An Enzyme Net Coating the Surface of Nanoparticles: A Simple and

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An enzyme net coating the surface of nanoparticles: a simple and efficient method for the immobilization of phospholipase D Binglin Li, Jiao Wang, Xiaoli Zhang, and Bin-xia Zhao Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b02192 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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Industrial & Engineering Chemistry Research

An enzyme net coating the surface of nanoparticles: a simple and efficient method for the immobilization of phospholipase D Binglin Li‡, Jiao Wang‡, Xiaoli Zhang*, Binxia Zhao

Dept. of Chemical Engineering, Northwest University, 229 North Taibai Road, Xi’an, 710000, Shaanxi, China

KEYWORDS: immobilization, enzyme net, phospholipase D, nanobiocatalyst, kinetics

ACS Paragon Plus Environment


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ABSTRACT: Phospholipase D (PLD) was immobilized in a simple and effective way by adsorption and precipitation of the enzyme, followed by chemical cross-linking to form an “enzyme net” covering the surface of non-porous silicon dioxide nanoparticles. For catalyzing transphosphatidylation to produce phosphatidylethanolamine (PE), at pH 6.0 and 35 °C (the optimum operational conditions), the specific activity of immobilized PLD reached 15872 U/gprotein, which was approximately 1.15 times higher than the maximum value of specific activity of free PLD (13813 U/gprotein). A kinetic study demonstrates immobilized PLD had increases in catalytic activity and enzymesubstrate affinity. In addition, the thermostability and pH tolerance were significantly enhanced compared with free PLD. The half-life of immobilized PLD was significantly increased from 30 to 70 days at 4 oC (approximately 2.3 times). This novel method has been proven to be suitable for the production of robust biocatalysts.


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1. INTRODUCTION

Enzyme as a highly efficient and green biocatalyst has been widely applied for the production of chemicals, food additives, nutrition, fuels, and polymers.1–6 However, the fragile nature, high cost, and high loadings required for the industrial production limit the application of the free enzyme.7 Therefore, the immobilization of enzymes becomes a requisite for commercial applications of these biocatalysts, since immobilization permits the ease recycling of the enzyme and simplifies the design and performance control of the bioreactors.8–10 Many investigations have been devoted to design a powerful biocatalyst to greatly improve enzyme performance in practical applications.11–14 For example, rigidification of the spatial position of enzymes prevents an aggregated enzyme forming when using anhydrous media or creation of favorable microenvironments surrounding the enzyme has been studied in many literatures.15,16 However, the employment of immobilized enzymes may be accompanied by a reduction of the enzymatic activity due to the change of the unique and native structure of enzymes during immobilization.

The two most widely used approaches for the immobilization of enzymes with a very high volumetric activity is cross-linked enzyme crystals (CLECs)17 or aggregates (CLEAs)18,19. The preparation process with respect to CLECs involves crystallization of the protein, which is never a simple issue even for expert companies. In this sense, the CLEAs preparation is a simplified technology.19,16 But the final particle size depends on various factors such as the stirring speed, the addition rate of precipitant,



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the amount of enzyme used, etc., making the preparation of a fully reproducible biocatalyst a challenging task for non-experts.20 The reuse of biocatalyst prepared by CLEAs is also complex, because the viscosity and the low density of some of the precipitated enzyme makes the collection of the immobilized enzyme difficult in practical applications. Mechanical resistance of CLEAs is another limitation for industrial applications; they are considered too soft for any kind of reactor configuration (except basket reactors).21,22 Aggregation and crystallization may keep the active center fully available, but in the cases that this did not occur, almost full loss of activity may be observed due to the internal diffusional problems. In an extreme example where substrate is a solid (a textile, cellulose), any enzyme buried in the center of aggregations will not be able to contact with the substrate; only the superficial enzymes may reach them.23

Therefore, the use of pre-existing non-porous solids instead of the “non-activity” and fragile core of aggregations of enzymes (Figure S1 in Supporting Information) seems to be the most advantageous solution, it is possible to improve the mechanical resistance, control the final particle size and reduce the difficulty of recovery by regulating the viscosity and density of immobilized enzymes.16 More importantly, the enzyme is immobilized on the outside surface of non-porous carriers, which can minimize internal diffusion problems. Considering that the specific surface area of the non-porous carrier is related to its diameter, the size of the carrier should be controlled in the nanometric scale to obtain a reasonable enzyme loading capacity. Nanoscale silicon dioxide is a kind of inexpensive nanoparticles (NPs) with unique properties


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including large surface area, suitable density, great mechanical properties, exceptional adsorptive affinity for organic macromolecules, etc16,24. Thus, the use of nanoscale silicon dioxide seems to be adequate for the core instead of the “non-activity” and fragile core of aggregations of enzymes. However, one of main challenges for the application of NPs in immobilization technology is that an enzyme, immobilized on the external surface of NPs, may be not protected by carriers and suffer inactivation caused by interfaces, which destabilizes electrostatic, hydrophobic and hydrogen bonds of the enzyme molecule, resulting in the irreversible denaturation of enzymes.25,26,27 From this viewpoint, we want to use the multipoint bond by intra and inter molecular cross-linked to form an “enzyme net” covering the surface of NPs producing a strong rigidification of the enzyme structure to prevent enzyme inactivation.

Phospholipase D (PLD) is chosen to demonstrate the simple immobilization technology and the potential application of immobilized enzymes. PLD catalyzes the interconversion of polar head groups of phospholipids (PLs), by a process called transphosphatidylation. It is widely used for the enzymatic synthesis of naturally less abundant PLs such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylglycerol (PG) from highly abundant ones such as phosphatidylcholine (PC).28–30 Synthesized PLs can be used as emulsifiers, components of cosmetics, medical formulations, food additives, nutrition, and for liposome preparations.31–35 For example, PE plays an important role in heart to prevent cell damage.36 Moreover, PE is an important material for the production of anandamide (N-arachidonoylethanolamine), which is extensively used in the pharmaceutical field.37,38 The immobilized PLD was



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systematically investigated including the process of immobilization and immobilized PLD-mediated transphosphatidylation for the production of PE. Further insight on the kinetics of the catalytic process has been explored. Operational, thermal and storage stabilities of immobilized PLD were also evaluated. The results indicated that this protocol could be successful in the application of PLD.

2.

MATERIALS AND METHODS

2.1. Materials. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Nanoscale silicon dioxide, 30nm of diameter, was purchased from Aladdin Ind. Co., Ltd. (China). Phospholipase D (EC 3.1.4.4), from Streptomyces sp., was purchased from Asahi Kasei Pharma Co. (Tokyo, Japan). PLD was diluted and stored in 0.2 M acetate buffer (1.38×104 U/gprotein, 3.30×10-4 gprotein/mL,4 oC, pH 5.5). All other reagents were of standard laboratory grade.

2.2. Phospholipase D immobilization. The immobilized PLD onto nanoscale silicon dioxide was prepared as follows. 0.65 mL PLD solution (1.38×104 U/gprotein, 3.30×10-4 gprotein/mL, adjusted to pH 8.0 using the 1 M NaOH solution) was mixed with 4.35 mL 0.2 M phosphate buffer (pH 8). 0.1 g of nanoscale silicon dioxide was added. The mixture was incubated at 200 rpm and room temperature for 4 hours. 20 mL water-free ethanol was slowly added into the above mixture to precipitate the enzyme for 20 min under gentle stirring at 0 oC, followed by injecting 0.87 mL of 25% glutaraldehyde and stirring for 1 hour. The resulting precipitate was washed for at least 3 times with acetate


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buffer (0.2 M, pH 6.0) and subjected to centrifugation. These collected immobilized PLD was resuspended and stored in acetate buffer (0.2 M, pH 6.0, 4 oC) with a protein content of 3.30×10-4 gprotein/mL prior to use and detect the transphosphatidylation activity via the method as described in 2.3. Immobilized yield (%) is defined as the difference obtained between the initial mass of the free PLD (g) before the immobilization and the mass of free PLD (g) after immobilization divided by the mass before immobilization (g).

Different precipitants such as methanol, ethanol, acetone, propanol, isopropanol, (NH4)2SO4 and peg6000 were compared to determine the optimal precipitant. 0.9 mL cooled precipitant was added slowly into 0.1 mL free PLD solution (1.38×104 U/gprotein, 3.30×10-4 gprotein/mL) to precipitate the enzyme for 20 min under gentle stirring at 0 oC. Then 19 mL fresh 0.2 M acetate buffer (pH 5.5) was added with stirring. 0.1 mL free PLD solution (1.65×10-6 gprotein/mL) was taken from the above mixture to detect the transphosphatidylation activity via the method as described in 2.3. Different volumes of ethanol were tested to determine the optimal amount of ethanol: 0-28 mL. To evaluate the influence of pH on PLD immobilization, the PLD solution (0.65 mL) where pH was adjusted by 0.2 M acetic acid or 1 M sodium hydroxide, was mixed with different fresh buffers: (i) acetate buffer 0.2 M for pH 5.0, 5.5, and 6.0; (ii) phosphate buffer 0.2 M for pH values of 7.0, 7.5 and 8.0; (iii) borate buffer 0.2 M for pH values of 8.5 and 9.0. To determine the optimum concentration of glutaraldehyde in crosslinking, PLD immobilization was carried out with different amounts of glutaraldehyde to regulate the concentration ranging from 0.6-1.2 %. Different ratios of PLD solution



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to fresh phosphate buffer (0.2 M, pH 8.0) were tested to determine the optimal amount of PLD at the optimum conditions: 0.4-0.7 mL PLD solution (corresponding to 1.322.31 mgprotein/gsilicon dioxide).

2.3. Measurements of free and immobilized PLD activity. The catalytic activity of free

or

immobilized

PLD

was

assayed

respectively

by

PLD-mediated

transphosphatidylation of PC with ethanolamine at the optimum operational condition (Figure S2 of Supporting Information). Free or immobilized PLD solution (0.1 mL, 3.30×10-4 gprotein/mL) was added into 0.7 mL acetate buffer (0.2 M, pH 5.5 for free PLD or 6.0 for immobilized PLD). The concentration of ethanolamine in the above mixture is 50 mM. The reaction was then initiated by adding 1.6 mL diethyl ether containing 10 mM PC, and incubated at 200 rpm and 30 (free PLD) or 35 (immobilized PLD) °C. The concentration of PE released was detected over 30 min to calculate the initial reaction rate (mM/min). One unit (U) of transphosphatidylation activity of PLD was defined as the amount of enzyme that produced 1 μmol of PE per minute under these assay conditions.

2.4. Characterization techniques. The morphology of nanoscale silicon dioxide before and after immobilization of PLD was investigated via a Carl Zeiss SIGMA (ZEISS, Germany) equipped with a field-emission gun operated at 5.0 kV. The size of the particles was calculated from the scanning electron microscopy (SEM) pictures using an average of 100 to 200 particles in almost all cases. Fourier transform infrared (FTIR) analysis was performed on a Frontier FT-IR Spectrometer (PerkinElmer, USA).


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2.5. Characterization of free and immobilized PLD. The immobilized PLD was prepared via the method as described in 2.2. 0.1 mL immobilized PLD solution (3.30×10-4 gprotein/mL) was used in transphosphatidylation as described in 2.3. Initial reaction rates of PE production were respectively measured over temperature ranging from 20 to 40 °C (at pH 6.0) and pH ranging from 4 to 8 (at 35 °C).

The control experiment was carried out using 0.1 mL free PLD solution (3.30×104

gprotein/mL). Initial reaction rates of PE production were respectively measured over

temperature ranging from 20 to 40 °C (at pH 5.5) and pH ranging from 4 to 8 (at 30 °C).

2.6. Determination of kinetic parameters for free and immobilized PLD. The assumptions employed in this work for the single-molecule kinetics were that the effect of ethanolamine could be neglected under the optimum concentration of ethanolamine, which was determined by a preliminary experiment. To determine the respective kinetic parameters of free and immobilized PLD at their optimum operational conditions., initial reaction rates were measured for concentrations of PC (in diethyl ether) ranging from 0 to 16 mM, according to the method as described in 2.3.

The parameters K

and V

were estimated via performing a non-linear

fit of the experimental values to the Michaelis–Menten kinetic model (Eq. (1)):

V=

[ ] [ ]


(1)


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where V (mM/min) is the catalytic reaction rate, V

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(mM/min) is the

maximum rate of the reaction, [S] (mM) is the concentration of PC in diethyl ether, and K

(mM) is the Michaelis–Menten constant.

2.7. Thermal and storage stabilities of free and immobilized PLD. The immobilized PLD was prepared via the method as described in 2.2. 0.1 mL free or immobilized PLD solution (3.30×10-4 gprotein/mL) was incubated at a varied temperature (0-60 °C) for 2 h, and the residual activity was determined by the method as described in 2.3.

The free or immobilized PLD solution (3.30×10-4 gprotein/mL) was stored at 4 °C, and then 0.1 mL was taken at different time to detect the residual activity by the method as described in 2.3.

2.8. Recycling of the immobilized PLD. The immobilized PLD was prepared via the method as described in 2.2. 0.1 mL immobilized PLD solution (3.30×10-4 gprotein/mL) was employed in transphosphatidylation described in 2.3. After one cycle of reaction finished (24 hours), the immobilized PLD was collected by centrifugation and washed for at least 3 times with acetate buffer (0.2 M, pH 6.0). These collected immobilized PLD was resuspended in 0.1 mL acetate buffer (0.2 M, pH 6.0, 4 oC), and used for the next batch under the same conditions.

In fact, the liquid-liquid system used in PLD-mediated transphosphatidylation provides the possibility of the reusability of free enzymes. Therefore, the control experiment was carried out using 0.1 mL free PLD solution (3.30×10-4 gprotein/mL).


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Other conditions were as described in 2.3. After one cycle of reaction finished (24 hours), the free PLD solution was collected, and then the concentration of ethanolamine was replenished to 50 mM. The obtained aqueous solution was used for the next batch.

2.9. High performance liquid chromatography (HPLC) analysis. The phospholipids PC, PE, and PA in the reaction mixture were analyzed via high performance liquid chromatography by using a Simadzu LC-20A HPLC (Tokyo, Japan) equipped with a Chromachem evaporative light-scattering detector (ELSD). The ELSD was operated at an evaporating temperature of 40 oC and a nebulizing temperature of 30 oC with air as the nebulizing gas. The column (purchased from GL Sciences, Inc. Japan) was an InertSustain C18 5 μm (4.8 × 150 mm), which was maintained at 40 oC. The eluting solvent was acetonitrile/methanol (15:85, v/v) with the flow rate of 1.25 mL/min. The relative concentrations of phospholipids were estimated from the peak area of the integrator. Each phospholipid peak was determined by the elution retention time using a standard phospholipid solution.

All data are the average value of triple experiments, and error bars represent standard error of the mean.

3.

RESULTS AND DISCUSSION

3.1. Variables affecting immobilization yield and activity of immobilized PLD. The rationale of immobilization of PLD onto nanoscale silicon dioxide was a three-step process (Figure 1). Firstly, PLD was physically adsorbed on the surface of the nonporous nanoscale silicon dioxide. Secondly, soluble PLD was further precipitated on



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the surface of nanoscale silicon dioxide. Thirdly, PLD deposited was cross-linked to form an “enzyme net” covering the surface of nanoscale silicon dioxide.

Previous studies indicated that the use of precipitants resulted in the loss of enzyme activity. Sometimes this loss of activity is irreversible. Even if the aggregation of PLD was redissolved, the relative enzyme activity could not reach 100 %. Figure 2a shows the activity retention for redissolving the PLD aggregation formed by using various precipitants. As can be seen, ethanol was the excellent candidate for precipitant with highest activity retention, about 91 %. It might be explained that the quaternary structure of PLD might be preserved well when the ethanol was used as precipitant. Similar finding was observed when ethanol was used as the precipitant on papain.39

After ethanol was confirmed as the most suitable precipitant, the amount of ethanol required was investigated systematically (Figure 2b). It was observed that for volumes of ethanol below 20 mL, the immobilization yield and the activity of immobilized PLD increased with the amount of ethanol, while for volumes above this value, a decrease in the activity was observed but the maximum of immobilization yield was not yet reached, indicating that the maximum of immobilization yield was not necessarily consistent with that of the activity of immobilized PLD.24,40 When the value of volume of ethanol was above 24.4 mL, PLD molecules adsorbed on the surface of NPs might be redissolved leading to a decrease in the immobilization yield. The enzyme structure and function is


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strongly dependent upon the dispersion of enzymes on the surface of nanoscale silicon dioxide. Figure 3 shows a schematic of the attachment of PLD of three levels of the surface coverage on nanoscale silicon dioxide. The edges of the approaching protein molecule were at a greater distance from the silica surface in the case of lower levels of the surface coverage, thus, less interaction between proteins for immobilization-induced rigidification, which improved the activity and the stability of structure of enzymes, would be expected, leading to a low catalytic activity.40,41,42 At high levels of surface coverages, the enzyme molecules were likely to be agglomerated leading to a mass transfer limitation for the substrate molecules to reach the active site of the enzyme. 24,43 In addition, the structural perturbation of the enzyme might be another reason of the decrease in the activity of immobilized PLD, because disruption of the hydrogen bonding network associated with the secondary structure is likely to be favored in highly crowded adsorption environments.42 It was noteworthy that the first point in Figure 2b also represented the immobilization of PLD on nanoscale silicon dioxide by adsorption and cross-linking (without precipitation). It was found that the specific activity of immobilized PLD was only 8156 U/gprotein, which was remarkably lower than that with precipitation (15391 U/gprotein). Obviously, precipitant plays a key in PLD immobilized onto nanoscale silicon dioxide with enhanced immobilized yield and catalytic performance.

Interestingly, the immobilized PLD under the optimum conditions showed a more active compared to the free PLD. As shown in Figure 2b, the dash line



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representing the activity of free PLD is provided to guide the eye only. After immobilization, the presence of a water/support interface might enhance the activity of PLD by interfacial activation, which was associated with a conformational change in enzyme leading to an “open” form, with an accessible active site.44,45 Also, transphosphatidylation was carried out in a water immiscible organic solvent-water (two liquid phase/biphasic) system, where PC and PLD needed to interplay in the biphasic interface. When the free enzyme contacted with organic solvent, an aggregation of enzyme might be formed by enzyme precipitation in that medium.41,46 The use of enzyme dispersed after immobilization was an advantage, as there was no possibility for undesired aggregations of enzymes that could make diffusional problems serious (Figure 4). Compared with free PLD, the chemical crosslinking of enzymes produced very high enzyme rigidification and prevented the dissociation of subunits of PLD enhancing the thermal stability of the resulting immobilized PLD47, viz. the immobilized PLD had a higher optimum operational temperature, which can improve the reaction rate of transphosphatidylation. According to the StokesEinstein and collision theory, nanoscale silica dioxide with a very small size can facilitate collisions between the substrate and enzyme to greatly decrease the boundary layer, which was main resistance for substrate availability, between the bulk solution and material surface particularly in biphasic system.48,49 Moreover, a more active enzyme form might be generated during the immobilization.41 The


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result was that an ‘‘improved’’ activity was observed in the immobilized PLD compared to the free PLD.

The effect of pH on the immobilization of PLD over nanoscale silica dioxide was assessed, by varying between 5.0 and 9.0 (Figure 5). Both immobilization yield and activity of immobilized PLD reach the maximum value at pH 8.0. It might be explained that pH 8.0 was close to the isoelectric point of PLD. PLD was easily denatured under the drastic experimental conditions with extreme pH values. After cross-linking, these denatured proteins could not play a role as the biocatalyst for transphosphatidylation.

The different concentrations of glutaraldehyde were tested to investigate the effect of the process of cross-linking on immobilization. As shown in Figure 6, the specific activity of immobilized PLD increased from 11687 to 15298 U/gprotein, with the glutaraldehyde concentration from 0.6 to 1.0 %. Beyond that, a decrease of activity was observed. It might be explained that a higher degree of polymerization of glutaraldehyde was formed on the amino group of the enzyme, which could alter the quarternary structure of the enzyme.50 In addition, glutaraldehyde was also an enzyme inactivation agent, which can penetrate into the protein active site and react with catalytically essential amino acid residues ,ultimately resulting in the loss of enzyme activity.51 Below that optimal glutaraldehyde concentration (1.0 %), fractional enzymes were not cross-linked with others leading to leakages of enzymes from matrix during the process of washing.



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The effect of the amount of PLD used on the immobilization was shown in Figure 7. Enzyme immobilization yield of above 80 % was achieved for all tested. Of all the examined mass ratios of PLD to nanoscale silicon dioxide, the highest activity of immobilized PLD has been obtained at a ratio of 2.15 mgprotein/gsilicon dioxide,

which was considered as the most suitable one for the immobilization.

While for mass ratios above this value, a decrease in activity of immobilized PLD was observed due to diffusional problems caused by multilayers of PLD deposited onto the surface of supports. At low loadings, only a small portion of the surface area was occupied and gave the PLD the opportunity to spread; it was hypothesized that the reduction in the activity of immobilized PLD at low loadings was due to a distortion of the active molecular conformation caused by the PLD maximizing its contact with the support as a result of its high affinity for the support surface, just like shown in Figure 343. The immobilized PLD using the technology of CLEAs was also carried out via the method as described in 2.2 (without nanoscale silicon dioxide addition). The observed specific activity of immobilized PLD was 9945 U/gprotein, which is 1.60 times lower than that prepared by the method proposed in this work (15872 U/gprotein). After one cycle of reaction finish (24 hours), the loss mass of proteins (wt %) caused by leakages of immobilized enzymes prepared by CLEAs (13.5 %) was approximately 10 times higher than that prepared by the method proposed in this work (1.32 %). As expected, the use of pre-existing non-porous solids instead of the “nonactivity” and fragile core of aggregations of enzymes improved significantly the


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enzymatic activity by minimizing internal diffusional problems, and the stability during the operation by enhancing the mechanical resistance.

3.2. Characteristics of immobilized PLD. Figure 8 shows the FT-IR spectra of the pure PLD (solid line), the naked nanoscale silicon dioxide (short-dash line), and PLD covering nanoscale silicon dioxide (dash line). Five main bands were observed in the pure PLD: (i) strong bands at 1520 and 1650 cm-1 attributed to CONH peptide linkage; (ii) a band peaking at 1250 cm-1 corresponding to CN stretching vibration of amines; (iii) weak bands at around 2800 cm-1 attributed to CH bonds; (iv) bands peaking at 1100 cm-1 due to COC groups; (v) typical band corresponding to OH and NH vibrations at 3300 cm-1. They were characteristic peaks of PLD. The band at 1090 cm-1 in the spectrum of nanoscale silica dioxide is typical to OH groups present on adsorbed water or phenol groups. After immobilization, the bands at 1250-1700 cm-1 ascribed to CONH peptide linkage obviously got more intense and the broad band at 3000–3800 cm-1 ascribed to OH vibration could be observed. Also bands at around 2800 cm-1 appeared, which was attributed to the presence of CH vibration from PLD. As a result, the PLD successfully covered the surface of nanoscale silica dioxide.

The SEM analysis of PLD-NPs hybrids shows the nanoscale silica dioxide, which seems to be covered by a layer of a distinct material (Figure 9). According to the SEM pattern (Figure S3 of the Supporting Information), particles of nanoscale silica dioxide were with a mean particle size of 26.6 nm (Figure 10a).



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The particles of PLD-NPs hybrids remained discrete and had the average diameter of 44.5 nm (Figure 10b), indicating a monolayer attachment of PLD on the surface of nanoscale silica dioxide might be likely to be formed considering the dimensions of the PLD (7.6 nm × 4.8 nm × 5.7 nm52). However, the formation of dimer or higher particles caused by cross-linking to bridge aggregated enzymes might not be completely avoided, which could give rise to the mildly broader size distribution. Compared to the nanoscale silicon dioxide prepared by flame synthesis and used as the matrix for immobilization in this paper, monodisperse and uniform-size silica nanoparticles may be better as the matrix for production of biocatalyst. However, so far, such nanoparticles have no value in practical applications due to its very expensive price which is approximately 650 times higher than that of NPs used in this paper. Therefore, non-porous silica nanoparticles prepared by flame synthesis actually have greater practical significance in the production of nanobiocatalysts.

3.3. Properties of immobilized PLD. The transphosphatidylation activities of free and immobilized PLD were measured at various temperatures, ranging from 20 to 40 o

C, and the results are shown in Figure 11a. The optimum operational temperature of

immobilized PLD (35 oC) was raised by 5 oC compared to the free PLD (30 oC). Besides, the immobilized PLD was less sensitive to the change of temperature than the free enzyme as the temperature ranged from 20 to 40 oC. For example, at a temperature of 40 oC the immobilized enzyme exhibited 3.08 mM/min initial reaction rate, which is approximately 1.5 times higher than the free enzyme (2.07 mM/min). Thus, the immobilized PLD exhibited a better thermos-resistance compared to its free one. It was


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explained that a more stable three-dimensional structure of PLD was obtained via crosslinking attached enzymes.

The pH dependence of transphosphatidylation for free and immobilized PLD at pH range between 4.0 and 8.0 was also investigated. As illustrated in Figure 11b, it was seen that a more alkaline pH was more suitable for the immobilized PLD due to the properties of the surface of nanoscale silica dioxide.53,54,55 Initial reaction rate using immobilized PLD of above free PLD was achieved for all test. The range of operational pH of the immobilized PLD (initial reaction rates of immobilized PLD ≥ the maximum initial reaction rate of free PLD) ranged from 5.0 to 7.0. After the immobilization, the matrix may play a role as an ionic exchanger, which may behave as a ‘‘solid’’ buffer, generating a pH inside the biocatalyst bead that may greatly differ from that in the reaction medium41. For nanoscale silica dioxide, the silanol groups (Si-OH) existing in its surface is exactly the ionic exchanger. They can ionize in the presence of water, and in a wide range of pH, SiO- and SiOH2+ coexist on the silica surface56.

3.3. Kinetic properties of free and immobilized PLD. The preliminary experiment was carried out to exclude the effect of rotational speeds on transphosphatidylation for free and immobilized PLD, shown in Figure S4 of the Supporting Information. 200 rpm was determined as the optimal speed. The concentration of ethanol amine was optimized to simplify the kinetic model (Figure S5 of the Supporting Information). When the concentration of ethanol amine was 50 mM, the reaction rate reached the



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maximum value for free and immobilized PLD. And a hypothesis was proposed that the effect of the inhibition of ethanol amine could be neglected when the concentration of ethanol amine was ≤ 50 mM. Therefore, the biochemical reactions in this paper was simplified to a single substrate following Michaelis–Menten kinetics.

The enzymatic kinetic studies were based on the evaluation of the kinetic parameters, K

and V

, obtained from the initial reaction rates of

transphosphatidylation at different PC concentrations, using free and immobilized PLD. The classical Michaelis–Menten model was employed to determine these kinetic parameters. Non-linear adjustment of the Michaelis– Menten equation (Eq. (1)) to the data for transphosphatidylation to convert PC to PE resulted in Figure 12, clearly confirming that the model is adequate.

The obtained V

and K

values were of 3.638 mM /min and 1.131 mM

for immobilized PLD and 3.290 mM/min and 2.600 mM for free PLD, respectively. These results obtained showed that free and immobilized PLD presented distinct behaviors; the immobilized PLD prepared by our proposed method seemed to be successfully used as an efficient biocatalyst with high activity and enzyme-substrate affinity in transphosphatidylation. Except the explanation as described in 3.1, another possibility with respect to the improvement of catalytic efficiency of PLD after immobilization is due to the interfacial activation57. During immobilization, PLD features were greatly altered, its activity and enzyme-substrate affinity were also altered. A certain


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hyperactivation of the enzyme might be formed by the closing/opening mechanism or just by distorting the flexible active center41,58,59. The improvement in the enzyme-substrate affinity arose from the use of enzymes dispersed on the surface of supports, which avoided aggregations and increased the effective contact area between enzymes and substrates41.

3.4. Thermal and storage stabilities of immobilized PLD. Thermal stability experiments were carried out for free and immobilized PLD, incubated at different temperatures ranging from 0 to 60 oC. The results showed that high temperatures (60 oC) led to almost inactivation of free PLD (Figure 13). Over the studied range of temperatures, immobilized PLD showed a better thermal stability. Even though immobilized PLD was incubated in 60 oC for two hours, the specific activity still remained 9021 U/gprotein. Meanwhile, the specific activity of free PLD was just 439 U/gprotein. The storage stability of the free and immobilized PLD was also investigated and the results are presented in Figure 14. The half-life of immobilized PLD was significantly increased (approximately 2.3 times) from 30 to 70 days at 4 oC.

The improved thermal and storage stabilities are due to the increasing stabilization of active center by multipoint bond by intra and inter molecular cross-linked50,60. After the immobilization, enzyme molecules were cross-linked with each other to form an “enzyme net” covering the surface of NPs, which could produce a strong rigidification of the enzyme structure to prevent enzyme



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conformational changes. It is expected that enzyme activity may be retained under distorting conditions41.

3.5. Operational stability of immobilized PLD. In industrial applications, the recycling of PLD is required to reduce the production costs. Thus, the operational stability of immobilized PLD was examined. The immobilized PLD were recovered by centrifugation after each batch reaction, followed by washing, and then added to fresh reactants. After 12 batches, the result showed that the initial reaction rate of immobilized PLD was >50% of that in the first batch (Figure 15), indicating that this novel immobilization method exhibits an excellent operational stability and that it is particularly suitable for the production of nanobiocatalysts. The immobilized PLD is highly stable, as demonstrated in the 13 days’ reactions during recycling experiment with acceptable loss of the enzyme activity. The activity loss may be due to enzyme leakage during washing and enzyme deactivation during repeated uses. The loss mass of proteins (wt %) caused by leakages after each run ranging from 1 to 2 % was minimum, which could be easily removed by liquid-liquid extraction avoiding the enzyme contamination of the product. In addition, the mass of products (PS, PA) and the substrate (PC) were respectively detected after each run (data not shown). The result indicated that the moles of disappeared substrate are equivalent to the moles of formed products, viz. no adsorption of PLs on nanoparticles or other used materials occurred.


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In fact, the liquid-liquid system also provides the possibility of recycling of free enzyme solution. Therefore, the operational stability of free PLD was investigated. Unfortunately, a sharp decrease in activity was observed. No activity of PLD has been detected even after its 7th repeated use. Except aggregation and dissociation of free enzymes as mentioned earlier, the byproduct, choline, could not be removed and accumulated in the aqueous solution, which has the inhibition for PLD-mediated transphosphatidylation.61

4.

CONCLUSION

We are always interested in exploring an original approach to develop highly effective biocatalysts. Enzymes were immobilized in a simple and effective way by adsorption and precipitation, followed by chemical cross-linking to form an “enzyme net” covering the surface of nanoparticles. This method provides a good reference for applications of heterogeneous reaction system. The maximum activity of immobilized PLD (measured under its optimum operational conditions) was 15872 U/gprotein, which is about 1.15 times higher than that of free PLD (13813 U/gprotein measured under the optimum operational conditions). Moreover, the immobilized PLD showed improved pH and thermal stability than its free counterpart. A kinetic study for both immobilized PLD and free PLD demonstrated that immobilized PLD had relatively higher catalytic efficiency and enzyme-substrate affinity. The obtained the maximum rate of the reaction and Michaelis-Menten constant were of 3.638 mM/min and 1.131 mM for



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immobilized PLD and 3.290 mM/min and 2.600 mM for free PLD. Operational stability of immobilized PLD over nanoscale silicon dioxide was observed over 13 cycles, which revealed the high capacity of reutilization of this nanobiocatalyst. The activity of immobilized PLD was maintained in 50% of its initial value for 70 days. On the basis of these results, we are willing to see this novel method can be used for further enzymes, increasing the library of immobilization methods.

FUNDING SOURCES

This work was supported by grants from Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2014JM2057).

ASSOCIATED CONTENT

Supporting Information

Figure S1: Schematic overview of the use of pre-existing non-porous solids instead of the “non-activity” and fragile core of aggregations of enzymes. Figure S2: Schematic overview of the reaction equation of transphosphatidylation. Figure S3: SEM micrographs of nanoscale silicon dioxide without immobilized enzyme. Figure S4. The effect of rotational speeds on transphosphatidylation for free and immobilized PLD. Figure S5: The effect of the concentration of ethanol amine on the reaction rate of transphosphatidylation for free and immobilized PLD. This material is available free of charge via the Internet at http:// pubs.acs.org.


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AUTHOR INFORMATION

Corresponding author

* TEL: ++86-29-88373052, ++86-13389200336, 15129937782. FAX: ++86-2988373052. Email: [email protected].

Author Contributions

‡

These authors contributed equally to this work.

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Figure 1. Schematic overview of immobilization of phospholipase D.

Figure 2. (a) Effect of different precipitants on the activity of PLD; (b) Effect of the volume of ethanol on the immobilization yield (blue) and the activity of immobilized PLD (black). The dash line representing the activity of free PLD (measured at its



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optimum operational conditions of pH 5.5 and 30 oC) is provided to guide the eye only.

Figure 3. Schematic of PLD on silica particles under three levels of the surface coverage.

Figure 4. Prevention of enzyme aggregations by immobilization.


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Figure 5. Effect of the immobilization pH on the immobilization yield (blue) and the activity of immobilized PLD (black). The dash line representing the activity of free PLD (measured at its optimum operational conditions of pH 5.5 and 30 oC) is provided to guide the eye only.

Figure 6. Effect of the glutaraldehyde concentration on the immobilization yield (blue) and the activity of immobilized PLD (black). The dash line representing the activity of free PLD (measured at its optimum operational conditions of pH 5.5 and 30 o

C) is provided to guide the eye only.



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Figure 7. Effect of the initial PLD concentration on the immobilization yield (blue) and the activity of immobilized PLD (black). The dash line representing the activity of free PLD (measured at its optimum operational conditions of pH 5.5 and 30 oC) is provided to guide the eye only.

Figure 8. FTIR-ATR spectra of PLD (solid line), nanoscale silicon dioxide (short-dash line) and PLD immobilized over nanoscale silicon dioxide (dash line).


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Figure 9. SEM micrographs of nanoscale silicon dioxide with immobilized PLD.

Figure 10. Particle size distributions measured by SEM: (a) nanoscale silicon dioxide; (b) nanoscale silicon dioxide with immobilized PLD.



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Figure 11. (a) Effect of temperature; (b) effect of pH on the initial reaction rate of free (black) and immobilized (blue) PLD


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Figure 12. Initial reaction rates for different concentrations of PC (from 0 to 16 mM) with free (black) and immobilized (blue) PLD. The solid line represents the fitting of Michaelis–Menten model to experimental data.

Figure 13. Thermal stability of free (black) and immobilized (blue) PLD.



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Figure 14. Storage stability of free (black) and immobilized (blue) PLD. The dash line representing the half of initial activity of free(black) and immobilized (blue) PLD is provided to guide the eye only.

Figure 15. Operational stability of free (gray) and immobilized (blue) PLD in subsequent 13 cycles of transphosphatidylation.


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Table of contents and abstract graphics


An Enzyme Net Coating the Surface of Nanoparticles: A Simple and

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