Immobilization and characterization of benzoylformate

Bioprocess Biosyst Eng DOI 10.1007/s00449-011-0516-0

ORIGINAL PAPER

Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier Stephanie Peper • Selin Kara • Wei Sing Long Andreas Liese • Bernd Niemeyer

•

Received: 3 November 2010 / Accepted: 12 January 2011 Ó Springer-Verlag 2011

Abstract If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460IF464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and S. Peper (&) W. S. Long B. Niemeyer Institute of Thermodynamics, Helmut-Schmidt-University Hamburg, Holstenhofweg 85, 22043 Hamburg, Germany e-mail: [email protected] S. Kara A. Liese Institute of Technical Biocatalysis, Hamburg University of Technology, Denickestrasse 15, 21073 Hamburg, Germany

from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme. Keywords Immobilization Benzoylformate decarboxylase Silicagel Enantioselectivity

Introduction The application of enzymes as biocatalysts is increasingly recognised in chemical and pharmaceutical industries. However, industrial application of biocatalysts is often hampered by the lack of long-term operational stability and difficult recovery and re-use. One approach for recycling and recovery of the biocatalyst is the immobilization on a heterogeneous support. In addition to a more convenient handling of the enzyme, immobilization provides an enhanced stability of the enzyme and a simpler separation from the product, thereby minimizing or eliminating protein contamination of the product. Both the improved enzyme performance and the enhanced stability result in a higher productivity of the biocatalyst and thereby in lower enzyme costs per kilogram of the product. More than 5,000 publications, including patents, have been published in the field of enzyme-immobilization [1], which reflects the increasing demand for stable and highly active immobilized enzymes and effective immobilization techniques, respectively. The manifold described techniques for enzyme immobilization can be categorized into a few different methods: adsorption, covalent bonding, entrapment, encapsulation, and cross-linking. Corresponding to the different variations of immobilization methods, many carriers of differing physical and chemical characteristics have been designed for a variety of protein-immobilizations [2–6]. Altogether,

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the performance of the carrier-bound enzymes depends on innumerable parameters such as the physical properties of the carrier, which include morphology, mechanical stability, swelling factor, pore- and particle size, tortuosity, hydrophilicity, as well as the chemical characteristics of the carrier such as composition, surface chemistry, binding chemistry, and the chemical microenviroment. The objective of this work was the preparation of mechanically stable immobilized benzoylformate decarboxylase (BFD) from Pseudomonas putida, which is used to synthesize a wide variety of chiral 2-hydroxyketones. Therefore, as carrier material, spherical silicagel was used. Silicagel is characterized by its high mechanical stability, which allows its use in different reactor types without restrictions. Contrary to diverse polymeric carriers, silica does not swell. Moreover, the use of small particles enables high mass transfer rates. These properties together with its availability in defined particle and pore sizes enable a scale-up from laboratory to production without problems. For the covalent immobilization of the protein under gentle conditions, an activation of the matrix is required. Starting with surface-bound hydroxyl, amino, thiol, aldehyde, or carboxyl groups, a number of activation protocols are available. Hydroxyl groups can be activated with cyanogens bromide [7], bisepoxirane [8], organic sulphonyl chlorides [9–11] or carbodiimidazole [12]; amino groups with glutaraldehyde, carboxyl groups and with N-hydroxysuccinimide [13]. In this work the activation of the carrier matrix was done with 2,2,2-trifluoroethanesulfonylchloride. Based on this activation, the immobilization occurs via reaction of the primary amino or thiol groups. The chosen reaction is fast, requires no additional chemicals, and runs under mild conditions (yield of linkage between 80 and 100% in the range of pH 7–9) which altogether ensures the preservation of the enzyme activity. The investigated enzyme benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) is a homotetrameric thiamine diphosphate dependent (ThDP) enzyme. BFD accepts a broad range of aldehydes as substrates to catalyze the enantioselective synthesis of 2-hydroxyketones, which are useful as synthons in organic chemistry [14–17]. These synthons are important intermediates in the production of antifungals [18] as well as of pharmaceuticals like WellbutrinÒ (treatment of depression) and ZybanÒ (smoking cessation) [19]. In this work, the synthesis of 2-hydroxypropiophenone (2-HPP) starting from benzaldehyde and acetaldehyde was catalyzed by BFD A460I-F464I. The focus is set on the question how far the immobilization influences the performance of the enzyme. Therefore, the carrier-bound enzyme was characterized in terms of its activity, stability, kinetics, and temperature as well as pressure dependency. Until now, the application of enzymes for chemical synthesis at high pressure is negligible because of the

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limited characterization of enzymes under these extreme conditions. Until date, only few investigations were published regarding the influence of pressure on the enantioselectivity of enzymes, whereas numerous results were documented in literature regarding the influence of pressure on the stability and activity of enzymes e.g. [25–29]. In general, the pressure is a thermodynamic acting parameter, which affects the enzyme as well as the solvent. In detail inter- and intramolecular interactions as well as the interactions between protein, substrate, and solvent are influenced by the pressure, including hydrogen bonds, ionic bonds, hydrophobic interaction as well as dissociation constants and solvation of charged groups accompanied by volume reduction resulting from electrostriction. The pressure effect on the catalytic activity and selectivity depends on the sign and magnitude of volume changes accompanying the binding and elementary chemical steps. Furthermore, a change of selectivity can be the result of steric effects through pressure-induced structural changes. In previous investigations, we found a significant pressure effect on the enantioselectivity of the BFD A460I-F464I variant [20]. Whereas the previous investigations were done with free enzyme, in this work the influence of pressure on the enantioselectivity of the carrier-bound enzyme was investigated.

Materials and methods All chemicals were purchased from Sigma-Aldrich (Schnelldorf, Germany) and Carl Roth GmbH (Karlsruhe, Germany). The carrier material, DAISOGEL SP-1000-20, was obtained from DAISO CO. LTD. (Osaka, Japan). Fermentation and enzyme purification Fermentation of transformed Escherichia coli SG13009 cells hosting the BFD plasmids was carried out as described in detail by Berheide et al. [20]. The enzyme was purified as described by Siegert et al. [21] but using a Q-Sepharose FF column (500 mL bed volume) at pH 7.5. Following anion-exchange chromatography, the enzyme solution was adjusted to pH 6.0, concentrated by ultrafiltration using a Millipore 30 kDa membrane, and desalted by gel filtration using a Superdex 200 column (300 mL bed volume). The enzyme was applied in biotransformations as freeze-dried preparations. Protein amounts in the solutions were determined by the standard Bradford method [22] using bovine serum albumin (BSA) as a standard. One unit of activity is defined as the amount of enzyme which catalyzes the formation of 1 lmol of 2-HPP in 1 min at 20 °C. Activity analysis for 2-HPP synthesis was performed with 40 mM benzaldehyde and 400 mM acetaldehyde in


50 mM phosphate buffer containing 0.5 mM ThDP and 2 mM MgSO4 at pH 7.5. Surface modification of silica-based matrix The surface modification of the pressure stable silica carrier was performed via a three-step modification prior to the enzyme immobilization: silanization of the silica carrier, coupling of a spacer and activation. Therefore, the spherical silica particles were covered with dried toluene before aminopropyltriethoxysilane was added. The mixture was heated up under weak agitation and reflux conditions for 1.5 h. The support and reagents were filtered, extracted for 4 h with acetone, and dried at 60 °C. The product was covered with water and reacted with 1,4-butanediol-diglycidyl ether. The dried material was activated with 2,2,2trifluoroethanesulfonylchloride under argon atmosphere and stored under HCl, at pH 3 [23]. The reaction diagram is depicted in Fig. 1.

reaction solution, as well as of the supernatant of four subsequent washing steps, whereby the supernatant of the last washing step was free from enzyme. The end-capping of excessive functional groups was carried out with 0.5 M Tris buffer at pH 7.5. Activity assay for immobilized enzyme Activity of immobilized BFD A460I-F464I was determined from the initial formation rate of 2-HPP monitored at 5–10% of benzaldehyde conversion. For the activity assay, reaction solution contained 4.5 mL buffer solution and 0.5 mL of immobilized enzyme solution (100 mg of dry carrier) rigorously mixed via magnetic stirrer at 550 rpm where the substrate concentrations were 40 mM benzaldehyde and 400 mM acetaldehyde. Reactions were carried out in 5 mL of total reaction volume for 60 min. The temperature was maintained at 20 °C. The amount of product (2-HPP) formed was analyzed as described in the section ‘‘Analytics’’.

Immobilization of the enzyme Analytics The immobilization procedure of the BFD A460I-F464I was accomplished in 0.5 M phosphate buffer, at pH 6.5 and at room temperature. The immobilization yield of covalentbound enzyme was measured by the determination of unbound enzyme concentration in the supernatant of the reaction solution, the supernatant of the end-capping

The reactions were quenched by addition of a stop-buffer (90% acetonitrile, 5% H2O, and 5% H3PO4) at the ratio of 2:1 (sample: stop-buffer, v/v) followed by intense mixing and centrifugation of the precipitate. The amount of product (2-HPP) formed was analyzed by HPLC (Agilent 1100,

Fig. 1 Synthesis pathway of the modified silica support: a silanized silica surface with aminopropyltriethoxysilane, b reacting with the spacer molecule 1,4-butane-dioldiglycidyl ether, c epoxide ring opening with hydrochloric acid, d activation of hydroxy groups with 2,2,2trifluoroethanesulfonylchloride, e immobilization of the enzyme BFD A460I–F464I

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Hewlett Packard) equipped with a LiChrosphere RP-8 column (Hypersil, 250 9 4 mm, Merck) using triethanolamine (0.2%, pH 3.5): methanol (50:50, v/v) as an eluent at a flow rate of 1.0 mL/min at 40 °C. Typical retention times are: 2-HPP 4.9 ± 0.1 min, benzaldehyde 5.7 ± 0.1 min and benzoin = 9.1 ± 0.1 min (remark: benzoin which is a self-ligation product of benzaldehyde was not detected in any type of the reaction carried out in this work). For the determination of the enantiomeric excess (ee), samples were extracted with isohexane and analyzed by HPLC using a chiral OD-H column with isohexane/isopropanol (98:2, v/v) as a mobile phase at a flow rate of 0.75 mL/min at 10 °C. Here typical retention times were: (S)-2-HPP = 17.2 ± 0.2 min and (R)-2-HPP = 20.9 ± 0.2 min. Mass transfer analysis For the investigation, if the film diffusion is the rate-limiting step in a fixed-bed flow reactor, two series of experiments with two different amounts of immobilized BFD A460I-F464I were carried out whereby the substrate flow rates were varied. Therefore, 1 g silica carrier and 2 g silica carrier, respectively, with immobilized enzyme were packed in a column (dimension 250 mm 9 4.6 mm). The flow rates were varied between 0.1 mL/min and 0.8 mL/ min for the series of experiments with the low amount of immobilized enzyme (1 g silica carrier), and between 0.3 mL/min and 1.6 mL/min for the series of experiments with the higher amount of immobilized enzyme (2 g silica carrier). The resulting conversion rates were plotted as a function of the time factor (ratio of amount of immobilized enzyme to substrate flow rate). Temperature dependence of immobilized BFD A460I–F464I To analyze the temperature dependency, reactions were carried out in a thermostated reactor. The reaction solution contained 4.5 mL buffer solution (100 mM phosphate buffer, 0.5 mM ThDP, 2 mM MgSO4 at pH 7.5) and 0.5 mL immobilized enzyme solution (dry carrier 50 mg) containing 40 mM benzaldehyde and 400 mM acetaldehyde. Following a reaction time of 20 min, the reactions were quenched followed by intense mixing and centrifugation of the precipitate. Stability of the silica-immobilized BFD A460I–F464I and analysis of the enzyme kinetics For the investigation of the stability of the carrier-bound BFD A460I–F464I, repetitive batch experiments were performed. Therefore, the reaction solution consisted of

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4.5 mL buffer solution and 0.5 mL immobilized enzyme solution (dry carrier 100 mg) containing 40 mM benzaldehyde and 400 mM acetaldehyde. Each batch was conducted for 60 min with four washing steps in between which again took 45 min. Each washing of collected immobilized enzyme was done with 1 mL of fresh buffer by intensive vortexing and the solution was centrifuged at 13,000 rpm for 4 min. Moreover, washed-out solutions were analyzed by Bradford assay where no leakage of enzyme was determined in any kind of reaction performed. Totally four repetitive batches were performed and the whole experiment was done for 6 h and 15 min. The reaction temperature was maintained at 20 °C. For the analysis of enzyme kinetics, two series of experiments were performed: (a) varying the concentration of benzaldehyde (1–40 mM) at constant acetaldehyde concentration (400 mM) and (b) varying the concentration of acetaldehyde (25–2,500 mM) at constant benzaldehyde concentration (40 mM). Each reaction was carried out in 5 or 2.5 mL of total reaction volume for 30 min. The temperature was maintained at 30 °C for all reactions. Immobilized enzyme solutions were added to the prepared substrate solutions with a volume of 0.5 mL containing 50 or 25 mg of dry carrier. High-pressure experiments The high-pressure experiments were performed in a highpressure fixed-bed reactor system described by Jansen and Niemeyer [30]; 1.5 g of immobilized BFD A460I–F464I (silica carrier) was packed in a column (dimension 200 mm 9 4.6 mm); the reaction was performed in a continuous flow mode. Samples were withdrawn at constant time intervals up a constant conversion rate.

Results and discussion The aim of this present work was the immobilization of BFD A460I–F464I from Pseudomonas putida on a mechanical stable carrier, followed by its characterization under atmospheric and high-pressure conditions. Generally, it is advantageous to use carriers with high surface area regarding the enzyme loading. However, high surface area is not necessarily correlated with a high activity relative to the mass of carrier, because only the accessible surface can be occupied. Therefore, the loading, activity retention, and performance of a carrier-bound enzyme are largely related to the pore size. Thus, in literature it is recommended that for efficient immobilization, pores need to be at least three times larger than the size of the enzymes [1]. It is because of that, a pore size of 100 nm was chosen, which is approximately one order of magnitude higher than


the mean diameter of the enzyme [mean diameter dm derived from the crystal dimension dm = 10.8 nm (PDB:1BFD)] and therefore big enough to ensure a high mobility and accessibility of the bound enzyme. Due to the large number of parameters influencing an enzymatic reaction, a comparison of different data is usually difficult and only conditionally convincing, in particular if the data are based on similar but not on the same enzyme/substrate/ solvent systems. The shown data from Hilterhaus et al. [5] refer to the same enzymatic reaction system but instead of the BFD A460I-F464I variant, a His-tagged wtBFD was investigated. While Hilterhaus et al. used Sepabeads EC–EA, which are spherical beads of a porous methacrylic polymer matrix, in this work spherical silicagel (Daisogel SP-1000-20P) was used. The properties of both carrier materials are summarized in Table 1. The activity of immobilized BFD A460I-F464I was determined to be 0.99 U/mg, which is 70% of the activity of free enzyme determined before immobilization. The result given for activity analysis is an average value based on values from independent duplicate assays. The ee of 2-HPP synthesized by immobilized enzyme is determined to be 43% (R) which is in good agreement with the ee yielded by free enzyme [44% (R)]. In Table 2, the results of the immobilization are summarized. For comparison of the performance of the silica-bound enzyme, Table 2 is extended with data from Hilterhaus et al. [5]. In general, the selectivity of an enzyme can be influenced by its immobilization. According to the source of the effect on the selectivity, one can basically differentiate between carrier-controlled and conformation-controlled selectivity [1]. However, often it is difficult to distinguish the different effects from each other because the selectivity is influenced by many factors, e.g., the microenvironment in the pores of the carrier, the pore size, and the structure of the carrier as well as the carrier-bound inert pendant groups, the carrier-bound active groups that participate in the binding and the used spacer. Therefore, depending on the type of binding and the number of attachments, a conformational change or a change in flexibility can be induced; thus, the geometry of the active center might be changed, leading to different selectivity. With the applied method of covalent binding on a surface provided with spacer molecules, the flexibility and the conformation of

the active site seems not be constricted: both here in the presented enzyme catalyzed reaction and in similar investigated systems, the enantioselectivity was not changed through immobilization. Furthermore, with the chosen immobilization technique, an acceptable amount of the applied enzyme could be bound on the carrier (50–60%). Polymeric carriers usually have significant larger specific surfaces compared to wide-pore silicagels as used in this work. Larger surfaces-to-volume ratios enable higher enzyme loadings, i.e., with the Sepabeads, a 20-fold enzyme loading compared to the applied silicagel could be reached. However, despite the enzyme loading of the Sepabeads being considerably higher than the loading of the silicagel, a significant lower retention of activity was reported. In the case of Sepabead-immobilization, the activity decreases from 11 U/mgenzyme (free enzyme) to 0.62 U/mgenzyme (immobilized enzyme). In contrast to this, the silica-immobilization results in an activity decrease from 1.4 to 0.99 U/mgenzyme that corresponds to an activity yield of 70% compared to 6% of immobilized enzyme on the Sepabead. Mass transfer analysis, namely, interparticle diffusion (film diffusion) and intraparticle diffusion (pore diffusion), is of prior importance in heterogeneous catalytic reaction systems. With mass transfer analyses, one may evaluate if an immobilization method is successful in terms of substrate accessibility to the immobilized enzyme and reaction rate related to free enzyme in homogeneous solutions. For the investigation, if the film diffusion is the rate-limiting step in a fixed-bed flow reactor, two series of experiments with two different amounts of immobilized BFD A460I– F464I were carried out whereby the substrate flow rates were varied. For each series of experiments, the conversion rates were plotted as a function of the time factor (ratio of amount of immobilized enzyme to substrate flow rate). If the conversion rates of both series of experiments are identical, one may assume that the heterogeneous catalyzed reaction is controlled only by the enzyme kinetics because the conversion rates are not dependent on the flow rate. Otherwise—if the two series of experiments result in two separate curves—film diffusion has an influence on the reaction [24]. Figure 2 shows the results of the film diffusion analysis. The conversion rates of the two series of experiments

Table 1 Properties of the silica carrier used for immobilization of the BFD A460I-F464I variant as well as properties of the Sepabead carrier used by Hilterhaus et al. for the immobilization of a His-tagged wtBFD [5] Carrier type

Mean pore size (nm)

Pore volume (cm3/g)

Particle diameter (lm)

Specific surface area (m2/g)

Daisogel SP-1000-20P

100

0.90

20

25

Sepabeads EC–EA

10–20

0.5–1

150–300

50–100

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Bioprocess Biosyst Eng Table 2 Comparison of the immobilization of wild-type BFD and BFD A460I-F464I on different carrier systems Enzyme

BFD variant A460I-F464I from His-tagged wtBFD from Pseudomonas putida Pseudomonas putida Daiso SP 1000-20 (spherical silica) Sepabeads EC-EA (polymethacrylat) Present work Reaction conditionsa Data from literature [5] reaction conditionsb

Carrier Activity of free enzyme (U/mgenzyme) Loading capacity of the carrier (mgenzyme/gdry

carrier)

1.4

11

5

100

Activity of immobilized enzyme (U/mgenzyme)

0.99

0.62

ee of 2-HPP synthesized by free enzyme/immobilized enzyme (ee %)

44 (R)/43 (R)

n.a.

Surface coverage (area %)

7

n.a

Percentage of enzyme bound to the carrier (ratio bound enzyme to applied enzyme) (%) Retention of activity of immobilized enzyme referred to free enzyme (%)

50–60

max. 43

70

6

n.a. Not available a

100 mM phosphate buffer pH 7.5 containing 40 mM benzaldehyde, 400 mM acetaldehyde, 0.5 mM ThDP and 2 mM MgSO4 at T = 20 °C

b

50 mM phosphate buffer pH 8 containing 50 mM benzaldehyde, 500 mM acetaldehyde, 0.5 mM ThDP and 1 mM MgSO4 at T = 50 °C

(series of data I and II) are identical in case of ratios of amount of immobilized enzyme to substrate flow rate lower than 2.5 g/(mL/min) which corresponds to flow rates higher than 0.4 mL/min. From this, it can be concluded that the reaction system is free from film diffusion at flow rates higher than 0.4 mL/min. At lower flow rates (higher ratios of amount of immobilized enzyme to substrate flow rate), the two series of experiments result in two separate curves, which is an indication that film diffusion influences the reaction system. Temperature dependency of immobilized BFD A460I–F464I The temperature dependency of the immobilized BFD A460I–F464I was determined as shown in Fig. 3. The maximum enzyme activity was observed at 45 °C (3.4 U/ mg) which is approximately 3.5 times higher than the standard analysis carried out at 20 °C. The same behavior for the BFD catalyzed synthesis of 2-HPP was described by Hilterhaus et al. [5]. They also found an activity maximum at about 50 °C for wtBFD immobilized on Sepabeads. Stability of the silica-immobilized BFD A460I–F464I and kinetic analysis For the investigation of the stability of the carrier-bound BFD A460I–F464I, repetitive batch experiments were performed at 20 °C. After each batch, the immobilized enzyme was removed from the reactor and washed four times with fresh buffer and intensive vortexing. In total

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Fig. 2 Analysis of film diffusion at two series of substrate flow rate ranges. Data series I contains 1.5 g immobilized BFD A460I–F464I; Data series II contains 1.0 g immobilized BFD A460I–F464I. The reactions were carried out with 50 mM phosphate buffer at pH 7.5 containing 40 mM benzaldehyde, 400 mM acetaldehyde, 0.5 mM ThDP and 2 mM MgSO4 at room temperature

four repetitive batches were performed (Fig. 4). The initial specific activity was found to be 1.1 U/mg BFD A460I– F464I and decreased within four batches to 0.1 U/mg. For the analysis of enzyme kinetics, two series of experiments were performed: (a) varying the concentration of benzaldehyde (1–40 mM) at constant acetaldehyde concentration (400 mM) and (b) varying the concentration of acetaldehyde (25–2.500 mM) at constant benzaldehyde


applied concentrations of acetaldehyde were not higher than 400 mM. v¼

Fig. 3 Analysis of the temperature dependency of immobilized BFD A460I–F464I activity. Reaction conditions: 40 mM benzaldehyde, 400 mM acetaldehyde in 5 mL 100 mM phosphate buffer, 0.5 mM ThDP, 2 mM MgSO4, at pH 7.5. Line is visual aid

concentration (40 mM). The results of kinetic investigations of the 2-HPP formation are shown in Fig. 5. The enzyme activity as a function of the benzaldehyde (BA) concentration follows a Michaelis–Menten type kinetic, which however is restricted by the solubility of benzaldehyde in aqueous solution (*50 mM). In the case of acetaldehyde (AA) concentration, the initial reaction rates increase with increasing concentration up to 400 mM. A further increase of the acetaldehyde concentration leads to a reduction of activity, which might be interpreted in terms of substrate surplus inhibition or a rapid inactivation of the enzyme. The kinetic parameters of the double-substrate Michaelis–Menten model (Eq. 1) were fitted to the data measured under initial reaction condition by means of a non-linear regression. Thereby, for the quantification of the kinetic parameters, the inhibition by surplus substrate of acetaldehyde was not taken into consideration since the

vmax cðBAÞ cðAAÞ KM;BA þ cðBAÞ KM;AA þ cðAAÞ

ð1Þ

KM,BA and KM,AA are the Michaelis–Menten constants for benzaldehyde and acetaldehyde, respectively; vmax is the maximal rate. In Table 3, the obtained kinetic parameters are summarized and compared with the kinetic parameters of the free enzyme determined by Berheide et al. [34]. The calculated values for the immobilized and the free enzyme are in the same order of magnitude. Here apparent (app) kinetic parameters for the immobilized enzyme were introduced since the mass-transfer limitations may affect the kinetic characteristics of the enzyme. It is because of the limited solubility of benzaldehyde in aqueous medium, the calculated value of vmax,app (immobilized enzyme) has to be considered as a theoretical value (KM,BA,app is equivalent to the solubility limit of about 50 mM). However, in conclusion, from the slightly lower vmax,app value of the immobilized enzyme compared to the free enzyme, the active sites of the immobilized enzyme are not completely exposed to the substrate and/or a few of them are blocked due to immobilization, which results in lower reaction rates. A similar tendency can be derived from the KM,BA values. The free enzyme shows 2.5 times lower KM,BA value than the immobilized enzyme. This implies a higher affinity of the free enzyme to the substrate benzaldehyde than the immobilized one. Conceivably the lower affinity of the immobilized enzyme can be referred to a lower flexibility and steric hindrances due to the immobilization. Whereby the lower flexibility and steric hindrances influence rather the accessibility of the active site for the substrate than the active site by itself as the mechanism in the active site and enantioselectivity respectively is not influenced by the immobilization.

Fig. 4 Repetitive batch experiments for the carboligation catalyzed by immobilized BFD A460I– F464I. Reaction conditions: 40 mM benzaldehyde, 400 mM acetaldehyde in 5 mL 100 mM phosphate buffer, 0.5 mM ThDP, 2 mM MgSO4, pH 7.5 at 20 °C. 100 mg of dry carrier (0.47 mg of BFD A460I–F464I) was used for the reactions. Line is visual aid

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Bioprocess Biosyst Eng Fig. 5 Determination of kinetic constants for the carboligation of benzaldehyde and acetaldehyde catalyzed by immobilized BFD A460I– F464I. Specific activity (U/mg) versus benzaldehyde plot obtained in the presence of a 400 mM acetaldehyde and b 40 mM benzaldehyde, respectively

Although the discussed values are ‘‘only’’ apparent values the conclusions derived from this parameter correspond to the found reduction of activity from 1.4 to 0.99 U/mg (Table 2) due immobilization of the enzyme. Influence of pressure on the enantioselectivity of the immobilized enzyme A significant pressure effect on the enantioselectivity of the BFD A460I-F464I variant was found in previous investigations. With increasing pressure, a general tendency towards higher amounts of (R)-2-HPP could be observed [20]. While the previous investigations were done with free enzyme, in this work the influence of pressure on the enantioselectivity of the carrier bound enzyme was investigated. In Table 4 the results of the investigations are summarized. The enantiomeric excess of the immobilized enzyme is in agreement with the enantiomeric excess obtained by the corresponding homogeneous enzyme at atmospheric pressure. Thereby the enantioselectivity of the enzymes (free and immobilized) shows significant pressure dependence: with increasing pressure the enantiomeric excess of (R)-2HPP increases from 44 to 76% for the free and from 43 to 66% for the immobilized enzyme (Table 4). To investigate whether this type of pressure-induced change in enantioselective product formation is reversible or not, the enzymes (free and immobilized) were incubated for 30 min at 250 MPa. After pressure incubation, the enzyme/buffer Table 3 Kinetic parameters for the 2-HPP formation catalyzed by immobilized BFD A460I–F464I (Michaelis–Menten double substrate kinetics) BFD A460I–F464I

Immobilized enzyme Free enzyme [34]

vmax,app (U/mg) vmax (U/mg)

KM,BA,app (mM) KM,BA (mM)

KM,AA,app (mM) KM,AA (mM)

9.9 ± 4.0

51.9 ± 20.9

515.9 ± 271.9

17.6 ± 3.4

19.4 ± 6.7

775 ± 235

App apparent kinetic parameters introduced for the immobilized enzyme

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solutions were depressurized and than analyzed under atmospheric pressure regarding to the enantioselectivity of product formation. No change could be observed compared to the untreated enzyme (Table 4). So the pressure dependent change in enantioselectivity is not being attributed to an irreversible pressure induced change of the enzyme performance. The final enantiomeric excess is a consequence of two competing reaction pathways, yielding the (S)- or the (R)product. In literature, possible mechanisms of the considered enantioselective carboligation catalyzed by the enzyme BFD at atmospheric pressure are discussed [31–33]. It was found that the residues in positions 460, 464 and 461 are important for the stereoselectivity of BFD whereby the latter mainly defines the so-called (S)-pocket responsible for the formation of the (S)-product [31, 32]. Thereby all residues are part of an a-helix located next to the cofactor ThDP in the active site [33]. A structural-based explanation for the pressure-induced change in stereoselectivity can be derived from modeling studies (atmospheric pressure) which show that (S)-2-HPP results from an antiparallel approach of acetaldehyde as the acceptor to the ThDP-bound benzaldehyde, whereas (R)-2-HPP is the product of a parallel approach [32]. If the structural elements in the active site like the a-helix alter, the size of the (S)-pocket may be decreased/changed, the (S)-path is disfavoured and the amount of (R)-2-HPP will increase, which was observed with increasing pressure. In this context, it is remarkable, that the immobilized enzyme shows the same enantioselectivity as the free enzyme. This allows the conclusion, that the chosen immobilization method does not affect the flexibility of the active site and the mechanisms in the active site, respectively.

Conclusion The immobilization of the synthetically important enzyme benzoylformate decarboxylase variant named BFD A460I– F464I from Pseudomonas putida has been investigated.

Bioprocess Biosyst Eng Table 4 Enantiomeric excess of free and carrier-bound BFD A460IF464I at atmospheric and high pressure. All reactions were carried out in 50 mM phosphate buffer at pH 7.5 containing 40 mM benzaldehyde, 400 mM acetaldehyde, 0.5 mM ThDP and 2 mM MgSO4 at room temperature Free enzyme

Immobilized enzyme

References

ee (R)-2-HPP (%)

Untreated enzymea

44

43

ee (R)-2-HPP (%)

Pressure treated enzyme (250 MPa)a Under high pressure (200 MPa)

46

43

76

66

ee (R)-2-HPP (%)

Acknowledgments We thank the Deutsch Forschungsgemeinschaft DFG for the financial support of the project. Prof. Dr. Martina Pohl (Institute of Molecular Enzyme Technology, Heinrich-Heine University of Du¨sseldorf, Research Center Ju¨lich) is gratefully acknowledged for kindly providing the variant BFD A460I-F464I.

a

Enantiomeric excess was determined at atmospheric pressure and at room temperature

The used wide-pore silica shows a good accessibility of the immobilized enzyme. Although the attained enzyme loading of the silica carrier is one order of magnitude lower than the loading of a polymeric carrier (Sepabeads, results from literature [5]), a significant higher retention of activity was obtained. In case of covalently immobilized BFD A460I–F464I variant the activity was determined to be 70% related to the activity of free enzyme. From the determined kinetic constants, it can be concluded that the active site of the immobilized enzyme is not completely exposed to the substrate and/or that a few of them are blocked due to immobilization, which results in slightly lower reaction rates. At atmospheric pressure the enantiomeric excess of 2-HPP synthesized by immobilized BFD A460I–F464I is in well agreement with thereof synthesized by free enzyme. This allows the conclusion that the chosen immobilization method does not affect the flexibility of the active site and the mechanisms in the active site, respectively. Both the free and the immobilized enzyme show a pressure induced change in stereoselectivity, whereby the pressure effect is lower for the immobilized enzyme than for the free enzyme. The pressure effect on the selectivity depends on the sign and magnitude of volume changes accompanying the binding and elementary chemical steps. Furthermore, a change of selectivity can be the result of steric effects through pressure induced structural changes. Following the structural based explanation for the stereoselectivity of the investigated enzyme, the immobilization seems to stabilize structural elements like the a-helix which results in a lower pressure induced change in enantioselectivity compared to the free enzyme. In summarizing, it can be stated that the used mechanical stable wide pore silica carrier, which allows its application in different reactor types without restrictions, together with the applied covalent binding of the synthetically important BFD results in an efficient and versatile biocatalyst.

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