exfoliated bentonite nanohybrids as highly efficient and recyclable biocatalysts in hydrolytic reaction

Journal of Molecular Catalysis B: Enzymatic 132 (2016) 41–46

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Novel enzyme/exfoliated bentonite nanohybrids as highly efficient and recyclable biocatalysts in hydrolytic reaction Jie Cao a , Yimin Li a,∗ , Ni Tu a , Ying Lv a , Qinqin Chen b , Huaping Dong a,∗ a b

College of Chemistry and Chemical Engineering, Shaoxing University, 508# Huancheng West Road, Shaoxing, Zhejiang 312000, PR China College of Medicine, Shaoxing University, 508# Huancheng West Road, Shaoxing, Zhejiang 312000, PR China

a r t i c l e

i n f o

Article history: Received 2 April 2016 Received in revised form 23 June 2016 Accepted 28 June 2016 Available online 29 June 2016 Keywords: Bentonite Enzyme immobilization Exfoliation Nanohybrid Recyclability

a b s t r a c t Bentonite exfoliation liberated the interlayer surface with large area for immobilization of enzyme at high enzyme loading. Upon the electrostatic attraction between positively charged bovine pancreatic lipase (BPL), Yarrawia lipolytica lipase (YLL), trypsin and negatively charged surface of unilaminar bentonite, three nanohybrids with a stably card-like structure named as BPL-B, YLL-B and TB were facilely produced. These nanohybrids showed higher activities in hydrolysis as 115.2% (BPL-B), 154.8% (YLL-B) and 138.2% (TB) of that of the correspondingly free enzyme. Furthermore, BPL-B, YLL-B and TB retained 80%, 90% and 82% of each original activity after repeated use for 10, 12 and 8 times, respectively. The recyclable and active nanohybrids are expected to be widely applied as industrial catalysts in the future. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Nowadays, enzymatic catalysis has been regarded as a green and sustainable technology in fine chemistry [1], pharmaceuticals [2], energy [3] and environmental technology [4]. However, the disadvantages of non-recycle, instability and prone to deactivation hampers the application of enzymes [5]. These drawbacks can be overcome through enzyme immobilization [1]. Nonetheless, enzyme immobilization generally leads to the decline of enzymatic activity arising from steric hindrance, enzyme structure distortion and mass transfer resistance on the support [1,6,7]. Thus, how to keep or improve the activity of immobilized enzymes has always been a difficult task for enzyme immobilization technology. During the past decades, nanobiocatalysts based on integration of enzymes on nanomaterials, such as silica nanoparticles [8,9], polymer biomimetic materials [10], nano-inorganic minerals [11] and self-assembled nanomaterials [12], have provided new options for enhancing the performance of immobilized enzymes in different applications, but their preparative processes are always difficult and time-consuming. Bentonite, a natural and biocompatible mineral with a lamellar structure [13], is also a good candidate for enzyme immobilization relying on the advantages of large specific surface area, strong

∗ Corresponding authors. E-mail addresses: [email protected] (Y. Li), [email protected] (H. Dong). http://dx.doi.org/10.1016/j.molcatb.2016.06.016 1381-1177/© 2016 Elsevier B.V. All rights reserved.

mechanical strength and adsorptive capacity, thermal stability and chemical inertness [14–16]. However, due to the larger dimension of enzyme than the interlayer spacings of original and modified bentonites (about 1.2–2.2 nm), previous studies mainly focused on the adsorption of enzymes onto the external surfaces of bentonites [17–19], which only occupied a small part of their total surface areas, causing low enzyme loading and activity. Furthermore, the weakly adsorbed enzymes were easily dissociated from bentonite’s external surface under stirring, resulting in the decrease of catalytic stability. Although the intercalation of excessive polymers could enlarge the interlayer spacing of layered clays for accommodation of protein molecules [20,21], the resulting steric hindrance and diffusion limitation would easily reduce enzymatic performance of immobilized enzymes. Therefore, preparing an efficient and recyclable immobilized enzyme on bentonite is too difficult to be accomplished. In this study, by using a green and facile method constituting of bentonite exfoliation and subsequent enzyme adsorption, three highly efficient nanohybrids of enzyme/exfoliated bentonite with a stably card-like structure were produced. The enzyme loading efficiencies, catalytic activities and stabilities were measured. The mechanisms for the high enzyme loading and superiorly catalytic performance of these immobilized enzymes were also clarified.

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J. Cao et al. / Journal of Molecular Catalysis B: Enzymatic 132 (2016) 41–46

Scheme 1. Schematic illustration for preparation of the enzyme/exfoliated bentonite nanohybrid.

2. Experimental 2.1. Materials Bovine pancreatic lipase (BPL), Yarrawia lipolytica lipase (YLL), trypsin and N-benzoyl-l-arginine ethyl ether (BAEE) were purchased from J&K chemicals in China. Bovine serum albumin, p-nitrophenyl palmitate (p-NPP) were purchased form SigmaAldrich. Sodium bentonite was obtained from Sanding technology Co. Ltd. in Zhejiang province. All other chemicals were of analytical grade.

by acetate buffer for three times before sample injection. The measurements for every sample were conducted in triplicate by using a Nano ZS90 particle-sizer (Malvern, Worcestershire, UK). 2.5. Enzymatic activity assays The hydrolysis of p-NPP by BPL and YLL, and the hydrolysis of BAEE by trypsin were used as the model reactions to determine the activities of these free and immobilized enzymes. The details were shown in Supplementary material. 2.6. Reusabilities of nanohybrids

2.2. Exfoliation of bentonite A certain amount of sodium bentonite was added into deionized water at concentration of 20 mg mL−1 . A 250 mL conical flask containing 100 mL of this suspension was placed in an untrasonic apparatus at room temperature, then the suspension was stirred at 2000 rpm and treated by ultrasonication at 40 KHz simultaneously. Therein, each run of ultrasonication treatment was conducted for 0.5 h and paused for 10 min subsequently. After 5 cycles, the whole exfoliation process was over, and the obtained exfoliated suspension was diluted into 1 mg mL−1 of colloidal solution with addition of acetate buffer (pH 4.0, 10 mmol L−1 ) for further use. 2.3. Preparation of nanohybrids on bentonite Three enzymes including BPL, YLL and trypsin were separately dissolved in acetate buffer (pH 4.0) at concentration of 1 mg enzyme mL−1 , and 50 mL of each enzyme solution was mixed with the exfoliated bentonite suspension (1 mg mL−1 in acetate buffer, pH 4.0) at the same volume. Every mixture was agitated at 200 rpm for 12 h under ambient condition, and three nanohybrids including BPL-B, YLL-B and TB were subsequently prepared after centrifugation (12,000 rpm, 10 min), washing and freeze drying. 2.4. Zeta potential measurement BPL, YLL and trypsin were respectively dissolved into acetate buffer (pH 4.0, 10 mmmol L−1 in ultrapure water) at concentration of 1 mg mL−1 , and the suspension of exfoliated bentonite at the same concentration was also prepared in pH 4.0 acetate buffer (10 mmmol L−1 in ultrapure water). The sample cell was washed

The reusabilities of these nanohybrids of BPL-B, YLL-B and TB were investigated by repetition of the corresponding activity assay as described in Supplementary material (Section 1). Between two consecutive assays, the nanohybrids were collected by centrifugation (10,000 rpm, 5 min) and washed with the buffer used in the activity assay twice. The residual activity was determined by comparison with the first running (activity defined as 100%). 2.7. Catalytic kinetics of nanohybrids and free enzymes The kinetic parameters, Km (mmol L−1 ) and Vmax (U g−1 enzyme min−1 ), of free and immobilized BPL and YLL were calculated from the Michaelis-Menten models via Lineweaver-Burk using varying concentrations of p-NPP from 0.01–0.2 mmol L−1 in the aqueous medium. By the same method, Km (mmol L−1 ) and Vmax (U mg−1 enzyme min−1 ) of free and immobilized trypsin were determined using varying concentrations of BAEE (0.05–0.3 mmol L−1 ). 3. Results and discussion 3.1. Preparation of enzyme/exfoliated bentonite nanohybrids The preparative process for enzyme/exfoliated bentonite nanohybrids were composed of the exfoliation of layered bentonite and the subsequent adsorption of enzyme on the unilaminar bentonite, as shown in Scheme 1. The unilaminar layers of bentonite with the length between 20 and 200 nm were obtained after exfoliation, which were explicitly shown in Fig. 1(A). The layered bentonite showed the diffraction peak (2␪ = 7.30◦ ) referring to its

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Fig 1. (A) TEM of the unilaminar bentonite and (B) enzyme loading on the solid phase and remaining in water phase.

(001) reflection, exhibiting that the interlayer spacing between the layers of bentonite was 1.25 nm (Fig. S1). This layered structure was broken by exfoliation due to the vanishing of diffraction peak at 7.30◦ (Fig. S1), confirming the exfoliation of the unilaminar layers from layered bentonite. In addition, the tyndall effect of exfoliated bentonite suspension also proved the production of unilaminar bentonite layers (Fig. S2). Three enzymes including BPL, YLL and trypsin were subsequently added into the exfoliated bentonite suspension (1 mg mL−1 , pH 4.0) at the same concentration (1 mg enzyme mL−1 ), respectively. After agitation for 12 h under ambient condition, three nanohybrids were produced. Fig. 1(B) showed that 97.2% of BPL, 95.8% of YLL and 96.3% of trypsin were separately loaded onto the nanohybrids of BPL-B, YLL-B and TB, and the exact enzyme loadings were correspondingly determined to be 243.5, 191.6 and 215.8 mg protein g−1 support on BPL-B, YLL-B and TB by Bradford method [22]. These loading efficiencies were much higher than those of enzymes adsorbed on the layered bentonites [15–18], due to the liberation of interlayer surface with larger area between the layers of bentonite than external surface for enzyme adsorption.

Fig. 2. TEM of enzyme molecules on the nanohybrids of (A) BPL-B, (B) YLL-B and (C) TB by negative stain.

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J. Cao et al. / Journal of Molecular Catalysis B: Enzymatic 132 (2016) 41–46

Fig. 3. Activities of free and immobilized enzymes.

To verify the driven force for enzyme immobilization on the unilaminar layers of bentonite, the zeta potential values of unilaminar bentonite and enzymes in the preparative medium (pH 4.0) were measured. The unilaminar bentonite was charged to be −34.8 mv, while BPL, YLL and trypsin were respectively charged as the values of 3.6, 4.2 and 6.7 mv. Therefore, the positively charged enzymes were gently adsorbed onto the negatively charged surface of unilaminar bentonite through electrostatic interaction. Due to the edge of bentonite was charged positively at pH 4 [23], which could bind to the surface of the bentonite to produce the nanohybrid with a stable card-like structure by self-assembly. The TEM images of BPL-B, YLL-B and TB by negative stain [6,24] distinctly confirmed the card-like structure of these nanohybrids (Fig. 2), which was different from the layered structure and protein/clay cluster of nanohybrids on the other inorganic materials such as LDH, phyllosilicate [25–27].

3.2. Activities of nanohybrids Owing to the preference of free enzyme for catalysis in the aqueous solution, and the mass transfer resistance on the support, the activity of immobilized enzyme always tends to be declined. However, the specific activities of three immobilized enzymes were improved to be 227.4 (BPL-B), 339.2 (YLL-B) U g−1 enzyme and 82.9 (TB) U mg−1 enzyme, equaling to 115.2%, 154.8% and 138.2% of that of the corresponding free enzyme (Fig. 3). This improvements were due to high dispersion of enzyme molecules on the bentonite surface (Fig. 2), minimizing the steric hindrance and mass transfer for enzymatic catalysis. By contrast, TEM image of BPL in the reaction medium (Fig. S3) showed that free enzyme molecules were induced to form the multimers with larger dimension by intermolecular interaction [28,29], increasing the steric hindrance and reducing the enzymatic activity. Furthermore, FTIR of these nanohybrids and their corresponding free forms showed the similar band positions and shapes of amide I (1600–1700 cm−1 , due to C O stretching vibration) and amide II (1510–1580 cm−1 , due to the N H bending and C N stretching vibration) peaks (Fig. 4), confirming that the secondary structures of enzymes generally remained unchanged after adsorption on bentonite. The higher intensities of both peaks of three nanohybrids than those of their free counterparts indicated that the catalytic center of enzyme on the support was probably activated to be more open for the access of substrate, by the selective recognition of the predetermined sites of unilaminar bentonite for enzyme molecules through electrostatic interactions, and hydrogen bonds between enzymes and Si-OH (or Al-OH) on the bentonite surface [12,16], enhancing the catalytic activities of these nanohybrids.

Fig. 4. FTIR of (A) BPL and BPL-B, (B) YLL and YLL-B, (C) trypsin and TB.

3.3. Reusabilities of nanohybrids Reusability is another important catalytic performance for immobilized enzyme. Fig. 5 showed that the nanohybrid of BPL-B kept 80% of its initial activity after ten runs for catalysis, and YLL-B even had little activity (≤10% of initial activity) to lose after twelve

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Table 1 Kinetic constants for free and immobilized enzymes. Biocatalysts

Km (mmol L−1 )

Vmax (U g−1 enzyme min−1 )

Vmax /Km b

Free enzymes

BPL YLL Trypsin

0.18 ± 0.02 0.21 ± 0.04 0.26 ± 0.02

47.62 ± 2.50 59.52 ± 3.00 13.05 ± 0.60a

264.56 283.43 50.19c

Nanohybrids

BPL-B YLL-B TB

0.52 ± 0.05 0.72 ± 0.03 1.10 ± 0.07

158.73 ± 5.40 256.41 ± 10.20 56.50 ± 4.00a

305.25 356.13 51.36c

a b c

U mg−1 enzyme min−1 . L kg−1 enzyme min−1 . L g−1 enzyme min−1 .

in previous studies [14–17,19]. This highly structural rigidity of nanohybrids promoted their application in wide ranges. 3.4. Catalytic kinetics of nanohybrids The catalytic kinetics for three nanohybrids and their free counterparts revealed that the maximum reaction rates (Vmax ) of nanohybrids were all higher than that of their free counterparts, as shown in Table 1. Michaelis constant (Km ) of each nanohybrid was higher than that of the corresponding free form, resulting from the lower transfer rate of substrate from bulk solution to the support at light concentration, which was improved gradually as the substrate concentration increasing. In addition, the ratio of Vmax /Km is an important parameter to describe the catalytic efficiency of enzyme. Table 1 showed that the value of Vmax /Km of each nanohybrid was higher that of the corresponding free enzyme, confirming the improved catalytic efficiencies of these nanohybrids by immobilization on bentonite with this method. 4. Conclusions The highly efficient and recyclable nanohybrids on lower-cost bentonite were firstly produced with a facile method in this study. In summary, the improvements in enzyme loading and catalytic performance of these nanohybrids were due to the following factor: (1) the liberation of interlayer surfaces of bentonite with large-area by exfoliation; (2) high dispersion of enzyme molecules with active conformation on the support; (3) protection of enzyme molecules from leakage by the card-like structure for repeated uses. These merits can make the recyclable and active biocatalyst supported on bentonite for a wide range of application. Acknowledgements This work was supported by the National Natural Science Foundation of China (21177088), the Natural Science Foundation of Zhejiang Province, China (LY16B070004), and the Research Project for Public Welfare of Zhejiang Province, China (2015C33229). Appendix A. Supplementary data

Fig. 5. Reusability of nanohybrids of (A) BPL-B, (B) YLL-B and (C) TB.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molcatb.2016.06. 016. Reference:

cycles. TB, which showed the lowest stability among these three nanohybrids, also retained about 82% of the initial activity after eight runs. These operational stabilities of nanohybrids were much higher than those of the other immobilized enzymes on bentonites

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