The Role of Buffer, Pyridoxal 5’‐Phosphate and Light on the Stability of the Silicibacter Pomeroyi Transaminase

Abstract

Transaminases are pyridoxal 5'-phosphate (PLP)-dependent enzymes that transfer amino-functions. The transaminase from Silicibacter pomeroyi (SpATA) exhibits a broad substrate spectrum. In this work we examined the effect of different conditions (light, buffer and PLP-concentration) on the stability of SpATA, as well as the causes for these effects. The enzyme was stored either in TRIS or CHES with 0-10 mM added PLP at 22 °C. The samples were either kept dark or they were exposed to light. The results show that invariably, all samples kept in darkness exhibited longer half-life times than the ones exposed to light. An increase in the half-life from 8 h to 720 h could be achieved solely by keeping the sample dark. Especially samples in CHES buffer inactivated faster in light the more PLP was present, due to the degradation of PLP. In TRIS however, an imine-bond between TRIS and PLP protects PLP from degradation.

Comparison of Four Immobilization Methods for Different Transaminases

“…The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/catal13020300/s1. Section S1: establishment of optimal immobilization conditions for each transaminase; Figure S1: establishment of optimal immobilization conditions Table S1: optimal immobilization conditions for each transaminase; immobilization parameters; Table S2: immobilization parameters; Section S2: methods and additional data on transaminase stability and activity; general setup of sample incubation [7,8,63,91,92]; Figure S2: stability and reactivity of ATA-Vfl, ATA-3FCR-5M, and ATA-Lsy under different conditions; Section S3: post-cross-linking and storage of dried beads; Figure S3: effect of post-cross-linking on specific activity of the immobilizates; Figure S4: effect of drying on specific activity of the immobilizates; Section S4: upscaled kinetic resolution catalyzed by the final immobilizates; Figure S5: chiral HPLC runs; Figure S6: exemplary NMR analysis of (S)-1-PEA. ; Figure S7: behavior of acetophenone and 1-phenylethylamine in bead-containing solutions; Section S5: cloning of transaminases; Table S3: components and concentrations of the add-on and OE-PCR [46]; Table S4: PCR program for the add-on and OE-PCRs; Section S6: nucleic acid sequences; open reading frames of ATA-Bmu, ATA-3FCR-5M, and ATA-Lsy in respective plasmids w/o aldehyde-tag; open reading frames of ATA-Bmu, ATA-3FCR-5M, and…”

Biosynthesis of Furfurylamines in Batch and Continuous Flow by Immobilized Amine Transaminases

“…4 min in a reactor volume of 236 µL (Figure 3). The column and feed solution were kept dark to avoid the inactivating effect of light on ATA-Spo [49,50]. The protein concentration in the flow-through was analyzed with the Bradford Assay, but no detectable enzyme leaching was observed.…”

“…The feed solution (50 mM HEPES buffer pH 8.0, 10 mM HMF, 0.1 mM PLP, and 500 mM amine donor (isopropylamine or L-alanine)) was pumped through the tube with a flow rate of 50 µL min −1 . The tube and the feed solution were wrapped in aluminum foil to exclude negative influences from light [49,50]. Samples of the flow were taken every 24 h and analyzed using HPLC.…”

“…For biocatalysis to become an attractive and competitive alternative to conventional chemical synthesis, a stable and easy-to-use enzyme is necessary [44]. Several research efforts have been made to increase the stability of enzymes, for example by genetic mutation or adapting the reaction environment [45][46][47][48][49][50]. One popular strategy is immobilization of the enzymes, which has shown promising results in increasing the stability of different transaminases under various conditions so far [51][52][53].…”

Biocatalytic cascade to polysaccharide amination

“…The enzyme was previously shown to be active on oxidized mono- and oligosaccharide substrates [ 13 ], however, its activity towards higher molecular weight oxidized polysaccharides is unknown. Previous studies showed that SpATA can accept bulky substrates including Jeffamines and halogenated prochiral ketones [ 25 , 26 ], which makes it a promising enzyme for polysaccharide amination. In this study, a new colorimetric assay was established to compare the yield of aminated polysaccharides produced by CvATA and SpATA.…”