Biochemical Engineering Journal 46 (2009) 186–192

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Biochemical Engineering Journal

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Immobilized transglucosidase in biomimetic polymer–inorganic hybrid capsules for efficient conversion of maltose to isomaltooligosaccharides

Lei Zhang, Yanjun Jiang, Zhongyi Jiang ∗ , Xiaohui Sun, Jiafu Shi, Wei Cheng, Qianyun SunKey Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, PR China

a r t i c l e i n f o a b s t r a c t

Article history:

Received 21 January 2009

Received in revised form 6 May 2009

Accepted 8 May 2009

Keywords:

Enzyme

Immobilized

Biocatalyst preparation

Kinetics

Biomimetic hybrid capsule

Isomaltooligosaccharide

Isomaltooligosaccharides (IMOs) are relatively new functional food ingredients which have great poten- tial to improve the quality of many foods due to their low calories, no cariogenicity and safety for diabetics. To convert maltose to IMOs efficiently, -transglucosidase was immobilized in a kind of alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP), which were prepared through a facile bio-inspired mineralization process. The surface morphology of Alg–Chi–CaP capsule and alginate–chitosan capsule (Alg–Chi) was characterized by scanning electron microscopy (SEM). Due to the presence of inorganic shell, immobilization efficiency of transglucosidase in Alg–Chi–CaP capsules was higher than that in Alg–Chi capsules. The optimal temperature (60 ◦ C) and pH (6.0) value for enzymatic conversion catalyzed by transglucosidase immobilized Alg–Chi–CaP capsules were identical to those catalyzed by free transglucosidase. As compared to the free enzyme, transglucosidase in Alg–Chi–CaP capsules exhibited significantly higher recycling stability and storage stability in a broader temperature and pH range.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

In recent years, considerable R&D efforts have been devoted to oligosaccharide engineering with the focus on exploring the func- tion of oligosaccharides for mammalian metabolic process [1–3]. Oligosaccharides are relatively new functional food ingredients which have great potential to improve the quality of many foods. Various oligosaccharides [4], including isomaltooligosaccharides (IMOs) [5], fructooligosaccharide [6] and soybean oligosaccharides [7], have been widely used for food or feed additives [8] and scaffold [9,10] due to their potential advantages such as bifidus- stimulating activity [11], low calorific value [12] and low cariogenic properties [13] etc. IMOs, comparing with other oligosaccharides, have received peculiar attention since they are very stable in acid solution, relatively low in price and have extensive sources [4]. Commercially available IMO is defined as saccharides that have 40% -(1-6) glucosidic linkages among the total linkages. IMOs with the degrees of polymerization (DP) ranging from 2 to 6 are pro- duced from corn starch by serial reactions of starch with -amylase and -amylase and transglucosidase [14]. -transglucosidase from Aspergillus niger [15,16] catalyzes the transglucosylation to the 6- OH of the accepting glucose unit and yielded the oligosaccharides

∗ Corresponding author. Tel.: +86 22 2350 0086; fax: +86 22 2350 0086.

E-mail address: zhyjiang@tju.edu.cn (Z. Jiang).

with an a-D-(1-6) linkage including isomaltose, panose and isomal- totriose. Compared to free enzyme, immobilized enzyme improved the operational stability and reusability, which could better meet requirement for industrial application. However, as far as we were concerned, immobilized transglucosidase has not yet been utilized for enzymatic production of IMOs.

The polymer–inorganic hybrid carrier have found increased application in enzyme immobilization due to its moderate hydrophilicity, controllable transport characteristics, and good physicochemical stability [17,18], which may create a benign microenvironment for enzymes [19]. At present, two kinds of con- figurations can be found for polymer–inorganic hybrid carriers: the mixed–matrix configuration [20] and the core–shell configu- ration [21]. Compared with the mixed–matrix configuration, the core–shell configuration could create a more nature-like envi- ronment for the immobilized enzyme. Chitosan and alginate are natural cationic or anionic polysaccharides, respectively, which have been successfully utilized in biomacromolecule encapsu- lation [22–25]. Furthermore, chitosan has been regarded as an efficient structure-directing agent for inorganic minerals [26]. Cal- cium phosphate, a principal component of hard tissues such as bone and tooth enamel [27,28], is of superior biocompatibility, insolubil- ity and mechanical stability, which have been demonstrated to be suitable for the enzyme immobilization [29].

In this work, alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) were employed to immobilize transglu- cosidase for efficient conversion of maltose to IMOs. The capsules

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were produced by a one-step method in which the deposition of a semi-permeable alginate–chitosan film around droplets of sodium alginate was coupled with in situ precipitation of calcium phos- phate. Ca2+ cross-linked alginate containing transglucosidase was first coated with chitosan and then coated with calcium phos- phate to format Alg–Chi–CaP capsules with enhanced mechanical strength and decreased enzyme leakage. The optimum catalytic condition, kinetic parameters, the recycling and storage stability of the immobilized transglucosidase were studied extensively.

2. Materials and methods

2.1. Materials

Transglucosidase (EC3.2.1.20, from A. niger) was obtained from megazyme. Chitosan (deacetylation degree 75–85%, viscosity20–200 cps) were obtained from Sigma–Aldrich. Sodium alginate

(average molecular weight, 6.27 × 105 ) was obtained from Shang- hai Tianlian, China. Maltose was obtained from Guangfu, China. All other reagents used were of analytical grade and were used without further purification.

2.2. Preparation of polymer–inorganic hybrid capsule

Sodium alginate and disodium hydrogen phosphate (100 mM) solution were mixed to a final concentration of 2.0% (w/v). Chi- tosan and calcium chloride solution (100 mM) were mixed to a final concentration of 1.0% (w/v). Alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) capsules were prepared by the drop- wise addition of alginate solution into a gently stirring chitosan solution, using a 5 ml syringe with a 0.9 mm diameter needle as shown in Fig. 1. The capsules formed were left in the chitosan solu- tion for 30 min, before being filtered off and rinsed in an excess of distilled water. Alginate–chitosan capsules (Alg–Chi) capsules were prepared following the same procedure, with the exception of alginate mixing in distilled water instead of HPO4

2− ions. All procedures were carried out at room temperature and pH 7.0.

2.4. Immobilization of transglucosidase

Sodium alginate [2.0% (w/v)] dissolved in HPO4 2− solution

con- taining 3 mg/ml transglucosidase was prepared. For one batch,1 ml of the mixture was added dropwise into a gently stirring

Ca2+ -containing chitosan solution [1.0% (w/v)] with a syringe. The capsules formed were left immersed in the chitosan solution for30 min, before being filtered off and rinsed in an excess of distilled water.

2.5. Activity assays of transglucosidase

The activities of free and immobilized transglucosidase were determined by the transglycosylation of maltose. The free or immo- bilized transglucosidase and 5 ml, 100 mg/ml maltose solution were mixed and the system was incubated in a water bath with con- stant shaking at different temperature for 10 min. The reaction was stopped by adding two times volume of acetonitrile reagent. Incu- bation was performed in a boiling water bath for 5 min. An enzyme activity unit (U) was defined as the amount of enzyme liberating1 mg maltose per minute under the assay conditions. Each result was an average of four or five separate experiments.

Maltose was quantified using high performance liquid chro- matography (HPLC) operating on an analytical column (Tsk-gel Amide80, 5 m, 4.6 mm id × 250 mm). After 10 min reaction, 60 l of the digested maltose solution was diluted with 140 l of ace- tonitrile. HPLC was used to analyse the samples. For the elution conditions; the mobile phase was 70% acetonitrile and 30% water.Temperature was kept constant at 50 ◦ C, with a flow rate of0.8 ml/min and an injection volume of 20 l. The detector was a Knauer Differential-Refractometer. In each set of experiment, a standard curve was plotted with maltose solutions of different con- centrations.

2.6. Immobilization efficiency

The immobilization efficiency of capsules was determined using the following equation.

immobilization efficiency (%)

[t r ansglucosidase] solution × V solution2.3. Characterizations of capsules = 100 − [transglucosidase]droplet ×Vdroplet

× 100 (1)

Intact capsules were observed with Zoom Stereo Microscope (Olympus SZ2-ILST). The morphology of capsule surface was observed with scanning electron microscopy (SEM, XL30, PHILIPS, Holand) using an accelerating voltage of 20 kV; before analyzing, the capsule was first freeze-dried and gold coated.

where [transglucosidase]solution and [transglucosidase]droplet are

the concentration of transglucosidase in the final solution and in the original liquid droplet, Vsolution and Vdroplet represent the

volume of the solution and liquid drop, respectively. The transglucosidase concentration was determined by the enzyme activity.

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Fig. 1. Schematic illustration of the enzyme immobilization process.

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2.7. Stirring speed

An experiment was done to choose the optimum stirring speed. The optimum stirring speed of reaction were determined by immersing transglucosidase-containing capsules into 5 ml,100 mg/ml of maltose at 50 ◦ C and pH 6.0 for 30 min, and the con-centration of transglucosidase changes were monitored at specified time intervals.

2.8. Leakage of transglucosidase from Alg–Chi and Alg–Chi–CaPcapsules

The leakage characteristics of capsules were determined by immersing capsules into 5 ml of deionized water at room temper- ature and 600 rpm/min, and the concentration of transglucosidase changes were monitored at specified time intervals. The leakage was calculated using the following equation:

[transglucosidase]solution × Vsolution

2.11. Recycling stability and storage stability

The recycling stabilities of the immobilized transglucosidase were evaluated by measuring the enzyme activity in each succes- sive reaction cycle, and were expressed by recycling efficiency. The immobilized transglucosidase were allowed to take effect in mal- tose solution with pH 6 and 50 ◦ C (optimum). The immobilized transglucosidase was then filtered off, rinsed with distilled water, and then, used in the next reaction cycle. The process was repeated for 15 times.

The activity found for each repetition was compared with the initial activity assuming it possessed 100% activity.

recycling efficiency (%)

enzyme activity on the storing for nth day= initial enzyme activity on the 1st cycle

× 100

(6)

Many batches of the free and immobilized transglucosidase were prepared and stored in glass vials at 4 ◦ C to determine the

leakage (%) = [transglucosidase]droplet ×Vdroplet× 100 (2)

storage efficiency. On the 1st, 2nd, 3rd, 5th, 7th, 10th, 13th, 15th,17th, 20th, 23rd, 27th and 30th day, 1 ml of each type of the free

2.9. Optimum conditions for enzyme activity

The optimum temperature of the free and immobilized trans- glucosidase was evaluated by adding the enzyme into the maltose solution for 10 min under different temperature condition. Temper-

and immobilized transglucosidase was added to 5 ml of maltose solution at pH 6 and under temperature of 50 ◦ C.

storage efficiency (%)

enzyme activity after storing for nth day

atures of 5, 25, 37, 50, 60, and 70 ◦ C were used for the experiment. =

The activity found at each temperature was compared with the activity at optimum temperature assuming it possessed 100% activity.

initial enzyme activity × 100 (7)

activity (%) =

enzyme activity at the specific temperature enzyme activity at optimum temperature

× 100

(3)

3. Results and discussion

3.1. Characterization of the capsules

As shown in Fig. 3, Alg–Chi–CaP capsules were prepared by the dropwise addition of phosphate-containing sodium alginate

The optimum pH of the free and immobilized transglucosidase was evaluated by adding the enzyme into the maltose solution for10 min. Maltose solutions with pH of 2, 3, 4, 5, 6, 7, 8 and 9 were used. The activity found at each pH was compared with the activity at optimum pH assuming it possessed 100% activity.

enzyme activity at the specific pH

solution into calcium-containing chitosan solution. Because of interfacial complexation of the oppositely charged polysaccharide, a thin chitosan film formed around the alginate droplets sponta- neously. Counter-diffusion of the oppositely charged ions across the polysaccharide interface results in the in situ precipitation of cal- cium phosphate. The capsules became much stiffer due to furthercross-linking between alginate and Ca2+ ions and the deposition ofactivity(%) =

enzyme activity at optimum pH × 100

(4)calcium phosphate [30].

The micrographs of Alg–Chi and Alg–Chi–CaP capsules were

2.10. Determination of Km and Vmax values

Activities of the free and immobilized transglucosidase were determined by using the classical Michaelis–Menten kinetics. In the graphical evaluation of Michaelis–Menten constants and maximum activities, Lineweaver–Burk plots obtained by plotting experimen- tal values were used.

shown in Fig. 2a and b, respectively. It could be observed that the Alg–Chi capsules were more jelly-like whereas the Alg–Chi–CaP capsules were noticeably harder. Fig. 2c and d showed that the Alg–Chi–CaP capsule had smooth and intact surface structure, in comparison, the Alg–Chi capsule had a wrinkled surface struc- ture, which was ascribed to the polymer shrunk during the freeze drying process. As shown in Fig. 2e and f, the internal surface of the capsule was Ca2+ -alginate hydrogel network and the external

1 Km 1 1 . (5) surface of the capsule was relatively smooth calcium phosphate

V =

Vmax ×

[S] +

Vmax

V and [S] are the initial reactive rate and initial substrate concentra- tion, respectively. Vmax is the maximum activity attained at infinite initial substrate concentration and Km is the Michaelis–Menten constant.

To determine Vmax and the Km , the activity assay was applied for different maltose concentrations (10, 12.5, 16.7, 25, 50 and100 mM). Activities of free and immobilized transglucosidase were all determined at the optimum conditions. The catalytic efficien- cies of both free and immobilized transglucosidase were calculated accordingly.

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layer.

3.2. Immobilization efficiency

The immobilization efficiency could be assessed by measur- ing the transglucosidase concentration in the solution during the immobilization. The immobilization efficiency was 92.6% for Alg–Chi–CaP capsules, whereas it was 70.5% for Alg–Chi capsules. Due to existence of the external inorganic layer, the leakage of transglucosidase was considerably reduced during the capsules for- mation process.

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Fig. 2. Optical micrographs of (a) Alg–Chi capsules and (b) Alg–Chi–CaP capsules; SEM image of (c) Alg–Chi capsules, (d) Alg–Chi–CaP capsules, (e) Alg–Chi–CaP capsules interior and (f) Alg–Chi–CaP capsules exterior.

3.3. Stirring speed

When stirring speed was greater than 400 rpm/min, it had trivial influence on reaction rate because the internal diffusion controlled the whole diffusion process as shown in Fig. 3. When the stirring

Fig. 3. Effects of stirring speed (50 ◦ C, pH 6.0, reaction time 30 min) on the maltose conversion ratio catalyzed by immobilized transglucosidases.

speed was less than 400 rpm/min, the reaction rate reached the highest, which was due to that the external diffusion constituted the control step for the whole diffusion process. In the following exper- iment, the stirring speed used was 400 rpm/min unless otherwise noted.

3.4. Leakage of transglucosidase

To assess the leakage transglucosidase from the Alg–Chi and Alg–Chi–CaP capsules, both capsules were dip in deionized water at 1000 rpm/min. As seen in Fig. 4, extended time studies indicated that over 80% of the immobilized transglucosidase were leaked from the Alg–Chi capsules to the surrounding solution within a period of 5 h. In contrast, capsules prepared with calcium phos- phate retained approximately 65% of the trapped transglucosidase after 7 h, indicating that in situ precipitation of calcium phosphate process was successful in significantly reducing the enzyme leakage from the capsules.

3.5. Thermal and pH stabilities of immobilized transglucosidase

The activity of free and immobilized transglucosidase was assayed at various temperatures (5–70 ◦ C) as shown in Fig. 5a. The results showed that optimum reaction temperature (50 ◦ C) was not affected by immobilization. The transglucosidase immobilized

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Fig. 4. The leakage of transglucosidase from Alg–Chi capsules and Alg–Chi–CaP

capsules.

in Alg–Chi–CaP capsules displayed the broadest temperature pro- file, indicating that immobilization could render enzyme with a more benign environment which protected enzyme from heat- induced denaturation and allowed the enzyme to become less

more in soluble form as compared to the immobilized state[31,32].

As shown in Fig. 5(b), the effect of pH on the activity of free and immobilized transglucosidase was studied (from pH 2.0 to 9.0.) and found that both free and immobilized transglucosi- dase were sensitive to the pH changes. The highest activity for immobilized transglucosidase was achieved at pH 6.0, completely consistent with that of free transglucosidase, which further sup- ported that the conformation of the transglucosidase was well preserved after immobilization. Transglucosidase immobilized in Alg–Chi–CaP capsules retained 77% of its maximum activity at pH2.0 and 53% at pH 9.0, whereas, free transglucosidase retained only 33% of its maximum activity at pH 2.0 and 29% at pH9.0, respectively. Such changes are generally analyzed as a result of immobilization, which greatly helped in the stabilization of enzyme at a wider pH range [31,33]. Additionally, it was noticed that enzyme-containing capsules preserved higher activity in acid condition than in alkaline condition. The strong resistance of immo- bilized transglucosidase against the acidic medium was tentatively explained by the buffering effect of the alginate capsule. According to the previous report, the pKa of

alginate ranges between 3.4 and4.4, which was attributed to the ionization of the unbound carboxylgroup (not bound to Ca2+ -ion) [34,35]. In acidic medium, the nega-

+temperature-depending. At 70 ◦ C, approximately 88% and 66% of

tively charged alginate core could attract and consume the H

ions,

the initial activity was lost for the free enzyme and immobi- lized enzyme, respectively. It is well established that thermal inactivation starts with the unfolding of the protein molecule which is followed by irreversible changes due to aggrega- tion and formation of scrambled structures which takes place

Fig. 5. (a) Effects of temperature (pH 6.0, reaction time 10 min) on the activity of free and immobilized transglucosidases. (b) Effects of pH value (50 ◦ C, reaction time

10 min) on the activity of free and immobilized transglucosidases.

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assisting in preventing H+ ion from diffusing into and contactingwith the enzymes [36].

3.6. Free and immobilized enzyme activity under optimal conditions

Under optimum condition (50 ◦ C and pH 6.0), the enzyme activ- ity of free and immobilized transglucosidase in Alg–Chi capsules and Alg–Chi–CaP capsules was investigated. As shown in Fig. 6, the bioconversion process was monitored by measuring the conversion of maltose with the lapse of time. The reaction rate and the final conversion rate using immobilized transglucosidase were slightly lower than that of free transglucosidase. The equilibrium conver- sion using free transglucosidase was obtained at 95.31% in 13 h, while that in the case of immobilized transglucosidase immobilized in Alg–Chi capsules was 96.99% in 13.83 h and in Alg–Chi–CaP cap- sules was 97.4% in 13.98 h. The enzyme activity unit was defined as the amount of transglucosidase needed to convert 1.0 mg of maltose/min at 50 ◦ C, pH 6.0. The specific activity of free trans- glucosidase was 1.22 U/mg, while specific activity of immobilized transglucosidase in Alg–Chi capsules and in Alg–Chi–CaP capsules was 1.17 and 1.18 U/mg, respectively. Lower specific activity of immobilized transglucosidase might be due to the additional dif- fusion resistance rather than enzyme denaturation.

Fig. 6. Maltose conversion with reaction time (50 ◦ C, pH 6.0).

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3.7. Kinetic studies

The Michaelis–Menten kinetics of free and immobilized trans- glucosidase was studied and the corresponding Michaelis constant (Km ) and maximum reaction rate (Vmax) were calculated from Lineweaver–Burk plots.

The value of the maximum reaction rate (Vmax ) and the Michaelis constant (Km ) evaluated from Lineweaver–Burk plots were shown. Both the Km and the Vmax values were changed by the immobiliza- tion processes. The Vmax value of the transglucosidase in Alg–Chi capsules (1.025 U/mg) and Alg–Chi–CaP capsules (1.165 U/mg) was found to be lower than that of free transglucosidase (1.239 U/mg). The Km value of the transglucosidase in Alg–Chi (16.015 mM) and Alg–Chi–CaP (22.37 mM) capsules were found to be higher than that of the free transglucosidase (9.094 mM). The decreased Vmax value of the immobilized transglucosidase could be due to the additional diffusion limitation to the substrate (maltose) and the product (IMOs) caused by the carrier, which induced the low accessibil- ity of the substrate to the active sites of the transglucosidase and consequently resulted in a lower possibility of enzyme–substrate complex formation. The Km value was known as the affinity of the enzymes toward substrates and the lower values of Km meant the higher affinity between enzymes and substrates. [37] The increase in Km after immobilization indicated a weaker binding between the maltose molecules and the immobilized transglucosidase.

3.8. Recycling stability and storage stability

Enzymes were very sensitive to environmental conditions and might lose their activities quite easily. Thus, it was meaningful to characterize their recycling stability and storage stability for industrial application. To determine the recycling stability of the immobilized enzyme, the activity of transglucosidase in capsules was measured sequentially 15 times at the optimum condition (50 ◦ C and pH 6.0). After one cycle, capsules containing enzyme were removed from the reaction medium and washed twice with distilled water.

As shown in Fig. 7, after the 7th reaction cycles, the transglucosi- dase in Alg–Chi capsule lost half of its initial activity, whereas in the Alg–Chi–CaP capsule, after the 15th cycles, it still retained 65% of its initial activity. The difference in recycling stability was due to the leakage of transglucosidase during the multiple soaking, sep- aration, and washing processes employed in the reaction cycles. Whereas, for the transglucosidase immobilized in Alg–Chi–CaP capsule, transglucosidase leakage was prevented during recycling process and the recycling stability was increased substantially. The pore size of the inorganic layer should meet two requirements: it must be big enough for the substrates and products to pass through freely but small enough to effectively prevent the enzyme from leaking. Since BET analysis showed the average pore diameter of the Alg–Chi–CaP capsule was slightly bigger than 3 nm [38], it could be concluded that Alg–Chi–CaP capsule was effective as it allowed for the substrates and products (0.6 nm) to pass through, and mean- while prevented transglucosidase which was larger than 3 nm from leaking from capsules.

In addition, the free and immobilized enzymes were stored without any buffer at 4 ◦ C and their activities were tested for 30 days. The storage stability of free and immobilized transglucosidasewas shown. Taking the initial activity level to be 100%, the relative activity of the free transglucosidase, transglucosidase immobilized in Alg–Chi and Alg–Chi–CaP capsules was decreased to 38%, 78%, and 85% after 30 days, respectively. The little differences in the storage stability of the Alg–Chi and Alg–

Chi–CaP capsule indicated that the calcium phosphate played trivial role in improving storage stability. It was reasonably believed that the immobilized trans- glucosidase would exhibit a distinct advantage over free enzyme in

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Fig. 7. (a) Recycling stability of immobilized transglucosidases in Alg–Chi

capsules and Alg–Chi–CaP capsules. (b) Storage stability of free and immobilized

transglu- cosidases.

long-time storage, owing to crowded microenvironment created by the biomimetic alginate capsule, which closely imitated the effects of crowding and confinement in a living cell. Alginate and trans- glucosidase (pI = 5.1) both bear net negative charges in a neutral pH environment. Therefore, the conformational transition of trans- glucosidase from a folded to an unfolded state was substantially inhibited by the electrostatic repulsion between transglucosidase and alginate molecules. Additionally, the biocompatible alginate helped the enzymes avoid the unfavorable attack possibly arising from the outside storage environment effectively.

4. Conclusions

A facile method for preparing alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP) as efficient transglu- cosidase immobilization carrier was proposed. The biocompatible alginate-core accommodated the suitable microenvironment for transglucosidase, the outer calcium phosphate shell of capsule ensured the facile accessibility of immobilized transglucosidase for substrates and prevent transglucosidase from leaking out effec- tively. Owing to the synergy effect of hydrophilic polymers and mechanically stable calcium phosphate, the immobilized transglu- cosidase displayed higher thermal stability and pH stability than that in the free form and retained more than 60% initial activity after15 repeated cycles. Additionally, compared to free transglucosidase, the immobilized transglucosidase exhibited improved storage sta- bility. The facile immobilization process and the enhanced stability set an encouraging example for

converting natural compounds into high value-added functional products.

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Acknowledgements

The authors thank the financial support from the National High- Tech Research and Development Plan (no. 2007AA10Z305), the Cross-Century Talent Raising Program of Ministry of Education of China, the program for Changjiang Scholars Innovative Research Team in University (PCSIRT) and the Programme of Introducing Talents of Discipline to Universities (no. B06006).

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