Ursula Mackfeld

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

(S)-Selective MenD variants from Escherichia coli provide access to new functionalized chiral α-hydroxy ketones.

We report the first rationally designed (S)-selective MenD from E. coli for the synthesis of functionalized α-hydroxy ketones. By mutation of two amino acids in the active site stereoselectivity of the (R)-selective EcMenD (ee > 93%) was inverted giving access to (S)-5-hydroxy-4-oxo-5-phenylpentanoate derivatives with stereoselectivities up to 97% ee.

Tailoring the S‐Selectivity of 2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate Synthase (MenD) from Escherichia coli

The thiamine diphosphate (ThDP)‐dependent enzyme 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase from Escherichia coli (EcMenD, E.C. 2.2.1.9) catalyzes the carboligation of α‐ketoglutarate (α‐KG) and various benzaldehyde derivatives with excellent chemo‐ as well as high R‐selectivity (enantiomeric excess (ee) >93 %) to yield chiral α‐hydroxy ketones. Based on the recently developed S‐pocket concept, we engineered S‐selective EcMenD variants by optimizing the steric properties and stabilization of the acceptor substrate in the S‐pocket. Moreover, the moderate S‐selectivity of the EcMenD variant I474A/F475G described recently for the carboligation of α‐KG and benzaldehyde (ee=75 %) could be improved by selective destabilization of the R‐pathway, which resulted in the variant I474A/F475G/R395Y (ee=85 % S). Subsequent investigation of the acceptor substrate range of this new variant revealed high S‐selectivity especially with meta‐substituted benzaldehydes, which gave access to 5‐hydroxy‐4‐oxo‐5‐arylpentanoates with excellent enantioselectivities of up to 99 % ee S. Thus, opening the S‐pocket and simultaneous destabilization of the R‐pathway provides a potential general new strategy to enhance the S‐selectivity of ThDP‐dependent enzymes.

Enantioselective, continuous (R)- and (S)-2-butanol synthesis: Achieving high space-time yields with recombinant E. coli cells in a micro-aqueous, solvent-free reaction system

The stereoselective production of (R)- or (S)-2-butanol is highly challenging. A potent synthesis strategy is the biocatalytic asymmetric reduction of 2-butanone applying alcohol dehydrogenases. However, due to a time-dependent racemisation process, high stereoselectivity is only obtained at incomplete conversion after short reaction times. Here, we present a solution to this problem: by using a continuous process, high biocatalytic selectivity can be achieved while racemisation is suppressed successfully. Furthermore, high conversion was achieved by applying recombinant, lyophilised E. coli cells hosting Lactobacillus brevis alcohol dehydrogenase in a micro-aqueous solvent-free continuous reaction system. The optimisation of residence time (τ) and 2-butanone concentration boosted both conversion (>99%) and enantiomeric excess (ee) of (R)-2-butanol (>96%). When a residence time of only τ=3.1 min was applied, productivity was extraordinary with a space-time yield of 2278±29g/(L×d), thus exceeding the highest values reported to date by a factor of more than eight. The use of E. coli cells overexpressing an ADH of complementary stereoselectivity yielded a synthesis strategy for (S)-2-butanol with an excellent ee (>98%). Although conversion was only moderate (up to 46%), excellent space-time yields of up to 461g/(L×d) were achieved. The investigated concept represents a synthesis strategy that can also be applied to other biocatalytic processes where racemisation poses a challenge.

Application of immobilized bovine enterokinase in repetitive fusion protein cleavage for the production of mucin 1.

Bovine enterokinase is a serine protease that catalyzes the hydrolysis of peptide bonds and plays a key role in mammalian metabolism. Because of its high specificity towards the amino acid sequence (Asp)(4)-Lys, enterokinase is a potential tool for the cleavage of fusion proteins, which are gaining more importance in biopharmaceutical production. A candidate for adaptive cancer immunotherapy is mucin 1, which is produced recombinantly as a fusion protein in CHO cells. Here, we present the first repetitive application of immobilized enterokinase for the cleavage of the mucin fusion protein. The immobilization enables a facile biocatalytic process due to simplified separation of the biocatalyst and the target protein. Immobilized enterokinase was applied in a maximum of 18 repetitive reactions. The enzyme utilization (total turnover number) was increased significantly 419-fold compared to unbound enzyme by both immobilization and optimization of process conditions. Slight enzyme inactivation throughout the reaction cycles was observed, but was compensated by adjusting the process time accordingly. Thus, complete fusion protein cleavage was achieved. Furthermore, we obtained isolated mucin 1 with a purity of more than 90% by applying a simple and efficient purification process. The presented results demonstrate enterokinase to be an attractive tool for fusion protein cleavage.

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids: Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL)

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. Whereas both plant enzymes are specific for phenylalanine, the bifunctional enzyme from Rhodosporidium toruloides shows KM-values for L-Phe and L-Tyr in the same order of magnitude and, compared to both plant enzymes, a 10-15-fold higher activity. At 30°C all enzymes were sufficiently stable with half-lives of 3.4days (PcPAL1), 4.6days (AtPAL2) and 9.7days (RtPAL/TAL). Very good results for the amination of various trans-cinnamic acid derivatives were obtained using E. coli cells as whole cell biocatalysts in ammonium carbonate buffer. Investigation of the substrate ranges gave interesting results for the newly tested enzymes from A. thaliana and R. toruloides. Only the latter accepts besides 4-hydroxy-CA also 3-methoxy-4-hydroxy-CA as a substrate, which is an interesting intermediate for the formation of pharmaceutically relevant L-Dopa. AtPAL2 is a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe. Such non-canonical amino acids are valuable building blocks for the formation of various drug molecules.

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2) : A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids.. Part II: Application in different reactor concepts for the production of (S)-2-chloro-phenylalanine

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) is in general a very good catalyst for the amination of fluoro- and chloro-cinnamic acid derivatives yielding halogenated (S)-phenylalanine derivatives with ≥85% conversion and excellent ee values >99%. We have studied the application of this enzyme as whole cell biocatalyst and immobilized on the cellulose carrier Avicel® for the production of the hypertension drug precursor (S)-2-chloro-phenylalanine using batch, fed-batch, as well as continuous membrane reactor and plug-flow reactor. For immobilization, a C-terminal fusion of the enzyme with a carbohydrate binding module (CBM) was produced, which selectively binds to Avicel® directly from crude cell extracts, thus enabling a fast and cheap immobilization, stabilization and recycling of the enzyme. 1g Avicel was loaded with 10mg enzyme. Best results were obtained with whole cells using the continuous membrane reactor (47gproduct/gDryCellWeight) and using the immobilized enzyme in a repetitive fed-batch (274gproduct/gimmobilized enzyme) or in a continuous plug-flow reactor (288gproduct/gimmobilize enzyme). Therewith the productivity of AtPAL2 outperforms the established fed-batch process at DSM using PAL from Rhodotorula glutinis in E. coli as whole cell biocatalyst with a productivity of 0.14gproduct/gWetCellWeight (ca. 0.7gproduct/gDryCellWeight) (de Lange et al., 2011; doi:10.1002/cctc.201000435).

Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production

Sustainable and eco-efficient alternatives for the production of platform chemicals, fuels and chemical building blocks require the development of stable, reusable and recyclable biocatalysts. Here we present a novel concept for the biocatalytic production of 1,5-diaminopentane (DAP, trivial name: cadaverine) using catalytically active inclusion bodies (CatIBs) of the constitutive L-lysine decarboxylase from E. coli (EcLDCc-CatIBs) to process L-lysine-containing culture supernatants from Corynebacterium glutamicum. EcLDCc-CatIBs can easily be produced in E. coli followed by a simple purification protocol yielding up to 43% dry CatIBs per dry cell weight. The stability and recyclability of EcLDCc-CatIBs was demonstrated in (repetitive) batch experiments starting from L-lysine concentrations of 0.1 M and 1 M. EcLDC-CatIBs exhibited great stability under reaction conditions with an estimated half-life of about 54 h. High conversions to DAP of 87–100% were obtained in 30–60 ml batch reactions using approx. 180–300 mg EcLDCc-CatIBs, respectively. This resulted in DAP titres of up to 88.4 g l−1 and space-time yields of up to 660 gDAP l−1 d−1 per gram dry EcLDCc-CatIBs. The new process for DAP production can therefore compete with the currently best fermentative process as described in the literature.

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

A structural model for thiamine‐diphosphate (ThDP)‐dependent transketolase (TK) was developed to analyse the effect of amino acid exchanges on the stereoselectivity of this synthetically important class of enzymes. In this study the carboligation of 3‐hydroxypyruvate as a donor and propanal, as well as pentanal, was studied. Based on literature data and additional mutagenesis studies using E. coli TK, a four‐state model was developed to explain the stereoselectivity of TKs by the relative orientation of donor and acceptor substrates in the active site prior to C−C‐bond formation. To enable a functional comparison of relevant amino acids of TKs from different species, a standard numbering scheme was developed. Using this concept, H26, H261, and F434 were identified as the key residues which mediate stereoselectivity, where two main factors influenced the arrangement of ThDP‐bound donor and acceptor prior to carboligation: the relative orientation of the substrate side chains and the orientation of the acceptor carbonyl group towards the donor hydroxy group. This model provides a first framework to understand the structure‐function relationships of TKs with respect to their stereoselectivity.