Covalent immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase onto modified silica nanoparticles

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Appl Microbiol Biotechnol (2011) 90:499–507 DOI 10.1007/s00253-011-3094-9

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Covalent immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase onto modified silica nanoparticles Ye-Wang Zhang & Manish Kumar Tiwari & Marimuthu Jeya & Jung-Kul Lee

Received: 9 October 2010 / Revised: 22 December 2010 / Accepted: 23 December 2010 / Published online: 19 January 2011 # Springer-Verlag 2011

Abstract Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t1/2 of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The Km of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into L-xylulose. A∼71% converElectronic supplementary material The online version of this article (doi:10.1007/s00253-011-3094-9) contains supplementary material, which is available to authorized users. Y.-W. Zhang : M. K. Tiwari : M. Jeya : J.-K. Lee (*) Department of Chemical Engineering, Konkuk University, Seoul 143-701, South Korea e-mail: [email protected] J.-K. Lee Institute of SK-KU Biomaterials, Konkuk University, Seoul 143-701, South Korea Y.-W. Zhang School of Pharmacy, Jiangsu University, Zhenjiang 212013, People’s Republic of China

sion and a productivity of 10.7 gh-1 l-1 were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to L-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production. Keywords Covalent immobilization . Functional groups . Nanoparticle . Silica . Stability . Xylitol dehydrogenase

Introduction Rare sugars and their derivatives have various applications in pharmaceutical, nutrition, and food industries as nucleoside analogs, precursor substances, no-calorie sweeteners, and bulk agents (Poonperm et al. 2008). L-xylulose, one of the rare sugars, is a ketopentose that exists in very low concentrations in nature. It has been used as an inhibitor of glycosidases and as a reliable indicator of patients with acute or chronic hepatitis or liver cirrhosis (Takata et al. 2010). Xylitol dehydrogenase (xylitol: NAD+-2-oxidoreductase; XDH; EC 1.1.1.9), a medium-chain dehydrogenase/reductase, is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol (Ehrensberger et al. 2006). It is capable of catalyzing the biotransformation of xylitol, ribitol, sorbitol, and erythritol to xylulose, ribulose, fructose, and erythrulose, respectively, and the reverse reactions (Ko et al. 2006a; Lima et al. 2006; Takata et al. 2010; Takamizawa et al. 2000; Yablochkova et al. 2003). Consequently, it has potential industrial application for production of various rare sugars. However, it is well known that the use of a free enzyme has some disadvantages

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such as poor stability, low reusability, and difficult product recovery. These drawbacks can be solved by immobilization. For enzyme immobilization, the important factors are the choice of immobilization method and the design of a suitable support (Hanefeld et al. 2009). Of all the available immobilization methods, covalent immobilization has been receiving increasing attention over the years because it is possible to get high stability in covalently immobilized enzymes. Mesoporous silica is a good immobilization material as it has a large surface area and pore volume, uniform and tunable pore size, is open to a wide range of chemical modifications, is conveniently reutilized, and is environmentally friendly. Over the past decades, biochemical production has become focused on nanostructured chemistry, and technology using highly selective enzymatic reactions and nanoparticles has proven to be a promising architecture for reproducing biological functions (Watanabe and Ishihara 2006). Of particular interest has been the use of nanoparticles as carriers for immobilization due to their advantages of high enzyme loading, minimum diffusional limitation, and maximal surface areas (Kim et al. 2006). In our previous work, a highly thermal stable XDH from Rhizobium etli CFN42 (ReXDH) was cloned, overexpressed in Escherichia coli and characterized (Tiwari et al. 2010). In the present study, we report the immobilization of this recombinant ReXDH on various carriers. Silica nanoparticles with four different functional groups were chosen for further investigation and compared for their effectiveness. Silica nanoparticles modified with an epoxy group showed the best immobilization performance. A 71% yield of L-xylulose was obtained when immobilized ReXDH was used as the dehydrogenase with xylitol as the substrate.

Materials and methods Materials Silica nanoparticles 4850HT, 4830HT, 4860MR, and 4806SF were purchased from Nanostructured and Amorphous Materials (Houston, TX). NADH, NAD+, glutaraldehyde (GA), γ-glycidoxypropyl trimethoxysilane (GT), Eupergit C, Sepabeads, Diaion HPA 25, Amberlite resins IRA 400, XAD16, XAD4, XAD7AH, and Duolite resins A568, A7 were from Sigma-Aldrich (St. Louis, MO). 1-Cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMT) was purchased from Kasei Kogyo (Tokyo). All the other reagents were of analytical or biotechnological grade. Cell culture and protein purification The recombinant E. coli harboring ReXDH was obtained as previously reported (Tiwari et al. 2010). Cells were cultured

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with induction using 0.1 mM of isopropyl-β-D-thiogalactopyranoside and harvested by centrifugation at 10,000×g for 15 min at 4 °C, then rinsed with phosphate-buffered saline. To purify the recombinant ReXDH, cell pellets were resuspended in 100 mM Tris–HCl buffer (pH 8) supplemented with 25 μg ml−1 DNase I. The cell suspension was incubated on ice for 30 min in the presence of 1 mg ml−1 lysozyme. Cell disruption was carried out by sonication at 4 °C for 5 min, and the lysate was centrifuged at 14,000×g for 20 min at 4 °C to remove the cell debris. The resulting crude extract was retained for purification. Purification using Glutathione Sepharose 4B was performed according to the manufacturer’s protocol (GE Healthcare). Enzyme assay The activity of free ReXDH was determined spectrophotometrically by monitoring the change in A340 upon oxidation or reduction of NADH at 25 °C. The XDH assay mixture for oxidation consisted of 0.5 mM NAD+, 400 mM xylitol, and enzyme solution 100 mM Tris–glycine–NaOH (pH 9.5). The reaction was started by the addition of substrate. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol NADH per minute under the assay condition. For the immobilized enzyme, the above-mentioned assay was adopted except that the NAD+ concentration was 10 mM, and a thermoreactor (Thermo Fisher Scientific, MA) with a stirred magnetic bar was used as the reaction vessel. Modification of silica nanoparticles to give different functional groups The modification to give aldehyde groups was proceeded according to the previous report (Lin et al. 2001). The typical protocol was included sonication of 1 g nanoparticles for 30 min in distilled water. Then, the particles were collected by centrifugation and resuspended in 1 M glutaraldehyde for 2 h at room temperature. Afterwards, the modified particles were washed five times with distilled water and separated via centrifugation (12,000×g) for 10 min. To modify the nanoparticles to give cyanogen group, the procedure was followed the report with minor modification (Singh et al. 2011, Zhang et al. 2009). The sonicated and washed nanoparticles were collected by centrifugation. Then, the washed silica was added into 600 μl of 2 M sodium carbonate and cooled the slurry to 0 °C, and followed by adding 160 μl of 0.45 gml-1 of CNBr dissolved in DMF and mixed this vigorously for 2 min and incubated for 20 min in shaker. The activated support material was washed with five volumes of cold distilled water. The modification of nanoparticles to give carbodiimide groups was carried out as follows. The sonicated and

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washed particles were suspended in 10 ml of 0.1 M sodium acetate buffer (pH 4.5). Then, 200 mg of 1-cyclohexyl-3-(2morpholinoethyl) carbodiimide metho-p-toluenesulfonate was added to the support slurry, and this was mixed for 4 h at room temperature, and the particles were washed with 500 ml of cold 0.1 M sodium phosphate buffer (pH 7.0). To cover the silica nanoparticles with epoxy groups, the modification reaction was carried out in a round flask with three necks (Liu et al. 2009). One gram of silica nanoparticles were added into 50 ml toluene, and then the reaction was started by adding 0.15 mL Et3N and 1 ml GT. The reaction mixture was continuously stirred and refluxed for 3 h. The modified silica was washed with acetone for three times, and dried at 60 °C for 24 h. The amount of the epoxy groups on the surface was determined using the titration method involving a reaction between the epoxy ring and sodium thiosulphate. The titration was typically performed with hydrochloric acid (0.1 M) after the activated and dried silica nanoparticles (0.5 g) reacted with 5 ml sodium thiosulfate solution (1.3 M) for 2 h. The loading of epoxide groups was determined to be 1.1 mmol g-particle-1. Immobilization of ReXDH Amberlite resins IRA 400, XAD16, XAD4, XAD7AH, Duolite resins A568, A7, Eupergit C, Diaion HPA 25, Sepabeads SP850, and silica nanoparticles 4850MR, 4860MR, 4830HT, and 4806SF were chosen as carriers for immobilization of ReXDH. Typically, 40 mg of modified carriers was washed with distilled water and then mixed with 0.4 mg of purified ReXDH at 50 mM pH 7.0 phosphate buffers and incubated 24 h at 4 °C. After immobilization, the beads were collected by centrifugation and washed three times with 50 mM phosphate buffer (pH 7.0). The protein concentration of washed solution was measured by Bradford method (Bradford 1976) and the activity of immobilized ReXDH was determined. The immobilization efficiency (Ye) and yield (Yy) were calculated as follows:Ye =100×(αi/αf); Yy =100× [(ρi−ρw−ρs)/ρi]. Where αi is the total activity of the immobilized enzyme and αf is the total activity of the free enzyme. ρi is the total protein content of the crude enzyme preparation; ρw and ρs are the protein concentration of wash solution and supernatant after immobilization, respectively.

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(3–9) in different buffers: citrate (100 mM, pH 3–4), sodium acetate (100 mM, pH 4–6), phosphate (100 mM, pH 6–8), and glycine (100 mM, pH 9). Characterization of immobilized ReXDH The effect of temperature on the activity of immobilized and free ReXDH was analyzed by assaying the enzyme samples over the range of 30–80 °C for 5 min. The optimum pH of ReXDH was determined using the standard assay conditions with three buffer systems, sodium acetate buffer (20 mM, pH 3.6–5.6), phosphate buffer (20 mM, pH 6.0–8.0), and Tris–HCl buffer (20 mM, pH 8.0–9.0). To determine kinetic parameters of immobilized ReXDH, the samples were incubated at 50 °C for 5 min with varying concentrations of xylitol (10 to 1,000 mM). Kinetic parameters (Km and Vmax) for substrates were obtained by using non-linear regression fitting. All assays were performed in triplicate. The kinetic data presented represent averages of statistically relevant measurements with their associated standard deviations. The thermal stability of the immobilized XDH was investigated by incubating the immobilized enzyme in 20 mM phosphate buffer (pH 9.5) at various temperatures from 30 °C to 70 °C. After 2, 4, 6, 8, and 10 h incubation, samples were withdrawn to measure the residual activity under standard assay conditions. Reusability of the immobilized ReXDH Reusability of the immobilized ReXDH was studied in a 5-ml thermo-reactor with a stirred bar. The xylitol and NAD+ concentration was 30 gl-1 and 10 mM, respectively. In order to start the reaction, 5 Uml-1 of immobilized ReXDH was added, and the samples were withdrawn and analyzed by high-pressure liquid chromatography Ultimate 3000 (Dionex, CA) equipped with a Shodex NH2P-50 4E column (Showa Denko, K. K., Kawasaki, Japan) and an evaporation light scattering detector (ESA6700, MA). The elution buffer was 70% acetonitrile at a rate of 1 ml min-1 and the elution was at 30 °C (column temperature), the evaporation and nebulizer temperature were 50 °C and 65 °C, respectively. The retention times of xylitol and xylulose were 13.3 and 5.7 min at the operation conditions, respectively.

Optimization of immobilization Temperature optimization of the immobilization procedure of ReXDH on silica nanoparticles was done achieved by carrying out the immobilization at different ratio of enzyme to support, and various temperatures (4 °C, 16 °C, 25 °C, 30 °C, and 37 °C). Similarly, for the pH optimization the immobilization procedure was carried out at different pH

Results Effect of support on the immobilization Various carriers, including ion exchange resins (Amberlite IRA400, Duolite A7 and A568), porous adsorption resins

(Amberlite XAD 7HP, XAD4, and XAD16), and silica nanoparticles modified with glutaraldehyde, were used for immobilization of ReXDH. The immobilization yields and efficiencies are shown in Fig. 1. The immobilization yield was higher than 80% for most of the selected carriers, with the exception of Amberlite IRA400 and silica nanoparticles 4806SF. The difference among nanoparticles with aldehyde groups is their particle size. Silica nanoparticles 4850MR, 4860MR, 4830HT, and 4806SF have particle sizes of 15, 30, 80, and 3,000 nm, respectively. The immobilization efficiency on silica 4830HT, 4850MR, 4860MR, and 4806SF was 72.5%, 67.5%, 48.1%, and 27.9%, respectively, while it was only 27.5% on Duolite A7 and less than 20% on the other resins. These results show that the immobilization efficiency of silica nanoparticles is much higher than that of resins. The adsorption isotherms on silica nanoparticles with various particle sizes are shown in Fig. 2. Silica nanoparticles 4830HT with a particle size of 80 nm displayed a higher binding capacity than did the other nanoparticles. The isotherms were fitted to the Langmuir model: q ¼ qm Ka C=ð1 þ Ka C Þ; where Ka is the Langmuir constant, C is the equilibrium concentration of ReXDH, qm is the adsorption capacity, and q is the amount of ReXDH adsorbed at concentration C. The Langmuir equation coefficients (Ka and qm) are summarized in Table 1. The adsorption capacity qm decreased sharply from 191 mg/g-support to 15 mg/gsupport when the particle size increased from 80 to 3,000 nm. Meanwhile, Ka, the rate of adsorption over the rate of desorption, which is related to the adsorption affinity, decreased from 11.8 to 2.96 ml mg-protein-1. Thus, the silica nanoparticles 4830HT, with particle size 80 nm, were chosen as the support for further immobilization.

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Adsorption amount (mg/g support)

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0 0.0

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Fig. 2 Fitting of Langmuir equation for immobilization of ReXDH on silica nanoparticles. Circle: 4850MR; uptriangle: 4860MR; square: 4830HT; downtriangle: 4806SF

Effect of functional groups on the immobilization The silica nanoparticles were modified to give different functional groups for immobilization of ReXDH (Fig. 3). All of the functional groups, including aldehyde, cyanogen, and epoxy, are common functional groups employed in the immobilization of proteins. Here, the immobilization yield and efficiency on different derivative silica nanoparticles were compared. The adsorption capacity of ReXDH on silica nanoparticles was high; more than 95% of purified ReXDH were adsorbed on the nanoparticles (Fig. 4). However, immobilization efficiency on silica nanoparticles without any chemical modification was only ∼20%. This means that protein adsorbed onto nanoparticles without covalent immobilization could be easily removed by washing with phosphate buffer. The immobilization efficiency on modified silica nanoparticles decreased as the amount of enzyme to support increased, and this can be easily explained by the inhibition of multiple-layer immobilization (Mateo et al. 2000). The immobilization efficiency on silica nanoparticles modified with GT, GA, CNBr, and CMT was 92%, 81%, 57%, and 43%, respectively. Thus, compared with the silica nanoparticles modified with CNBr and CMT, the immobilization efficiency on silica nanoparticles modified with GT and GA were significantly higher. Optimization of pH and temperature for the immobilization

Fig. 1 Effect of carrier on the immobilization yield and efficiency. Blue column: immobilization yield (%); red column: immobilization efficiency (%)

GT modified silica nanoparticles were chosen as the candidate carrier for immobilization of ReXDH as they showed the highest immobilization efficiency. Immobilization conditions, including pH and temperature, were optimized as shown in Fig. 5. The immobilization efficiency decreased from 92% to 30% as temperature increased from 4 °C to

Appl Microbiol Biotechnol (2011) 90:499–507 Table 1 Properties of silica nanoparticles and Langmuir isotherm coefficients of ReXDH adsorption

APS average particle size, SSA specific surface area

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Nanoparticles

APS (nm)

SSA (m2/g)

Ka (mL/mg)

qm (mg/g)

4850MR 4860MR 4830HT 4806SF

15 30 80 3000

640 160 440 5

11.6 7.02 11.8 2.96

138 103 191 15

Characterization of the immobilized ReXDH

37 °C (Fig. 5a). Low temperature was favorable for immobilization of ReXDH. The efficiency was improved from 2% to 90% when pH was changed from 4.0 to 7.0. However, the immobilization yield at all the investigated conditions was higher than 90%.

ReXDH immobilized onto silica nanoparticles was characterized and compared with its free counterpart. The immobilized and free ReXDH activities increased

A

B H

O

O

H 3

OH + O

3

H

OH

+ O

H2N

XDH

N

O 3

XDH

OH

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D

Fig. 3 Scheme for the modification of silica nanoparticles with different functional groups. a Modified silica with CMT; b modified silica with GA; c modified silica with CNBr; d modified silica with GT

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A Immobilization efficiency (%)

Immobilization efficiency (%)

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3

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Temperature (oC)

Enzyme ratio to support (mg/g)

Production of L-xylulose and the reusability of immobilized ReXDH The immobilized ReXDH was employed to produce Lxylulose, as shown in Fig. 8. The conversion of xylitol to Lxylulose reached 71% after 2 h of reaction time. A volumetric productivity of 10.7 gl-1 h-1 was obtained when the substrate concentration was 30 gl-1. The reusability was also investigated, as shown in the inset of Fig. 8. The

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Immobilization yield (%)

with increases in pH over the investigated pH range. A higher activity was seen for the immobilized ReXDH than for the free enzyme (Fig. 6a), which means the immobilized enzyme has a better resistance to pH changes; it showed the highest activity at pH 10.0. After immobilization, the optimal temperature shifted from 40 °C to 50 °C (Fig. 6b). The activity of immobilized ReXDH improved twofold over the temperature range from 50 °C to 75 °C compared with the free enzyme. The thermostability increased significantly after immobilization (Fig. 7). The t1/2 was 41.8, 39.8, and 7.2 h at 30 °C, 40 °C, and 50 °C, which are 14, 16, and 14 times the values for the free ReXDH, respectively. Even at 60 °C and 70 °C, the t1/2 values of the immobilized ReXDH were still as long as 2.2 and 0.4 h, respectively. When the effects of substrate concentration were investigated, no significant change was noted after immobilization as the Km, xylitol was 19.2 mM for the immobilized enzyme and 17.9 mM for the free enzyme. The catalytic efficiency kcat/ Km was decreased from 0.25 s-1 mM-1 to 0.08 s-1 mM-1, probably due to the limitation in mass transfer after immobilization.

B Immobilization efficiency (%)

Fig. 4 Effect of functional groups of the nanoparticles on the immobilization efficiency of ReXDH. Filled square: modified with GT; filled circle: modified with GA; filled uptriangle: modified with CNBr; filled diamond: modified with CMT; open square: without modification

0 4.0

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10.0

pH

Fig. 5 Effect of pH and temperature on the immobilization of ReXDH nanoparticles derivatives. Filled circles: immobilization yield; open squares: immobilization efficiency

immobilized ReXDH retained a residual activity of 81% after eight batches of conversion.

Discussion L-Xylulose

has received wide attention because it is a worthy target for various applications in industry and in the laboratory. However, the supply is inadequate even for laboratory use because it is very costly in the market. The production from xylitol by catalysis by XDH has proved to be the most effective production method (Takata et al. 2010). Although several XDHs from various organisms have been cloned and expressed (Tiwari et al. 2010; Annaluru et al. 2007; Poonperm et al. 2007; Ko et al. 2006b; Lima et al. 2006), no research regarding immobilization of XDH has yet been reported. Compared with the biotransformations catalyzed by whole cells, the immobilized enzyme catalyzed reactions have many advantages

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A

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Relative acitivity (%)

Relative activity (%)

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10.0

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Fig. 7 Thermal stability of immobilized ReXDH nanoparticles derivatives. Square: 30 °C; circle: 40 °C; uptriangle: 50 °C; reverse uptriangle: 60 °C; diamond: 70 °C

B

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0 30

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Temperature (oC)

Fig. 6 Effect of pH (a) and temperature (b) on the activity of immobilized ReXDH nanoparticles modified with GT. Square: acetate buffer; circle: Tris–glycine–NaOH buffer. Filled and open symbols represent immobilized and free ReXDH, respectively

such as simple purification, no formation of byproducts, no sterilization needed, short reaction time, higher reaction temperature, and easy control. Thus, in the present study, several immobilization methods, including adsorption, ion exchange binding, and covalent immobilization, were tested to investigate the immobilization of recombinant ReXDH on various carriers. Among these carriers, activated silica nanoparticles had the best immobilization efficiency and yield due to the large surface area and stable covalent bond formed during the immobilization. The size of the silica nanoparticles influences the adsorption of enzyme significantly (Wu and Narsimhan 2008; Vertegel et al. 2004). Among the investigated silica nanoparticles, 4830HT with 80 nm particle size was found to be a suitable support for immobilization of ReXDH. The 80-nm particles most likely offer a better integration and

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Relative activity (%)

80

provide more efficient interactions between the ReXDH, substrate, and cofactor. Most of the ReXDH immobilized by adsorption could be easily removed by washing with phosphate buffer, and covalent bonds were more important for the stability of immobilized ReXDH (Fig. 4). Four different functional groups were prepared for the immobilization of ReXDH on silica nanoparticles. A high immobilization efficiency of 92% was obtained on silica nanoparticles containing epoxy groups. The epoxy-silica supports activated by GT can perform an easy immobilization of protein via multipoint covalent attachment. Moreover, epoxy groups are stable enough to perform long-term incubations of immobilized enzymes under alkaline or neutral conditions (Cesar Mateo

Conversion (%)

Relative activity (%)

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cycles 0

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Fig. 8 Production of L-xylulose with immobilized ReXDH. Inset is the reusability of immobilized ReXDH in the batch bioconversion experiments

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et al. 2007). In contrast, immobilization on silica nanoparticles modified by CMT (carbodiimide groups) gave only 43% immobilization efficiency. Enzymes contain several functional groups capable of covalently binding to supports. Of these functional groups, –NH2 and –COOH (Novick and Rozzell 2005) in lysine, arginine, glutamic acid, and aspartic acid residues are the main functional groups for immobilization. There are 15 arginine, 16 lysine, 21 glutamic acid, and 17 aspartic acid residues that could be used for immobilization of the ReXDH. Compared with the other residues, lysine is a better residue for immobilization when stability is considered (Abian et al. 2004; Grazú et al. 2010), and the increased lysine content is likely to increase the immobilization efficiency (Ryan and Ó'Fágáin 2007). Most of the lysine residues are located on the surface of ReXDH, which is favorable for the immobilization (supplementary Fig. 1). Thus, the immobilization efficiencies on GA- (aldehyde groups) and GT-activated (epoxy groups) silica nanoparticles were higher because of the multiple point covalent attachment. For immobilized ReXDH on CNBr derivatives, the stability at acidic or neutral pH is lower than with the other two activated carriers. Similar results were also reported for the immobilization of an alcohol dehydrogenase from horse liver (Bolivar et al. 2006). High immobilization efficiency was achieved at pH 6.0– 8.0, 4 °C (Fig. 5). More than 90% immobilization efficiency was obtained by immobilization of ReXDH on epoxy-silica nanoparticles activated by GT. The recombinant ReXDH was highly thermostable due to the increased formation of hydrogen bonds and salt bridges compared to other XDHs (Tiwari et al. 2010). More than tenfold higher thermostability of this enzyme was obtained by immobilization onto silica nanoparticles. The optimal temperature was also improved from 40 °C to 50 °C and the optimum pH range become broader. These changes probably result from strong enzymesupport interactions, similar to those seen with the immobilization of lipase, glucose oxidase, and other enzymes on mesoporous silica (Blanco et al. 2004; Cui et al. 2010; Huang et al. 2010). The difference in kinetic constants between the free and immobilized enzyme may be a consequence of either the conformational integrity of the immobilized enzyme or lower accessibility of substrate to the active sites of the immobilized enzyme (Bai et al. 2006). XDH can catalyze several reactions including the conversions of xylitol, ribitol, sorbitol, and erythritol to xylulose, ribulose, fructose, and erythrulose, respectively. Consequently, the immobilized XDH with broader pH and better thermostability can be used for production of a wide range of rare sugars. The immobilized ReXDH showed the highest thermal stability among the known XDHs. When the reusability of the immobilized ReXDH was studied with respect to the bioconversion of xylitol into L-xylulose, 81% activity was retained after eight cycles of the reaction. When the substrate

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and NAD+ concentrations were 30 gl-1 and 10 mM, respectively, a volumetric productivity of 10.7 g l-1 h-1 was obtained, which is twofold higher than that reported previously (Usvalampi et al. 2009). These properties of the immobilized ReXDH show its promise in practical application for the industrial production of rare sugars. Acknowledgment This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science and Technology, Republic of Korea. It was also supported by grant (code PJ007449201006) from the Biogreen 21 Program, Rural Development Administration, Republic of Korea.

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