much less. Another trend that should be noticed was that the swelling degree increased on prolonged gelation time at various CaCl2 concentrations. This might be due to the inhomogeneous dense-to-loose structure of the membrane which had been clearly observed by SEM (Figure 2a,b). The longer

0.05 20 99

0.10 30 88

0.20 60 58

the gelation time lasted, the thicker the membrane containing more alginate with loose structure, leading to higher water uptake. 3.1.5. Mechanical Strength. The mechanical strength is an important factor for encapsulated enzymes in consideration of practical application. Here, the compression intensity of the capsules was tested (Table 1). It is easy to understand that the capsules prepared with higher CaCl2 and SA concentration as well as longer gelation time had higher compression intensities, owing to the denser structure and the growing membrane thickness. 3.2. Bioconversion Reaction and Enzyme Activity. 3.2.1. Effect of CaCl2 Concentration on Enzyme Activity. The CaCl2 concentration in the liquid core decreased with the ongoing gelation process. When gelation was stopped, there might have been some unreacted CaCl2 left in the core. No literature, to the best of our knowledge, has reported on the influence of the residual CaCl2 concentration on the enzyme (GUS) activity. If the residue amount of CaCl2 had no influence on the enzyme activity, the enzyme activity would have been reduced to some extent with the increase of gelation time since the additional diffusion resistance for substrate and product molecules became larger with the growth of membrane thickness. However, in fact, a contrary phenomenon was observed in this study. For capsules prepared at the conditions 1.0% w/v SA and 0.20 mol/L CaCl2, the relative activity of encapsulated GUS increased from 19% to 58% when the gelation time was prolonged from 10 to 60 min. It was, therefore, suspected that the amount of the residual CaCl2 might also be an influencing factor for the GUS activity. To verify the above suspicion, the free GUS activity was examined in buffer with different CaCl2 concentrations. The remaining activities of free GUS in CaCl2containing buffers compared to that in pure buffer without CaCl2 were shown in Figure 7. It was clearly observed that CaCl2 concentration had a great influence on enzyme activity. The free enzyme activity decreased rapidly and was even totally lost when the CaCl2 concentration was higher than 5 mmol/L. So, for the sake of retaining enzyme activity, a lower CaCl2 concentration should be used for capsule preparation. The optimal CaCl2 concentration and gelation time for high relative activity of encapsulated GUS were summarized in Table 2. To determine the final optimum conditions for enzyme encapsulation, both the physical property as discussed in section 3.1 and enzymatic catalysis performance as discussed in this section should both be taken into account. Since in most cases these two factors are not always changing in the same trend, a compromise is often needed to be made. As shown in Table 2,

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Figure 8. Productivity of baicalein with reaction time (37 °C, pH 7.0).

Figure 9. Typical Lineweaver-Burk plots for free and encapsulated GUS (37 °C, pH 7.0).

the highest relative activity was achieved at the conditions 0.05 mol/L CaCl2 and 20 min gelation time. However, the capsule prepared under the conditions 0.10 mol/L CaCl2 and 30 min gelation time showed much better physical properties while also possessing an acceptable relative activity (88%). Therefore, the latter conditions were suggested to be the optimum ones. The GUS-containing alginate capsules prepared under the optimum conditions (1.0% w/v SA, 0.10 mol/L CaCl2, and 30 min gelation time) exhibited an encapsulation efficiency of 77%, a swelling degree of 38%, a compression intensity of 17.3 N, and a relative activity of 88%. The following further studies were carried out with capsules prepared under the optimum conditions. 3.2.2. Bioconversion of Baicalin by Free or Encapsulated GUS. The bioconversion reaction catalyzed by either free or encapsulated GUS was conducted in a sealed beaker and a small amount of Na2SO3 was used as antioxidant to prevent the oxidation of baicalein produced as shown and compared in Figures 1b and 1c. As shown in Figure 8, the bioconversion process was monitored by measuring the productivity of baicalein with the lapse of time. The reaction rate and the final productivity using encapsulated GUS were both lower than those using free GUS. The equilibrium productivity using free GUS was obtained at 80% in 2.5 h, while that in the case of encapsulated GUS was 65% in 4 h. The slower conversion rate was due to the lowered activity of the encapsulated enzyme and the additional diffusion resistance. The specific activities of free and encapsulated enzyme were 28 and 18 U/mg GUS, respectively. 3.2.3. Kinetic Parameters. The bioconversion reaction of baicalin to baicalein followed the Michaelis-Menten kinetics and the corresponding Michaelis constant (Km) was calculated from Lineweaver-Burk plots as shown in Figure 9. Kinetic parameters involving the Michaelis constant (Km) and the maximum reaction rate (Vmax) were measured for both free

Figure 10. Effects of (a) temperature (pH 7.0, reaction time 40 min) and (b) pH value (37 °C, reaction time 60 min) on the activity of free and encapsulated GUS. Table 3. Kinetic Parameters for Free and Encapsulated GUS (37 °C, pH 7.0) GUS

Km (mmol/L)

Vmax (µmol/min‚mg GUS)

free encapsulated

0.19 0.36

1.70 0.84

and encapsulated GUS and presented in Table 3. The maximum reaction rate Vmax was lowered about twofold after encapsulation. This was owing to the additional diffusion resistance to the substrate baicalin and the product baicalein molecules caused by the alginate membrane and the viscous CMC liquid core. The increase in Km after encapsulation indicated a weaker binding between the baicalin molecules and the encapsulated GUS. 3.2.4. Optimum Conditions for Enzyme Activity. The enzyme activity changes of both free and encapsulated GUS with reaction temperature and pH value were studied and shown in relative activities when their respective specific activity at optimal temperature and pH value was taken as 100%. As shown in Figure 10, the optimal temperature (60 °C) and pH value (7.0) for highest GUS activity were both unchanged after encapsulation. This indicated that the encapsulation carrier did not cause deactivation to the enzyme inside. The encapsulated enzyme displayed higher relative activity than its free counterpart under the same reaction temperature, indicating a higher thermal stability was achieved by encapsulation. In addition, the pH stability was also significantly enhanced by encapsulation. The free GUS lost all its activity at basic pH 8.0 and acidic pH 4.0 conditions whereas the encapsulated GUS could retain almost 100% and 50% relative activity under the same pH value, respectively. 3.2.5. Storage Stability. The storage stabilities of free and encapsulated GUS were compared in Figure 11. The activity of free GUS decreased sharply to 81% of its original activity after 5-day storage and continued falling down thereafter. On

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4. Conclusions

Figure 11. Storage stability of free and encapsulated GUS.

Baicalin was efficiently converted into baicalein by β-glucuronidase (GUS) encapsulated biomimetically in liquid-core alginate capsules. High encapsulation efficiency, optimized physical properties, and catalytic activities of the encapsulated enzyme could be achieved by modulating the preparation conditions including SA, CaCl2 concentration, and gelation time. CaCl2 concentration was a key parameter in the GUS encapsulation process and also had a significant effect on GUS activity. 1.0% w/v SA, 0.1 mol/L CaCl2, and 30 min gelation time were selected to prepare GUS-containing alginate capsules and a high relative activity of 88% was obtained. The productivity of baicalein in one reaction batch catalyzed by free and encapsulated GUS reached 80% and 65%, respectively. The encapsulated GUS had the same optimum temperature and pH conditions for enzyme activity as that in the free form and was stable for four repeated cycles without any appreciable loss in activity. In addition, encapsulated GUS exhibited improved storage stability compared to free GUS, and 90% of the initial activity was preserved after storage for 26 days at 4 °C. Reducing the viscosity of the inner core and introducing hybrid materials as outer shell to enhance the bioconversion efficiency and stability are under further investigation. Acknowledgment

Figure 12. Recycling stability of encapsulated GUS.

the 11th day of storage, the storage efficiency of free GUS had reduced to 74%. In contrast, the storage stability of GUS was significantly improved after encapsulation. No loss in activity was found during the first 10 days, and the remaining activity was kept as high as 90% of its initial activity in 26 days. Considering that the initial relative activity of encapsulated GUS was 88%, a promising result could be deduced that the activity of encapsulated GUS surpassed that of free GUS by 7% on the 5th day of storage and remained higher thereafter. It was reasonably believed that the encapsulated GUS would exhibit a distinct advantage over free enzyme in long-time storage, owing to the liquid core-membrane shell structure and special microenvironment provided by the biomimetic alginate capsule. For lysosome, the internal surface of its membrane is negatively charged, which helps to keep the hydrolases inside free. As for the alginate capsule, not only the alginate membrane but also the CMC core carries net negative charge under the neutral pH environment. Therefore, the conformational transition of GUS from the folded to unfolded state that may likely result in the denaturalization can be effectively inhibited by the electrostatic repulsion between the GUS, CMC, and alginate molecules. In addition, the biocompatible alginate membrane itself not only had no harmful influence on the enzymes but also helped the enzymes effectively avoid the unfavorable influence possibly rising from the outside storage environment. 3.2.6. Recycling Stability. The reuse capability of the encapsulated GUS was examined and illustrated in Figure 12. No loss in activity was found for four cycles. About 40% and 80% of the initial activity were lost after the fifth and sixth batch, respectively. It should be mentioned that the mechanical strength of the capsules after four repeated cycles became much weaker and some of them began to break during filtration. The leakage of enzyme due to the loosened polymer network was an important factor in the loss of activity.

The authors acknowledge the financial support from the Natural Science Foundation of Tianjin (No. 06YFJMJC10600), the Cross-Century Talent Raising Program of Ministry of Education of China, the program for Changjiang Scholars and Innovative Research Team in University (PCSIRT), the National Science Foundation of China (No. 20576096), and the Program of Introducing Talents of Discipline to Universities (No. B06006). Literature Cited (1) Ma, Z.; Otsuyama, K. I.; Liu, S. Baicalein, a component of Scutellaria radix from Huang-Lian-Jie-Du-Tang (HLJDT), leads to suppression of proliferation and induction of apoptosis in human myeloma cells. Blood 2005, 105, 3312. (2) Liu, J. J.; Huang, T. S.; Cheng, W. F. Baicalein and Baicalin are Potent Inhibitors of Angiogenesis: Inhibition of Endothelial Cell Proliferation, Migration and Differentiation. Int. J. Cancer 2003, 106, 559. (3) Rossi, M.; Meyer, R.; Constantinou, P. Molecular Structure and Activity Toward DNA of Baicalein, a Flavone Constituent of the Asian Herbal Medicine “Sho-saiko-to”. J. Nat. Prod. 2001, 64, 26. (4) Cheng, K. T.; Hou, W. C.; Huang, Y. C. Baicalin Induces Differential Expression of Cytochrome C Oxidase in Human Lung H441 Cell. J. Agric. Food Chem. 2003, 51, 7276. (5) Zhao, Y. L.; Li, H. L.; Gao, Z. H. Effects of flavonoids extracted from Scutellaria baicalensis Georgi on hemin-nitrite-H2O2 induced liver injury. Eur. J. Pharmacol. 2006, 536, 192. (6) Xing, J.; Chen, X. Y.; Zhong, D. F. Absorption and enterohepatic circulation of baicalin in rats. Life Sci. 2005, 78, 140. (7) Lai, M. Y.; Hsiu, S. L.; Tsai, S. Y. Comparison of metabolic pharmacokinetics of baicalin and baicalein in rats. J. Pharm. Pharmacol. 2003, 55, 205. (8) Che, Q. M.; Huang, X. L.; Li, Y. M. Studies on metabolites of baicalin in human urine. China J. Chin. Mater. Med. 2001, 11, 769. (9) Chen, X.; Mei, Y. C. A method for preparing baicalein. Chinese Patent CN 1673222 A, 2005. (10) Pan, F. S., Ye, B. A method for preparing high purity baicalein. Chinese Patent CN 1683356 A, 2005. (11) Che, Q. M. A method for preparing baicalein. Chinese Patent CN 1398862 A, 2003. (12) Gao, W. Y.; Deng, Y. Y. A method for preparing baicalein from baicalin. Chinese Patent CN 1594305 A, 2005. (13) Wandrey, C.; Liese, A.; Kihumbu, D. Industrial Biocatalysis: Past, Present, and Future. Org. Process Res. DeV. 2000, 4, 286.

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ReceiVed for reView October 14, 2006 ReVised manuscript receiVed February 3, 2007 Accepted February 7, 2007 IE0613218