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Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747
Journal of Biotechnology 92 (2001) 179– 186 www.elsevier.com/locate/jbiotec
Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747 Kerstin Ragnitz, Markus Pietzsch *, Christoph Syldatk Institute of Biochemical Engineering, Uni6ersity of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany Received 13 June 2000; received in revised form 25 September 2000; accepted 13 October 2000
Abstract The immobilization procedure of the two industrially important hydantoin cleaving enzymes— hydantoinase and from Arthrobacter aurescens DSM 3747— was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B. © 2001 Elsevier Science B.V. All rights reserved. L-N-carbamoylase
Keywords: Hydantoinase; L-N-carbamoylase; Immobilization; Stabilization
1. Introduction
Abbre6iations: DEAE, diethylaminoethyl; HPLC, high-performance liquid chromatography; IMH, 5-indolylmethylhydantoin; N-CTrp, N-carbamoyl tryptophan; TCA, trichloroacetic acid. * Corresponding author. Present address: Institute of Biochemistry and Biotechnology, Technical University of Braunschweig, Spielmannstraße 7, D-38106 Braunschweig, Germany. Tel.: +49-531-391-5731; fax: +49-531-391-5763. E-mail address: [email protected] (M. Pietzsch).
The ‘hydantoinase method’ can be used for the production of a variety of proteinogenic and nonproteinogenic amino acids in optically pure form (Pietzsch and Syldatk, 1995). Depending on the enzymatic system, either D- or L-amino acids can be produced with 100% theoretical yield. For this hydrolytic reaction, two enzymes are involved in the stereoselective cleavage of D,L-5-monosubstituted hydantoins and N-carbamoyl amino acids, respectively.
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In contrast to the enzymes used in the preparation of D-amino acids starting from hydantoins, which are usually absolutely D-specific, the hydantoinase has only limited enantioselectivity in case of the enzymes obtained from Arthrobacter aurescens DSM 3747 and 3745 for a range of interesting products (May et al., 1998). Therefore, the key step in the synthesis of optically pure products is represented by the second enzyme, an N-carbamoyl-L-amino acid amidohydrolase (L-Ncarbamoylase) which therefore is absolutely necessary, in contrast to the D-process. Unfortunately, in resting cell biotransformations using A. aurescens DSM 3747, especially this second enzyme was found to be relatively unstable, which can be explained by the fast proteolysis observed for this enzyme (Siemann et al., 1993). Whole cell biocatalysts therefore are not reusable and a production process economically not feasible with the wildtype strain. For this reason, the immobilization and stabilization of the hydantoinase and the L-N-carbamoylase were of major importance for the development of a process for the production of unnatural L-amino acids from D,L-5-monosubstituted hydantoins. In a previous contribution, we were able to show that both isolated enzymes (crude preparations) could significantly be stabilized by several immobilization methods. However, so far the yield of enzymatic activity immobilized to the support was too low to fulfill industrial requirements (Pietzsch et al., 1998). In the present paper, we report on our results obtained during the optimization of covalent coupling methods of the hydantoinase and the L-Ncarbamoylase. The influence of the support, the protein concentration and the crosslinking agent (including glutaraldehyde which was recently used for the immobilization of a recombinant D-N-carbamoylase (Nanba et al., 1999)) on the yield of activity are discussed. For the L-N-carbamoylase, both, the crude wild-type as well as the recombinant enzyme purified after expression in Escherichia coli (Wilms et al., 1999; Pietzsch et al., 2000) were used for the optimization experiments.
2. Materials and methods
2.1. General All chemicals were of analytical grade and purchased from Fluka Chemie AG, Buchs, Switzerland. Immobilization supports were a gift from Ro¨ hm GmbH (Darmstadt, Germany: Eupergit C, Eupergit C 250 L) or purchased from Pharmacia Biotech (Freiburg, Germany: EAH Sepharose 4B).
2.2. Enzyme preparation (A) To obtain the hydantoinase and the L-Ncarbamoylase wild-type enzymes, A. aurescens DSM 3747 was cultivated in a 100-l bioreactor under conditions as previously reported (Syldatk et al., 1990) using 0.3 g l − 1 N-3-methylene-D,LIMH as inducer. Cell disruption was carried out continuously under optimized conditions according to the results presented elsewhere (Bunge et al., 1992) using a cooled (− 20 °C) 600 ml disintegration cell and the Dyno-Mill KDL (Willy A. Bachofen, Basel, Switzerland). Unleaded glass beads (480 ml, diameter 0.3 mm) were agitated at an agitator speed of 2500 rpm. An ice-cooled suspension of bio-wet-mass (30% w/v) suspended in 0.2 M TRIS–buffer, containing 1 mM MnCl2, pH 7.0 was pumped three times through the cell with a flow rate of 80 ml min − 1 (crude extract). The enzyme fraction used for the immobilization experiments was obtained by ammonium sulfate precipitation at 4 °C. Therefore, a precooled, saturated (NH4)2SO4-solution (105 ml) was continuously added with a flow rate of 0.34 ml min − 1 (Watson Marlow, Falmouth, GB) to the crude extract (70 ml) while mechanical stirring (Heidolph, Kelheim, Germany). The hydantoinase and the L-N-carbamoylase precipitated at a (NH4)2SO4-concentration of 60%. After centrifugation (Beckman, GB) of the suspension with 24 700× g for 20 min at 4 °C, the supernatant was decanted and the pellet was stored at − 20 °C. (B) To obtain the pure recombinant L-N-carbamoylase, E. coli W3110 pAW178-2 was cultivated in 400 ml batch cultures in LB-medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.5)
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containing 0.1 mg ml − 1 ampicillin according to the results presented elsewhere (Wilms et al., 1999). At OD500 = 0.5 the enzyme activity was induced by adding rhamnose (2 g l − 1). Cell disruption of an ice-cooled suspension of 5 g of bio-wet-mass suspended in 11 ml of 0.1 M TRIS– buffer, containing 0.1 mM MnCl2, pH 7.0 was carried out using a French pressure cell (AMINCO, Urbana, IL). The enzyme fraction for the immobilization experiments was obtained by two chromatography steps. The crude extract (13 ml) was diluted with 10 ml of 10 mM TRIS – buffer, pH 7.0 and applied to a Streamline diethylaminoethyl column (Pharmacia; column volume 20 ml) equilibrated with buffer A (50 mM TRIS, pH 7.0) and eluted by applying a linear gradient between buffer A and B (50 mM TRIS, 1 M NaCl, pH 7.0) within 200 ml. The active fractions were collected (54 ml) and concentrated by ultrafiltration (Filtron, Northborough; 30 kDa). The concentrate (20 ml) was diluted with 20 ml 10 mM TRIS – buffer, pH 7.0 and applied to a Mono Q column (Pharmacia) equilibrated with buffer A and eluted by applying a linear gradient between buffer A and B within 200 ml. The active fractions were collected (volume 10 ml; protein concentration 3.4 g l − 1; enzyme activity 4.3 U ml − 1) and stored in portions of 1 ml at −20 °C.
2.3. Preparation of Eupergit C ( NH2) and Eupergit C 250 L ( NH2) For the immobilization via the carbodiimide method (Verhoeven et al., 1994), NH2-groups were introduced into Eupergit C and Eupergit C 250 L. Ammoniolysis of 5 g dry carrier was carried out in closed containers for 48 h at 45 °C using 40 ml aqueous ammonia (2.5%). Afterwards the excess of NH3 was washed off with deionized water until the pH was neutral again. Finally, the modified carrier was equilibrated with phosphate buffer (0.1 M, pH 6.5) and stored at 4 °C.
2.4. Immobilization of the crude hydantoinase and L -N-carbamoylase to Eupergit C ( NH2), Eupergit C 250 L ( NH2) and EAH Sepharose 4B The pellet from enzyme preparation A was resuspended in 150 ml deionized water containing
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1 mM MnCl2. After centrifugation at 24 700× g for 20 min at 4 °C, the clarified supernatant containing both enzymes was diluted with deionized water containing 1 mM MnCl2 to a final protein concentrations of 0.6 (Eupergit C (NH2), Eupergit C 250 L (NH2)) and 1.2 g l − 1 (EAH Sepharose 4B). Eight milliliters of the diluted enzyme solution was added to 1 g of wet carrier (Eupergit C (NH2), EAH Sepharose 4B) or 0.5 g of dry Eupergit C 250 L (NH2). While manually shaking from time to time, 500 ml of a N-(dimethylaminopropyl)-N%-ethylcarbodiimide (EDC) stock solution in deionized water (1.7 M, pH 5.0, adjusted with 1% HCl) were added dropwise to the respective ice-cooled suspensions which were afterwards shaken overhead for 18 h at 4 °C. The immobilized enzymes were filtered off and washed three times each with 20 ml 0.1 M TRIS containing 0.5 M NaCl, pH 8.5. Before storage at 4 °C, the immobilized enzymes were washed three times with 20 ml 0.1 M TRIS, pH 8.5.
2.5. Immobilization of the crude hydantoinase and L -N-carbamoylase to Eupergit C and Eupergit C 250 L The pellet from enzyme preparation A was resuspended in 30 ml of a 1 M phosphate buffer, pH 7.5 and centrifuged at 24 700× g for 20 min at 4 °C. After diluting the clarified supernatant to a final concentration of 3 g l − 1, 4 and 6 ml, respectively, were added to 1 g of dry Eupergit C and Eupergit C 250 L. The suspension was gently shaken by horizontal rotation for 20 h at 4 °C. To hydrolyze excess oxirane groups, the immobilized enzyme was filtered off and washed three times each with 25 ml of 0.1 M TRIS–buffer, pH 8.5 before storage at 4 °C for at least 48 h prior to use.
2.6. Immobilization of the pure recombinant L -N-carbamoylase to Eupergit C ( NH2), Eupergit C 250 L ( NH2) and EAH Sepharose 4B The thawed Mono Q fraction (enzyme preparation B) was diluted 1:80 with deionized water containing 1 mM MnCl2. Varying amounts of the respective carrier were added to 4 ml of the
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diluted enzyme solution. For the investigation of the influence of the EDC concentration 444 ml of a 10 mM EDC stock solution, 1 ml of a 100 mM EDC stock solution and 444, 545, 651, 762 and 878 ml of a 500 mM EDC stock solution were added dropwise to each portion (final concentrations 1, 10, 20, 50, 60, 70, 80 and 90 mM). The suspensions were shaken overhead for 17 h at 4 °C. To hydrolyze excess activated esters, the immobilized enzymes were filtered off and washed three times each with 25 ml of 0.2 M TRIS –buffer containing 0.5 M NaCl, pH 8.5. For storage, the immobilized enzymes were washed three times each with 25 ml 0.1 M TRIS –buffer, pH 8.5 and stored at 4 °C.
2.7. Analytical methods Protein concentrations were determined according to the method of Bradford (1976). The test kit was purchased from BioRad (Munich, Germany). High-performance liquid chromatography (HPLC) analysis was carried out as described elsewhere (May et al., 1998). Retention times: Trp, 10 min; 5-indolylmethylhydantoin (IMH), 15.4 min; and N-carbamoyl tryptophan (N-CTrp), 17.3 min.
2.8. Determination of the acti6ity of free enzymes Fifty microliters of the enzyme solution were added to 800 ml of the preincubated (37 °C) substrate solution (0.4 g l − 1 IMH or 0.4 g l − 1 N-C-L-Trp, respectively, in 0.1 M TRIS– buffer, pH 8.5). The reaction was stopped after 5 min by adding 400 ml trichloroacetic acid (TCA) (12%) and substrate as well as product concentrations were analyzed by HPLC after centrifugation.
2.9. Determination of the acti6ity of the immobilized enzymes Eight hundred microliters of the preincubated substrate solution (see above) were added to 100 mg of wet immobilized enzyme and shaken for 10 min at 37 °C. The reaction was stopped by adding 400 ml of TCA and the conversion was analyzed by HPLC after centrifugation.
3. Results and discussion For the immobilization of proteins, several methods including adsorptive, covalent and inclusion techniques have been investigated and published (Tischer, 1995; Fa´ ga´ in, 1997). Usually, covalent immobilization is of major interest because the bonds formed are much more stable than those formed by electrostatic interactions or by adsorption. Because the carrier cannot be regenerated, for industrial purposes it is required that covalently bound biocatalysts have a high stability. For the hydantoin cleaving enzymes from A. aurescens, covalent coupling to different types of Eupergit (via oxirane groups) and via amino groups to EAH Sepharose and to modified Eupergit C and C 250 L (using water-soluble carbodiimide, see Table 1 for a characterization of the various supports used) was investigated and the procedure of immobilization was optimized. In order to provide a technically useful method, the crude hydantoinase and L-N-carbamoylase obtained after cell disruption and ammonium sulfate precipitation were used as starting materials for the immobilization procedures at first. Covalent coupling of the hydantoinase using carbodiimide resulted in coupling yields between 10% (Eupergit C (NH2)) and 60% (EAH Sepharose 4B (Fig. 1). The lower yield obtained with modified Eupergit C and C 250 L in comparison to EAH Sepharose 4B may be explained by the more hydrophilic matrix of the Sepharose based support. The direct immobilization of the crude hydantoinase to epoxy activated Eupergit C and C 250 L led to activity yields of 20 and 90%, respectively. Obviously, Eupergit C 250 L is favorable for the immobilization of the hydantoinase in active form, which may be explained by the larger pore size reported for this carrier (Ro¨ hm, 1995). For the wild-type L-N-carbamoylase it was previously reported that the covalent coupling via oxirane groups which are known to react with amino, thiol and hydroxy groups of the enzyme resulted in only low activity yields (Pietzsch et al., 1998). During the present optimization, it turned out that crosslinking of the L-N-carbam-a
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Table 1 Functional groups and corresponding immobilization methods for different types of supports Density of groups (mmol g−1 dry support)
Immobilization method
Eupergit C
\600a
Oxirane
Eupergit C 250 L
\200a
Oxirane
530b 370b 440b
Carbodiimide Carbodiimide Carbodiimide
Support
Modified Eupergit C Modified Eupergit C 250 L EAH Sepharose 4B a b
Functional group
NH2 NH2 NH2
Data from supplier (Ro¨ hm, 1995). Measured according to Halling and Dunhill (1979).
oylase with preactivated supports carrying aldehyde groups (e.g. glutaraldehyde method according to Bryjak et al. (1993)) resulted in an almost complete loss of activity, too (results not shown). Only the carbodiimide method – crosslinking carboxylic acid groups of the enzyme and amino groups of a carrier – was applicable for the immobilization of the L-N-carbamoylase. However, initial experiments of covalently coupling the wild-type L-N-carbamoylase by carbodiimide resulted in low activity yields (Fig. 4). In order to investigate whether the reasons for these results were related to contaminating proteins or to a generally inactivating modification of amino acid residues essential for activity, the recombinant L-N-carbamoylase expressed in E. coli was purified (Wilms et al., 1999) and the pure enzyme was used for the immobilization experiments. Interestingly, coupling via carbodiimide resulted in significant amounts of active L-N-carbamoylase bound to the carrier. Also, it was proven for the purified recombinant enzyme that coupling via glutaraldehyde was not possible, even if pure enzyme was used. Therefore, covalent coupling of the L-N-carbamoylase seems only to be possible via carboxylic groups. In order to optimize the activity yield for the carbodiimide-mediated immobilization of the
purified L-N-carbamoylase, the influence of the concentration of N-(3-dimethylaminopropyl)-N%ethylcarbodiimide (EDC), the protein to carrier relation and the type of support were investigated.
Fig. 1. Immobilization of L-hydantoinase from A. aurescens DSM 3747. Influence of different supports and methods on the yield of activity.
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Fig. 2. Immobilization of the purified, recombinant L-N-carbamoylase from E. coli. Dependence of the activity yield on the carbodiimide concentration after adsorption of the L-Ncarbamoylase to modified Eupergit C 250 L (NH2). Protein concentration 0.04 g l − 1, reaction volume 4 ml, 0.5 g wet carrier.
For direct comparison of the oxirane and the carbodiimide coupling results, amino groups were introduced to Eupergit C and Eupergit C 250 L by reaction with aqueous ammonia. As shown in Fig. 2 the activity yield versus EDCconcentration plot shows a typical saturation profile with a slight decrease of activity yield at higher EDC concentrations. At a protein concentration of 0.04 g l − 1 the optimum concentration of EDC was determined to be between 50 and 60 mM. Almost 20% of the initial activity were immobilized. The activity yield could be further enhanced by variation of the protein to carrier relation. As can be seen from Fig. 3 the activity yield of the purified L-N-carbamoylase coupled to Eupergit C 250 L (NH2) increased from 20 to : 30% at higher surface to protein ratios. Interestingly, 90% of the total protein amount were bound to the carrier during these experiments but only 30% were active. The reasons for this difference are still unknown, but may be caused by the formation of multiple layers of the enzyme and by adsorption effects accompanied with conformational changes of the adsorbed protein which may result in changed catalytic
efficacy or even complete inactivation (Soderquist and Walton, 1980; Yongli et al., 1999). Under optimized conditions, the influence of the type of support, varying in the basic matrix and the density of reactive groups (Table 1) was investigated. As can be seen from Fig. 4 and Table 2, the activity yield could be significantly enhanced by immobilizing the purified, recombinant enzyme from E. coli. Although the density of amino groups presented by the carrier is higher for modified Eupergit C (NH2) (Table 1) the enzyme was coupled to EAH Sepharose with nearly quantitative yield of activity. On the other hand, Eupergit based supports are obtained by bead polymerization of the monomers methacrylamide, N,N%-methylene-bis-methacrylamide, glycidyl methacrylate and allyl glycidyl ether. Thus, Eupergit supports are in general more hydrophobic than EAH Sepharose. Obviously, the interactions between the hydrophilic EAH Sepharose and the L-N-carbamoylase are favorable for the retention of enzymatic activity.
Fig. 3. Immobilization of the purified L-N-carbamoylase from recombinant E. coli using water-soluble carbodiimide (60 mM EDC): Influence of the protein to carrier relation on the activity yield. Protein concentration 0.04 g l − 1, reaction volume 4 ml.
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Fig. 4. Immobilization of the L-N-carbamoylase (crude enzyme from A. aurescens DSM 3747 and purified enzyme from recombinant E. coli ). Comparison of different supports with respect to the activity yield after immobilization via water-soluble carbodiimide (60 mM EDC, 0.04 mg ml − 1 protein concentration).
4. Conclusions Whereas the immobilization of the hydantoinase from A. aurescens turned out to be relatively simple in performance, the immobilization of the L-N-carbamoylase required a lot of optimization experiments. Since this enzyme catalyzes the stereospecific conversion of the N-carbamoyl
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amino acid produced by the unspecific hydantoinase, it is essential for the production of optically pure L-amino acids. As could be shown by the immobilization of the purified recombinant enzyme, the activity yield of the L-N-carbamoylase was deteriorated by contaminating proteins. Furthermore, covalent immobilization of the L-Ncarbamoylase was only possible via the carbodiimide method, i.e. coupling via enzymes carboxylic groups to support carrying primary amino groups. Additionally, a hydrophilic carrier such as EAH Sepharose was favorable in immobilizing the L-N-carbamoylase in active form. Using the optimized protocol, the activity yield could be enhanced from 4 to :100%. Coupling via amino groups by water-soluble carbodiimide had to be carried out at a concentration of between 50 and 60 mM of EDC to ensure a sufficient coupling yield without loss of activity, which might be caused by crosslinking of enzyme molecules prior to the covalent coupling to the support. Although the high activity yields obtained in the present paper and the operational stabilities observed in a previous one (Pietzsch et al., 1998) represent important improvements towards an industrially feasible biocatalyst, the specific activities obtained so far still are too low to fulfill economical requirements. Since the proteins could almost completely be immobilized in all experiments, there should be some reason for the differences between the activity yields and the coupling yields. Further experiments should reveal, if there
Table 2 Specific activity, activity yield and protein yield after immobilization of the hydantoinase and the carbodiimide method
L-N-carbamoylase
by the
Enzyme
Support
Specific activity (U g−1)
Activity yield (%) Protein (mg g−1)
Protein yield (%)
Hydantoinase Hydantoinase Hydantoinase L-N-Carbamoylase L-N-Carbamoylase L-N-Carbamoylase
EAH Sepharose 4B Eupergit C (NH2) Eupergit C 250 L (NH2) Eupergit C (NH2) Eupergit C 250 L (NH2) EAH Sepharose 4B
0.2 0.14 0.11 0.005 0.014 0.032
49 15 12 19 54 100
95 48 73 99 98 99
3.23 3.63 5.63 0.25 0.24 0.25
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is an influence of initial adsorption processes on the amount of active proteins, which are then fixed by covalent bonds. In continuing experiments, genetically modified proteins have been created and purified (Pietzsch et al., 2000). For example, a L-N-carbamoylase carrying an aspartate-tag has been prepared in order to investigate if an N-terminal fusion of carboxylic groups to the wild-type enzyme is accompanied with a positive influence on the amount of actively coupled enzyme during carbodiimide-mediated immobilization.
References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Bryjak, J., Trochimczuk, A., Noworyta, A., 1993. Effect of polymer matrix on penicillin acylase immobilization on copolymers of butyl acrylate and ethylene glycol dimethacrylate. J. Chem. Tech. Biotechnol. 57, 73 –78. Bunge, F., Pietzsch, M., Mu¨ ller, R., Syldatk, C., 1992. Mechanical disruption of Arthrobacter sp. DSM 3747 in stirred ball mills for the release of hydantoin-cleaving enzymes. Chem. Eng. Sci. 47, 225 – 232. ´ ., 1997. Stabilizing Protein Function. Springer, Fa´ ga´ in, O Berlin. Halling, P.J., Dunhill, P., 1979. Improved nonporous magnetic supports for immobilized enzymes. Biotechnol. Bioeng. 21, 393– 416. May, O., Siemann, M., Pietzsch, M., Kiess, M., Mattes, R., Syldatk, C., 1998. Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterisation as new member of cyclic amidases. J. Biotechnol. 61, 1 –13. Nanba, H., Ikenaka, Y., Yamada, Y., Yajima, K., Takano, M., Ohkubo, K., Hiraishi, Y., Yamada, K., Takahashi, S., 1999. Immobilization of thermotolerant N-carbamyl-Damino acid amidohydrolase. J. Mol. Catal. B Enzym. 6, 257 – 263.
Pietzsch, M., Oberreuter, H., Petrovska, B., Ragnitz, K., Syldatk, C., 1998. Immobilization of hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747 – effect of the coupling method on the stability of the L-N-carbamoylase. In: Ballesteros, A., Plou, F.J., Iborra, J.L., Halling, P. (Eds.), Stability and Stabilization of Biocatalysts. Elsevier, Amsterdam, pp. 517 – 522. Pietzsch, M., Syldatk, C., 1995. Hydrolysis and formation of hydantoins. In: Drauz, K., Waldmann, H. (Eds.), Enzyme Catalysis in Organic Synthesis. VCH, Weinheim, pp. 409 – 431. Pietzsch, M., Wiese, A., Ragnitz, K., Wilms, B., Altenbuchner, J., Mattes, R., Syldatk, C., 2000. Purification of recombinant hydantoinase and L-N-carbamoylase from Arthrobacter aurescens expressed in Escherichia coli: comparison of wild-type and genetically modified proteins. J. Chromatogr. B 737, 179 – 186. Ro¨ hm, 1995. Eupergit: immobilization of enzymes on Eupergit C and Eupergit C 250 L. Product Inform. Siemann, M., Syldatk, C., Wagner, F., 1993. Detection and comparison of strains with selective L-hydantoin cleaving activity using polyclonal antibodies. Biotechnol. Tech. 7, 361 – 366. Soderquist, M.E., Walton, A.G., 1980. Structural changes in proteins adsorbed on polymer surfaces. J. Coll. Interface Sci. 75, 386 – 397. Syldatk, C., Mackowiak, V., Ho¨ ke, H., Groß, C., Dombach, G., Wagner, F., 1990. Cell growth and enzyme synthesis of a mutant of Arthrobacter spec. (DSM 3747) used for the production of L-amino acids from D,L-5-monosubstituted hydantoins. J. Biotechnol. 14, 345 – 362. Tischer, W., 1995. Immobilization of enzymes. In: Drauz, K., Waldmann, H. (Eds.), Enzyme Catalysis in Organic Synthesis. VCH, Weinheim, pp. 73 – 87. Verhoeven, M., Cahalan, P., Cahalan, L., Hendriks, M., Foache, B., 1994. Biomolecule immobilization with carbodiimide. 0 608 095 A1, Medtronic, Inc., USA. Wilms, B., Wiese, A., Syldatk, C., Mattes, R., Altenbuchner, J., Pietzsch, M., 1999. Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli. J. Biotechnol. 68, 101 – 113. Yongli, C., Xiufang, Z., Yandao, G., Nanming, Z., Tingying, Z., Xinqi, S., 1999. Conformational changes of fibrinogen adsorption onto hydroxyapatite and titanium nanoparticles. J. Coll. Interface Sci. 214, 38 – 45.
Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747 Kerstin Ragnitz, Markus Pietzsch *, Christoph Syldatk Institute of Biochemical Engineering, Uni6ersity of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany Received 13 June 2000; received in revised form 25 September 2000; accepted 13 October 2000
Abstract The immobilization procedure of the two industrially important hydantoin cleaving enzymes— hydantoinase and from Arthrobacter aurescens DSM 3747— was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B. © 2001 Elsevier Science B.V. All rights reserved. L-N-carbamoylase
Keywords: Hydantoinase; L-N-carbamoylase; Immobilization; Stabilization
1. Introduction
Abbre6iations: DEAE, diethylaminoethyl; HPLC, high-performance liquid chromatography; IMH, 5-indolylmethylhydantoin; N-CTrp, N-carbamoyl tryptophan; TCA, trichloroacetic acid. * Corresponding author. Present address: Institute of Biochemistry and Biotechnology, Technical University of Braunschweig, Spielmannstraße 7, D-38106 Braunschweig, Germany. Tel.: +49-531-391-5731; fax: +49-531-391-5763. E-mail address: [email protected] (M. Pietzsch).
The ‘hydantoinase method’ can be used for the production of a variety of proteinogenic and nonproteinogenic amino acids in optically pure form (Pietzsch and Syldatk, 1995). Depending on the enzymatic system, either D- or L-amino acids can be produced with 100% theoretical yield. For this hydrolytic reaction, two enzymes are involved in the stereoselective cleavage of D,L-5-monosubstituted hydantoins and N-carbamoyl amino acids, respectively.
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In contrast to the enzymes used in the preparation of D-amino acids starting from hydantoins, which are usually absolutely D-specific, the hydantoinase has only limited enantioselectivity in case of the enzymes obtained from Arthrobacter aurescens DSM 3747 and 3745 for a range of interesting products (May et al., 1998). Therefore, the key step in the synthesis of optically pure products is represented by the second enzyme, an N-carbamoyl-L-amino acid amidohydrolase (L-Ncarbamoylase) which therefore is absolutely necessary, in contrast to the D-process. Unfortunately, in resting cell biotransformations using A. aurescens DSM 3747, especially this second enzyme was found to be relatively unstable, which can be explained by the fast proteolysis observed for this enzyme (Siemann et al., 1993). Whole cell biocatalysts therefore are not reusable and a production process economically not feasible with the wildtype strain. For this reason, the immobilization and stabilization of the hydantoinase and the L-N-carbamoylase were of major importance for the development of a process for the production of unnatural L-amino acids from D,L-5-monosubstituted hydantoins. In a previous contribution, we were able to show that both isolated enzymes (crude preparations) could significantly be stabilized by several immobilization methods. However, so far the yield of enzymatic activity immobilized to the support was too low to fulfill industrial requirements (Pietzsch et al., 1998). In the present paper, we report on our results obtained during the optimization of covalent coupling methods of the hydantoinase and the L-Ncarbamoylase. The influence of the support, the protein concentration and the crosslinking agent (including glutaraldehyde which was recently used for the immobilization of a recombinant D-N-carbamoylase (Nanba et al., 1999)) on the yield of activity are discussed. For the L-N-carbamoylase, both, the crude wild-type as well as the recombinant enzyme purified after expression in Escherichia coli (Wilms et al., 1999; Pietzsch et al., 2000) were used for the optimization experiments.
2. Materials and methods
2.1. General All chemicals were of analytical grade and purchased from Fluka Chemie AG, Buchs, Switzerland. Immobilization supports were a gift from Ro¨ hm GmbH (Darmstadt, Germany: Eupergit C, Eupergit C 250 L) or purchased from Pharmacia Biotech (Freiburg, Germany: EAH Sepharose 4B).
2.2. Enzyme preparation (A) To obtain the hydantoinase and the L-Ncarbamoylase wild-type enzymes, A. aurescens DSM 3747 was cultivated in a 100-l bioreactor under conditions as previously reported (Syldatk et al., 1990) using 0.3 g l − 1 N-3-methylene-D,LIMH as inducer. Cell disruption was carried out continuously under optimized conditions according to the results presented elsewhere (Bunge et al., 1992) using a cooled (− 20 °C) 600 ml disintegration cell and the Dyno-Mill KDL (Willy A. Bachofen, Basel, Switzerland). Unleaded glass beads (480 ml, diameter 0.3 mm) were agitated at an agitator speed of 2500 rpm. An ice-cooled suspension of bio-wet-mass (30% w/v) suspended in 0.2 M TRIS–buffer, containing 1 mM MnCl2, pH 7.0 was pumped three times through the cell with a flow rate of 80 ml min − 1 (crude extract). The enzyme fraction used for the immobilization experiments was obtained by ammonium sulfate precipitation at 4 °C. Therefore, a precooled, saturated (NH4)2SO4-solution (105 ml) was continuously added with a flow rate of 0.34 ml min − 1 (Watson Marlow, Falmouth, GB) to the crude extract (70 ml) while mechanical stirring (Heidolph, Kelheim, Germany). The hydantoinase and the L-N-carbamoylase precipitated at a (NH4)2SO4-concentration of 60%. After centrifugation (Beckman, GB) of the suspension with 24 700× g for 20 min at 4 °C, the supernatant was decanted and the pellet was stored at − 20 °C. (B) To obtain the pure recombinant L-N-carbamoylase, E. coli W3110 pAW178-2 was cultivated in 400 ml batch cultures in LB-medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.5)
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containing 0.1 mg ml − 1 ampicillin according to the results presented elsewhere (Wilms et al., 1999). At OD500 = 0.5 the enzyme activity was induced by adding rhamnose (2 g l − 1). Cell disruption of an ice-cooled suspension of 5 g of bio-wet-mass suspended in 11 ml of 0.1 M TRIS– buffer, containing 0.1 mM MnCl2, pH 7.0 was carried out using a French pressure cell (AMINCO, Urbana, IL). The enzyme fraction for the immobilization experiments was obtained by two chromatography steps. The crude extract (13 ml) was diluted with 10 ml of 10 mM TRIS – buffer, pH 7.0 and applied to a Streamline diethylaminoethyl column (Pharmacia; column volume 20 ml) equilibrated with buffer A (50 mM TRIS, pH 7.0) and eluted by applying a linear gradient between buffer A and B (50 mM TRIS, 1 M NaCl, pH 7.0) within 200 ml. The active fractions were collected (54 ml) and concentrated by ultrafiltration (Filtron, Northborough; 30 kDa). The concentrate (20 ml) was diluted with 20 ml 10 mM TRIS – buffer, pH 7.0 and applied to a Mono Q column (Pharmacia) equilibrated with buffer A and eluted by applying a linear gradient between buffer A and B within 200 ml. The active fractions were collected (volume 10 ml; protein concentration 3.4 g l − 1; enzyme activity 4.3 U ml − 1) and stored in portions of 1 ml at −20 °C.
2.3. Preparation of Eupergit C ( NH2) and Eupergit C 250 L ( NH2) For the immobilization via the carbodiimide method (Verhoeven et al., 1994), NH2-groups were introduced into Eupergit C and Eupergit C 250 L. Ammoniolysis of 5 g dry carrier was carried out in closed containers for 48 h at 45 °C using 40 ml aqueous ammonia (2.5%). Afterwards the excess of NH3 was washed off with deionized water until the pH was neutral again. Finally, the modified carrier was equilibrated with phosphate buffer (0.1 M, pH 6.5) and stored at 4 °C.
2.4. Immobilization of the crude hydantoinase and L -N-carbamoylase to Eupergit C ( NH2), Eupergit C 250 L ( NH2) and EAH Sepharose 4B The pellet from enzyme preparation A was resuspended in 150 ml deionized water containing
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1 mM MnCl2. After centrifugation at 24 700× g for 20 min at 4 °C, the clarified supernatant containing both enzymes was diluted with deionized water containing 1 mM MnCl2 to a final protein concentrations of 0.6 (Eupergit C (NH2), Eupergit C 250 L (NH2)) and 1.2 g l − 1 (EAH Sepharose 4B). Eight milliliters of the diluted enzyme solution was added to 1 g of wet carrier (Eupergit C (NH2), EAH Sepharose 4B) or 0.5 g of dry Eupergit C 250 L (NH2). While manually shaking from time to time, 500 ml of a N-(dimethylaminopropyl)-N%-ethylcarbodiimide (EDC) stock solution in deionized water (1.7 M, pH 5.0, adjusted with 1% HCl) were added dropwise to the respective ice-cooled suspensions which were afterwards shaken overhead for 18 h at 4 °C. The immobilized enzymes were filtered off and washed three times each with 20 ml 0.1 M TRIS containing 0.5 M NaCl, pH 8.5. Before storage at 4 °C, the immobilized enzymes were washed three times with 20 ml 0.1 M TRIS, pH 8.5.
2.5. Immobilization of the crude hydantoinase and L -N-carbamoylase to Eupergit C and Eupergit C 250 L The pellet from enzyme preparation A was resuspended in 30 ml of a 1 M phosphate buffer, pH 7.5 and centrifuged at 24 700× g for 20 min at 4 °C. After diluting the clarified supernatant to a final concentration of 3 g l − 1, 4 and 6 ml, respectively, were added to 1 g of dry Eupergit C and Eupergit C 250 L. The suspension was gently shaken by horizontal rotation for 20 h at 4 °C. To hydrolyze excess oxirane groups, the immobilized enzyme was filtered off and washed three times each with 25 ml of 0.1 M TRIS–buffer, pH 8.5 before storage at 4 °C for at least 48 h prior to use.
2.6. Immobilization of the pure recombinant L -N-carbamoylase to Eupergit C ( NH2), Eupergit C 250 L ( NH2) and EAH Sepharose 4B The thawed Mono Q fraction (enzyme preparation B) was diluted 1:80 with deionized water containing 1 mM MnCl2. Varying amounts of the respective carrier were added to 4 ml of the
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diluted enzyme solution. For the investigation of the influence of the EDC concentration 444 ml of a 10 mM EDC stock solution, 1 ml of a 100 mM EDC stock solution and 444, 545, 651, 762 and 878 ml of a 500 mM EDC stock solution were added dropwise to each portion (final concentrations 1, 10, 20, 50, 60, 70, 80 and 90 mM). The suspensions were shaken overhead for 17 h at 4 °C. To hydrolyze excess activated esters, the immobilized enzymes were filtered off and washed three times each with 25 ml of 0.2 M TRIS –buffer containing 0.5 M NaCl, pH 8.5. For storage, the immobilized enzymes were washed three times each with 25 ml 0.1 M TRIS –buffer, pH 8.5 and stored at 4 °C.
2.7. Analytical methods Protein concentrations were determined according to the method of Bradford (1976). The test kit was purchased from BioRad (Munich, Germany). High-performance liquid chromatography (HPLC) analysis was carried out as described elsewhere (May et al., 1998). Retention times: Trp, 10 min; 5-indolylmethylhydantoin (IMH), 15.4 min; and N-carbamoyl tryptophan (N-CTrp), 17.3 min.
2.8. Determination of the acti6ity of free enzymes Fifty microliters of the enzyme solution were added to 800 ml of the preincubated (37 °C) substrate solution (0.4 g l − 1 IMH or 0.4 g l − 1 N-C-L-Trp, respectively, in 0.1 M TRIS– buffer, pH 8.5). The reaction was stopped after 5 min by adding 400 ml trichloroacetic acid (TCA) (12%) and substrate as well as product concentrations were analyzed by HPLC after centrifugation.
2.9. Determination of the acti6ity of the immobilized enzymes Eight hundred microliters of the preincubated substrate solution (see above) were added to 100 mg of wet immobilized enzyme and shaken for 10 min at 37 °C. The reaction was stopped by adding 400 ml of TCA and the conversion was analyzed by HPLC after centrifugation.
3. Results and discussion For the immobilization of proteins, several methods including adsorptive, covalent and inclusion techniques have been investigated and published (Tischer, 1995; Fa´ ga´ in, 1997). Usually, covalent immobilization is of major interest because the bonds formed are much more stable than those formed by electrostatic interactions or by adsorption. Because the carrier cannot be regenerated, for industrial purposes it is required that covalently bound biocatalysts have a high stability. For the hydantoin cleaving enzymes from A. aurescens, covalent coupling to different types of Eupergit (via oxirane groups) and via amino groups to EAH Sepharose and to modified Eupergit C and C 250 L (using water-soluble carbodiimide, see Table 1 for a characterization of the various supports used) was investigated and the procedure of immobilization was optimized. In order to provide a technically useful method, the crude hydantoinase and L-N-carbamoylase obtained after cell disruption and ammonium sulfate precipitation were used as starting materials for the immobilization procedures at first. Covalent coupling of the hydantoinase using carbodiimide resulted in coupling yields between 10% (Eupergit C (NH2)) and 60% (EAH Sepharose 4B (Fig. 1). The lower yield obtained with modified Eupergit C and C 250 L in comparison to EAH Sepharose 4B may be explained by the more hydrophilic matrix of the Sepharose based support. The direct immobilization of the crude hydantoinase to epoxy activated Eupergit C and C 250 L led to activity yields of 20 and 90%, respectively. Obviously, Eupergit C 250 L is favorable for the immobilization of the hydantoinase in active form, which may be explained by the larger pore size reported for this carrier (Ro¨ hm, 1995). For the wild-type L-N-carbamoylase it was previously reported that the covalent coupling via oxirane groups which are known to react with amino, thiol and hydroxy groups of the enzyme resulted in only low activity yields (Pietzsch et al., 1998). During the present optimization, it turned out that crosslinking of the L-N-carbam-a
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Table 1 Functional groups and corresponding immobilization methods for different types of supports Density of groups (mmol g−1 dry support)
Immobilization method
Eupergit C
\600a
Oxirane
Eupergit C 250 L
\200a
Oxirane
530b 370b 440b
Carbodiimide Carbodiimide Carbodiimide
Support
Modified Eupergit C Modified Eupergit C 250 L EAH Sepharose 4B a b
Functional group
NH2 NH2 NH2
Data from supplier (Ro¨ hm, 1995). Measured according to Halling and Dunhill (1979).
oylase with preactivated supports carrying aldehyde groups (e.g. glutaraldehyde method according to Bryjak et al. (1993)) resulted in an almost complete loss of activity, too (results not shown). Only the carbodiimide method – crosslinking carboxylic acid groups of the enzyme and amino groups of a carrier – was applicable for the immobilization of the L-N-carbamoylase. However, initial experiments of covalently coupling the wild-type L-N-carbamoylase by carbodiimide resulted in low activity yields (Fig. 4). In order to investigate whether the reasons for these results were related to contaminating proteins or to a generally inactivating modification of amino acid residues essential for activity, the recombinant L-N-carbamoylase expressed in E. coli was purified (Wilms et al., 1999) and the pure enzyme was used for the immobilization experiments. Interestingly, coupling via carbodiimide resulted in significant amounts of active L-N-carbamoylase bound to the carrier. Also, it was proven for the purified recombinant enzyme that coupling via glutaraldehyde was not possible, even if pure enzyme was used. Therefore, covalent coupling of the L-N-carbamoylase seems only to be possible via carboxylic groups. In order to optimize the activity yield for the carbodiimide-mediated immobilization of the
purified L-N-carbamoylase, the influence of the concentration of N-(3-dimethylaminopropyl)-N%ethylcarbodiimide (EDC), the protein to carrier relation and the type of support were investigated.
Fig. 1. Immobilization of L-hydantoinase from A. aurescens DSM 3747. Influence of different supports and methods on the yield of activity.
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Fig. 2. Immobilization of the purified, recombinant L-N-carbamoylase from E. coli. Dependence of the activity yield on the carbodiimide concentration after adsorption of the L-Ncarbamoylase to modified Eupergit C 250 L (NH2). Protein concentration 0.04 g l − 1, reaction volume 4 ml, 0.5 g wet carrier.
For direct comparison of the oxirane and the carbodiimide coupling results, amino groups were introduced to Eupergit C and Eupergit C 250 L by reaction with aqueous ammonia. As shown in Fig. 2 the activity yield versus EDCconcentration plot shows a typical saturation profile with a slight decrease of activity yield at higher EDC concentrations. At a protein concentration of 0.04 g l − 1 the optimum concentration of EDC was determined to be between 50 and 60 mM. Almost 20% of the initial activity were immobilized. The activity yield could be further enhanced by variation of the protein to carrier relation. As can be seen from Fig. 3 the activity yield of the purified L-N-carbamoylase coupled to Eupergit C 250 L (NH2) increased from 20 to : 30% at higher surface to protein ratios. Interestingly, 90% of the total protein amount were bound to the carrier during these experiments but only 30% were active. The reasons for this difference are still unknown, but may be caused by the formation of multiple layers of the enzyme and by adsorption effects accompanied with conformational changes of the adsorbed protein which may result in changed catalytic
efficacy or even complete inactivation (Soderquist and Walton, 1980; Yongli et al., 1999). Under optimized conditions, the influence of the type of support, varying in the basic matrix and the density of reactive groups (Table 1) was investigated. As can be seen from Fig. 4 and Table 2, the activity yield could be significantly enhanced by immobilizing the purified, recombinant enzyme from E. coli. Although the density of amino groups presented by the carrier is higher for modified Eupergit C (NH2) (Table 1) the enzyme was coupled to EAH Sepharose with nearly quantitative yield of activity. On the other hand, Eupergit based supports are obtained by bead polymerization of the monomers methacrylamide, N,N%-methylene-bis-methacrylamide, glycidyl methacrylate and allyl glycidyl ether. Thus, Eupergit supports are in general more hydrophobic than EAH Sepharose. Obviously, the interactions between the hydrophilic EAH Sepharose and the L-N-carbamoylase are favorable for the retention of enzymatic activity.
Fig. 3. Immobilization of the purified L-N-carbamoylase from recombinant E. coli using water-soluble carbodiimide (60 mM EDC): Influence of the protein to carrier relation on the activity yield. Protein concentration 0.04 g l − 1, reaction volume 4 ml.
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Fig. 4. Immobilization of the L-N-carbamoylase (crude enzyme from A. aurescens DSM 3747 and purified enzyme from recombinant E. coli ). Comparison of different supports with respect to the activity yield after immobilization via water-soluble carbodiimide (60 mM EDC, 0.04 mg ml − 1 protein concentration).
4. Conclusions Whereas the immobilization of the hydantoinase from A. aurescens turned out to be relatively simple in performance, the immobilization of the L-N-carbamoylase required a lot of optimization experiments. Since this enzyme catalyzes the stereospecific conversion of the N-carbamoyl
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amino acid produced by the unspecific hydantoinase, it is essential for the production of optically pure L-amino acids. As could be shown by the immobilization of the purified recombinant enzyme, the activity yield of the L-N-carbamoylase was deteriorated by contaminating proteins. Furthermore, covalent immobilization of the L-Ncarbamoylase was only possible via the carbodiimide method, i.e. coupling via enzymes carboxylic groups to support carrying primary amino groups. Additionally, a hydrophilic carrier such as EAH Sepharose was favorable in immobilizing the L-N-carbamoylase in active form. Using the optimized protocol, the activity yield could be enhanced from 4 to :100%. Coupling via amino groups by water-soluble carbodiimide had to be carried out at a concentration of between 50 and 60 mM of EDC to ensure a sufficient coupling yield without loss of activity, which might be caused by crosslinking of enzyme molecules prior to the covalent coupling to the support. Although the high activity yields obtained in the present paper and the operational stabilities observed in a previous one (Pietzsch et al., 1998) represent important improvements towards an industrially feasible biocatalyst, the specific activities obtained so far still are too low to fulfill economical requirements. Since the proteins could almost completely be immobilized in all experiments, there should be some reason for the differences between the activity yields and the coupling yields. Further experiments should reveal, if there
Table 2 Specific activity, activity yield and protein yield after immobilization of the hydantoinase and the carbodiimide method
L-N-carbamoylase
by the
Enzyme
Support
Specific activity (U g−1)
Activity yield (%) Protein (mg g−1)
Protein yield (%)
Hydantoinase Hydantoinase Hydantoinase L-N-Carbamoylase L-N-Carbamoylase L-N-Carbamoylase
EAH Sepharose 4B Eupergit C (NH2) Eupergit C 250 L (NH2) Eupergit C (NH2) Eupergit C 250 L (NH2) EAH Sepharose 4B
0.2 0.14 0.11 0.005 0.014 0.032
49 15 12 19 54 100
95 48 73 99 98 99
3.23 3.63 5.63 0.25 0.24 0.25
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is an influence of initial adsorption processes on the amount of active proteins, which are then fixed by covalent bonds. In continuing experiments, genetically modified proteins have been created and purified (Pietzsch et al., 2000). For example, a L-N-carbamoylase carrying an aspartate-tag has been prepared in order to investigate if an N-terminal fusion of carboxylic groups to the wild-type enzyme is accompanied with a positive influence on the amount of actively coupled enzyme during carbodiimide-mediated immobilization.
References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Bryjak, J., Trochimczuk, A., Noworyta, A., 1993. Effect of polymer matrix on penicillin acylase immobilization on copolymers of butyl acrylate and ethylene glycol dimethacrylate. J. Chem. Tech. Biotechnol. 57, 73 –78. Bunge, F., Pietzsch, M., Mu¨ ller, R., Syldatk, C., 1992. Mechanical disruption of Arthrobacter sp. DSM 3747 in stirred ball mills for the release of hydantoin-cleaving enzymes. Chem. Eng. Sci. 47, 225 – 232. ´ ., 1997. Stabilizing Protein Function. Springer, Fa´ ga´ in, O Berlin. Halling, P.J., Dunhill, P., 1979. Improved nonporous magnetic supports for immobilized enzymes. Biotechnol. Bioeng. 21, 393– 416. May, O., Siemann, M., Pietzsch, M., Kiess, M., Mattes, R., Syldatk, C., 1998. Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterisation as new member of cyclic amidases. J. Biotechnol. 61, 1 –13. Nanba, H., Ikenaka, Y., Yamada, Y., Yajima, K., Takano, M., Ohkubo, K., Hiraishi, Y., Yamada, K., Takahashi, S., 1999. Immobilization of thermotolerant N-carbamyl-Damino acid amidohydrolase. J. Mol. Catal. B Enzym. 6, 257 – 263.
Pietzsch, M., Oberreuter, H., Petrovska, B., Ragnitz, K., Syldatk, C., 1998. Immobilization of hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747 – effect of the coupling method on the stability of the L-N-carbamoylase. In: Ballesteros, A., Plou, F.J., Iborra, J.L., Halling, P. (Eds.), Stability and Stabilization of Biocatalysts. Elsevier, Amsterdam, pp. 517 – 522. Pietzsch, M., Syldatk, C., 1995. Hydrolysis and formation of hydantoins. In: Drauz, K., Waldmann, H. (Eds.), Enzyme Catalysis in Organic Synthesis. VCH, Weinheim, pp. 409 – 431. Pietzsch, M., Wiese, A., Ragnitz, K., Wilms, B., Altenbuchner, J., Mattes, R., Syldatk, C., 2000. Purification of recombinant hydantoinase and L-N-carbamoylase from Arthrobacter aurescens expressed in Escherichia coli: comparison of wild-type and genetically modified proteins. J. Chromatogr. B 737, 179 – 186. Ro¨ hm, 1995. Eupergit: immobilization of enzymes on Eupergit C and Eupergit C 250 L. Product Inform. Siemann, M., Syldatk, C., Wagner, F., 1993. Detection and comparison of strains with selective L-hydantoin cleaving activity using polyclonal antibodies. Biotechnol. Tech. 7, 361 – 366. Soderquist, M.E., Walton, A.G., 1980. Structural changes in proteins adsorbed on polymer surfaces. J. Coll. Interface Sci. 75, 386 – 397. Syldatk, C., Mackowiak, V., Ho¨ ke, H., Groß, C., Dombach, G., Wagner, F., 1990. Cell growth and enzyme synthesis of a mutant of Arthrobacter spec. (DSM 3747) used for the production of L-amino acids from D,L-5-monosubstituted hydantoins. J. Biotechnol. 14, 345 – 362. Tischer, W., 1995. Immobilization of enzymes. In: Drauz, K., Waldmann, H. (Eds.), Enzyme Catalysis in Organic Synthesis. VCH, Weinheim, pp. 73 – 87. Verhoeven, M., Cahalan, P., Cahalan, L., Hendriks, M., Foache, B., 1994. Biomolecule immobilization with carbodiimide. 0 608 095 A1, Medtronic, Inc., USA. Wilms, B., Wiese, A., Syldatk, C., Mattes, R., Altenbuchner, J., Pietzsch, M., 1999. Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli. J. Biotechnol. 68, 101 – 113. Yongli, C., Xiufang, Z., Yandao, G., Nanming, Z., Tingying, Z., Xinqi, S., 1999. Conformational changes of fibrinogen adsorption onto hydroxyapatite and titanium nanoparticles. J. Coll. Interface Sci. 214, 38 – 45.