Hendrik Mallin

Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications

In this review we analyse structure/sequence-function relationships for the superfamily of PLP-dependent enzymes with special emphasis on class III transaminases. Amine transaminases are highly important for applications in biocatalysis in the synthesis of chiral amines. In addition, other enzyme activities such as racemases or decarboxylases are also discussed. The substrate scope and the ability to accept chemically different types of substrates are shown to be reflected in conserved patterns of amino acids around the active site. These findings are condensed in a sequence-function matrix, which facilitates annotation and identification of biocatalytically relevant enzymes and protein engineering thereof.

A self-sufficient Baeyer–Villiger biocatalysis system for the synthesis of ɛ-caprolactone from cyclohexanol

In order to establish a new route for ɛ-caprolactone production from the corresponding cyclohexanol with an internal cofactor recycling for NADPH, a recently redesigned thermostable polyol dehydrogenase (PDH) and the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus were combined. First, the expression of PDH could be improved 4.9-fold using E. coli C41 with co-expression of chaperones. Both enzymes were also successfully co-immobilized on glutaraldehyde-activated support (Relizyme™ HA403). Cyclohexanol could be converted to ɛ-caprolactone (ɛ-CL) with 83% conversion using the free enzymes and with 34% conversion using the co-immobilized catalysts. Additionally, a preparative scale biotransformation of ɛ-caprolactone starting from cyclohexanol was performed using the soluble enzymes. The ɛ-CL could be isolated by simple extraction and evaporation with a yield of 55% and a purity of >99%.

Immobilization of two (R)-Amine Transaminases on an Optimized Chitosan Support for the Enzymatic Synthesis of Optically Pure Amines

Two (R)‐selective amine transaminases from Gibberella zeae (GibZea) and from Neosartorya fischeri (NeoFis) were immobilized on chitosan as a carrier to improve their application in the biocatalytic synthesis of chiral (R)‐amines. An (S)‐selective enzyme from Vibrio fluvialis (VfTA) was used for comparison. After improving the immobilization conditions, all enzymes could be efficiently immobilized. Additionally, the thermal stability of GibZea and NeoFis could be improved and also a slight shift of the pH optimum was observed for GibZea. All enzymes showed good activity in the conversion of α‐methylbenzylamine. In the asymmetric synthesis of (R)‐2‐aminohexane from the corresponding ketone, a 13.4‐fold higher conversion (>99 %) was found for the immobilized GibZea compared to the free enzyme. Hence, the covalent binding with glutaraldehyde of these enzymes on chitosan beads resulted in a significant stabilization of the amine transaminases investigated.

Efficient Biocatalysis with Immobilized Enzymes or Encapsulated Whole Cell Microorganism by Using the SpinChem Reactor System

Nowadays, biocatalysis is an established method for the enzymatic synthesis of chiral building blocks for organic compounds and pharmaceuticals, compounds for the flavor and fragrance industry, the production of bulk chemicals, and the modification of lipids for the food industry. Biocatalysis has become highly competitive with classical (asymmetric) chemical routes that use transition-metal catalysts, especially in combination with new methods for enzyme discovery and protein engineering, as recently shown for the synthesis of the drug Sitagliptin. The cost-effective application of enzymes, in particular for the synthesis of cheap products, requires immobilization of the biocatalyst (or the encapsulation of whole cells) to enhance their long-term stability 5] and facilitate their reuse. At the same time, immobilization of the biocatalyst should enable the use of established reactor setups, such as fixed-bed reactors (FBRs), instead of simple stirred-tank reactors (STRs, Figure 1). 6] FBRs are used, for instance, for the large-scale production of chiral amines or emollient esters for the cosmetic sector by using lipase catalysts. However, several disadvantages are encountered with FBRs, which depend on, for example, the length, diameter, and particle size in the reactor, the flow rate, the pressure drop within the column, and reactant and pH gradients, as well as inactivation profiles after extended use. In contrast, the more operationally simple STR encounters mechanical challenges for the carrier, which results in abrasion of the biocatalyst material and severe damage of encapsulated whole cells beside the fact that the recycling of the immobilized biocatalyst is rather laborious. Herein, we have investigated the use of an alternative setup for the application of immobilized enzymes and encapsulated whole cells. This SpinChem reactor (SCR; SpinChem is a registered trademark by Nordic ChemQuest AB, Ume , Sweden) enables the simultaneous stirring and efficient percolation of a liquid through packed particle beds, which is implemented by a hollow stirring device that allows the solid reaction chamber to be located inside the stirring element itself. The SCR can be seen as an evolution of the standard basket reactor. 10] The basket reactor, first published by Carberry in 1964, is a setup in which four baskets rotate inside a well for gas/solid reactions. This concept was later developed as the “annular spinning basket reactor” by Mahoney et al. in 1978. However, in the SpinChem reactor, the solid phase (such as an immobilized enzyme) is present in the stirring element itself in up to four separate compartments, which provides greater mixing and flexibility compared to the basket reactors. Figure 1. Top: Schematic representation of the three reactor setups that were investigated. Bottom: Photograph of the SpinChem device (reflux cooler and oxygen supply only for BVMO reaction).

Immobilization of (R)- and (S)-amine transaminases on chitosan support and their application for amine synthesis using isopropylamine as donor

Transaminases from Aspergillus fumigatus ((R)-selective, AspFum), Ruegeria pomeroyi ((S)-selective, 3HMU) and Rhodobacter sphaeroides 2.4.1 ((S)-selective, 3I5T) were immobilized on chitosan with specific activities of 99, 157, and 163U/g and acceptable yields (54, 21, and 23%, respectively) for glutaraldehyde (GA) immobilization. Besides GA, also divinylsulfone was used as linker molecule leading to a similar efficient immobilization for two enzymes, GibZea and NeoFis, whereas GA was superior in the other cases. Storage of the GA-immobilized enzymes for one month resulted in increased relative activities between 120 and 180%. The thermal stability was improved, especially for the GA-immobilized AspFum compared to the free enzyme after incubation for 4h at 60°C (10% vs. 235% residual activity). Especially after incubation of AspFum (free or immobilized) for 2h at 50°C a strongly increased activity was observed (up to 359% of the initial activity). This effect was studied in more detail, revealing that one heat activation prior and one after immobilization increased the overall immobilization efficiency. Recycling of the immobilized ATAs resulted only in a small reduction of activity after four batches. Asymmetric synthesis of (R)- or (S)-1-methyl-3-phenylpropylamine from the prostereogenic ketone using isopropylamine (IPA) as amino donor was applied with conversions up to 50% (AspFum) or 75% (3HMU). Except for NeoFis, all immobilized ATAs showed higher conversions compared to the free enzyme.

Protein engineering of a thermostable polyol dehydrogenase.

The polyol dehydrogenase PDH-11300 from Deinococcus geothermalis was cloned, functionally expressed in Escherichia coli and biochemically characterized. The enzyme showed the highest activity in the oxidation of xylitol and 1,2-hexanediol and had an optimum temperature of 45 °C. The enzyme exhibited a T⁶⁰₅₀-value of 48.3 °C. The T⁶⁰₅₀ is the temperature where 50% of the initial activity remains after incubation for 1h. In order to elucidate the structural reasons contributing to thermostability, the substrate-binding loop of PDH-11300 was substituted by the loop-region of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scope of this chimera basically represented the average of both wild-type enzymes, but surprisingly the T⁶⁰₅₀ was noticeably increased by 7 °C up to 55.3 °C. Further mutations in the active site led to identification of residues crucial for enzyme activity. The cofactor specificity was successfully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD⁺ binding cleft, without influencing the catalytic properties of the dehydrogenase.

Upregulation of an Artificial Zymogen by Proteolysis.

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.

Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology

Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d.

Streptavidin-Enzyme Linked Aggregates for the One-Step Assembly and Purification of Enzyme Cascades

Herein, we report enzyme aggregates assembled around covalently cross‐linked streptavidin tetramers. The streptavidin oligomeric matrix (SavMatrix) is produced by using SpyTag/SpyCatch technology and binds tightly to fusion proteins bearing a streptavidin‐binding peptide (SBP). Fusing the SBPs to different enzymes leads to precipitation of the streptavidin–enzyme aggregates upon mixing the complementary components. This straightforward strategy can be applied to crude cell‐free extracts, allowing the one‐step assembly and purification of catalytically active aggregates. Enzyme cascade assemblies can be produced upon adding different SBP‐fused enzymes to the SavMatrix. The reaction rate for lactate dehydrogenase (LDH) is improved tenfold (compared with the soluble enzyme) upon precipitation with the SavMatrix from crude cell‐free extracts. Additionally, the kinetic parameters are improved. A cascade combining a transaminase with LDH for the synthesis of enantiopure amines from prochiral ketones displays nearly threefold rate enhancement for the synthesis of (R)‐α‐methylbenzylamine compared with the free enzymes in solution.

Streptavidin as a Scaffold for Light-Induced Long-Lived Charge Separation

Long-lived photo-driven charge separation is demonstrated by assembling a triad on a protein scaffold. For this purpose, a biotinylated triarylamine was added to a RuII -streptavidin conjugate bearing a methyl viologen electron acceptor covalently linked to the N-terminus of streptavidin. To improve the rate and lifetime of the electron transfer, a negative patch consisting of up to three additional negatively charged amino acids was engineered through mutagenesis close to the biotin-binding pocket of streptavidin. Time-resolved laser spectroscopy revealed that the covalent attachment and the negative patch were beneficial for charge separation within the streptavidin hosted triad; the charge separated state was generated within the duration of the excitation laser pulse, and lifetimes up to 3120 ns could be achieved with the optimized supramolecular triad.