Production of l -theanine using glutaminase encapsulated in carbon-coated mesoporous silica with high pH stability

Biochemical Engineering Journal 68 (2012) 207–214

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Production of l-theanine using glutaminase encapsulated in carbon-coated mesoporous silica with high pH stability Tetsuji Itoh a,∗ , Yasuto Hoshikawa c , Shun-ichi Matsuura a , Junko Mizuguchi a , Hiroyuki Arafune a , Taka-aki Hanaoka a , Fujio Mizukami a , Akari Hayashi b , Hirotomo Nishihara c , Takashi Kyotani c,∗ a

National Institute of Advanced Industrial Science and Technlogy (AIST), Nigatake 4-2-1, Miyagino-ku, Sendai 983-8551, Japan Kyusyu University, International Research Center for Hydrogen Energy & International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), 744 Motooka, Nshi-ku, Fukuoka819-0395, Japan c Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan b

a r t i c l e

i n f o

Article history: Received 23 April 2012 Received in revised form 6 July 2012 Accepted 12 July 2012 Available online 8 August 2012 Keywords: Carbon Mesoporous silica Enzyme immobilization

a b s t r a c t A new enzymatic process for production of l-theanine was presented using glutaminase combined with immobilization technique on carbon-coated mesoporous silica (carbon/SBA-15). For the attempt to enhance the durability against high pH conditions, the pore surface of mesoporous silica SBA-15 was coated with a thin layer of carbon. Carbon coating was done by addition of 2,3-dihydroxynaphthalene (DN) on the pore surface, followed by a dehydration reaction between surface silanol groups in SBA-15 and hydroxyl groups of DN molecules, and further carbonization of DN. A glutaminase was confined in nanospace (about 5 nm) of carbon/SBA-15. The resulting product, glutaminase/carbon/SBA-15, successfully catalyzed the reaction for production of l-theanine under high pH conditions. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In terms of green and sustainable chemistry, utilization of enzymes is considered a suitable alternative to industrial catalysts that fabricate products for chemical, pharmaceutical and food industries. However, there are still issues on their low operational stability and reusability in the industrial processes. Their immobilization on mesoporous silicas (MPS) is an effective technique to improve these problems [1–4]. There have been successful reports on the application of techniques for enzymatic processes [5–18]. We have also reported that the encapsulation of enzymes in mesoporous silica not only enhances the stability of enzymes but also results in new capabilities such as selectivity in reactions [19–25]. In this work, we have focused on production of an amino acid, ␥-glutamylethylamide, particularly l-theanine, which is known as a useful food additive [26]. Currently, l-theanine is industrially produced by a biological process using a microorganism, but this method has a disadvantage such as no reusability of catalysts. For the alternative way of producing l-theanine, Tachiki et al. have reported a ␥-glutamyl transfer reaction, as shown in Scheme 1 [27]. The reaction catalyzed by glutaminase (Enzyme

∗ Corresponding authors. E-mail addresses: [email protected] (T. Itoh), [email protected] (T. Kyotani). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.07.012

Commission (EC) number 3.5.1.2) has an advantage of a higher reusability than microorganisms. Further encapsulation of enzymes into such as MPS is important for successful industrial processes with high stability of enzymes, but there is a drawback to this application because the reaction proceeds under high pH conditions (pH = 10), which causes deterioration of the MPS. Here, we rather have an interest on carbon materials with nanostructure besides MPS. They have been paid attention as enzyme supports for selective organic synthesis since carbon supports have advantages of high surface area, in addition to high thermal, chemical, and mechanical stabilities [28,29]. Among a variety of nanostructured carbons, in particular, ordered mesoporous carbon (OMC) is highly interesting because of tunable pore sizes and pore structures using appropriate silica materials as hard templates [30–32]. The most common synthetic route for OMC involves preparation of ordered mesoporous silica (OMS) templates, impregnation of the OMS pores with carbon precursors, carbonization, and removal of templates. In this process, an extra step is necessary to prepare a silica template before the multisteps of syntheses. The obtained materials result in ordered arrays of carbon nanorods with reverse structures to the parent mesoporous structure of silica. We believe that size control of mesopore’s wall thickness is much harder than that of mesopores themselves in OMS templates, so that means, controlling of pore size of classic OMC is not easy. Therefore, we have attempted to improve the applicability of mesoporous silica surface with coating of an extremely thin

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T. Itoh et al. / Biochemical Engineering Journal 68 (2012) 207–214

O

H2N

O

EtHN

EtNH2

pH 10

NH3

Glutaminase H2N

CO2H

L-glutamine

H2N

CO2H

L-theanine

Scheme 1. ␥-Glutamyl transfer reaction of l-glutamine by glutaminase.

carbon layer, comprising only 1–2 graphene sheets [33]. In this case, the template removal process is not needed, which is very advantageous from an economical point of view, and it is also easy to control the pore size by just changing kinds of surfactants (CTAB, P123, F127, etc.). In the present study, we report the preparation of carbon coated MPS (SBA-15), which is stable under high pH conditions, and encapsulation of glutaminase into such nanospace. Accordingly, we describe successful production of l-theanine using glutaminase encaged in the nanopores of carbon coated SBA-15 under high pH. 2. Materials and methods 2.1. Materials Glutaminase was isolated from Pseudomonas nitroreducens IFO 12694 by a previously described way [34]. Mesoporous silica, SBA15, was synthesized according to a method reported formerly [35,36]. Carbon coated SBA-15 was prepared the synthetic procedure reported by Nishihara et al. as follows [33]. The 4.0 g of 2,3-dihydroxynaphthalene (DN) was dissolved in 4.0 g (5.1 ml) of acetone, and the resulting solution was poured into 1.0 g of dried SBA-15 in vacuo. The mixture was stirred for 1 h at room temperature, followed by the evaporation of acetone at 368 K for 12 h under N2 flow. The resulting solid mixture of DN and SBA-15 was heat-treated at 573 K for 1 h under N2 flow. During this step, liquid state DN was allowed to react with SBA-15 through a dehydration reaction between silanol groups on the pore surface of SBA-15 and hydroxyl groups of DN [37,38]. The unreacted DN was then washed away many times (ca. 20 times) with an excess amount of acetone to obtain DN-coated SBA-15 (DN/SBA-15). Finally, the obtained DN/SBA-15 was heat-treated under N2 flow at 1073 K for 4 h to carbonize DN in the silica pores. The obtained carbon-coated SBA-15 is referred to as carbon/SBA-15 hereafter. 2.2. Characterization Powder X-ray diffraction (XRD) data were obtained using an M21X diffractometer (MAC Science Co. Ltd., Japan) with a curved graphite monochromator (Cu K␣ radiation) operated at 45 kV and 250 mA. The ordered mesoporous structures were directly observed by using a Hitachi S-5200 high-resolution field emission scanning electron microscope (FE-SEM) and scanning transmission electron microscopy (STEM) at acceleration voltage of 1.0–30.0 kV without any metal deposition treatment for the reduction of charge build-up. DTA-TG curves were collected on a Thermo plus EVO TG8120 thermal analyzer (Rigaku Co. Ltd., Japan) at a heating rate of 10 K/min in air (100 ml/min). Brunauer–Emmett–Teller (BET) surface areas for samples were derived from nitrogen adsorption and desorption measurements at 77 K with samples degassed at 423 K below 10−3 mmHg for 4 h using a Belsorp-MAX (Japan BEL Co. Ltd., Japan). The pore diameters were estimated using the Barrett–Joyner–Halenda (BJH) method. The amount of the products were evaluated by a high pressure liquid chromatography (HPLC) apparatus (Alliance HPLC system, Waters Co. Ltd., Japan) with a UV detector.

2.3. Stability of SBA-15 and carbon/SBA-15 under high pH conditions The stability of carbon/SBA-15 was examined by stirring the carbon/SBA-15 or SBA in varied pH solutions for 7 days. The experiments were conducted with 50 mg of carbon/SBA-15 in 5 ml of 50 mM Tris–HCl buffer (pH 8.5, 9.5, and10.5). 2.4. Labeling glutaminase with amine-reactive fluorescent dyes To visualize individual glutaminase encapsulated in mesoporous materials using a fluorescence microscope, the glutaminase was chemically modified with amine-reactive fluorescent dyes, Alexa Fluor 488 (Abs/Em = 495 nm/519 nm; molecular probes), at some positions on lysine residues. Fluorescent labeling is known to damage enzymes, sometimes seriously, because of binding of dyes to the active center of glutaminase. To avoid this problem, glutaminase was modified with a minimum degree of labeling: approximately one dye molecule was bound to one enzyme molecule. The labeling reaction was carried out by combining 10 mg of enzyme powder with 1 ml of 20 mM MES buffer (pH 6) containing an appropriate amount of fluorescent dye. The sample solution was gently shaken using a rotator for 1 h at 4 ◦ C in the dark. The labeled glutaminase was purified with gel filtration using a NAP-5 Column (GE Healthcare) equilibrated with a 20 mM MES buffer (pH 6). The labeled glutaminase concentration was determined using the Bradford method with bovine serum albumin as a standard. Fluorescence intensities were measured, with and without mesoporous materials, using a fluorescence spectrophotometer (F-4500; Hitachi). 2.5. Encapsulation of glutaminase and labeled glutaminase in SBA-15 and carbon/SBA-15 Encapsulated glutaminase in SBA-15(Glu/SBA-15) and carbon/SBA-15 (Glu/carbon/SBA-15) was prepared as follows. Carbon/SBA-15 powder (100 mg) was stirred with glutaminase (0–60 mg) in 5 ml of ethylamine/ethylamine hydrochloride aqueous solution (200 mM, pH = 10) at 277 K overnight. For comparison, the reaction was also performed using SBA-15 or carbon/SBA-15. The amounts of glutaminase in solutions before and after the immobilization were evaluated by the bicinchoninic acid (BCA) method [39]. We prepared a labeled-glutaminase/SBA-15 or carbon/SBA-15 in MES buffer (20 mM, pH = 6.0) with the similar manner to that described for preparing Glu/SBA-15 or carbon/SBA15. We tested whether leaching of enzymes occurred or not, by washing the materials with pH 6 buffer solution several times. As a result, we did not observe any leaching. 2.6. Microscopic observation by epi-fluorescence and differential interference contrast The labeled Glu/carbon/SBA-15 (1.0 mg: labeled enzyme 0.028 mg) and labeled Glu/SBA-15 (1.0 mg: glutaminase 0.01 mg) were observed using an inverted fluorescence microscope (Eclipse TE2000-U; Nikon) equipped with a 100×, 1.49 numerical aperture (NA) oil-immersion objective lens. The excitation and emitted light were selected with filter sets GFP-BP (suitable for Alexa 488: EX480/40, DM505, BA535/50) produced by Nikon. Fluorescent images of the individually labeled-enzyme, marked with fluorescent dyes, were made visible using a high-sensitivity EMCCD camera (ImagEM; C-9100-13, Hamamatsu Photonics) and recorded with an image processor (AQUACOSMOS analysis software; Hamamatsu Photonics). Simultaneously, the corresponding fluorescence spectra of the labeled glutaminase over an area of 10 ␮m diameter on the fluorescence images were obtained with a

T. Itoh et al. / Biochemical Engineering Journal 68 (2012) 207–214

microspectroscope. Mesoporous materials, used as the capsule for the enzyme, were independently observed, by differential interference contrast (DIC) using the above instruments. Combining Rayleigh’s formulas [22,40,41] for the lateral resolution x (1) and for the depth of focus z (2) we get:  NA

(1)

n (NA)2 2

(2)

z =

The lithographic factor k has the standard or conventional metric for estimating the difficulty of lithographic processes in semiconductor manufacturing, where  is the wavelength of illumination, NA is the numerical aperture of the optical system, and n is the index of refraction. The numerical aperture is defined as NA = n sin ˛, where ˛ is the angle, along with resolution (x) and depth of focus, DOF(z). 2.7. Production of l-theanine Glu/carbon/SBA-15 (2 mg: glutaminase 0.5 mg), Glu/SBA-15 (5 mg: glutaminase 0.52 mg) or native Glu (0.52 mg) were suspended with 29.2 mg (20 mmol) of l-glutamine in 10 ml of ethylamine/ethylamine hydrochloride aqueous solution (200 mM, pH = 10). The solution was incubated at 298 K. At a given time, an aliquot was taken out and was added to perchloric acid to stop the reaction. This resulting sample was injected to a chromatography in water/methanol/trifluoroacetic acid (980:20:1) at room temperature. 3. Results and discussion 3.1. Characteristics of carbon/SBA-15 Fig. 1 shows the small-angle XRD pattern of SBA-15 (a) and carbon/SBA-15 (b). SBA-15 shows three well-resolved peaks (Fig. 1a) that are indexable as (1 0 0), (1 1 0), and (2 0 0) reflections associated with two-dimensional (2D) hexagonal symmetry. The intense (1 0 0) peak reflects a d spacing of 9.9 nm. On the other

(100)

(200) (110)

hand, the XRD pattern (Fig. 1b) of carbon/SBA-15, obtained by heat-treatment of DN/SBA-15 under N2 flow at 1073 K for 4 h to carbonize DN in the silica pores, shows that the two-dimensional (2D) morphology is preserved although the peaks appear at slightly larger 2 values, with d(1 0 0) = 9.6. The reason why the relative intensity of peak decreased has not been clarified yet. It is probably due to the slightly reduced order of homogeneity of the pores with carbon coating. Others have also reported similar phenomena that after only modified surface of SBA-15, the relative intensity of peak (1 0 0) is decreased [42,43]. Four XRD peaks are still observed, confirming that hexagonal carbon/SBA-15 is thermally stable. A similarly high degree of mesoscopic order (four XRD peaks) is observed for hexagonal carbon/SBA-15 [35]. However, it should be noted that all the three peaks in SBA-15 are shifted to a higher angle in carbon/SBA-15. This shift is due to the thermal shrinkage during the heat-treatment (1073 K, 4 h) in the carbonization step. Fig. 2 shows the TG/DTA curves of carbon/SBA-15 under air. The curve shows a large weight loss between 770 and 900 K, owing to the loss of coated carbon. The corresponding DTA curves show an exothermic peak around 860 K (Fig. 2, dashed line). In this case, the total carbon content results in ca. 22 wt.%. Fig. 3 shows the electron microphotographs (SEM and STEM) of the SBA-15 and carbon/SBA-15 samples. It is observed from Fig. 3a and b that there is no morphological change in a silica particle even after the carbon coating. The high-magnification images of the particles observed in Fig. 3a and b are shown in each inset, and the ordered mesoporous structure confirmed by the XRD pattern (Fig. 1, curve B) is also observed in both samples. Although the carbon/SBA-15 contains an amount of carbon as high as 22 wt.%, we did not observe any carbon deposition on the external surface of the ordered pores. With further observation on STEM (Fig. 3c and d), the nanochannel structure was determined as 2D-hexagonal structure. Also, we found the pore size of SBA-15 and carbon/SBA-15 was 7.0 nm and 5.0 nm, respectively (Fig. 3c and d inset). The N2 adsorption–desorption isotherms for SBA-15 and carbon/SBA-15 are shown in Fig. 4a, where clear hysteresis loops are observed in the pressure range of 0.4–0.8, indicating the development of mesoporosity in the samples. The BET surface areas and total pore volumes were determined from the isotherms. As expected, the amount of N2 adsorption decreased when carbon was loaded on the pristine SBA-15. However, the loaded samples (carbon/SBA-15) still retained considerable mesoporosity. The mesopore size distribution curves were calculated by using the BJH method (Fig. 4a (inset)). The distribution was shifted to the smaller pore size owing to carbon coating on the surface of pores, but sharpness of the distribution remained almost intact. In other words, despite the decrease in the pore size, the size stays uniform. Thus, it is expected that the carbon layer completely covers the silica surface, and nature of mesoporous silica changes to carbon-like. For estimating the thickness of such a

(210) X 10

(100)

B Weigtloss / wt%

(110)

(200)

1

80 90

60 40

80

20 70

X5

0

100

100

DTA / μ V mg-1

x = k ×

209

0

A 2

3

4

5

(2θ) Fig. 1. XRD patterns of SBA-15 (a) and carbon/SBA-15 (b).

200

400

600

800

1000

Temperature / K Fig. 2. TG/DTA curves of carbon/SBA-15. TG curve: solid line and DTA curve: dashed line.

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Fig. 3. Typical SEM images: SBA-15 (a) and carbon/SBA-15 (b). Typical STEM image: SBA-15 (c) and carbon/SBA-15 (d).

500 400 300

300 250 200 150 100 50 0 0

C A

D

5 10 15 Pore size (nm)

B 200 100 0

0

0.2

0.4

0.6

P/P0

0.8

amounts of mesopores in this sample. It is thus very likely that the pore surface of SBA-15 is coated with the carbon layer. 3.2. Stability of carbon/SBA-15 under high pH conditions In order to evaluate the effects of carbon coating against high pH, we compared the porosities of carbon/SBA-15 and SBA-15 before and after stirring in varied pH solutions for 7 days. Fig. 5 presents the pore size distributions of the materials, derived from the N2 adsorption isotherms, before and after stirring in varied pH solutions at 298 K for 7 days. In the case of SBA-15, the pore size was shifted from 7 nm to 8 nm under high pH condition (Fig. 5a and Table 1). Furthermore, the peak at 7 nm decreased under pH 10.5, followed by appearance of the browed peak toward larger pores. This indicates that the micropores are destroyed and pore wells of SBA-15 are dissolved followed by enlargement of pores under the high pH condition. On the other hand, the main peak (5 nm) of carbon/SBA-15 little changed before and after (Fig. 5b) although the peak of micropores appeared. As the micropores appeared, the

Adsorbed amount/cm3 g-1 (STP)

600 dv/dr

Adsorbed amount/cm3g-1 (STP)

carbon layer, we roughly calculated by subtracting the actual mesopore size (ca. 5.0 nm) from the silica pore size (ca. 7.0 nm), which is about 1 nm and close to the thickness of 1–2 graphene sheets. The mesopore size, which was determined from the peaks of the distribution curves, was in good agreement with the corresponding sizes observed in the FE-SEM and STEM images (Fig. 3). If such a carbon coating is achieved, carbon/SBA-15 should show strong hydrophobic properties. Next, we examined the completeness of the carbon coating by a water-vapor adsorption experiment. Fig. 4b shows the water-vapor adsorption isotherms. The isotherms of SBA-15 and carbon/SBA-15 are characterized by rapid uptake at a relative pressure of 0.78 and 0.8, respectively, owing to the capillary filling of water into the mesopores. In the case of SBA-15 (pressure of 0.78), the adsorbed water fills ca. 80% of the pore volume. On the other hand, when a relative pressure is 0.8, the adsorbed water fills only ca. 17% of the pore volume of carbon/SBA15. Thus, carbon/SBA-15 rarely adsorbs water vapor, revealing its hydrophobic characteristic, although the SEM observations and the N2 adsorption experiments clearly indicate the presence of large

1.0

800

600

400

A

200

B

0 0.2

0.4

0.6

0.8

P/P0

Fig. 4. (a) N2 adsorption–desorption isotherms of SBA-15 (curve A) and carbon/SBA-15 (curve B) measured at 77 K. Inset shows a pore size distribution plot of SBA-15 (curve C) and carbon/SBA-15 (curve D) calculated by BJH. (b) H2 O adsorption isotherms of SBA-15 (curve A) and carbon/SBA-15 (curve B) at 298 K.

T. Itoh et al. / Biochemical Engineering Journal 68 (2012) 207–214

b

SBA-15

350

300 Carbon-SBA-15

SBA-15

250

250

pH 8.5 pH 9.5

200

dv/drp

dv/drp

Carbon/SBA-15

pH 10.5

150

50

50 10

15

20

25

pH 9.5 pH 10.5

150 100

5

pH 8.5

200

100

0

Amount of glutaminase adsorbed materials (mg/m2)

a

0

5

10

15

Pore size (nm)

Pore size (nm)

surface area and pore volume of carbon/SBA-15 increased. The reason for increased micropores under high pH has not been clarified yet, but it is probably due to dissolution of partly non-carbon coated silica at the high pH condition. The deterioration could be caused by the high pH conditions as there is a considerable increase in the solubility of amorphous silica from pH 9 to pH 10.7 [44]. Consequently, the stability of SBA-15 toward high pH has successfully increased by coating it with carbon. 3.3. Encapsulation of glutaminase into the pores of carbon/SBA-15 (Glu/carbon/SBA-15) For the investigation on a new enzymatic process with carbon/SBA-15, glutaminase (1 unit; 5–6 nm) [45], which catalyze production of l-theanine in high pH condition, was chosen. The adsorption amounts of glutaminase into carbon/SBA-15 powder in the solution (pH 10) were measured with respect to the equilibrium concentration of glutaminase (Fig. 6). The adsorptions of glutaminase into the carbon/SBA-15 (pore: ca. 5.0 nm) and SBA15 (pore: ca. 7.0 nm) are shown by A and B, respectively. When the pore surface of mesoporous silica SBA-15 was coated by carbon, the adsorption proceeded efficiently with an increase in the equilibrium concentration of glutaminase (curve A). The 24.3 mg (0.8 mg/m2 ) out of 60 mg of glutaminase were adsorbed onto carbon/SBA-15 (100 mg) in ethylamine/ethylamine hydrochloride aqueous solution (200 mM, pH = 10). On the other hand, the amount of glutaminase adsorbed onto SBA-15 was smaller than that of carbon/SBA-15 (curve B), even though its pore size was larger than that of carbon/SBA-15. Pore volumes and BET surface areas were then examined by the nitrogen adsorption isotherm of glutaminase into carbon/SBA-15 (Table 2 and Fig. S1). With an increase in the amount of glutaminase (0, 7 and 16 mg) adsorbed into carbon/SBA-15 (100 mg), both the pore volume and specific surface Table 1 Physicochemical properties of materials against high pH.a Materials

a

pH

Pore diameter (nm)

Specific surface area (m2 g−1 )

Pore volume (cm3 g−1 )

SBA-15

– 8.5 9.5 10.5

7 8 8 8

826 451 480 476

0.80 0.80 1.10 1.13

Carbon/SBA-15

– 8.5 9.5 10.5

5 5 5 5

304 427 491 734

0.36 0.45 0.53 0.69

Specific surface areas were calculated by the BET method using adsorption data and the pore size distributions were determined by analyzing the adsorption branch by the BJH method.

0.8

A 0.6

0.4

B 0.2

0

Fig. 5. Stability of SBA-15 and carbon/SBA-15 against different pH. Symbols show the curves of original (䊉), at pH 8.5 (), at pH 9.5 (), and at pH 10.5 (), respectively.

211

2

4

6

8

Equilibrium concentration of glutaminase (mg/ml) Fig. 6. Equilibrium concentrations of glutaminase and Glu/SBA-15 or Glu/carbon/SBA-15 against adsorption of glutaminase into carbon/SBA-15 (curve A) and SBA-15 (curve B) in ethylamine/ethylamine hydrochloride aqueous solution (200 mM, pH = 10) at 277 K. Adsorption was measured by the bicinchoninic acid (BCA) method.

area decreased (compare A–C in Table 1 and Fig. S1a), indicating that glutaminase was smoothly introduced into the pores of carbon/SBA-15 (Fig. S1b). In the case of C (Table 2 and Fig. S1), the pore volume of Glu/carbon/SBA-15 became only ca. 36% of initial pore volume of carbon/SBA-15 (Fig. 7). 3.4. Direct observation of the enzyme encapsulated in mesoporous materials Using Alexa 488 dye-labeled glutaminase enables us to directly observe the encapsulation of glutaminase in the pores of SBA-15 and carbon/SBA-15. Resolution and depth of focus (DOF) of the fluorescence microscope was 174 nm and 117 nm, respectively. The resolution of the fluorescence microscope was not sufficient to distinguish each enzyme. It could, however, directly visualize the enzyme adsorbed into the mesoporous silica particle because the DOF is smaller than the silica particle. Fig. 8a and b are DIC images of the conjugate particles (10–100 ␮m in length) for dye-labeled Glu/SBA-15 and Glu/carbon/SBA-15, respectively. Fig. 8c and d are the corresponding fluorescent images of these same particles observed in the DIC, dye-labeled Glu/SBA-15 and Glu/carbon/SBA15, respectively, The fluorescent image and fluorescent spectrum of the dye-labeled Glu/SBA-15 have shown green-colored particles having the same size and morphology as the particles observed in the DIC ones (Fig. 8c and e), because the Alexa 488 dye is green fluorescence dye. On the other hand, the fluorescent image and fluorescent spectrum of the dye-labeled Glu/carbon/SBA-15 does not show the green emission (Fig. 8d and f), indicating that glutaminase molecules are adsorbed specifically in pore of the carbon/SBA-15 particles. Only when the labeled enzyme is adsorbed mainly on Table 2 Physicochemical properties of the carbon/SBA-15 conjugate.a Adsorbed protein (mg/100 mg carbon-SBA-15)

Pore volume (cm3 g−1 )

Specific surface area (m2 g−1 )

A B C

0.36 0.22 0.13

304 171 85

0.80

826

SBA-15 a

0 7 16

Specific surface areas were calculated by the BET method using adsorption data and the pore size distributions were determined by analyzing the adsorption branch by the BJH method.

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Fig. 7. Direct observation of Glu/SBA-15 and Glu/carbon/SBA-15: DIC images of mesoporous materials (a and b), corresponding fluorescence images of Alexa 488-labeled glutaminase (c and d) and corresponding fluorescence spectra of area with 10 ␮m diameter (white circle) on fluorescence images (e and f) are for Glu/SBA-15 and Glu/carbon/SBA-15, respectively.

synthesis during the industrial process. On the other hand, in the case of Glu/carbon/SBA-15 (curve C), although the production of l-theanine slowly progressed over time, l-theanine was not lost by enzymatic hydrolysis. The amount of l-theanine increased over the incubation time. After 48 h of incubation, the amount of ltheanine was 1400 ppm. The conversion rates from l-glutamine to l-theanine using Glu/carbon/SBA-15 were approximately 50%. This result strongly suggests that carbon/SBA-15 properly functioned as a support even in high pH. In 1998s, Tachiki et al. [27] have reported a ␥-glutamyl transfer reaction as an alternative process. However, the method using a microorganism requiring plant for biological waste disposal has still been used in current industrial processes. That is because we have three important subjects to overcome in order

1600

Fig. 8 presents production of l-theanine from glutamine and ␥-glutamyl, through ␥-glutamyl transfer reaction (Scheme 1), using Glu/carbon/SBA-15, Glu/SBA-15 or native (Glu) in ethylamine/ethylamine hydrochloride aqueous solution (200 mM, pH = 10). The amount of l-theanine produced using native (curve A) or Glu/SBA-15 (curve B) increased with the incubation time at 298 K. After 2–4 h of incubation, the amount of l-theanine formed by native and Glu/SBA-15 were 1420 and 1100 ppm, respectively. The conversion rates from l-glutamine to l-theanine using native and Glu/SBA-15 were approximately 50% and 40%, respectively. However, the amount of l-theanine reversed the upward trend and became 90 ppm after 144 h in both cases. Decrease of product in later incubation period with Glu/SBA-15 or native might be due to its enzymatic hydrolysis. This is the one of the reasons for why native enzymes have been neglected as catalysts in organic

L-theanine (ppm)

3.5. Production of l-theanine using Glu/carbon/SBA-15

C

1400 1200

40

1000

30

800 600 400 200 0 0

50

B A

20 10

Conversion (%)

surfaces of the particles of carbon/SBA-15, fluorescent enhancement would be obtained from the particles, because carbon/SBA-15 cannot transmit light. These results indicate that the pores of carbon/SBA-15 are occupied by glutaminase. Moreover, despite the slightly larger size of glutaminase (1 unit; 5–6 nm) than the pore size (ca. 5 nm) of carbon/SBA-15, glutaminase probably makes slight change in the shape and fits into the pores of carbon/SBA-15. In the previous study, we have indicated that pores of materials, which are smaller than the protein size, can encapsulate the protein owing to high affinity relationship between proteins and surface of materials [22]. Hydrophobic carbon also attracts the hydrophobic portion of glutamine. Similar effect was reported by Vijayaraj et al. [46]. We assume the glutaminase has changed somewhat to be slimmer near the carbon/SBA-15 pore entrance due to its structural flexibility, and thus entered the pores.

0 20 40 60 80 100 120 140 160

Incubation time (h) Fig. 8. Synthesis of l-theanine and the conversion rates for native (Glu) (A), Glu/SBA-15 (B), and Glu/carbon/SBA-15 (C). (A) Native Glu (0.52 mg), (B) Glu/SBA15 (5 mg: glutaminase 0.52 mg) or (C) Glu/carbon/SBA-15 (2 mg: glutaminase 0.5 mg) were suspended with 29.2 mg (20 mmol) of l-glutamine in 10 ml of ethylamine/ethylamine hydrochloride aqueous solution (200 mM, pH = 10) at 298 K.

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to use glutaminase for the industrial theanine synthesis. Important points are the separation of enzymes and synthetic substance, temperature control, and stability in high pH. Regarding to the separation of enzymes and synthetic substance, without separation further hydrolysis process results in decomposition of theanine, and so the process is important but costly. Theanine should be synthesized at 277 K to suppress hydrolysis process [27], which leads to the decrease in reaction rate and the increase in cost for temperature control. Low stability of support against high pH is estimated to cause leaching of enzymes and further decrease in production of theanine through hydrolysis process. Here, our technique of enzyme fixation on carbon coated pore surface of MPS has an advantage on the separation of enzymes and synthetic substance. Furthermore, l-theanine is successfully produced without decomposition even at 298 K, and carbon/SBA-15 is very stable under high pH condition. The cost spent for the separation of enzymes and synthetic substance or temperature control can be mostly reduced in our system. Thus, these results certainly suggest the industrial application of MPS as an enzyme support with effective l-theanine production. 4. Conclusions We successfully synthesized carbon coated SBA-15, introduced glutamine to carbon/SBA-15, and showed successful catalytic activity of Glu/carbon/SBA-15 for l-theanine synthesis. The technique of carbon coating on the pore surface of MSP opens up the industrial application of MPS as an enzyme support, since fixation of enzymes on carbon/SBA-15 has an advantage on the separation of enzymes and synthetic substance and the stability in high pH. Our method has a possibility of applying to an industrial process for l-theanine production and moreover to various chemical processes especially with enzymes. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2012.07.012. References [1] S. Hudson, J. Cooney, E. Magner, Proteins in mesoporous silicates, Angew. Chem. Int. Ed. 47 (2008) 8582–8594. [2] M. Hartmann, Ordered mesoporous materials for bioadsorption and biocatalysis, Chem. Mater. 17 (2005) 4577–4593. [3] E. Ruiz-Hitzky, M. Darder, P. Aranda, K. Ariga, Advances in biomimetic and nanostructured biohybrid materials, Adv. Mater. 22 (2010) 323–336. [4] E. Ruiz-Hitzky, P. Aranda, M. Darder, M. Ogawa, Hybrid and biohybrid silicate based materials: molecular vs. block-assembling bottom-up processes, Chem. Soc. Rev. 40 (2011) 801–828. [5] M. Hartmann, D. Jung, Biocatalysis with enzymes immobilized on mesoporous hosts: the status quo and future trends, J. Mater. Chem. 20 (2010) 844–857. [6] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Catalytic activity in organic solvents and stability of immobilized enzymes depend on the pore size and surface characteristics of mesoporous silica, Chem. Mater. 12 (2000) 3301–3305. [7] J. Deere, E. Magner, J.G. Wall, B.K. Hodnett, Adsorption and activity of cytochrome c on mesoporous silicates, Chem. Commun. 5 (2001) 465–466. [8] J. Deere, E. Magner, J.G. Wall, B.K. Hodnett, Adsorption and activity of proteins onto mesoporous silica, Catal. Lett. 85 (2003) 19–23. [9] A. Vinu, V. Murugesan, O. Tangermann, M. Hartmann, Adsorption of cytochrome c on mesoporous molecular sieves: influence of pH, pore diameter, and aluminum incorporation, Chem. Mater. 16 (2004) 3056–3065. [10] J. Fan, J. Lei, L. Wang, C. Yu, B. Tu, D. Zhao, Rapid and high-capacity immobilization of enzymes based on mesoporous silicas with controlled morphologies, Chem. Commun. 17 (2003) 2140–2141. [11] Y. Wang, F. Caruso, Mesoporous silica spheres as supports for enzyme immobilization and encapsulation, Chem. Mater. 17 (2005) 953–961. [12] Y. Wang, F. Caruso, Macroporous zeolitic membrane bioreactors, Adv. Funct. Mater. 14 (2004) 1012–1018.

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