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1 Chinese Journal of Catalysis 34 (213) 催化学报 213 年第 34 卷第 4 期 available at journal homepage: Article Catalytic performance and stability of C C bond hydrolase BphD immobilized onto single wall carbon nanotubes SHEN E, QU Yuanyuan *, ZHOU Hao, KONG Chunlei, MA Qiao, ZHANG Xuwang, ZHOU Jiti Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 11624, Liaoning, China A R T I C L E I N F O A B S T R A C T Article history: Received 21 December 212 Accepted 27 February 213 Published 2 April 213 Keywords: C C bond hydrolase Single wall carbon nanotube Immobilization Molecular simulation Physical adsorption Covalent bonding Single wall carbon nanotubes (SWCNTs) possess unique mechanical properties and extraordinary thermal conductivity, and were used as the matrix for immobilized biocatalysts. The C C bond hydrolase BphD was immobilized on SWCNTs by physical adsorption and covalent bonding. The relative activity, stability, and reusability of the immobilized enzyme were investigated. BphD immobilized by physical adsorption retained 52.5% of the activity of free BphD, and BphD thermal stability and denaturant resistance were also improved. Covalently bound BphD exhibited the activity nearly the same as that of free BphD (99.7% of initial activity), but its stability showed no significant improvement over that of free BphD. 213, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved. 1. Introduction Single wall carbon nanotubes (SWCNTs) are one dimensional nanomaterials widely used in etching, photoelectrocatalysis, biosensors, and high resolution imaging, because of their unique mechanical, electrical, and chemical properties [1]. Enzymes including horse radish peroxidase (HRP) and lipase have recently been immobilized on SWCNTs, demonstrating SWCNTs' potential as an immobilization matrix for biocatalysts [2 5]. Asuri et al. [6] adsorbed soybean peroxidase and α chymotrypsin on SWCNTs, with enzyme loadings reaching 6 mg/g. They also covalently immobilized HRP on SWCNTs, with the loading amount reaching 1.3 mg/g and HRP retaining 4% of its initial activity. Pedrosa et al. [7] covalently immobilized parathion hydrolase on SWCNTs and multi wall carbon nanotubes (MWNTs), obtaining biosensors for monitoring the presence of parathion. Immobilized parathion hydrolase retained 75% of its initial activity, even after storage for several months. While such SWCNT immobilized enzymes retained high loading amounts and activities, some issues require further attention. First, the enzyme types immobilized on SWCNTs have been relatively limited, and the immobilization of other enzymes should be investigated. Second, the detailed effect of physical adsorption and covalent bonding on loading amounts and relative activities remains unclear. Last, the SWCNT binding site and enzyme binding mechanism remain unclear. C C bond hydrolases are involved in the meta cleavage degradation pathway of aromatic compounds. They catalyze the C C bond hydrolysis of meta cleavage products. This step requires high energy and is the rate limiting process of the overall degradation [8]. Researchers have recently demonstrated the unique catalytic promiscuity of C C bond hydrolases, i.e. catalyzing different reactions using the same active site. * Corresponding author. Tel: ; E mail: qyy7@126.com This work was supported by the National Natural Science Foundation of China (517854, ). DOI: 1.116/S (12) Chin. J. Catal., Vol. 34, No. 4, April 213

2 724 SHEN E et al. / Chinese Journal of Catalysis 34 (213) For example, the C C bond hydrolase MhpC can catalyze the hydrolysis of C C, C O bond, S O and even the reverse synthesis of C C bonds, so has potential biosynthesis applications [9]. We cloned the genes encoding the C C bond hydrolase BphD from the strain Dyella ginsengisoli LA 4 and expressed in recombinant E. coli [1]. In this study, we immobilized BphD on SWCNTs, and compared the catalytic performance and stability of enzymes immobilized by physical adsorption and covalent bonding. A molecular simulation was used to account for differences in residual activity. 2. Experimental 2.1. Materials The strain was recombinant E. coli BL21(DE3) containing the gene bphd, which encoded the C C bond hydrolase BphD. Lysogeny broth (LB) medium was used, which contained NaCl 1. g/l, tryptone 1. g/l and yeast extract 5. g/l. The ph was adjusted to 7.. SWCNTs (95+%) were purchased from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China). The SWCNT length was 5 15 μm, and their diameter was < 2 nm. 2 (N Morpholino) ethanesulfonic acid (MES), N hydroxysuccinimide (NHS) and N ethyl N (3 (dimethylamino)propyl) carbodiimide hydrochloride (EDC) were purchased from Sinopharm (Shanghai, China). Kanamycin sulfate, isopropyl β D 1 thiogalactopyranoside (IPTG), and p nitrophenyl benzoate were purchased from J&K Chemical (Beijing, China). All other chemicals were of analytical grade and were used without further purification Culture of bacteria Bacterial liquid (2 μl) was transferred into 25 ml of LB medium containing kanamycin sulfate (3 mg/l), and incubated at 3 C and 15 r/min for 12 h. 2 ml of incubated bacterial liquid was transferred into ml of LB medium containing kanamycin sulfate (3 mg/l), and incubated at 37 C and 15 r/min until the optical density values reached.4.6. IPTG (1 mmol/l) was then added for 3 h to induce the expression of BphD. The broth was centrifuged at 4 C and r/min for 5 min. The precipitate was washed twice with sodium phosphate buffer (PBS) and stored at 8 C in a freezer until use Modification of SWCNTs Step 1: Carboxylation. To functionalize with carboxyl groups, SWCNTs (.1 g) were suspended in 4 ml of 3:1 v/v H2SO4: HNO3, and sonicated at 3 C for 5 h. The suspension was diluted with MilliQ water and filtered through a.22 μm polycarbonate membrane. The nanotube film was resuspended into MilliQ water and sonicated again to remove any residual acid. The suspension was centrifuged and the precipitate dried at 5 C under vacuum for 8 h. The solid obtained was characterized by Fourier transform infrared spectroscopy (FTIR). Step 2: Carboxyl group activation. The acidified SWCNTs were dispersed in a MES buffer, and an equal volume of 4 mmol/l NHS was added. The suspension was sonicated for 3 min. The addition of 2 mmol/l EDC initiated the coupling of NHS to the carboxylic groups, and the mixture was stirred at 15 r/min for 1 h. Excess NHS and EDC were separated through a.22 μm polycarbonate membrane. The precipitate was dried at 8 C under vacuum for 8 h and named CM SWCNT Purification of BphD BphD was purified based on the affinity of the His tag for nickel nitrilotriacetic acid (Ni NTA) metal affinity chromatography. E. coli cells harvested above were resuspended in 2 mmol/l PBS and lysed by sonication for 4 min. After centrifugation at 22 r/min for 2 min, the supernatant was separated and used as the crude extract for further purification. Crude extracts were bound to the resin in the equilibrating buffer ( mmol/l phosphate buffer containing.5 mol/l NaCl and 2 mmol/l imidazole, ph 7.4), and the target His tagged BphD was eluted by the buffer containing 325 mmol/l imidazole. Fractions were collected, dialyzed and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) Immobilization of BphD The optimal immobilization ph was tested in a 3 ml reaction volume. The concentration of BphD was 3 μg/ml and the SWCNT mass was.5 mg. The activity of immobilized BphD was monitored by absorption at 45 nm using a UV visible spectrophotometer. The ph values were set to 6, 7, 8, 9, and 1 using different buffers. Tubes were incubated at 3 C and 15 r/min. After ensuring the optimal ph was achieved, SWCNTs and CM SWCNTs were each used to immobilize BphD. The concentrations of BphD were set as 15, 3, 6, and 9 μg/ml. SWCNTs or CM SWCNTs (1 mg/ml), protein and a corresponding volume of PBS (ph 7.) were mixed and incubated at 3 C and 15 r/min. The relative activity and loading amount were monitored by absorption of 45 and 595 nm, respectively [11]. Sample (6 μl) was removed after a specific interval to determine the residual protein amount using the Bradford method [12] Characterization of immobilized BphD The reaction volume for characterizing immobilized BphD was 2 ml, which contained 4 μl of immobilized BphD, 4 μl of substrate and 192 μl of PBS. One unit of enzymatic activity was defined as the quantity of enzyme required to produce 1 μmol/l of p nitrophenol. Effect of temperature on the activity of immobilized BphD. Immobilized and free BphD were each incubated at 3, 4, 5, 6, and 65 C for 15 min. The activity was measured when the solution was cooled to room temperature. Thermostability of immobilized BphD. Immobilized and free BphD were each incubated at 6 C, and the activity deter

3 SHEN E et al. / Chinese Journal of Catalysis 34 (213) mined every 5 min. The initial activity was normalized as %, and the change in relative activity was recorded. Effect of denaturing agent on the activity of immobilized BphD. Immobilized and free BphD were each incubated in guanidine hydrochloride (GdmCl) for 15 min, and the activity of the enzyme was then recorded. The concentrations of GdmCl used were,.2,.4,.8, and 1.6 mmol/l. Reusability of immobilized BphD. After one cycle of use, immobilized BphD was collected by centrifuging at 12 r/min for 5 min, and resuspended in the same volume of PBS. The activity of the resuspended immobilized enzyme was determined, and the change in relative activity was recorded Homology modeling, docking and prediction of the covalent binding site The three dimensional structure of BphD was modeled using the online server SWISS MODEL program ( The BphD protein sequence was set to Automated Mode [13]. Molecular docking of a SWCNT and BphD was performed using Patchdock and Firedock [14]. The covalent binding site was predicted using the method provided by Pamela et al. [15]. The pka values for residues containing amino groups, i.e. for the N terminal residue, Lys and Tyr, were calculated using the online server PROPKA. The LIG Re value of each amino group was then calculated using: LIG Re = 1 (ph pka) When LIG Re was <.1, the amino group was considered to have low activity. When LIG Re was.1 1 and > 1, it had moderate and high activity, respectively. 3. Results and discussion 3.1. Modification and characterization of SWCNTs To covalently attach protein molecules, the SWCNT surface was functionalized with carboxyl groups that were active to NHS. The modification is shown in Fig. 1. FT IR spectra of pristine, carboxylated and acylated SWCNTs are shown in Fig. 2. The characteristic C=O stretching vibration at ~172 cm 1 indicating carboxyl group modification [16]. The band at 1645 cm 1 in the NHS/EDC modified SWCNT sample was due to the amide C=O stretching vibration. Transmittance 3.2. Optimal ph for immobilization The main force determining the interaction between BphD and SWCNTs was electrostatic. The protein and SWCNT charges were both affected by ph, so it was important to determine the optimal ph for immobilization. Relative activities and loading amounts of immobilized BphD were compared. Figure 3 shows the effect of ph on the relative activities of free and immobilized BphD. BphD SWCNT, BphD CM SWCNT, and BphD all had similar optimal ph values at around 7.. BphD SWCNT exhibited a slightly higher relative activity than the other two samples at ph 6., while BphD CM SWCNT had a slightly higher relative activity at ph >7.5. SWCNT and CM SWCNT could each (3) (2) (1) Wavenumber (cm 1 ) Fig. 2. FT IR spectra of pristine SWCNTs (1), H2SO4/HNO3 oxidized SWCNTs (2), and NHS/EDC modified SWCNTs (3). BphD ph Fig. 3. Effect of ph on the relative activities of free and immobilized BphD. NH2 H 2 SO 4 HNO 3 EDC NHS Protein Fig. 1. Covalent modification process of SWCNT.

4 726 SHEN E et al. / Chinese Journal of Catalysis 34 (213) be fully loaded with the added protein molecules. The following experiments were performed at ph Adsorption kinetics of BphD The adsorption kinetics of BphD on the surface of SWCNTs and CM SWCNTs were determined at different protein concentrations. The curves displayed in Fig. 4 show the relationship between adsorption percentage and time. Figure 4(a) shows that when the protein concentration was low (15 and 3 μg/ml), adsorption reached equilibrium within 2 h, and the adsorption percentage was nearly %. At higher concentrations (6 and 9 μg/ml), loading equilibrium was reached within 3 h, but the adsorption percentage only reached 6%. The adsorption kinetics of BphD on CM SWCNT were similar to that on SWCNT, but the time required to reach adsorption equilibrium was typically much longer (for a constant protein concentration). This result could be explained by the interaction between BphD and CM SWCNT being more specific. According to calculations based on adsorption in Fig. 4, maximum loadings were obtained at a protein concentration of 9 μg/ml. Therefore, the following experiments were all performed at this concentration. Under these conditions, the loading amounts of SWCNT and CM SWCNT were 255 and 518 mg/g, respectively. BphD SWCNT retained 52.5% of its initial activity, while BphD CM SWCNT retained 99.7% of its initial activity. The loading amount and relative activity of BphD CM SWCNT were both higher than those of BphD SWCNT, which may have been due to covalent bonding retaining the BphD conformation of its free form. The result where CM SWCNTs exhibited a larger loading amount than pristine SWCNT was consistent with Asuri et al. [6] SEM observations of morphology of immobilized BphD The surface morphology of immobilized BphD was characterized by scanning electron microscopy (SEM). Figures 5(a) Percentage (%) g/ml 3 g/ml 6 g/ml 9 g/ml Time (h) Fig. 4. Adsorption of BphD on SWCNT (a) and CM SWCNT (b). and 5(c) show the morphology of SWCNTs and CM SWCNTs, respectively. SWCNTs and CM SWCNTs both existed as clusters, but the size of the CM SWCNT clusters was more variable. Figures 5(b) and 5(d) show the morphology of BphD SWCNT and BphD CM SWCNT, respectively. Amorphous particles appeared among nanotubes and could have been crystalline phosphate TEM observations of morphology of immobilized BphD Protein molecules cannot be observed using SEM, so immobilized enzymes were characterized using transmission electron microscopy (TEM). The morphologies of SWCNTs and CM SWCNTs were not significantly different, as shown in Figs. 6(a) and 6(b), respectively. Figures 6(c) and 6(d) show the morphologies of BphD SWCNT and BphD CM SWCNT, respectively. These both appeared to show colloidal materials covering the nanotubes, which may have been protein molecules. (a) (b) (a) (b) (c) (d) Fig. 5. SEM images of SWCNT (a), BphD SWCNT (b), CM SWCNT (c), and BphD CM SWCNT (d). (a) (b) (c) (d) Fig. 6. TEM images of SWCNT (a), CM SWCNT (b), BphD SWCNT (c), and BphD CM SWCNT (d).

5 SHEN E et al. / Chinese Journal of Catalysis 34 (213) BphD BphD Temperature ( o C) Fig. 7. Effect of temperature on the activity of free BphD and BphD CNT. More colloidal material was apparent in the TEM image of BphD CM SWCNT, consistent with the higher loading amount observed earlier Characterization of immobilized BphD BphD SWCNT and BphD CM SWCNT were each characterized, and the effects of temperature, ph, and denaturant on enzyme activity were investigated. The thermal stability and reusability of immobilized BphD were also determined Effect of temperature on BphD activity Figure 7 shows the effect of temperature (3, 4, 5, 6, and 65 C) on the relative activities of the enzymes. The highest relative activity of free BphD and BphD SWCNT were both observed at 5 C, while BphD CM SWCNT exhibited its highest activity at 4 C. Basha et al. [17] immobilized an invertase enzyme from thermophilic fungus using phenyl sepharose, and found the optimal temperature for immobilized invertase was lowered by 1 C. This was due to a conformation change of the enzyme during immobilization, which enhanced the product inhibition at higher temperatures Effect of ph on BphD activity The effect of ph on the activity of BphD immobilized at ph 7. was investigated. Figure 8 shows that the relative activity changed with ph, and that the optimal ph for both BphD SWCNT and BphD CM SWCNT was 7.. At lower ph, the relative activity of immobilized BphD was significantly higher than that of free BphD. At ph 6., 62% of BphD activity was retained for BphD SWCNT, while only 25% was retained for free BphD. This indicated that the tolerance for changing ph could be enhanced through immobilization and the change in microenvironment Thermostability of immobilized BphD The thermostability of immobilized BphD was investigated at 6 C, and the result is shown in Fig. 9. After incubation at 6 C for 4 min, both immobilized and free BphD lost at least part of their activity. Free BphD lost all activity within 25 min, while BphD SWCNT exhibited improved thermostability compared ph Fig. 8. Relative activity of free and immobilized BphD at different ph values. with free BphD. The activity of BphD CM SWCNT decreased more quickly than that of free BphD, the reason for which may be similar to the decreased optimal temperature Effect of denaturant on BphD activity GdmCl is a commonly used protein denaturant, and can denature protein molecules by capturing bound water necessary for function. Figure 1 shows the effect of different concentrations of GdmCl on the relative activity of BphD. BphD SWCNT exhibited an improved tolerance to GdmCl compared with the other two samples. When the GdmCl concentration reached 1.6 mmol/l, BphD SWCNT still retained 33% of its initial activity. BphD CM SWCNT did not exhibit any improvement to denaturant compared with free BphD Reusability of immobilized BphD The reusability of BphD SWCNT and BphD CM SWCNT was tested over 1 cycles. Figure 11 shows that BphD SWCNT retained 9% of its initial activity after five cycles, while BphD CM SWCNT lost all activity. The stability of a covalently bound enzyme is usually greater than that of a physically adsorbed enzyme. However, in the current situation the reusability of BphD CM SWCNT was worse than BphD SWCNT. This may have resulted from few ionized amino groups being present in BphD at ph 7.. According to the calculation method BphD Time (min) Fig. 9. Thermostability of free and immobilized BphD at 6 C.

6 728 SHEN E et al. / Chinese Journal of Catalysis 34 (213) BphD Concentration (mmol/l) Fig. 1. Effect of denaturant concentration on the activity of free and immobilized BphD. provide by Torres Salas et al. [15], the pka values of the N terminal residues, Lys and Tyr in BphD were estimated. The N terminal residue possessed moderate activity (.1 < LIG Re < 1), while those of the Lys and Tyr residues possessed low activities. This could explain the lower reusability observed for BphD CM SWCNT Homology modeling and molecular docking Because of limited available experimental techniques, it was not possible to directly observe the binding site of the enzyme and SWCNT, so the binding site was modeled through molecular docking. The three dimensional structure of BphD was first constructed by SWISS MODEL, using the crystal structure of BphD from Burkholderia xenovorans LB4 as a template (sequence identity was 88.73%). The BphD model is shown in Fig. 12, and suggested that BphD existed as a homotetramer. The model quality was evaluated by the PROCHECK program in the online server UCLA DOE ( mbi.ucla.edu/). The Ramachandran plot showed a reasonable conformation of the main chain. 9.7% of the residue dihedral angles φ and ψ were located in favor of the allowed region. Therefore, the model was suitable for further docking studies [18]. The binding mode of BphD and SWCNT was generated by Patchdock and Firedock. All possible binding sites of SWCNT were far removed from the active site of BphD (Fig. 13(a)). The Fig. 12. Three dimensional structure of BphD generated from homology modeling. decreased catalytic efficiency of BphD SWCNT could not have resulted from the insertion of the SWCNT into the BphD active site. When an inhibitor is bound at the interface of adjacent subunits, it can decrease catalytic efficiency through disrupting the interaction between subunits. We identified the interaction force between subunits. Taking subunits B and C as an example, two salt bridges, Glu717 Lys438 and Lys72 Glu435, were important for the interaction of subunits. Figure 13(b) shows the interaction site of SWCNT and BphD. When the size of the SWCNT was.5 nm, it could insert into the subunit interface and disrupt the salt bridges, decreasing the catalytic efficiency of BphD SWCNT. Zuo et al. [19] investigated the toxic effect of (a) (b) Reuse number Fig. 11. Reusability of immobilized BphD. Fig. 13. Interaction between BphD and SWCNT, showing an overview (a) and enlargement of the interaction site (b).

7 SHEN E et al. / Chinese Journal of Catalysis 34 (213) Graphical Abstract Chin. J. Catal., 213, 34: doi: 1.116/S (12) Catalytic performance and stability of C C bond hydrolase BphD immobilized onto single wall carbon nanotubes SHEN E, QU Yuanyuan*, ZHOU Hao, KONG Chunlei, MA Qiao, ZHANG Xuwang, ZHOU Jiti Dalian University of Technology Physical adsorption Higher stability Higher activity The C C bond hydrolase BphD was immobilized on single wall carbon nanotubes by physical adsorption and covalent bonding. The former imparted higher stability while the latter imparted higher activity on the immobilized enzyme. Immobilization of C C hydrolase BphD SWCNTs on the WW domain of the human Yes associated protein using molecular dynamics. They suggested that SWCNTs could disrupt and block the active sites, thus reducing the possibility of direct binding of the ligand. The active site of BphD was buried in a hydrophobic core not easily accessed by the SWCNT. The binding site of CM SWCNT could not be predicted through molecule docking, because the covalent interaction was much stronger than intermolecular interactions (i.e. hydrogen bonding, salt bridges and Van der Waals forces). The most probable binding site was the N terminal residue of BphD, but because this residue had only moderate activity, the BphD conformation may not have changed during covalent bonding. This could explain why CM SWCNT BphD had a comparable catalytic efficiency to that of free BphD. 4. Conclusions BphD immobilized by covalent modification (BphD CM SWCNT) had a higher activity than that immobilized by physical absorption (BphD SWCNT). BphD CM SWCNT retained 99.7% of the activity of free BphD, compared with 52.5% retained by BphD SWCNT. Meanwhile, BphD SWCNT exhibited improved stability to thermal treatment, ph changes and denaturant exposure, while BphD CM SWCNT did not exhibit any significant improvement. Molecular docking suggested that the decreased catalytic efficiency of SWCNT BphD resulted from the disruption of salt bridges between adjacent subunits. Therefore, single wall carbon nanotubes could be used for immobilizing C C bond hydrolase, thus provide an alternative matrix for immobilization. References [1] Karajanagi S S, Vertegel A A, Kane R S, Dordick J S. Langmuir, 24, 2: [2] Feng W, Ji P J. Biotechnol Adv, 211, 29: 889 [3] Lee Y M, Kwon O Y, Yoon Y J, Ryu K. Biotechnol Lett, 26, 28: 39 [4] Tan H Sh, Feng W, Ji P J. Bioresour Technol, 212, 115: 172 [5] Wang L, Wei L, Chen Y, Jiang R R. J Biotechnol, 21, 15: 57 [6] Asuri P, Bale S S, Pangule R C, Shah D A, Kane R S, Dordick J S. Langmuir, 27, 23: [7] Pedrosa V A, Paliwal S, Balasubramanian S, Nepal D, Davis V, Wild J, Ramanculov E, Simonian A. Colloids Surf B, 21, 77: 69 [8] Seah S Y K, Ke J Y, Denis G, Horsman G P, Fortin P D, Whiting C J, Eltis L D. J Bacteriol, 27, 189: 438 [9] Li Ch, Hassler M, Bugg T D H. ChemBioChem, 28, 9: 71 [1] Zhou H, Qu Y Y, Kong Ch L, Wu Y G, Zhu K, Yang J, Zhou J T. Biotechnol Lett, 212, 34: 117 [11] Zhou H, Qu Y Y, Bu Y B, Li X L, Kong Ch L, Ma Q, Zhang Q, Zhang X W, Zhou J T. ChemPlusChem, 212, 77: 293 [12] Bradford M M. Anal Biochem, 1976, 72: 248 [13] Arnold K, Bordoli L, Kopp J, Schwede T. Bioinformatics, 26, 22: 195 [14] Mashiach E, Schneidman Duhovny D, Andrusier N, Nussinov R, Wolfson H J. Nucleic Acids Res, 28, 36: W229 [15] Torres Salas P, del Monte Martinez A, Cutiño Avila B, Rodriguez Colinas B, Alcalde M, Ballesteros A O, Plou F J. Adv Mater, 211, 23: 5275 [16] Qu Z H, Wang G J. J Nanosci Nanotechnol, 212, 12: 15 [17] Basha S Y, Palanivelu P. World J Microbiol Biotechnol, 2, 16: 151 [18] Qu Y Y, Zhou H, Li A, Ma F, Zhou J T. Appl Microbiol Biotechnol, 211, 89: 655 [19] Zuo G H, Huang Q, Wei G H, Zhou R H, Fang H P. ACS Nano, 21, 4: 758 单壁碳纳米管对 C C 键水解酶 BphD 的固定化性能 沈娥, 曲媛媛 *, 周豪, 孔春雷, 马桥, 张旭旺, 周集体大连理工大学环境学院, 工业生态与环境工程教育部重点实验室, 辽宁大连 摘要 : 采用修饰与未修饰单壁碳纳米管固定 C C 键水解酶 BphD, 并对固定化酶的相对活性 稳定性 重复使用性进行了考察. 结果表明, 未修饰单壁碳纳米管固定的 BphD 相对活性为游离态的 52.5%, 其热稳定性和在变性剂中的稳定性均有所提高, 且重复使用 1 次仍可保持初始活力的 9%. 修饰单壁碳纳米管固定的 BphD 相对活性可达 99.7%, 但其稳定性没有明显提高. 同源模建及分

8 73 SHEN E et al. / Chinese Journal of Catalysis 34 (213) 子对接分析结果显示, 未修饰的单壁碳纳米管对 BphD 亚基之间的联系可能存在干扰作用, 从而对其活性产生影响. 关键词 : C C 键水解酶 ; 单壁碳纳米管 ; 固定化 ; 分子模拟 ; 物理吸附 ; 共价结合 收稿日期 : 接受日期 : 出版日期 : * 通讯联系人. 电话 : (411) ; 电子信箱 : qyy7@126.com 基金来源 : 国家自然科学基金 (517854, ). 本文的英文电子版由 Elsevier 出版社在 ScienceDirect 上出版 ( 1. 前言 作为新兴的一维纳米材料, 单壁碳纳米管 (SWCNT) 具有独特的力学 电学和化学性能, 因而广泛应用于纳米刻蚀 纳米光电催化 纳米传感以及高解析度成像等领域 [1]. 近年来, 研究者陆续将辣根过氧化物酶 脂肪酶等成功地固定在 SWCNT 上, 表明 SWCNT 在生物催化剂固定领域同样具有应用价值 [2~5] [6]. Asuri 等首先采用物理吸附法在 SWCNT 上固定豌豆过氧化物酶和糜蛋白酶, 负载量均可达到 6 mg/g, 但固定化后酶二级结构的改变导致其活性的保留程度存在很大差异. 随后, 他们又利用共价修饰法将辣根过氧化物酶固定在 SWCNT 上, 负载量为 1.3 mg/g, 并能保留 4% 左右的初始活性. Pedrosa 等采用共价修饰法将对硫磷水解酶分别固定在单 [7] 壁和多壁碳纳米管上, 并制备用于检测对硫磷的生物传感器. 结果表明, 经过共价修饰的固定化对硫磷水解酶存放 7 个月后, 仅损失 25% 的活性. 尽管利用 SWCNT 固定酶研究可以获得较高的负载量和保留活性, 但仍然存在着一些亟待解决的问题 : (1) 利用 SWCNT 固定化的酶种类较少, 应探索 SWCNT 作为更多类型酶固定化载体的可行性 ; (2) 酶分子与 SWCNT 通过物理吸附或共价作用结合对于负载量和保留活性的具体影响 ; (3) SWCNT 与酶具体结合位点及结合机制尚不明确. C C 键水解酶来源于芳香化合物间位开环降解途径, 催化儿茶酚类物质间位开环产物中高能 C C 键的水解反应, 是整个芳香化合物间位开环降解途径中的限速步骤 [8]. 近年来的研究表明, C C 键水解酶具有独特的催化混杂性, 即利用同一活性位点能催化多种反应, 如 C C 键 C O 键 S O 键的水解以及 C C 键的可逆合成, 因而, 具有潜在的生物催化应用前景 [9]. 本课题组从菌株 Dyella ginsengisoli LA-4 中克隆得到了一段编码 C C 键水解酶的基因 bphd, 并在大肠杆菌中成功地异源表达 [1]. 本文利用 SWCNT 对 C C 键水解酶 BphD 进行固定化, 比较直接物理吸附和共价修饰后得到的固定化酶的活性和稳定性, 并利用分子模拟手段说明两者活性保留的差异. 2. 实验部分 2.1. 材料本文所用菌株为含有携带编码 C C 键水解酶 BphD 基因 bphd 的重组大肠杆菌 BL21 (DE3). 培养基为 Lysogeny broth (LB) 培养基 (g/l): NaCl 1., 蛋白胨 1., 酵母浸粉 5., ph = 7.. SWNT 购自深圳市纳米港有限公司, 其长度为 5~15 μm, 直径小于 2 nm, 纯度 >95%; 2-(N- 吗啉基 ) 乙磺酸 (MES) N- 羟基丁二酰亚胺 (NHS) 1-(3- 二甲基氨丙基 )-3- 乙基碳二胺 (EDC) 购自国药集团化学试剂有限公司 ; 卡那霉素 异丙基硫代半乳糖苷 (IPTG) 对硝基苯基苯甲酸酯等购自百灵威公司, 其余试剂均为国产分析纯 菌株的培养将 2 μl 菌液转接至含 25 ml LB 培养基的锥形瓶 (5 ml) 中, 加入 3 mg/l 的卡那霉素, 置于摇床中培养 12 h 作为种液 (3 C, 15 r/min). 取 2 ml 种液转接至含 ml LB 培养基的锥形瓶 (25 ml) 中, 并加入 3 mg/l 的卡那霉素, 在 37 C, 15 r/min 的条件下培养至 OD 6 =.4~.6, 加入 1 mmol/l IPTG 诱导 3 h 后, 在 4 C, r/min 条件下离心 5 min 收集菌体, 并用磷酸盐缓冲溶液清洗两次, 放置于 8 C 冰箱中保存备用 碳纳米管的修饰第一步修饰 ( 羧基化 ): 称.1 g SWCNT 溶于 4 ml 浓硫酸和浓硝酸的混合物 (V:V = 3:1) 中, 在 3 C 超声 5 h. 冷却至室温后小心将其倒入去离子水中, 混合均匀进行抽滤, 将滤饼放入去离子水中超声振荡. 将振荡后的混合液离心 (22 r/min, 2 min), 收集沉淀于 5 C 真空干燥 8 h. 干燥后的固体采用红外光谱 (FT-IR) 仪表征特征官能团. 第二步修饰 ( 羧基活化 ): 将上述 SWCNT 加入 MES 缓冲溶液 (5 mmol/l, ph = 6.2) 中, 加入等体积的 NHS 4 mmol/l. 超声 3 min 后加入终浓度为 2 mmol/l 的 EDC ( 固体 ), 反应混合液置于摇床中振荡 (3 C, 15 r/min, 1 h). 利用.22 μm 膜过滤洗涤除去多余的 NHS 和 EDC. 得到的 SWCNT 在超纯水中超声 2 min 后悬起, 离心之后置

9 SHEN E et al. / Chinese Journal of Catalysis 34 (213) 于真空干燥箱中 8 C 下干燥 8 h 后, 室温下储存备用. 修饰后的 SWCNT 命名为 CM-SWCNT BphD 的纯化从 8 C 冰箱中取出菌体, 用磷酸盐缓冲溶液悬浮, 得到的悬浮液利用超声破碎仪破碎 4 min, 得到的样品离心 2 min (22 r/min, 4 C), 上清液即为 BphD 的粗酶液. 纯化采用 Ni 柱亲和层析法, 65% 咪唑梯度 (325 mmol/l) 下洗脱得到的组分即为 BphD 纯酶液, 蛋白纯度采用聚丙烯酰氨凝胶电泳确定 BphD/ 碳纳米管的固定化实验中采用 3 ml 反应体系进行固定化最优 ph 摸索, 其中 BphD 浓度为 3 μg/ml, 碳纳米管含量为.5 mg. 缓冲溶液 ph 值分别为 6, 7, 8, 9 和 1. 将反应体系放入摇床中振荡 (15 r/min, 3 C) 使其混合均匀, 利用紫外 - 可见分光光度法在 45 nm 下测定固定化前后反应产物的吸光度值变化. 实验中将不同浓度的 BphD 分别用 SWCNT 和 CM-SWCNT 进行固定化. 两组反应体系中 BphD 浓度分别为 15, 3, 6 和 9 μg/ml, 在 5 ml 锥形瓶中加入初始浓度为 1 mg/ml 的碳纳米管材料, 一定量 BphD 及相应体积 ph = 7. 的磷酸盐缓冲溶液, 最终反应体系为 6 ml. 将反应体系放入摇床中振荡 (15 r/min, 3 C). 每隔一定时间取出 6 μl 样品, 离心 (12 r/min, 2 min) 后测定上清液中蛋白的残余量, 利用紫外分光光度计法对 BphD 固定化过程进行吸附动力学的考察, 并计算不同蛋白浓度下固定化酶的相对活性及负载量 [11]. 蛋白质定量采用 Bradford 法 [12] 固定化 BphD 特性考察实验本实验反应体系包括 4 μl 固定化酶, 4 μl 底物, 192 μl 磷酸盐缓冲溶液 (ph = 7.). 单位酶活的定义为每分钟生成 1 μmol/l 对硝基苯酚所需的酶量. 为考察温度对固定化酶活性的影响, 分别将固定化酶和游离酶置于恒温水浴锅中, 温度分别设定为 3, 4, 5, 6 和 65 C. 15 min 后取出, 待其冷却至室温后进行活性的测量. 对于固定化酶热稳定性考察, 分别取 1 组固定化酶和游离酶置于 6 C 恒温水浴锅中, 每隔 5 min 取样, 待其冷却至室温进行活性的测量. 以初始的活性为 %, 计算活性随时间的变化趋势. 对于变性剂对固定化酶活性的影响, 分别取 4 μl 固定化酶和游离酶于 192 μl 不同浓度盐酸胍溶液中放置 15 min 后, 进行活性的测量. 盐酸胍溶液浓度分别为,.2,.4,.8 和 1.6 mmol/l. 对于固定化酶重复使用性的考察, 将固定化酶离心 (12 r/min, 5 min), 弃去上清液, 用同样体积的磷酸缓冲溶液 (ph = 7.) 将固定化酶悬起, 测量每次悬浮后固定化酶的活性. 重复上述过程多次. 以初始活性为 %, 计算活性随时间的变化趋势 同源模建 分子对接及共价结合位点预测利用在线服务器对蛋白质进行三维结构的构建. 通过 访问 SWISS-MODEL 服务器, 在自动模式 (Automated Mode) 中输入通过 NCBI 数据库中得到的菌株 Dyella ginsengisoli LA-4 中 C C 键水解酶 BphD 的蛋白质序列, 构建 C C 键水解酶 BphD 的三级结构模型 [13]. 碳纳米管与 BphD 的分子对接利用 Patchdock 以及 Firedock 进行 [14]. 针对共价结合, 采用 [15] Pamela 等的方法进行计算, 首先利用在线程序 PROPKA 计算具有氨基的残基 (N- 端残基, Lys 和 Tyr) 的 pka 值, 再利用下式计算 LIG Re 值 : LIG Re = 1 (ph pka) 当 LIG Re <.1 时, 该氨基被认为是低活性的 ; 当.1< LIG Re <1 时, 该氨基被认为是中等活性的 ; 当 LIG Re>1 时, 该氨基被认为是高活性的. 3. 结果与讨论 3.1. 碳纳米管的修饰及表征结果为了实现蛋白质分子在碳纳米管上的共价结合, 需要对碳纳米管进行修饰, 在其表面添加经过 NHS 活化的羧基, 修饰过程原理如图 1 所示. 图 2 为修饰前后碳纳米管的 FT-IR 谱. 由图可见, 经混酸处理的碳纳米管在 172 cm 1 附近均有一个比较强的峰, 归属于羧基的 C=O 伸缩振动峰, 表明经过混酸修饰后的碳纳米管表面含有羧基 [16]. 经 NHS/EDC 处理的碳纳米管在 1645 cm 1 左右有一个比较强的峰, 表明材料表面的羧基得到活化, 对应的 C=O 伸缩振动峰受新形成的 N O 键的影响而出现红移 BphD 固定化最优 ph 值 BphD 与 SWCNT 结合时, 主要作用力为静电作用. 蛋白质所带电荷受 ph 值影响, 因此 ph 值是二者结合过程中的重要影响因素. 本文考察了不同 ph 条件下固定化酶的负载量及相对活性, 其中相对活性随 ph 值的变化趋势见图 3. 可以看出, 两种固定化酶的最适 ph 值与游离酶类似, 均在 ph = 7 时具有最高的比活力. 在 ph 值较低 (ph = 6) 时具有较高的相对活性 ; 而 在 ph = 7.5~8. 时, 比游离酶

10 732 SHEN E et al. / Chinese Journal of Catalysis 34 (213) 具有更高的相对活性. 当 ph = 7 时, BphD 在两种碳纳米 管上都能达到 % 的吸附率. 因此, 下文选取 ph = 7 的 缓冲溶液来进行固定化 BphD 固定化过程的吸附动力学 对不同浓度的 BphD 与碳纳米管的固定化过程进行 了吸附动力学考察, 测定了蛋白质吸附百分比随时间的 变化曲线, 如图 4 所示. 由图 4(a) 可见, 对于 复合体系, 当蛋白浓度较低 (15 或 3 μg/ml) 时, 吸附 2 h 后可以达到平衡, 吸附率几乎为 %; 而蛋白浓度较高 (6 或 9 μg/ml) 时, 3 h 后吸附可以达到 平衡, 吸附率为 6% 左右. 由图 4(b) 可见, 复合体系所呈现的吸附趋势与 的基本相同, 但吸附达平衡所需时间均有 所增加. 这可能是因为共价修饰后的 SWCNT 与蛋白质 分子是特异性的结合. 通过计算吸附达到平衡时不同蛋白浓度下 SWCNT 和 CM-SWCNT 的负载量可知, 当蛋白浓度为 9 μg/ml 时, 负载量达最大值. 因此, 以下实验蛋白浓度均为 9 μg/ml. 此时 复合体系负载量为 255 mg/g, 相对活性为游离酶的 52.5%, 而 复合 体系分别为 518 mg/g 和 99.7%. 可见 BphD 与 CM-SWCNT 结合时, 负载量及相对活性均高于. 这可 能是因为 BphD 与 CM-SWCNT 属于特异性结合, 使得活 性得以较高程度的保留. 而共价修饰可提高固定化酶负 [6] 载量的实验结果也与 Asuri 等的研究结果类似 固定化 BphD 扫描电子显微镜 (SEM) 结果 利用 SEM 对碳纳米管及碳纳米管 -BphD 复合物的表 面形貌进行了表征, 结果示于图 5. 可以看出, 修饰前后 的碳纳米管均以团簇形式存在, 但 CM-SWCNT 的长短 更不均一 ; 而在修饰前后的碳纳米管 -BphD 复合物中, 除了碳纳米管外, 还有部分无定形的固定颗粒, 可能为 磷酸盐的结晶体 固定化 BphD 透射电子显微镜表征结果为了进一步观察碳纳米管上蛋白质分子的分布情况, 利用透射电镜对单壁碳纳米管 - 蛋白质复合物的形貌进行了表征. 图 6(a) 和 (b) 分别为修饰前后的 SWNT, 其形貌并未观察到明显的变化. 比较图 6(c) 和 (d) 可以发现, 当 BphD 与 CM-SWCNT 结合时, 表面的蛋白质覆盖的更为致密, 这也与 CM-SWCNT 上 BphD 的负载量更多的实验结果相吻合 固定化 BphD 的基本特性考察了 BphD 与碳纳米管两种复合物的基本特性, 包括温度 ph 值及变性剂盐酸胍对其活性的影响 固定化酶的热稳定性和重复利用性 温度的影响图 7 为温度对固定化酶相对活性的影响. 由图可知, 5 C 时游离 BphD 和 复合物的相对活性均达最高 ; 而 复合物在 4 C 时最高. [17] Basha 等利用苯基琼脂糖固定了来源于真菌的转移酶, 发现固定化后的转移酶催化反应的最适温度比游离酶的低 1 C, 其原因可能是固定化后酶的构象发生改变, 使得温度较高时对产物的抑制作用增加, 从而导致固定化酶最适温度下降 ph 值的影响图 8 是两种固定化酶 (ph = 7 时制得 ) 在不同 ph 值时的相对活性. 可以看出, 和 BphD-CM- SWCNT 仍在 ph = 7 时活性达最高 ; 但经过固定化后的 BphD 在较低 ph 值下的活性保留明显要高于游离的 BphD. 其中当 ph = 6 时, 具有 62% 的相对活性, 远远高于游离酶的 25%. 这表明通过固定化后酶对 ph 的耐受能力得到强化, 可能与酶所处的微环境有关 酶的热稳定性图 9 为游离酶和固定化酶的热稳定性. 由图可知, 在 6 C 恒温孵化 4 min 后, 酶的相对活性均有所下降. 25 min 后游离 BphD 的活性完全丧失 ; 复合物的热稳定性较游离酶有所提高 ; 但 复合物热稳定性不如游离酶. 这可能与 最适温度下降的原因类似 变性剂的影响盐酸胍是一种常用的蛋白质变性剂, 通过夺取蛋白质分子中的结合水使得蛋白质变性. 图 1 为盐酸胍浓度对游离 BphD 以及两种固定化 BphD 相对活性的影响. 由图可知, 在不同浓度盐酸胍下 复合物的相对活性较游离酶均有所提高 ; 当盐酸胍浓度高达 1.6 mmol/l 时, 复合物仍能保持 33% 的初始活性, 而 复合物与游离酶的类似, 几乎完全丧失 酶的重复使用性图 11 为两种固定化酶分别重复使用 1 次的相对活性. 由图可知, 复合物重复使用 1 次后, 其相对活性仍保持在 9% 以上 ; 而 复合物重复使用 5 次后, 其活性完全丧失, 固定化酶效果不理想. 通常来说, 通过共价修饰的酶一般具有比游离酶

11 SHEN E et al. / Chinese Journal of Catalysis 34 (213) 更高的稳定性, 但是利用修饰后碳纳米管固定的 BphD 稳定性降低. 这可能是由于 ph = 7 时 BphD 可供与活化 [15] 羧基结合的游离氨基较少有关. 根据 Torres-Salas 等 的方法, 我们计算了 BphD 的 N- 端残基 Lys 和 Tyr 的 pka 值, 发现只有 N- 端残基所带的氨基在 ph = 7 时具有中等 活性 (.1< LIG Re <1), 主链上 Lys 和 Tyr 的氨基均活性较 低. 这可以解释 重复使用性不如 的原因 同源模建与分子对接 通过现有的实验手段, 我们无法直接观察到酶与纳 米材料的结合位点. 因此, 通过引入分子模拟手段, 可以对结合位点进行合理的预测. 首先, 通过 SWISS- MODEL 构建了菌株 LA-4 中 BphD 的三级结构模型, 采用的模板蛋白为 Burkholderia xenovorans LB4 中的 BphD, 两者的序列一致性达 88.73%, 可以用于同源模建, 构建结果如图 12 所示, 表明 BphD 可能为同源四聚体. 采用 UCLA-DOE 服务器 ( 的 PROCHECK 对模型进行质量检测, PROCHECK 中的 Ramachandran Plot 结果显示, 模建结构蛋白的主链结构合理 : 主链二面角 φ, ψ 落于中心区域和可接受区域的残基占 9.7%, 可应用于进一步的分子对接 [18]. 利用 Patchdock 以及 Firedock 获得的对接结果表明, BphD/ 碳纳米管复合物中可能的结合模式都是远离活性位点的. 因此, 复合物活性降低可能不是由于 SWCNT 进入活性位点内部所致, 如图 13(a) 所示. 而当抑制剂结合在亚基界面, 可以通过干扰亚基间的相互作用而抑制酶的活性. 因此, 我们对亚基之间的相互 作用残基进行了识别, 以亚基 B, C 为例, 两者之间通过两对盐桥进行结合 (Glu717-Lys438, Lys72-Glu435). 如图 13(b) 所示, 当 SWCNT 的直径为.5 nm 时, 它可以深入到两个亚基的界面处, 破坏这两对盐桥的作用, 从而导 [19] 致 BphD-SWNT 活性降低. Zuo 等利用分子动力学模拟研究了碳纳米管对双色氨酸功能域的毒性效应, 得到可能的作用机制是碳纳米管进入活性口袋, 并阻止正常的配体与双色氨酸功能域的结合. 在本文中, 由于 BphD 的活性位点深埋在疏水内核之中, 因而不太可能通过直接作用于活性位点而使得酶活性降低. 需要说明的是, CM-SWNT 与 BphD 的结合位点并不能通过对接进行预测, 因为共价作用要远远强于分子对接所考虑的分子间相互作用力. 正如上文所述, CM-SWNT 与 BphD 最有可能的结合位点是 BphD 的 N- 末端游离氨基. 由于两者之间的结合力并不是特别强, 因此共价结合对于 BphD 本身构象的影响不大, 从而保留了大部分的活性. 4. 结论 修饰单壁碳纳米管固定化的 BphD 能更好地保持游离酶的活力, 其相对活性可达 99.7%; 而未修饰单壁碳纳米管固定化的 BphD 仅能保留 52.5% 的相对活性, 但其稳定性较游离酶有所提高, 而修饰单壁碳纳米管固定化的变化不大. 分子对接结果表明, 未修饰单壁碳纳米管固定化的 BphD 活性的降低可能与单壁碳纳米管破坏了 BphD 亚基间相互作用有关. 由此可见, 单壁碳纳米管可用作生物催化剂的载体, 为酶固定化载体的选择提供新的思路.