Lipase B from Candida antarctica Immobilized on Epoxy-functionalized Hollow Silica Microspheres : Efficient Biocatalysts for Enantiomer Selective Acylation of Alcohols and Amines

Hollow silica microspheres with promising physical properties (MAT540TM) as support for enzyme immobilization and biocatalyst were investigated in this study. The amine-functionalized MAT540TM was activated by six bisepoxides inclosing different spacers and used as epoxy-functionalized carrier for immobilization of lipase B from Candida antarctica (CaLB). The novel, covalently fixed CaLB biocatalysts were compared in kinetic resolution (KR) of racemic 1-phenyethanol rac-1 and five racemic amines rac-3a-e using shaken flasks and continuous-flow packed-bed microreactors. Mechanic stability, re-usability and the effect of temperature (0–90 °C) on productivity and enantiomer selectivity of the covalently immobilized CaLB were investigated. The best performing CaLB biocatalyst showed good mechanic stability after 24 h operation time in continuous-flow mode at 60 °C and provided in KRs of racemic 1-phenyethanol rac-1 with vinyl acetate and of five racemic amines with isopropyl 2-ethoxyacetate as acylating agent the non-reacted (S)-alcohol [(S)-1] or (S)-amines [(S)-3a-e] and the forming (R)-ester [(R)-2] or (R)-amide [(R)-4a-e] in good yields with high enantiomeric excess (ee > 99 %, for all).


Introduction
Chirality is a key property of organic fine chemicals in the field of agrochemical [1], pharmaceutical [2,3] and food industries [4,5].Consequently, there is an ever increasing demand for optically pure final products especially by active pharmaceutical ingredients [6][7][8] where particularly hydroxyl [9,10] and amine [11] functions or their derivatives [12] are bound to the center of asymmetry.Asymmetric techniques, such as aldol reaction [13], epoxide opening [14], ketone reduction [15] or kinetic resolution of racemic mixtures [16] are well known methods to produce pure enantiomers of alcohols.On the other hand, synthesis of enantiopure amines can rely on asymmetric synthesis catalyzed by chiral metal-ligand complexes [17], differential crystallization of their diastereomeric salts with chiral ligands [18,19] and diverse resolution methods [20].It is important to note that most of asymmetric chemical catalysts have their biopair in form of enzymes and applied as biocatalyst such as oxidoreductases [21,22], transferases [23], hydrolases [24] or lyases [25].In this context, lipases (triacylglycerol ester hydrolases, EC 3.1.1.3)[26] have gained popularity in laboratory and industrial scale as well since they can perform kinetic resolution [27] both of alcohols and amines in aqueous [28] or in organic medium [29] and do not require any cofactors during the catalytic process.
Covalent attachment of enzymes onto solid supports is based on a stable bound formation, therefore it requires the activation of enzyme or support (or both) [48,49].This kind of enzyme immobilization requires several steps including modification and activation of support surface and enzyme coupling [50].Covalent bond formation between aldehyde functions on the support -usually obtained by activation of aminated surfaces with glutaraldehyde -and amine functions of the enzyme is one of the most commonly used coupling methods [33,42], involving Schiff's base formation between surface exposed nucleophilic amino groups (mostly terminal amino functions of Lys) and the aldehyde moieties.This hydrolytically unstable Schiff's base type bond is often reduced by sodium borohydride treatment to prevent further enzyme release and to transform the residual aldehydes on the carrier to less active hydroxyl groups [51].Besides aldehydes, epoxy-activated carriers are also applied to immobilize enzymes under mild conditions (e.g.pH = 7.0).Besides being epoxy-carriers storable for long terms, they can provide biocatalysts of improved stability after the ring opening reactions due to the non-hydrolysable nature of the formed covalent bonds with the protein (secondary amine, ether, thioether structures may be formed) [52][53][54][55][56][57][58][59][60][61].In addition, mixed-functionalization of surfaces by two or more functions involving reactive and inert moieties allows fine tuning the properties of support (such as polarity and functional group density) [62][63][64].Two methods are widely applied for surface functionalization of the carriers, the direct co-condensation synthesis applying epoxy-containing monomers and the post-synthesis surface grafting methods [65].Surface grafting is a process for modification of surface by covalently linking species with reactive groups.The most conspicuous advantage is the good preservation of carrier before or even after the surface functionalization [66].In case of carrier-bound enzymes, the quality of solid support is another key factor to create a productive immobilized biocatalyst.In this context, high protein binding-capacity, high surface-to-volume ratio, insolubility in application medium, high mechanical, thermal, chemical stability contributing to efficient recoverability are crucial factors in support selection.Carriers can be divided into two groups according to their porosity, the porous and nonporous carriers and each have their own limits and benefits [67].In porous carriers, enzyme could be fixed mostly within the pores of the system resulting in high enzyme loading capacity due to the high specific area of carrier.However, porous systems suffer from considerable mass transfer limitations depending on the porosity, particle diameter and shape (pore size and volume) which all can influence diffusion rate [68] and the kind of reactors where the biocatalyst may be utilized [69,70].
In this study, hollow silica microspheres (M540: MATSPHERES ® series 540; Materium Innovations) were chosen as promising carrier for immobilization of lipase B from Candida antarctica (CaLB).M540 microspheres were developed for bio applications such as support to enhance microorganism's growth, immobilization of enzymes, and additives for concentration or purification of biomolecules.Accordingly, M540 microspheres have promising properties for enzyme immobilization: high surface-to-volume ratio (> 100 m 2 /g), proper particle size for batch and packed-bed continuous-flow bioreactor applications (10-30 µm), dispersibility in water and in organic solvents as well and easy functionalisability owing to the presence of amine groups on the surface.
Herein, we present a detailed study on application of M540 silica carriers for covalent immobilization of CaLB and biocatalytic application (batch and continuous-flow modes) of the resulted biocatalysts.

Experimental Section 2.1 Materials
Isopropyl 2-ethoxyacetate was synthesized as described previously [47], all other solvents and chemicals were purchased from the following companies Sigma Aldrich (Saint Louis, MO, USA), Alfa Aesar Europe (Karlsruhe, Germany), Merck (Darmstadt, Germany) and used as received.Lyophilized Lipase B from Candida antarctica was purchased from c-LEcta (Leipzig, Germany).

Analytical instrumentation and methods
TLC was carried out using Kieselgel 60 F254 (Merck) sheets.Spots were visualized under UV light (Vilber Lourmat VL-6.LC, 254 nm) or after treatment with 5 % ethanolic phosphomolybdic acid solution and heating of the dried plates.
The NMR spectra were recorded in CDCl 3 on a Bruker DRX-300 spectrometer operating at 300 MHz for 1 H and 75 MHz for 13 C, and signals are given in ppm on the δ scale.
Optical rotation was measured on Perkin-Elmer 241 polarimeter at the D-line of sodium.The polarimeter was calibrated with measurements of both enantiomers of menthol.
The morphology of different immobilized enzyme preparations was analyzed by a JEOL JSM-5500LV scanning electron microscope (SEM) in high vacuum at accelerating voltage 25 kV.For better imaging, samples were coated with a gold nano-film layer by a vacuum nebulizer.
Elemental analysis was carried out on a VarioEL 15.5.6 instrument in CHN mode measuring two parallel of each sample.

Immobilization of CaLB on different supports
Aldehyde-modification: M540-GA was prepared by reacting of M540 silica (1000 mg) in the solution of glutaraldehyde (592 mg, 5.93 mmol) and ethanol (16 mL) in shaken flask (350 rpm) at 60 °C for 24 h.The M540-GA was filtered off from the reaction mixture, washed with ethanol (2 × 20 mL) and dried in vacuum (RT, 24 h).Then M540-GA was afforded as an off-white solid.
The batch mode kinetic resolution reactions of racemic amines rac-3a-e were stopped after 4 h and biocatalyst was filtered off on glass filter (G4) and washed with toluene (2 × 5 mL) and ethyl acetate (2 × 5 mL).Hydrochloric acid (10 mL, 20 %) was added to the combined organic phase (filtrate and washing solvents) and mixture was stirred for 20 min at rt.After separating the phases, the aqueous phase was extracted with CH 2 Cl 2 (2 × 10 mL) and the organic phases were unified and washed with saturated brine (2 × 10 mL) and dried over Na 2 SO 4 .The solvents and other volatiles were subsequently removed in vacuum and (R)-2-ethoxyacetamides (R)-4a-e were afforded as light yellow liquids.

Kinetic resolution of alcohol rac-1 and amines rac-3a-e in continuous flow mode
Kinetic resolutions in the packed-bed bioreactors filled with the CaLB biocatalysts in continuous-flow mode were performed in a laboratory scale flow reactor which comprised a HPLC pump (Knauer, Azura 4.1S), packedbed column filled with CaLB biocatalyst within an in house made thermostated aluminum metal block holder with precise temperature control (Lauda, Alpha RA8).Before usage, the actual CaLB-filled column was washed with the corresponding solvent (for rac-1: 2:1 mixture of hexane and MTBE for 1 phenylethanol; for rac-3a-e: toluene; 0.5 mL min -1 , 30 min).At each set of reaction parameters (temperature, substrate concentration, flow rate), samples were analyzed by GC every 10 min up to 40 min after the start of the experiment.After the stationary mode of operation has been established (40 min after the start of the experiment) samples were collected (20 µL sample was diluted with ethanol to 1 mL) and analyzed by TLC and GC.After a series of experiments, the actual CaLBfilled column was washed with the corresponding solvent (0.5 mL min -1 , 30 min) and stored in refrigerator (4 °C).

Results and discussion 3.1 Covalent immobilization of lipase B from Candida antarctica onto hollow silica microspheres and kinetic resolution of 1 phenylethanol rac-1
By the aid of amine groups on surface of M540 hollow silica microspheres as support, six bisepoxide-activated carriers (M540-A-F) were prepared having different length and hydrophobicity in the spacer region of the activating bisepoxide (Fig. 1).The carriers were characterized by FT-IR, elemental analysis and scanning electron microscopy (SEM).As reference, the unmodified M540 was used to compare adsorptive immobilization and the glutaraldehyde (GA)-activated form of M540 (M540 GA) was also investigated as further reference for covalent binding.For comparison, the commercially available epoxy-functionalized polyacrylic carrier CV T2-150 (particle size 300-700 µm) was also included in immobilization experiments.With the three reference carriers and the six bisepoxide-activated M540 carriers in hand, immobilization of CaLB was performed using lyophilized form of CaLB in phosphate buffer (100 mM, pH 7.5) at room temperature (24 h).
To distinguish between the catalytic activity of the physically adsorbed and the covalently bound enzymes, a post-treatment with Triton X 100 non-ionic detergent solution was performed after the immobilization step which could remove those enzyme molecules which were only adsorbed by physical interactions onto the surface.
Catalytic performance of the resulted biocatalysts was tested in kinetic resolution (KR) of racemic 1-phenylethanol rac-1 in batch mode (Fig. 2) before and after the Triton X-100 wash to provide information of enzyme immobilization efficiency (i.e.washing resistance) of supports (Table 2).
As it was expected, the CaLB attached to unmodified M540 carrier retained the least activity after post-treatment (Table 2, Entry 1, 39 %) while the covalently fixed biocatalysts retained at least 83 % residual activity    2, Entries 2-9) after detergent washing.This fact strongly support that the non-activated M540 carrier could not establish covalent attachment but only ionic and physical interactions to fix enzyme molecules on the surface.
In the case of M540-A-CaLB, an interesting effect was observed.Instead of decreasing the enzyme activity upon Triton X-100 washing a 52 % activation was observed.This difference could be explained by assuming activation due to the bioimprinting effect of surfactant [33,[75][76][77].Compared to the adsorptive CaLB-M540 preparation, catalytic performance of the GA-activated biocatalyst (M540-GA-CaLB) was three times higher while conversion values of epoxy-activated M540-X-CaLB (X = A-F) biocatalysts were at least ten times higher in KR of rac-1 indicating the beneficial nature of bisepoxide-activation over the widely used GA-derivatization or adsorptive immobilization.These results could be attributed to the enhanced covalent bond formation capability of epoxy containing supports compared to aldehyde-activated carriers under mild conditions (approximately neutral pH, RT) [54].
Moreover, inspection of the length and lipophilicity of epoxy-spacers (M540-A-F) revealed that flexibility and hydrophobicity of sidechain affected the immobilization efficiency of the bisepoxide-activated carriers.This could be related to the first event of immobilization on epoxy supports, since a physical adsorption of enzymes onto the surface always preceded the covalent bond formation by epoxide ring-openings.Analyzing the activities of covalently attached CaLB on pre-activated carriers indicated the role of functionalization and the quality of spacers, where the aldehyde-and any epoxy-modification exceeded the catalytic performance of untreated silica carrier M540.Amongst the investigated bisepoxide-activated carriers, M540-FE with a poly(ethylene glycol) spacer proved to be the most suitable to immobilize CaLB in active form [78].In addition, the enantiomeric purity of formed product (R)-1 phenylethylacetate (R)-2 was excellent (ee (R)-2 ≥ 98.6 %) when KR was catalyzed by covalently immobilized forms of CaLB.

Continuous-flow mode kinetic resolution of 1-phenylethanol rac-1 catalyzed by covalently attached silica CaLB preparations -substrate concentration and temperature effects
Immobilization of enzymes plays an indispensable and crucial role in the application of biocatalysts with natural origin in batch and continuous-flow bioreactors [45][46][47]79].The operation temperature could substantially influence the productivity and selectivity of lipase-catalyzed hydrolytic and acylation reactions of chiral and non-chiral substrates [73,80].Accordingly, two series of experiments were performed to study the effect i) of substrate concentration rac-1 (1-86 mg mL -1 ) and ii) of reaction temperature (0-90 °C, in 15 °C steps).The reactions were carried out in heat and pressure resistant stainless steel packedbed columns operated within a lab reactor system with precise temperature and flow rate control.The three most effective CaLB preparations immobilized covalently onto hollow silica microspheres (M540-C-CaLB, M540-D, M540-F-CaLB CaLB) were selected as biocatalysts in packed-bed columns for the continuous flow kinetic resolution studies (Fig. 2).
During the substrate concentration dependence study, the solution of substrate rac-1 in different (1-86 mg mL -1 ) and the vinyl acetate acylating agent in hexane and MTBE was pumped through the CaLB-filled columns thermostated to 30 °C at a constant flow rate (0.20 mL min -1 ).
The productivity, characterized by the specific reaction rate (r f , μmol min -1 g -1 ) of enzyme preparations was investigated as a function of substrate concentration (Fig. 3, panel A).The specific reaction rates (r f ) with the three selected, covalently immobilized CaLB biocatalysts were quite similar at lower concentrations (1-48 mg mL -1 ), while in higher concentration range (48-86 mg mL -1 ), the M540-F-CaLB surpassed the performance of the two other selected CaLB forms, similarly as in case of batch mode KRs.In addition, the ee (R)-2 values of formed (R)-1phenylethyl acetate (R)-2 (Fig. 3, panel B) were significantly higher in continuous-flow mode (ee (R)-2 = 99.2-99.4%) than in batch mode (ee (R)-2 = 98.8-98.9%), due to the shorter contact time between the biocatalyst and the racemic substrate.The highly enantioselective KR process with these CaLB preparations immobilized covalently onto hollow silica microspheres also showed the prosperous technological benefit of continuous-flows systems over the batch reactors.
Next, the thermal behavior of immobilized CaLB preparations on epoxy-activated carriers (M540-C-, M540-D-, M540-F-CaLB) was investigated to gain information about their catalytic performance at different temperatures.For these series of experiments, the former system (used for the substrate concentration dependence studies) was applied at the highest substrate concentration (86 mg mL -1 ), while the temperature was varied between 0-90 °C in 15 °C steps at a constant flow rate (0.20 mL min -1 ).
First, as a function of temperature, the productivity of enzyme preparations (r f ) was monitored (Fig. 3c, panel C).
As shown, the productivity-temperature curves in the lower temperature range (0-45 °C) were quasi-linear in case of all the three selected CaLB preparations.In the higher temperature range (45-90 °C), however, the M540-D-CaLB started to lose its activity and the productivitytemperature curve descended, while the M540-C-CaLB and M540-F-CaLB biocatalysts kept the linearly increasing tendency until 90 °C.
The influence of the temperature on the enantiomeric excess of the product (ee (R)-2 ) was also studied.It was apparent, that the overall shapes of the ee (R)-2 -temperature curves were quite similar with all the three CaLB preparations (Fig. 3, panel D) in the entire temperature range.After a short ascending region (0-30 °C), local maxima were found by the ee (R)-2 values between 30 °C and 60 °C after which a slightly decreasing region was detected at elevated temperature (> 60 °C).According to these results, the most productive and selective operating temperature was at around 60 °C.

Kinetic resolution of racemic amines rac-3a-e with M540-F-CaLB
After the catalytic performance and stability of CaLB bound to hollow silica microspheres were investigated in KR of alcohol rac-1, the best performing M540-F-CaLB was further studied in KRs of five racemic amines rac-3a-e.Isopropyl 2-ethoxyacetate as acylating agent in KR of 1-phenylethylamine rac-3a [47] surpassed the productivity and selectivity of the previously best performing isopropyl 2-methoxyacetate [81] in terms of both productivity (2-3-fold) and selectivity (ee (R)-4a ≥ 98.3 %).Accordingly, this acyl donor was selected for our study on KRs of racemic amines rac-3a-e performed in batch and continuous-flow bioreactors at 60 °C (Fig. 4) which was the thermal optimum of M540-F-CaLB with alcohol rac-1.As the rate of product formation is not a linear function of conversion [73], productivity (i.e. the specific reaction rate, r) of a continuous-flow reaction (r f ) and of the corresponding one in batch mode (r b ) were compared at similar degrees of conversions (> 49 %).As shown in Table 3, in KRs of amines 1.5-2 higher r flow values could be achieved in packed-bed microreactors as compared to the r batch values in batch system (in accordance with the known benefit of continuous-flow systems compared to batch reactors [82]).

Reusability, mechanical and thermal stability of covalently immobilized CaLB preparations
Possibly the most beneficial advantage of immobilized enzyme preparations is the capability of biocatalyst recycling thus making the process more economical and environment friendly.Therefore, beside the three best proven bisepoxide-activated biocatalysts (M540-C-, M540-D-, M540-F-CaLB), the covalent standard (CV T2-150-CaLB) and the widely applied commercially available adsorptive Novozyme® 435 (N435-CaLB) were selected for studying the stability of enzyme preparations during re-cycling over 10 cycles using the KR of rac-1 (reported in the former sections) as reference reaction.Between each cycles, the biocatalysts were filtered off from the reaction mixture, washed with hexane and MTBE to remove organic compounds and dried to determine their exact quantity.
As shown in Fig. 5, every biocatalyst presented decreasing catalytic activity cycle-by-cycle whereas M540-F-CaLB has proven the least vulnerable and retained 54 % of its initial effectivity after the 10 th cycle while CV T2-150-CaLB kept only 23 %.This result agreed with our previous experiences, where the carriers with hydrophobic characteristic surface enhanced the immobilization efficiency of CaLB and herein improved the capability of enzyme preparations to keep their activity for many catalytic cycles.
Besides the biocatalytic performance, mechanical stability of immobilized enzymes is also a crucial parameter which is strongly related to the durability of support material quality.Thus, samples were taken from the biocatalysts during recycling study -the best performing silica microspheres (M540-F-CaLB) and two macroporous acrylic resins (CV T2-150-CaLB and N435-CaLB) -and their morphologies were analyzed by scanning electron microscopy (SEM).As the SEM pictures in Fig. 6 illustrate, appreciable fractions were observed in acrylic polymer based biocatalysts (CV T2-150 and N435) even after the first cycle.The high level of fractionations after the tenth cycle could be related to their low residual activity.In case of the silica-based M540-F-CaLB, fractionation was not a typical event but aggregated particles appeared.Aggregation could account for the descending activity due to increasing diffusion resistance owing to the longer pore length and the lower approachable surface area.
As the productivity-operation time curves show (Fig. 7, panel A), at all temperatures a linearly decreasing tendency with diverse gradients was noticed.It was not surprising that the biocatalyst started to lose its activity over 60 °C after elongated reaction time.In accordance with our previous results in KRs of secondary alcohols [73], increasing temperature resulted in lowered of ee (R)-2 values (Fig. 7, panel B).Nevertheless, M540-F-CaLB kept more than 96 % of its catalytic activity after 24 hours uninterrupted operation at 60 °C while 4 052 mg (25 mmol) of (R)-2 was formed (17-fold of the catalyst mass applied) with excellent enantiomeric purity (ee (R)-2 = 99.1 %).
SEM picture of M540-F-CaLB applied in the packedbed reactor operated in continuous-flow mode revealed no noticeable aggregates or fractions (Fig. 8) -in contrast to the same biocatalyst utilized in batch mode (Fig. 6) -even after a series of experiments (concentration, thermal and continuous operational stability tests).This observation could account for the higher stability of M540-F-CaLB in packed-bed columns as compared to the reactions performed in shaken flasks.

Conclusion
Our study focused on surface activation of MAT540 TM hollow silica microspheres as support using six bisepoxides with different spacers allowing covalent immobilization of CaLB.To study the biocatalytic performance of the six novel CaLB preparations, operation conditions, stability and recyclability of biocatalysts were investigated in  The biocatalytic usefulness of M540-F-CaLB was further demonstrated with kinetic resolutions of five different racemic amines rac-3a-e using isopropyl 2 ethoxyacetate as acylating agent under the optimal reaction temperature (60 °C).The formed 2 ethoxyacetamides (R)-4a-e could be isolated in good yields (≥ 42 %) and with high enantiomeric excess (ee (R)-4a-e ≥ 98.3 %).The bisepoxide activated MAT540 TM carriers allow general and smooth immobilization of a broad spectrum of enzymes thus creating biocatalysts applicable under continuous-flow conditions with high operational stability.

Fig. 8
Fig. 8 SEM image of M540-F-CaLB biocatalyst after the concentration, thermal and continuous operational stability tests