Highly efficient covalent immobilization of catalase on titanate nanotubes

Biochemical Engineering Journal 83 (2014) 8–15

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Regular Article

Highly efficient covalent immobilization of catalase on titanate nanotubes Qinghong Ai a , Dong Yang a,c , Yuanbing Li a , Jiafu Shi b,c , Xiaoli Wang b , Zhongyi Jiang b,c,∗ a Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 30072, China b Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China c Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China

a r t i c l e

i n f o

Article history: Received 28 August 2013 Received in revised form 12 November 2013 Accepted 28 November 2013 Available online 12 December 2013 Keywords: Enzymes Immobilization Titanate nanotubes Chelation Enzyme technology Biocatalysis

a b s t r a c t In this study, titanate nanotubes (TNTs) with desirable biocompatibility and hydrophilicity have been synthesized by a facile and cost-effective alkaline hydrothermal method, and used to immobilize the enzyme. The characterization results reveal that the prepared TNTs have a regular tubular morphology with a length about 100–180 nm and an outer diameter about 10 nm, and a BET specific surface area of 305.4 m2 g−1 . Catalase (CAT), as the model enzyme, was pre-modified by 3-(3,4-dihydroxyphenyl) propionic acid (3,4-diHPP) via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry, and then covalently immobilized on the TNTs surface by the chelation of catechol groups with Ti4+ ions. It is found that TNTs exhibits excellent performances as the immobilized supporter of enzyme: the enzyme loading is as high as 820 mg g of support−1 ; the relative activity of immobilized enzyme is about 60% of that of free enzyme; the immobilized CAT demonstrates enhanced storage and recycling stability. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Nanobiocatalysis, which refers to the incorporation of enzymes into nanostructured materials, has attracted much attention based on the nanotechnology progress in recent years [1–5]. Nanostructured materials have been recognized excellent enzyme scaffolds, because they offer the special and fascinating characteristics for balancing the key factors that determine the biocatalyst efficiency, including high specific surface area, minimized mass transfer resistance, and effective enzyme loading over their bulk counterparts [1,4]. Till now, various nanostructures, such as nanoparticles [6], nanofibers [7], nanotubes [8], nanoporous media [9], and graphene [10] have been explored as supporters for enzyme immobilization, demonstrating superior and unique application potentials. Among all of the nanostructured materials, much effort has been dedicated to the research and development of nanotubes, especially carbon nanotubes (CNTs), as the enzyme immobilization supporter due to their exceptional properties, for instance, large aspect

∗ Corresponding author at: Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 30072, China. Tel.: +86 22 2740 6642; fax: +86 22 2740 6642. E-mail address: [email protected] (Z. Jiang). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.11.021

ratio, extraordinary mechanical and thermal properties [11–14]. Wang et al. [15] immobilized NADH oxidase on the CNTs surface based on the specific interaction between His-tagged NADH oxidase and functionalized single-walled CNTs, which demonstrated high enzyme loading capacity and stabilities. Zhang et al. [16] investigated the interactions between adsorbed catalase and CNTs with different morphologies and surface functionalities, indicating that the enzyme activity was significantly influenced by the unique curvature of the nanomaterials. However, unmodified CNTs incline to exhibit high hydrophobicity and severe aggregation, and thus difficult to be compatible with organic or biological materials. One of the most popular approaches for functionalizing CNTs involves the introduction of carboxylic groups onto their surfaces via the strong acid treatment, nevertheless the chemical inertness of CNTs dramatically hampers their potential applications [17]. In comparison, titanate nanotubes (TNTs) which possess considerable specific surface area, demonstrate attractive biocompatibility, hydrophobicity and special chelation interactions with catechol groups [18,19]. Although TNTs have been recently applied in the bio-sensing [20–22], there are few reports concerning their utilization in enzyme immobilization. Furthermore, the functional hydroxyl groups on the TNTs surface are expected to provide an aqueous-like environment, and stabilize the structure of immobilized enzymes.

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In this study, TNTs were employed as the supporter for pursuing the efficient enzyme immobilization. TNTs were synthesized by a facile and cost-effective alkaline hydrothermal method, and utilized for the covalent immobilization of enzyme for the first time. TEM, XRD, XPS, Raman, BET and FTIR analysis methods were used to determine the morphology and chemical characteristics of the as-prepared TNTs. Catalase (CAT) as the model enzyme was modified by 3-(3,4-dihydroxyphenyl) propionic acid (3,4-diHPP) by EDC/NHS method, and then immobilized on the TNTs surface through the chelation of catechol groups with Ti4+ ions. The enzyme loading efficiency and corresponding catalytic activity were measured, and the thermal, storage, and recycling capability of immobilized CAT were also experimented. 2. Materials and methods 2.1. Materials Catalases (EC 1.11.1.6 from bovine liver; 2.48 × 104 U mg of protein−1 ); 2-(N-morpholino) ethanesulfonic acid sodium salt (MES) and tris (hydroxymethyl) aminomethane (Tris) were purchased from Sigma–Aldrich Chemical Co. Ltd. 3(3,4-Dihydroxyphenyl) propionic acid (98+%) was purchased from Alfa Aesar. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Medpep Co. Ltd. Hydrogen peroxide (30.0%) was purchased from Guangfu Co. Ltd. (Tianjin, China). Sodium hydroxide was purchased from Jiangtian Co. Ltd. (Tianjin, China). Rutile TiO2 powder (99.8%, 60 nm) was purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). Other chemicals were of analytical grade. The water used in all experiments was prepared in a Millipore Milli-Q purification system. 2.2. TNTs synthesis Regular titanate nanotubes were prepared by a hydrothermal method described by Geng et al. [23]. In a typical procedure, nanosized rutile TiO2 powders (2 g) were firstly dispersed in 85 mL NaOH solution (10 mol L−1 ). Then, the suspension was transferred into a sealed Telfon-lined container, and statically heated for 72 h at 130 ◦ C. The white precipitate was obtained after centrifuged, washed with excess deionized water, and soaked in abundant HCl (0.1 mol L−1 ) for 10 h, followed by washing with deionized water until pH 7.0. Finally, alcohol was used to disperse the white precipitate in order to displace water on the TNTs surface and enable the formation of aggregation-free nanotubes. 2.3. Covalent immobilization of CAT on the TNTs surface As shown in Scheme 1, CAT was modified by grafting 3,4-diHPP coupled with EDC and NHS as activators, and then immobilized on the TNTs surface based on the chelation interaction between Ti4+ and catechol group of 3,4-diHPP. Briefly, 3,4-diHPP (20 mg), NHS (36 mg) and EDC (60 mg) were added orderly in an MES solution (pH 6.5, 15 mL) under continuous magnetic stirring. Then, a CAT solution was added in the above mixed solution, reacting for 4 h at 4 ◦ C. Through the dehydration–condensation reaction between COOH and NH2 radicals, 3,4-diHPP was grafted on the CAT molecules. Subsequently, 50 mg TNTs were dispersed in 5 mL MES buffer solution (50 mmol L−1 , pH 6.5) under ultrasonic treatment for 15 min, and then mixed with the above modified CAT solution for 30 min under stirring vigorously. Finally, CAT immobilized on the TNTs surface (TNTs-CAT) was obtained after centrifugation and thoroughly washing with deionized water for removing free CAT adsorbed physically on the TNTs surface.

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2.4. Characterization The morphology of the prepared TNTs was performed on a transmission electron microscopy (TEM) (IEM-100CX II) instrument. X-ray diffraction (XRD) pattern was obtained with a Rigaku D/max 2500 v/PC X-ray diffractometer in the range of 5–40◦ at the speed of 5◦ min−1 (Cu K␣, 40 kV, 200 mA). The Raman spectrum of TNTs was recorded by a Bruker FS100 FT-Raman spectrometer with a liquid N2 -cooled super InGaAs detector. The spectrum was excited with a diode pumped YAG laser (532 nm) with a power of 100 MW. The specific surface area and pore volume were recorded by nitrogen absorption–desorption isotherm measurements performed on a Micromeritics Tristar 3000 gas adsorption analyzer. The pore size distribution was determined by the nitrogen isotherms based on the Barret–Joyner–Halenda (BJH) method. The element-mapping analysis was carried out by an energy dispersive X-ray spectroscope (EDS), which was directly connected to the TEM. X-ray photoelectron spectroscopy (XPS) was conducted to examine surface elemental composition of TNTs. FTIR spectra were acquired on a Nicolet560 spectrometer. UV–vis spectra were acquired using a UV spectrophotometer (Hitachi U3010). Thermogravimetric analysis (TGA) was recorded on a Perkin–Elmer thermogravimetric analyzer. 2.5. Enzyme activity The catalytic activity of CAT was determined spectrophotometrically at 240 nm by using H2 O2 as the substrate. Briefly, 0.1 mL of free CAT or as-prepared TNTs-CAT (0.1 mg mL−1 ) was added to 20 mL H2 O2 (20 mmol L−1 ) solution prepared by a Tris–HCl buffer (50 mmol L−1 pH 7.0) under stirring for 3 min. The absorbance decrease at 240 nm caused by H2 O2 decomposition was recorded after CAT or TNTs-CAT being added in the above solution for 3 min. One unit of catalytic activity was defined as the decomposition of 1 ␮mol H2 O2 per min at 25 ◦ C and pH 7.0. The concentration of CAT was determined by Brandford method, and the catalytic activity of free CAT and TNTs-CAT were given as U mg of protein−1 . The relative activity (%) of TNTs-CAT was calculated by comparing with the free CAT under equal enzyme amount under the same conditions (Eq. (1)). Relative activity % =

activity of TNTs-CAT × 100 activity of free CAT

(1)

2.6. Immobilization efficiency, kinetic parameter and stability of TNTs-CAT 2.6.1. Immobilization efficiency CAT solutions with different initial concentrations were modified to covalently bind on the surface of prepared TNTs. The amount of CAT immobilization was determined by the TGA analysis, and the corresponding relative activity of TNTs-CAT with different loading capacities was also calculated. 2.7. Leakage rate Fifty milligram TNTs-CAT with a loading efficiency of 820 mg g of support−1 was incubated in 20 mL Tris–HCl buffer (50 mmol L−1 , pH 7.0) with vigorous magnetic stirring for 72 h. Then, the supernatant was collected by centrifugation, and the concentration of CAT in supernatant was examined by the Bradford’s method. The leakage rate of TNTs-CAT was calculated by the following equation: Leakage ratio % =

Ci Vi × 100 MT

(2)

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Scheme 1. Illustration of CAT modification and immobilization on the surface of TNTs (not drawn to scale). All procedures were conducted in an MES buffer solution (50 mmol L−1 , pH 6.5).

where Ci , Vi and MT represent the concentration of CAT released in the buffer solution, the buffer solution volume, and the initial amount of CAT covalently immobilized on TNTs. 2.8. Kinetic parameters The kinetics parameters, maximum reaction rates Vmax (mmol (L min)−1 ) and Michaelis–Menten constants Km (mmol L−1 ) for free CAT and TNTs-CAT, were investigated by using the classical Michaelis–Menten kinetics. Activities of free CAT and TNTs-CAT were measured at different H2 O2 concentrations (5–35 mmol L−1 , 50 mmol L−1 Tris–HCl buffer, and pH 7.0) as the substrate. The Lineweaver–Burk plot was adopted to analyze the experimental dates: 1 1 1 Km + × = V Vmax Vmax [S]

(3)

where V (mmol (L min)−1 ) and [S] (mmol L−1 ) were the initial reactive rate and initial substrate concentration, respectively.

The storage stability of free CAT or TNTs-CAT was determined by selectively measuring the residual activity of free CAT (TNTs-CAT) after stored for a certain period of time at 4 ◦ C. The relative activity (%) was calculated from the ratio of its activity after stored for a period of time to the initial activity under optimum condition (Eq. (5)) Relative activity % =

residual activity after storage × 100 initial activity

(5)

The recycling stability of TNTs-CAT was determined by measuring the residual activity after each successive reaction cycle at room temperature and neutral pH. The TNTs-CAT after each reaction cycle were collected by centrifugation accompanied by thoroughly rinsed with Tris–HCl solution (50 mmol L−1 , pH 7.0), and then reused in the next reaction cycle. In all the stability experiments, the initial activity of TNTs-CAT was assumed as 100%, while other activities were the relative values through comparison with the initial activity. Each result was obtained by averaging three individual experiments.

2.9. Stability The stability experiments of TNTs-CAT included three parts, i.e. the thermal, storage and recycling stability, and were carried out by using TNTs-CAT with a loading amount of 470 mg g of support−1 as a model. The thermal stability of free CAT or TNTs-CAT was determined by measuring the residual activity of free CAT or TNTs-CAT after incubated at different temperatures (30–60 ◦ C) in a Tris–HCl solution (50 mmol L−1 , pH 7.0) for 2 h. The relative activity was calculated from the ratio of its activity at each specific temperature to the activity at the optimum conditions (Eq. (4)) Relative activity % =

activity at the specific temperature × 100 activity at the optimum temperature (4)

3. Results and discussion 3.1. Characterization of as-synthesized TNTs Fig. 1 shows the characterization results of the as-synthesized TNTs including TEM, XRD, Raman spectrum and nitrogen adsorption–desorption isotherms. From a typical low magnification TEM image (Fig. 1a), it is found that all obtained TNTs almost possess homogeneous nanotube structure. According to the statistical information of a large number of TNTs, the length of 73% TNTs ranges from 100 to 180 nm. As exhibited in the HRTEM micrograph of a TNT (the inset of Fig. 1a), the as-synthesized TNT is a multi-walled nanotube with open ends, and its inside and outside diameter are around 4 and 10 nm, respectively.

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The crystal structure of the as-synthesized TNTs was monitored by X-ray diffraction (XRD), and shown in Fig. 1b. The diffraction peaks in the XRD pattern of TNTs are much broader than those obtained from normal crystals, which can be explained as the nanometer size of TNTs and the bending of some atom planes [24]. The peaks at 9.8◦ , 24.5◦ , 28◦ and 48.1◦ are attributed to (2 0 0), (1 1 0), (2 1 1), and (0 2 0) plane of protonic trititanate (H2 Ti3 O7 , Joint Committee on Powder Diffraction Standards (JCPDS) card No. 36-654), respectively, in agreement with the literatures [24–26]. In order to further confirm the composition of the as-synthesized TNTs, Raman spectrum was conducted (Fig. 1c). There are six peaks in the Raman spectrum of TNTs. The peaks at 191 and 275 cm−1 can be attributed to lattice modes and Ti–O stretching vibrations, respectively. The peak at about 449 cm−1 is assigned to the internal vibrations of the robust TiO6 octahedra. The band at about 680 cm−1 is due to the Ti–O–Ti stretch of edge-shared TiO6 , and the bands around 830 and 930 cm−1 are assigned to a Ti–O–H symmetrical stretching mode with a very short Ti–O distance and four-coordinate Ti–O vibration in the titanate structure, respectively [27–31]. This result suggests that the structure of the synthesized TNTs is H2 Ti3 O7 nanosheets, which is consistent with the XRD result. The nitrogen adsorption–desorption isotherm and pore size distribution of TNTs produced from a N2 desorption curve using the BJH algorithm are shown in Fig. 1d. The isotherm has an obvious hysteresis loop at P/P0 = 0.7–1.0, which indicates the presence of mesopores in the materials. Two peaks exhibit in the inserted pore size distribution plot: a sharp peak located at 3.5 nm is considered as the inner diameter of TNTs, which corresponds to the observation by HRTEM; while the other broad peak ranges from 10.7 to 14.9 nm can be explained as the interspaces produced by the TNTs aggregation [32]. In addition, the BET specific surface area is calculated to be 305.4 m2 g−1 , which suggesting the sufficient enzyme immobilization sites. Based on the above results, it is confirmed that the composition of as-synthesized TNT is H2 Ti3 O7 -type. Generally, Na+ ions are inserted into the spaces among TiO6 octahedra, and layered sodium titanate nanosheets are formed as intermediates during the alkaline hydrothermal process, which are then folded into tubular structures. Further ion exchange of Na+ by H+ takes place during the washing and the HCl treatment, resulting in the formation of H2 Ti3 O7 nanotubes [26,33]. 3.2. Characterization of TNTs-CAT

Fig. 1. The characterization of prepared TNTs: (a) TEM image (the inset is a typical HRTEM image of a single TNT; (b) XRD pattern; (c) Raman spectrum; and (d) nitrogen adsorption–desorption isotherms (the inset is BJH pore size distribution).

Direct covalent attachment possesses the favorite potential for the development of commercial enzyme immobilization due to the stability of the resultant bond. It is a popular and highly versatile method for the covalent immobilization of enzyme to activate carboxylic acids to a relative intermediate, which is susceptible to nucleophilically attack by amines on free lysine chains of the enzyme using EDC and NHS [34,35]. The process of the modification and immobilization of CAT on the TNTs surface is illustrated in Scheme 1. All procedures are conducted in MES buffer solution (50 mmol L−1 , pH 6.5) at room temperature, in favor of keeping the enzyme activity. 3,4-DiHPP is at first activated by EDC and NHS, and then bind CAT with amide bonds. Catechol groups on the modified CAT can chelate with Ti4+ on the TNTs surface, once the ultrasonically dispersed TNTs are immersed in the modified CAT solution. Since EDC and NHS should be much excessive during the modification process, the cross-linked CAT aggregates are also produced [36] and immobilized on the TNTs surface. In order to testify whether CAT is successfully immobilized on the TNTs surface, the immobilized product is characterized by HRTEM and XPS. As shown in Fig. 2a, a layer of substances spread on the TNTs surface homogeneously. The corresponding

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Fig. 2. The characterization of prepared TNTs-CAT: (a) TEM image of TNTs-CAT; (b) EDS element mapping of N (green) and Ti (red) in the TNTs-CAT; and full survey scan XPS spectra of prepared TNTs and TNTs-CAT. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.)

3.3. Immobilization efficiency

1- TNTs-CAT 2- TNTs

a Intensity

1540

1 1650

467 1630

2 3400

3600

3000

2400

1800

1200

600

Wave number /cm-1 2.0

1- TNTs-CAT 2- TNTs 3- 3,4-diHPP

b

1.5

Absorbance

EDS element mapping of N and Ti demonstrates that the N element mainly distributes on the TNTs surface, indicating that these substances should be the enzyme (Fig. 2b). Furthermore, the full scan XPS spectrum of the immobilized CAT has an apparent N1s peak and a weak S2p peak (Fig. 2c), which are the further evidence for the successful immobilization of CAT on the TNTs surface. FTIR and UV–vis spectra of TNTs-CAT were performed to obtain the covalent linking information between TNTs and CAT, and shown in Fig. 3a. Before immobilization, there are only three broad absorption bands centered at 3400, 1630 and 467 cm−1 in the FTIR spectrum of TNTs, which are assigned to O–H stretching mode for interlayer water, hydrogen group and H–O–H bending for water on the TNTs surface, and O–Ti–O stretching vibration, respectively [37,38]. After CAT immobilization, two new absorption bands at 1650 cm−1 and 1540 cm−1 appear, which are attributed to the Amide I band and the Amide II band of the immobilized CAT, respectively [39]. As shown in Fig. 3b, the UV–vis spectrum of 3,4-diHPP exists only a peak at 280 nm, which is assigned to the catechol group; while a new broad absorption band spanning the whole visible range appears in the UV–vis spectrum of TNTs-CAT, indicating the formation of surface titanate–catechol complexes [40,41]. Based on above results, it is concluded that CAT molecules can be immobilized on the TNTs surface through the chelation between Ti4+ ions and catechol groups on the modified CAT under mild conditions.

1.0 1

0.5 2

0.0 3

The CAT loading ratio was measured by the thermogravimetric analysis (TGA) after TNTs-CAT was treated at 100 ◦ C for 2 h to remove the adsorbed water. As illustrated in Fig. 4, there are three weight loss stages at room temperature −200 ◦ C, 200–520 ◦ C and 520–800 ◦ C, respectively. For the TNTs-CAT, the first stage is

100

200

300

400

500

600

700

800

Wavelength /nm Fig. 3. (a) FTIR spectra of TNTs and TNTs-CAT and (b) UV–vis spectra of TNTs, TNTsCAT and 3,4-diHPP.

Q. Ai et al. / Biochemical Engineering Journal 83 (2014) 8–15

120

a

90

Weight /%

Relative Activity /%

100

80 70 a b c d

60 50 0

b

Modified TNTs TNTs-CAT(2mg/mL) TNTs-CAT(4mg/mL) TNTs-CAT(6mg/mL)

150

300

c

600

750

a

Free CAT Immobilized CAT

100 80 60 40 20

d

450

13

0 30

900

40

Temperature / C Fig. 4. TGA scans of TNTs modified by 3,4-diHPP (a) and TNTs-CAT with different initial CAT concentrations (b–d).

750 60 600 50 450 40

300 150 2

3

4

60

70

5

6

120

Relative Activity /%

b 100 80 60 40 20 TNTs-CAT Free CAT

0 0

10

20

30

40

50

60

Storage Time /d 120

c

Relative Activity /%

105 90 75 60 45 30 15 0

0

1

2

3

4

5

6

7

8

9

10 11

Recycle Number Fig. 6. The thermal (a), storage (b) and recycling (c) stabilities of TNTs-CAT.

zero-leakage of the enzyme on the TNTs surface may find a promising application in the industrial biocatalysis.

70

900

Relative Activity /%

Loading Efficiency /mg g support-1

attributed to the structural water; the second stage is assigned to the decomposition of 3,4-diHPP and CAT [37,42]; the third stage is originated from the transformation of TNTs to TiO2 crystals. As for the TNTs modified by 3,4-diHPP, the total weight loss is caused by the structure water, the decomposition of 3,4-diHPP and the transformation of TNTs to TiO2 crystals. Therefore, by comparing TGA scans of TNTs-CAT with TNTs modified by 3,4-diHPP, the extra weight loss of TNTs-CAT should be attributed to the decomposition of immobilized CAT, and thus the loading ratio of CAT can be calculated. It can be observed from Fig. 5, the loading ratio almost increases linearly as the initial CAT concentration increases. When the initial CAT concentration is 6 mg mL−1 , the loading ratio can reach as high as 820 mg g of support−1 , which is higher than most of the published values [36,43,44]. Since the maximum monolayer loading efficiency of CAT on the TNTs surface is calculated to be about 628 mg g of support−1 , multilayer cross-linked CAT aggregates should exist. However, the relative activity of TNTsCAT calculated according to Eq. (1) decreases gradually from 60% to 46.8% with the enhancement of the CAT loading ratio, which is caused by the burying of some enzyme active centers due to the accumulation of abundant protein molecules on the TNTs surface. The chemical modified CAT maintains 95% catalytic activity of its native form, indicating that the three-dimensional conformation and active sites of the enzymes are well preserved. In addition, the leakage experiment demonstrated that few enzymes fell off from TNTs after TNTs-CAT incubated in Tris–HCl buffer was continuously stirred for 72 h. The highly efficient immobilization and

1

50

Temperature /oC

o

30

[CAT] /mg mL-1

Fig. 5. CAT loading efficiency and relative activity of TNTs-CAT with different initial CAT concentrations.

3.4. Kinetic parameters It is well known that Vmax and Km are important kinetic parameters for the enzyme. Vmax is defined as the highest possible rate when the enzyme is saturated with the substrate, which reflects the intrinsic catalytic character of the enzyme. Km , is defined as the substrate concentration that gives a reaction rate of 1/2 Vmax reflects the affinity between enzyme and substrate [45]. In this study, Vmax and Km are calculated based on the Linerweave–Burk plots. The Vmax values of free CAT and TNTs-CAT are 4.34 × 104 and 1.43 × 104 mol H2 O2 (mg protein min)−1 , respectively. The Vmax decrease for the immobilized enzyme is a universal phenomenon in the enzyme immobilization field, which may be caused by structure changes of CAT [46–48]. The Km value of TNTs-CAT is 74.2 mmol L−1 , which is approximately 1.7 times of that of free CAT. The Km increase

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means the affinity reduction between enzyme and substrate, which is caused by either the CAT conformation changes introduced by either the CAT conformation changes by the immobilization procedure and thus interactions, resulting in a lower possibility forming a substrate-enzyme complex, or a less accessibility of the substrate to the active site of TNTs-CAT [48,49]. 3.5. Stability Since the stability is very important for various biotechnological and industrial applications, the TNTs-CAT stabilities including thermal, storage and recycling stability were studied. As demonstrated in Fig. 6a, the free CAT and TNTs-CAT have the similar thermal stability curve, i.e. the enzyme activity decreases gradually as the temperature increases from 30 to 70 ◦ C. The TNTs-CAT only exhibits a slightly better thermal stability than the free CAT, which is related to the enzyme exposed directly in the reaction environment. The storage stability of TNTs-CAT was evaluated by determining the residual activities after keeping a period of storage time at 4 ◦ C (Fig. 6b). Compared to the free CAT, the storage stability of TNTs-CAT is significantly enhanced. After sixty-day storage, the free CAT only keeps 44% of its initial activity, while TNTs-CAT preserves 92% of its initial activity, which is much higher than that of other immobilized CAT reported in literatures [36,43,44]. It is reasonably believed that TNTs can afford CAT suitable microenvironment, and remain most of the catalytic activity. The operational stability of TNTs-CAT was determined by measuring the residual activity after several successive reusing (Fig. 6c). TNTs-CAT retains more than 50% of the initial activity after nine successive cycles, which is in the average level of immobilized CAT [43,50,51]. 4. Conclusions In this study, a novel kind of nanostructured supporter with large specific surface area and high hydrophilicity, TNT, was synthesized by an alkaline hydrothermal method and applied for the covalent immobilization of enzyme. The as-synthesized TNT is a multi-walled nanotube with open ends, and its inside and outside diameter are around 4 and 10 nm, respectively. Meanwhile, the BET specific surface area of TNTs was determined to be 305.4 m2 g−1 , suggesting the sufficient enzyme immobilization sites. CAT, as a model enzyme, was firstly pre-modified by 3,4diHPP, and then covalently immobilized on the TNTs surface by the chelation between Ti4+ ions and catechol groups. The maximum loading ratio of CAT could reach as high as 820 mg g of support−1 ; while the relative activity of immobilized CAT remain as high as 60% of the free CAT, when the loading efficiency was 238.3 mg g of support−1 . Furthermore, the immobilized CAT exhibited high storage and recycling stability compared to the free CAT, i.e. TNTs-CAT preserved 92% of its initial activity after sixty-day storage and more than 50% of its initial activity after nine successive cycles. Hopefully, TNTs can become promising nanostructured supporters for enzyme immobilization, biosensor and biochip. Acknowledgments This research is supported by National Science Fund for Distinguished Young Scholars (21125627), the National Basic Research Program of China (2009CB724705) and the Program of Introducing Talents of Discipline to Universities (No. B06006). References [1] P. Wang, Nanoscale biocatalyst systems, Curr. Opin. Biotechnol. 17 (2006) 574–579.

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