Comparison of Four Immobilization Methods for Different Transaminases

Abstract

Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HAGA-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from Luminiphilus syltensis. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChemTM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability.

Co‐Immobilization of a Multi‐Enzyme Cascade: (S)‐Selective Amine Transaminases, l‐Amino Acid Oxidase and Catalase

“…Glutaraldehyde-functionalized amine beads (HA GA ) were prepared as described previously. [31] Lyophilized horseradish peroxidase (HRP) was obtained from SERVA. Rac-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine (rac-1) and 1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)-ethan-1-one (2) was provided by Xiang et al [49] 13 mm rac-1 was dissolved in 1 % DMSO and 100 mm 2 in pure DMSO.…”

“…For the application of the enzyme cascade in an immobilized form, optimal conditions for the immobilization of each enzyme have to be established, which was already done previously for ATA-Vfl. [31] Thus, comprehensive studies of the immobilization of hcLAAO4 and hCAT on HA-, HA GA -, and EP-beads were performed with varied temperatures, pH values, enzyme amounts, and durations (Figure S1). Importantly, for the siteselective immobilization on the HA-beads, hcLAAO4 [18] and hCAT (generated in this study, see section S7-S8) with aldehyde-tag were used after FGE-conversion, whereas the enzymes without aldehyde-tag were applied for multi-attachment on EP-and HA GA -beads.…”

“…Cloning of ATA-Vfl-8M. The coding sequence of the engineered ATA-Vfl-8M was cloned from the plasmid pRSFduet-1-ATA-Vfl-8M [49] into the pET24b-ATA-Lsy- [31] (ATA-Lsy-sequence was substituted by the ATA-Vfl-8M-sequence) and into the pET-24b-X-CTPSR-His 6 - [60] plasmid to obtain an expression plasmid for the enzyme without aldehyde-tag (pET24b-ATA-Vfl-8M) and with aldehyde-tag (pET24b-ATA-Vfl-8M-AH) ATA-Vfl-8M, respectively. Add-on and overlapextension PCR were therefore used, and the corresponding primers, PCR programs, nucleic acids, and further information are provided in the supporting information (section S8).…”

“…~72-hFGE was produced as described previously. [31] The enzymes (ATAs, hcLAAO4, hCAT) with and without aldehyde-tag were produced as described Figure 3. Kinetic resolution of rac-1 by ATA-Vfl-8M co-immobilized with the co-substrate recycling system.…”

“…Finally, 1-PEA was extracted from the pooled reaction solution (i. e., 120 mL) and enantiomeric excess was determined by chiral HPLC measurements, both as described previously. [31] Kinetic resolution of rac-1. Kinetic resolution of 10 mm rac-1 and 1 mol % co-substrate in 2 mL reaction solution (50 mm Tris pH 7.0, 1.2 % or 10 % DMSO, 0.1 mm PLP, 0.1 mm l-norleucine) was catalyzed in 2 mL reaction tubes by the co-immobilized enzyme cascade at 37 °C for 7 d in duplicates each under rotation.…”