Remarkably enhanced activity and substrate affinity of lipase covalently bonded on zwitterionic polymer-grafted silica nanoparticles

Accepted Manuscript Remarkably enhanced activity and substrate affinity of lipase covalently bonded on zwitterionic polymer-grafted silica nanoparticles Chunyu Zhang, Xiaoyan Dong, Zheng Guo, Yan Sun PII: DOI: Reference:

S0021-9797(18)30187-5 https://doi.org/10.1016/j.jcis.2018.02.039 YJCIS 23311

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

1 December 2017 8 February 2018 12 February 2018

Please cite this article as: C. Zhang, X. Dong, Z. Guo, Y. Sun, Remarkably enhanced activity and substrate affinity of lipase covalently bonded on zwitterionic polymer-grafted silica nanoparticles, Journal of Colloid and Interface Science (2018), doi: https://doi.org/10.1016/j.jcis.2018.02.039

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Remarkably enhanced activity and substrate affinity of lipase covalently bonded on zwitterionic polymer-grafted silica nanoparticles

Chunyu Zhang,a Xiaoyan Dong,a Zheng Guo,b Yan Sun*,a a

Department of Biochemical Engineering and Key Laboratory of Systems

Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300354, China b

Department of Engineering, Aarhus University, DK-8000 Aarhus, Denmark

∗ Corresponding author: Tel.: +86 22 27403389; Fax: +86 22 27403389. E-mail address: [email protected] (Y. Sun)

1

ABSTRACT: Enzymes are promising biocatalysts for the production or degradation of chemical compounds, but low stabilities of free enzymes restrict their industrial applications. Therefore, development of effective immobilization methods to maintain or increase enzyme activity and stability remains a challenge. In this work, a novel support made of zwitterionic polymer-grafted silica nanoparticles (p-SNPs) was fabricated and Candida rugosa lipase (CRL) was covalently attached onto the p-SNPs. The zwitterionic polymer was a product of the reaction between poly(maleic anhydride-alt-1-octadecene) and N,N-dimethylenediamine and contained a cetane side chain. The hydrolytic activity, reaction kinetics, thermal stability, pH tolerance, storage stability and reusability of the immobilized CRL (SNPs-CRL) were investigated. It revealed that the specific activity of SNPs-CRL was two to four times higher than the free CRL in the temperature range of 25 ℃ to 60 ℃. It is considered mainly due to the interfacial activation effect regulated by the cetane side chains of the zwitterionic polymer. Kinetic studies revealed remarkable improvement of the enzymatic reaction efficiency by the immobilized enzyme as demonstrated by the significant increases of the reaction rate constant and the decreases of Michaelis constant (i.e., increase of enzyme-substrate affinity) determined with two different substrates (p-nitrophenyl acetate and p-nitrophenyl palmitate). Moreover, the immobilization improved the enzyme stabilities and SNPs-CRL displayed good reusability. Finally, the SNPs-CRL was proven to catalyze the hydrolysis of methyl mandelate to produce mandelic acid at an activity three times higher than the free enzyme. The results indicate that zwitterionic polymers deserved further development for enzyme immobilization.

Keywords: zwitterionic polymer; silica nanoparticles; lipase; immobilization; interfacial activation; enzymatic hydrolysis

2

1. Introduction As biocatalysts with high specificity and efficiency, enzymes have been widely used in the processing of food, medicine, cosmetics, biofuels and many other products [1-4]. However, industrial applications of natural enzymes are often hampered by their drawbacks including high costs, instability and difficulty in recycled use [5,6]. One of the most effective methods for overcoming these disadvantages is immobilization [7,8]. Enzymes can be immobilized onto/inside solid supports through physical entrapment or encapsulation [9,10], adsorption [2,11] and covalent attachment [12,13]. The structure and properties of support materials are essential for keeping high performance of immobilized enzymes. Among various supports, nanostructured materials are attracting increased attention because of their high specific surface area, high surface reaction activity and high mechanical strength [14,15]. Therefore, various nanomaterials, such as nanofibers [16], carbon nanotubes [17], cellulose nanocrystals [1] and nanoparticles [18,19], have been used for enzyme immobilization. However, problems remain for some of these materials. For example, the low biocompatibility of some nanomaterials often led to rapid denaturation and sharply decreased the activity of surface-bound enzymes [11,15,20]. To increase the biocompatibility of supports for enzyme immobilization, it is necessary to modify the supports with hydrophilic materials containing functional groups. Zwitterions contain both anionic and cationic groups, resulting in positive and negative charge balance and overall electrical neutrality. Due to the unique charging structure, zwitterions can bind large amounts of water molecules to form a hydration layer via ionic solvation [21,22]. This hydration layer based on electrostatic interaction endows zwitterionic materials with excellent biocompatibility and resistance to nonspecific protein adsorption [23,24]. Moreover, previous studies have indicated that zwitterionic polymers are capable of stabilizing the spatial structure of proteins and maintaining protein stability and bioactivity [25-27]. However, recent applications

of

zwitterion-based

materials

such

as

phosphorylcholine,

poly(sulfobetaine) and poly(carboxybetaine) have mostly focused on their biomimetic antifouling properties, widely studied in biomaterials engineering, drug delivery and 3

biosensors [28-30]. Although efforts were made to explore the effects of zwitterionic compounds on enzyme activity and stability [25,26,31], there are still no reports on covalent immobilization of enzymes on zwitterionic polymer-modified supports. Therefore, this work is proposed to design a novel support, a zwitterionic polymer-grafted nanoparticle, for enzyme immobilization. This design is expected to develop a biocompatible nanoparticle surface that affords three-dimensional spatial immobilization of enzyme molecules for enhanced enzyme activity and stability. The zwitterionic polymer used for nanoparticle modification was that recently synthesized by the ring-opening reaction between poly(maleic anhydride-alt-1-octadecene) and N,N-dimethylenediamine, denoted as p(MAO−DMEA), which was shown to possess good antifouling ability, hemocompatibility and biocompatibility [32]. Because p(MAO−DMEA) has abundant carboxylic groups, it is convenient to covalently bond amino

groups

of

proteins

by

reactions

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

and

via

the

conventional

N-hydroxysuccinimide

(EDC/NHS) chemistry [27,33,34]. Therefore, p(MAO−DMEA)-modified materials would be promising for enzyme immobilization because of their dual-functional properties. The polymer was grafted onto silica nanoparticles (SNPs). We used an important and widely studied enzyme, lipase from Candida rugosa [7,35,36], to test the new support. The support was characterized by thermal gravimetric analysis and transmission electron microscopy. The properties of immobilized lipase in terms of hydrolytic activity, enzymatic kinetics, thermal stability, pH tolerance, storage stability and reusability were investigated. In addition, the immobilized enzyme was further used in the hydrolysis of methyl mandelate to produce mandelic acid, a valuable chemical that has been widely employed in the medical community for antibacterial treatment and as building blocks in organic synthesis [37,38]. The results obtained in the research are expected to provide a reliable basis for further exploration of the immobilization material and method.

2. Materials and methods 2.1. Materials 4

Lipase from Candida rugosa (CRL, type VII), N-hydroxysuccinimide (NHS), 3-aminopropyltriethoxysilane

(APTES),

N,N-dimethylethylenediamine

(DMEA),

p-nitrophenyl bovine

serum

palmitate albumin

(p-NPP),

(BSA)

and

poly(maleic anhydride-alt-1-octadecene) (MAO, average Mn 30,000-50,000) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bradford reagent, gum Arabic and TritonX-100 were obtained from Dingguo Biotech (Tianjin, China). Sodium dihydrogen phosphate, dibasic sodium phosphate and tris (hydroxymethyl) aminomethane (Tris) were of analytical grade from Sangon Biotech (Shanghai, China). Citric acid and trisodium citrate dihydrate were of analytical grade from Kewei (Tianjin, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was obtained from Alfa Aesar (Ward Hill, MA, USA). 2-Morpholino-ethane sulfonic acid (MES) was obtained from Genview (Calimesa, USA). Silica nanoparticles, dicyclohexyl carbodiimide (DCC) and p-nitrophenyl acetate (p-NPA) were from Aladdin (Los Angeles, USA). Methyl mandelate and mandelic acid were from Tianjin Heowns Biochemical Technology (Tianjin, China). Acetonitrile was of guarantee reagent from Tianjin Hope Biotech (Tianjin, China). Tetrahydrofuran (THF), chloroform (CHCl3), isopropanol and other reagents were of analytical grade from Yuanli Chemical Technology (Tianjin, China).

2.2. Synthesis of zwitterionic polymer p(MAO−DMEA) was synthesized using the method described previously [32]. A schematic for the preparation of the polymer is presented in Fig. S1. MAO and DMEA were mixed in THF at 10 wt% MAO and 40 wt% DMEA. After reaction at room temperature for 20 min, the resultant polymer was collected by centrifugation at 5000 g for 30 min, washed three times with THF, and finally dried under vacuum and stored at 4 ℃ for further use. The molecular structure of p(MAO−DMEA) was characterized by 1H NMR spectra with a Varian Inova 500 MHz NMR spectrometer (Varian, California, USA) at 500MHz.

2.3. Amine-functionalization of silica nanoparticles and p(MAO−DMEA)-grafting 5

Surface modification of SNPs was performed as described by Liu et al. [39]. SNPs were first activated with 1 mol/L hydrochloric acid for 36 h and then rinsed with water and ethanol. Next, 10.0 g of the activated SNPs was dispersed in 200 mL of ethanol via ultrasonication for 20 min, followed by adding 4 mL of APTES in the mixture. This amine-functionalization reaction was continued at 60 ℃ by refluxing for 12 h. The functionalized product (SNPs-NH2) was successively washed with ethanol and deionized water for next step reaction. Before grafting reactions (Fig. 1), the carboxylic groups of p(MAO−DMEA) were activated with DCC/NHS [34]. A certain amount of the polymer was added to 20 mL of equimolar DCC/NHS solution (90% v/v chloroform and 10% v/v ethanol). The reaction solution was stirred gently for 2 h at room temperature. After activation, 0.5 g of SNPs-NH2 was added into the above solution and ultrasonically dispersed for reaction in a shaking incubator at 25 ℃ and 170 rpm for 12 h. Finally, the product (p-SNPs) was separated from the mixture by centrifugation at 7425 g and successively washed with N,N-dimethylformamide, ethanol and deionized water.

2.4. Lipase immobilization Chemical reactions of lipase immobilization are showed in Fig. 1. A predetermined amount of the support (100 mg) was dispersed in 2.5 mL of 50 mM MES buffer (pH 5.5). An EDC/NHS solution in 2.5 mL of 50 mM MES buffer (pH 5.5) were added to obtain final EDC and NHS concentrations of 50 mg/mL and 15 mg/mL, respectively. The mixture was kept for stirring at room temperature for 40 min. The suspension was then centrifuged and the precipitate was washed three times with 50 mM phosphate buffer (pH 7.4) to remove excess EDC, NHS and by-product (urea). To prepare immobilized lipase, the EDC/NHS-treated p-SNPs were mixed with 10 mg/mL lipase solution in 50 mM phosphate buffer (pH 7.4) and the mixture was incubated at 4 ℃ for 12 h. The resulting CRL-immobilized p-SNPs (SNPs-CRL) were recovered by centrifugation at 7425 g and washed three times with the same phosphate buffer. The loading amount of lipase on SNPs-CRL was determined by measuring the initial and the final concentrations of the protein in the lipase solutions 6

and the washings following the Bradford method using BSA as the standard [40]. The immobilization efficiency (%) of CRL was calculated from the ratio of immobilized enzyme to added enzyme in the immobilization. The specific activity of immobilized lipase was calculated from the ratio of immobilized enzyme activity to the immobilized protein. Then, the relative specific activity (RSA) was defined as the ratio of the specific activity of immobilized lipase to that of free one.

2.5. Characterization of SNPs-NH2, p-SNPs and SNPs-CRL Thermal gravimetric analyses (TGA) of SNPs-NH2 and p-SNPs were performed using a thermal analyzer TGA Q50 V20.13 Build 39 (TA Instruments-Waters LLC, Shanghai, China). The samples were heated from 25 ℃ to 800 ℃ at 10 ℃/min under a nitrogen atmosphere. SNPs before and after immobilizing lipase were observed by transmission electron microscopy (TEM) (JEM-2100F, Tokyo, Japan). A drop (20 μL) of a well-dispersed nanoparticle suspension was kept on a carbon support film (Beijing Xinxing Braim Technology, Beijing, China) and dried at ambient atmosphere.

2.6. Assays of lipase activity Two different substrates, p-NPA and p-NPP, were used in CRL activity assays. With p-NPA, the activities of free and SNPs-CRL were determined by the colorimetric method [41] with minor modifications. With p-NPA as the substrate, the substrate solution was composed of 0.5 mL of acetonitrile containing 10 mM p-NPA and 2.4 mL of 20 mM phosphate buffer (pH 7.0). The reaction was commenced by addition of 0.1 mL of enzyme sample and incubated at 37 ℃ for 10 min. After that, 3 mL of the phosphate buffer was added to dilute the reactants and the absorbance value was measured immediately at 400 nm. One unit of enzyme activity (U) was defined as the quantity of lipase that liberated 1 μmol p-nitrophenol (p-NP) per minute under the assay condition. Lipase activity assay using p-NPP as substrate followed that reported previously [42]. In brief, 30 mg of p-NPP was dissolved in 10 mL of isopropanol by sonication 7

and then added with stirring to 90 mL of 50 mM phosphate buffer (pH 7.0) containing 1.25% Triton X-100 and 0.1% gum Arabic, which resulted in a final p-NPP solution of 790 μM. In the activity assay, 50 μL of an enzyme sample was added to 2.95 mL of the substrate solution, mixed and afterwards was monitored at 400 nm to detect the production of p-NP. Definition of one unit (U) lipase activity is the amount of enzyme that released 1 μmol p-NP by the hydrolysis of p-NPP per min under the assay condition. Effects of temperature on CRL activity were determined using the two substrates in the temperature range of 25-60 ℃. The highest specific activity of the free enzyme at certain temperature was taken as 100%.

2.7. Determination of kinetic parameters The enzymatic reaction kinetics with free and immobilized CRLs was investigated by measuring the initial catalytic velocity as a function of substrate concentration. The assays were performed at 37 ℃ in 20 mM phosphate buffer (pH 7.0) with p-NPA and 25 ℃ in 50 mM phosphate buffer (pH 7.0) with p-NPP. In the reactions, protein (enzyme) concentrations of free and immobilized CRLs were kept the same, 0.602 and 0.307 μg/mL, respectively, or 1.0×10-5 and 0.51×10-5 mM, respectively, for the hydrolyses of p-NPA and p-NPP, as calculated from the molecular weight of CRL (60 kDa) [43].

2.8. Stability investigations Thermal stabilities of free and immobilized CRLs were determined by incubating the same amount of free and immobilized enzymes in 20 mM phosphate buffer (pH 7.0) at 50 ℃ for 3 h. Aliquots of the enzyme samples were withdrawn at different time intervals and the residual activities were measured as described above. The thermal stability was also investigated by incubating free and immobilized CRLs in 20 mM phosphate buffer (pH 7.0) for 20 min in the range of 25-60 ℃. Afterwards, the residual activity of the enzyme samples was determined at 37 ℃ with p-NPA as substrate. To examine the stabilities at different pH values, a certain amount of SNPs-CRL 8

or free lipase was incubated at 25 ℃ in different buffer solutions (50 mM citrate buffers for pH 4.0 and 5.0; 50 mM phosphate buffers for pH 6.0, 7.0, and 8.0; 50 mM Tris–HCl buffer for pH 9.0) for 4 h and the residual activity of the enzyme sample was measured using the two substrates as described above. In the above stability assays, the enzyme activity before an incubation was appointed as 100% for representing the residual activities. Finally, long-term storage stability was tested by keeping the free and immobilized CRLs in 20 mM phosphate buffer (pH 7.0) at 4 ℃ for 10 weeks. The residual activity was compared to the initial enzyme activity defined as 100%.

2.9. Reusability In the investigation of the reusability of the immobilized lipase, it was repeatedly used to catalyze the hydrolysis of p-NPA. The hydrolysis reaction was carried out in a thermostatic water bath at 37 °C and 170 rpm. After 10 min reaction, samples of the reaction mixture were withdrawn to determine the enzyme activity and the remaining mixture was allowed to react for additional 20 min before being centrifuged. The recovered immobilized CRL was washed twice with 20 mM phosphate buffer (pH 7.0) and applied in the next reaction cycle with a fresh substrate solution.

2.10. Enzymatic hydrolysis of methyl mandelate The hydrolysis of methyl mandelate was performed

by adding 7.0 mg of

immobilized CRL or 15.8 μg of free CRL to 1 mL of 50 mM phosphate buffer (pH 7.0) containing methyl mandelate (final concentration 5-50 mM) at 37 °C under continuous stirring. The degree of hydrolysis was analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) with a Waters C18 reversed-phase column (Waters, Milford, MA, USA) connected to an Agilent 1100 (Agilent Technologies, Santa Clara, CA) and UV detection at 230 nm. In the RP-HPLC, 20 μL of samples were injected and eluted at 1.0 mL/min with a mobile phase composed by 20% (v/v) acetonitrile and 80% (v/v) of 10 mM ammonium acetate buffer at pH 3.2. Retention times for mandelic acid and methyl mandelate were 3.2 and 7.5 min, 9

respectively. One unit of activity was defined as the amount of enzyme needed for produce one μmol of mandelic acid per hour. Activity was determined for at least three times with a maximum conversion of 10–20%, and data are reported as average values with standard deviations.

3.

Results and discussion

3.1. Characteristics of p(MAO−DMEA), SNPs-NH2 and p-SNPs Fig. S2 shows the NMR spectra of the zwitterionic polymer. The peaks at 2.27 and 0.90 ppm corresponded to the methyl protons held by a nitrogen atom and the terminal methyl protons of the alkyl chain, respectively. The data are consistent with those reported in the literature [32]. TGA plots of weight loss versus temperature (Fig. S3) provides an estimate of p(MAO-DMEA) quantity grafted on SNPs-NH2 surface. A weight loss of 2.39% at temperatures below 125 ℃ was due to the removal of physically adsorbed water molecules on surface of SNPs [44]. The further decline from 125 to 260 ℃ resulted from the thermal decomposition of 3-aminopropyl groups. Thereafter, a sharp decrease in weight (19.3%) over a temperature range from 260 to 660 ℃ was observed in p-SNPs, which was attributed to the decomposition of p(MAO-DMEA). In contrast, SNPs-NH2 showed a mass loss of 2.31% over the same temperature range. This means that p(MAO-DMEA) accounted for 17.0% of the mass of p-SNPs. The grafting density of the polymer was thus estimated to be 0.20 g/g SNPs, and the density of carboxylic groups was estimated to be 0.47 mmol/g SNPs. Fig. S4 shows the TEM images of SNPs-NH2, p-SNPs and SNPs-CRL. The aggregative appearance could be clearly observed in all cases, which was mainly due to the aggregation-prone nature of the nanoparticles [45,46].

3.2. Remarkably enhanced specific activity of immobilized CRL Table 1 provides the conditions for lipase immobilization and the preliminary properties of the immobilized enzyme preparations. It can be seen that both the loaded protein and activity of immobilized CRL obtained in lower protein concentrations 10

(0.09 and 0.18 mg/mL) increased with increasing the initial protein concentration. The specific activities of SNPs-CRL-1 and SNPs-CRL-2 were about twice that of the free enzyme. This could be explained by the unique properties of lipase as well as zwitterionic polymer on which the enzyme was covalently bonded. Lipases are serine hydrolases and catalyze both the hydrolysis and formation of ester bond [35,47]. The active sites in most lipases are covered by an α-helix structure, composed of amino acids with amphiphilic properties, namely “lid” or “flap” [36,43]. The hydrophobic internal face of the “lid” interacts with the hydrophobic protein chains surroundings the active center [48]. In the presence of a hydrophobic interface, the “lid” could shift from a closed and inactive structure to an open and active structure, the so called interfacial activation [49-51]. In the present immobilized CRL, there should be hydrophobic interactions between the hydrophobic cetane side chains of p(MAO-DMEA) (Fig. S1) and the hydrophobic domains around the lipase active site, leading to the stabilization of the opening of the molecular lid over the active site of CRL, which favored the active site accessibility to substrates [49]. On the other hand, p(MAO-DMEA) is a zwitterionic polymer with a large amount of tertiary amino groups and carboxyl groups. The positive and negative charges might regulate the conformational flexibility of the enzyme, which might be helpful to accelerate the induced-fit of enzyme to substrate, resulting in improved catalytic activity of the immobilized enzyme. In contrast, polymers with only positive or negative charges tend to interact with the opposite charges on the native conformation of the enzyme protein, disrupting the native conformation and therefore decreases in activity for enzymes immobilized onto cationic or anionic-functionalized carriers were often observed [52-54]. However, it can be seen from Table 1 that the specific activity of immobilized CRL became lower than free lipase when the initial protein concentration was increased to 0.89 mg/mL, which resulted in over two times higher loaded protein on the support than SNPs-CRL-2. It is considered that the high loaded protein was likely to result in the crowding or agglomeration of enzyme molecules onto the surface of p-SNPs, or in other words, multilayer immobilization of CRL on the support (Fig. 2). 11

This would result in a steric hindrance effect that could limit the mass transfer of substrate, and consequently the reduction of apparent catalytic activity [18,55]. More importantly, the multilayer bonding of protein molecules to those already bonded on the surface of p-SNPs led to the inaccessibility of the outer enzymes to contact with the zwitterionic polymer, and thereby failed to be activated by the hydrophobic interactions (Fig. 2). Thus, it is of importance to control modification conditions so as to achieve an optimal enzyme load that displays a high specific activity. In the work, SNPs-CRL-2 suited to this criterion and was selected for detailed investigations on the enzymatic properties, reaction kinetics and stability of the immobilized enzyme. Fig. 3 shows a comparison of the relative activities of free and immobilized CRLs as a function of temperature. Here, the relative activities for free and immobilized CRLs were defined as the relative percentages to the maximal free lipase activity obtained at the optimal temperature. For p-NPA hydrolysis, the maximum specific activity of free CRL was observed at 45 ℃, while it was at 35 ℃ for the immobilized CRL (Fig. 3a). This was different from the results reported in literatures, in which immobilizations often resulted in a right-shift in the optimum temperature [47,55]. The activation effect of the zwitterionic polymer-modification would be the primary cause, because temperature influences hydrophobic interactions [56-58]. In the p-NPP hydrolysis (Fig. 3b), however, both the free and immobilized CRLs exhibited the maximal activities at 50 ℃. Additionally, the immobilized CRL kept high activities over a wide high temperature range. Then, the relative specific activity of immobilized CRL to that of free CRL as a function of temperature is represented for the two substrates (Fig. S5). It can be seen that the ratios were over unity in the entire temperature range; it was as high as over 4 for p-NPP at 25 ℃ and decreased with increasing temperature. Overall, the activation effect of CRL immobilization on the zwitterionic polymer was less significant with p-NPA as substrate, and it almost vanished at temperatures higher than 50 ℃. This means

that

the

activation

effect

on

the

immobilized

CRL was

more

temperature-sensitive with the small substrate p-NPA, which led to the presence of optimal temperature at a lower value (Fig. 3a). p-NPP (Mw, 377.52) has longer 12

hydrocarbon chains than p-NPA (Mw, 181.15) and the long hydrophobic chains might facilitate the substrate binding to the enzyme by hydrophobic interactions, resulting in the higher hydrolytic activities with p-NPP as substrate (Fig. S5). Generally, enzyme immobilization via amino groups could enhance its structural rigidity and lead to higher activation energies for enzyme molecules to bind substrates and to adjust conformations [55]. However, the activation effect of the hydrophobic side chains on the zwitterionic polymer would greatly change the active site structure of the enzyme, and it is evident that the substrate p-NPP was more suited to the activation than p-NPA at a wide temperature range.

3.3. Catalytic kinetics Fig. S6 shows the kinetic data of the free and immobilized CRLs for the hydrolysis of p-NPA and p-NPP. As seen in Fig. S6a, for both the free and immobilized CRLs, the enzymatic reaction rate displayed a decrease at higher p-NPA concentrations, indicating the presence of substrate inhibition at high p-NPA concentrations. The substrate inhibition effect for immobilized CRL was weaker than for the free CRL. Similar results were reported by Pimentel et al. [59] for CRL immobilized on ferromagnetic azide polyethyleneterepthalate. Therefore, the kinetics were analyzed at the low substrate concentrations with the Michaelis-Menten equation [60],

v

Vmax [ S ] K m  [S ]

(1)

where v is the reaction rate (mM/s) at the substrate concentration [S] (mM), Vmax the maximum reaction rate (mM/s) and Km the Michaelis constant (mM). Because of the low solubility of p-NPP, the reaction could not be done at concentrations higher than 3.0 mM p-NPP. The kinetic parameters (Km and Vmax) were determined by the Lineweaver-Burk plot (Fig. S7) [61],

1 Km 1 1    v Vmax [ S ] Vmax

(2) 13

The reaction rate constants (kcat) with free and immobilized CRLs were calculated from,

kcat 

Vmax [E 0 ]

(3)

where [E0] is enzyme concentration (mM) in the reaction medium, which were 1.0×10-5 and 0.51×10-5 mM, respectively, for the hydrolyses of p-NPA and p-NPP. The kinetic parameters obtained fitting the experimental data to Eq. (2) are listed in Table 2. It can be seen from the table that the Vmax value of SNPs-CRL was 1.6 times greater than that of free enzyme for the hydrolysis of p-NPA, and 3.6 times for the hydrolysis of p-NPP. At the same time, the Km value of the enzyme decreased after immobilization; the Km ratios for the SNPs-CRL to free CRL was 0.59 for p-NPA and 0.82 for p-NPP. The kcat increased by 1.6-fold and 3.6-fold after immobilization, respectively, for the hydrolysis of p-NPA and p-NPP. The corresponding kcat/Km values were improved 2.7-fold for p-NPA and 4.4-fold for p-NPP. These changes in kinetic constants indicate the remarkable enhancement of enzymatic reaction efficiency due to the significant increases of enzymatic activity and enzyme-substrate affinity after immobilization. Besides, the Km value was smaller for the longer chain substrate (p-NPP), consistent with the literature data [47,62], indicating that p-NPP has higher affinity for the enzyme than p-NPA does. The higher affinities of the substrates for the immobilized CRL than for free lipase could be explained as follows. First, the enzyme was covalently attached to the zwitterionic polymers coupled on the particle surface, so the diffusive mass transfer resistance was minimized [45,63]. This means that mass transfer-mediated Km increase, which was often observed for immobilized enzymes, was minimized. Second, the zwitterionic polymer is of high biocompatibility and hydrophilicity, so it could affect the hydration degree of enzyme surface and create a local environment that suited the interactions between the enzyme molecules and the substrates [26]. Third and most importantly, the hydrophobic side chains of p(MAO-DMEA) could induce the “lid” to open and increase the accessibility of the substrate toward the active site of the immobilized lipase, as observed in immobilization of Pseudomonas 14

cepacia lipase on mesoporous silica [49]. The kinetic parameters of the free and immobilized CRLs are compared to literature data with p-NPP as substrate, as listed in Table 3. The decrease in Vmax values and increase in Km values were generally due to protein structure changes, active site hindrance effects as well as diffusive mass transfer limitations by the supports used for immobilization [11,64-66]. In the case that both the Vmax and Km of immobilized lipases were smaller than those of free enzymes [1,12,61], it was considered due to the conformational changes of the immobilized enzymes, which resulted in the higher accessibility of the substrate to the active site of immobilized lipase. In this work, the most favorable behavior, the increase in Vmax and decrease in Km occurred after immobilization. In the literature, only immobilized lipase on gigaporous polystyrene microspheres showed similar results [67], probably because the support shares a common nature with the present work on surface hydrophobicity.

3.4. Stability and reusability of immobilized CRL Fig. 4 shows thermal stabilities of free and immobilized CRLs at 50 ℃. It is observed that the thermal stability of immobilized CRL was much better than the free one. Moreover, Fig. 5 presents thermal stabilities of free and immobilized CRLs as a function of temperature. The activity losses for the free and immobilized CRLs increased with increasing temperature, but the temperature tolerance of immobilized CRL was higher than the free one in the entire temperature range. Fig. 6 is a comparison of pH tolerances of free and immobilized CRLs as a function of buffer pH. The immobilized CRL maintained high residual activity (>74% for p-NPA (Fig. 6a) and >85% for p-NPP (Fig. 6b), while the activity loss of free CRL significantly increased with pH at pH>6 and the residual activity was 10% and 56% at pH 9.0 when using p-NPA and p-NPP as substrates, respectively. Fig. 7 shows the storage stabilities of free and immobilized CRLs. The activity of SNPs-CRL decreased more slowly than that of the free CRL. After 70 days of storage, SNPs-CRL retained 90% of its original specific activity whereas the corresponding value for free CRL was only 52%. 15

The enhanced lipase stability after immobilization could be attributed to the covalent attachment itself as well as the material used for immobilization. Under harsh conditions such as high temperature, acidic and basic solutions, and long-term storage, conformational transitions of enzyme molecules would occur, leading to the exposure of more hydrophobic patches to the enzyme surface, and finally to the unfolding and inactivation of the enzyme. Covalent attachment of an enzyme to solid supports could enhance the enzyme rigidity and improve the conformational stability, preventing conformational transitions of the enzyme and protecting it from unfolding [12,64,68,69]. In addition, the structure of the zwitterionic polymer used in this work would be beneficial in the stabilization of the enzyme due to the following reasons. First, compared with neat ionic compounds with only one type of charge, zwitterionic polymer is electrostatically neutral and has negligible electrostatic effect on the bonded enzyme, which is conducive to the conformational stability of the protein [70,71]. Second, the tertiary amino and carboxyl groups in p(MAO-DMEA) could dissociate into cations and anions respectively in a broad pH range, which could reduce the negative effects of acidity and alkalinity on the lipase conformation to a certain extent and then improve pH tolerance of the immobilized enzyme. Third, the hydrophobic side chains of p(MAO-DMEA) could bind to the hydrophobic patches of the enzyme, thus increasing the hydration degree at the enzyme surface and stabilizing the enzyme structure [72,73]. Therefore, it is considered that the zwitterionic polymer used for immobilization contributed greatly to the stability increase. Fig. 8 presents the reusability of SNPs-CRL. It is seen that SNPs-CRL maintained around 50% of the initial activity after nine consecutive enzymatic reactions. Two causes might lead to the activity decrease in the recycling. One would be the substantial activity decrease as observed in Figs. 4 to 7, and the other would be the detachment of enzyme molecules from the support in repeated use [65,68].

3.5. Enzymatic hydrolysis of methyl mandelate The as-prepared SNPs-CRL was also employed as an efficient biocatalyst for 16

hydrolysis of methyl mandelate. As listed in Table 4, increasing methyl mandelate concentration led to the increase in hydrolysis efficiency. By comparing the hydrolytic activity data, it is evident that the immobilized form of the enzyme was more potent. The activity of SNPs-CRL was about threefold higher than that of the free enzyme in the range of substrate concentration studied. The result was similar with the above two substrates, p-NPA and p-NPP.

4. Conclusions In this work, a zwitterionic polymer-grafted silica nanoparticle support was developed for CRL immobilization via covalent attachment. The as-prepared SNPs-CRL exhibited 2 to 4 times increase in specific activity as compared to free CRL when using p-nitrophenyl acetate and p-nitrophenyl palmitate as substrates. The activity improvement of the immobilized CRL was considered due to the interfacial activation by the zwitterionic polymer with long hydrocarbon side chains (cetane groups) that could interact with the enzyme to let the lipase “lid” shift from a closed and inactive structure to an open and active structure. Kinetic studies with the two substrates further proved that the immobilization resulted in significantly higher enzymatic reaction efficiency and enzyme–substrate affinity than the free enzyme. It was previously shown by various researches that enzyme immobilization resulted in reduced catalytic activity and affinity to the substrate molecules due to the conformational changes or blocking of the active sites of the enzymes [52,53,66]. Our results demonstrated that the zwitterionic polymer was capable of increasing both the enzyme activity and the enzyme–substrate affinity of the immobilized lipase. In addition, SNPs-CRL showed distinctly higher stability in comparison with its free counterpart, similar to those reported for immobilized enzymes onto/inside other supports [12,50,64,68]. Finally, SNPs-CRL was successfully applied for the hydrolysis of methyl mandelate with threefold higher activity than the free lipase. Therefore, it is convincing that the zwitterionic polymer, p(MAO-DMEA), and possibly many other materials of similar structures, are of great potential for development as biocompatible supports for biocatalyst immobilization. 17

Acknowledgments This work was funded by the National Natural Science Foundation of China (Nos. 21561162005, 21236005 and 21621004).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/xxxxxx

18

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21

Biophys.

Caption to Figures: Fig. 1. Schematic for the preparation of zwitterionic polymer-grafting onto the support and lipase immobilization. Fig. 2. Schematic representation of CRL immobilized on zwitterionic polymer-grafted silica nanoparticles at lower and higher CRL loads. The “lid” opening and closing of CRL is illustrated in the low panel. Fig. 3. Effect of temperature on the activities of free and immobilized CRLs. The substrates were (a) p-NPA and (b) p-NPP. Fig. 4. Thermal stabilities of free and immobilized CRLs in phoshate buffer (pH7.0) at 50 ℃. The substrates were (a) p-NPA and (b) p-NPP. Fig. 5. Thermal stabilities of free and immobilized CRLs as a function of temperature. Activity was determined using p-NPA as substrate. Fig. 6. pH tolerances of free and immobilized CRLs. The substrates were (a) p-NPA and (b) p-NPP. Fig. 7. Storage stability of free and immobilized CRLs at 4 ℃. Activity was determined using p-NPA as substrate. Fig. 8. Stability of the immobilized CRL in repeated use. p-NPA was the substrate for determination of lipase activity.

22

23

24

25

26

27

28

29

30

Table 1. Results of lipase immobilization. CRL

Initial protein

Loaded

Immobilization

Lipase activity

Specific activity

preparation

concentration (mg/mL)

protein (mg/g)

efficiency (%)

(U/g support) a

(U/mg protein) a

SNPs-CRL-1

0.09

1.43±0.50

43.4±5.0

16.8±4.2

12.1±1.4

2.07±0.33

SNPs-CRL-2

0.18

2.43±0.18

34.6±1.9

33.6±10.5

15.9±3.8

2.36±0.26

SNPs-CRL-3

0.89

5.28±1.00

15.7±2.7

21.1±3.1

4.01±0.23

0.88±0.19

a

RSA (-)

The activities of the enzyme samples were assayed by using p-NPA as substrate in 20 mM phosphate buffer (pH7.0) at 37 ℃.

31

Table 2. Kinetic parameters of the free and immobilized CRL for the two substrates p-NPA Free

a

p-NPP

Immobilized

Free

Immobilized

Vmax ×104 (mM s-1)

5.16

8.11

4.14

15.1

Km (mM)

11.4

6.75

2.48

2.04

kcat (s-1)

51.6

81.1

81.2

296

kcat/Km (s-1 mM-1)

4.5

12

33

145

Vmaxi/Vmaxf a

1.6

3.6

Kmi/Kmf a

0.59

0.82

Subscripts ‘i’ and ‘f’ represent immobilized enzyme and free enzyme, respectively.

32

Table 3. Kinetic parameters for the enzymatic reactions involved in this work and comparison with literature data. Enzyme

Vmax

Km

Free CRL

4.14×10-4 b

2.48

Immobilized CRL

15.1×10-4 b

2.04

PCL

0.246 b

37.9

PCL@MCNC

0.123 b

12.4

Free lipase

46.4 c

0.45

Immobilized lipase

26.2 c

1.08

ANL

0.157 b

28.7

ANL@PANI/Ag/GO-NC

0.103 b

15.8

Free CALB

10.5×10-4 b

14.5

PGMA-CALB

2.72×10-4 b

1.57

Free LCS

1.49 b

1.37

LCS@DPMS

1.03 b

2.26

Free lipase

46.4 c

0.45

Immobilized lipase

21.2 c

1.43

Vmi/Vmf

Kmi/Kmf

Reference

3.6

0.82

This work

0.50

0.33

[1]

0.57

2.4

[11]

0.66

0.55

[12]

0.26

0.11

[61]

0.69

1.7

[64]

0.46

3.2

[65]

33

a

Free lipase

3.46 c

0.51

IL-S1

3.19 c

0.92

lipase from B. cepacia

11.7×10-4 b

0.44

PST-300-lipase

14.6×10-4 b

0.40

0.92

1.8

[66]

1.2

0.91

[67]

a

p-NPP was the substrate for determination of the kinetic parameters.

b

Unit in mM s-1.

c

Unit in U mg-1.

34

Table 4. Hydrolysis of methyl mandelate catalyzed by lipase preparations from Candida rugosa at pH 7 and 37 ℃ Methyl mandelate

Free CRL

Immobilized CRL RSA (-)

concentration (mM)

a

Activity

a

Activity

a

5

0.20±0.05

0.57±0.02

2.85

10

0.35±0.01

1.01±0.03

2.89

20

0.40±0.01

1.17±0.05

2.93

50

0.43±0.03

1.34±0.05

3.12

Specific activity was defined as μmol/(h·mg protein).

35

Graphical abstract

36