Investigating recombinant transaminase stability with cofactor variations and enzyme immobilization

Amine transaminases are valuable biocatalysts in the pharmaceutical industry for the production of chiral amines. Their high regioselectivity and inherent regeneration of cofactor pose them as a greener alternative to the chemical synthesis. However, their application is hampered by their limited stability and narrow substrate scope and product inhibition. Recently, a stable tetrameric amine transaminase from Burkholderia multivorans CGD1, ATA-10, was identified and characterised. ATA-10 bears many characteristics attractive for industrial application. However, expression of ATA-10 by our group initially showed only poor recombinant expression in Escherichia coli. In this work, the improvement of stability of the ATA-10 is examined. Firstly, supplementation of the co-factor, pyridoxal 5’–phosphate, was employed. Cultures with PLP supplementation at the beginning of the growth phase and before the induction phase at concentrations 0.01-1 mM were tested. Supplementation of the cofactor during cultivation didn’t alter the target protein yield in the soluble protein fraction, which remained around 60% across all conditions. The effect of the cofactor was evident for the folding stability of the enzyme, as the activity was elevated when PLP was supplemented in the growth medium. This activity was linearly increased to the increase of PLP concentration, and the effect was more pronounced when PLP was added in the beginning of the culture. A potential inhibitory effect of high PLP concentrations was observed only during the induction phase, where 10 mM PLP resulted to halved growth. Highest activity was observed for ATA-10 produced in the presence of 1 mM PLP supplemented from the beginning of cultivation. Purification of ATA-10 was achieved but complicated because of interference of PLP with the UV signal. To improve the stability of the ATA-10 for production processes, immobilization on hydrophilic carriers was carried out. Among the carriers, polyacrylic beads demonstra
Enzymes are nature’s most versatile biocatalysts. These biomolecules act like tiny, specialized machines, each designed to perform a specific task with incredible precision and speed. In industrial processes, enzymes are important because they make chemical reactions happen faster and under milder conditions, saving energy and reducing the need for harsh chemicals. This makes the process like making medicines, cleaning products or even biofuels more effective, environmentally friendly and cost-effective. In drug development, the high specificity of the enzymes is used to produce the right structure of molecules that delivers the desired effect, as the mirror-image of that molecule can be harmful. One such enzyme important in the pharmaceutical industry is transaminase. Transaminases facilitate the transfer of amino groups between molecules. This simple reaction is particularly valuable to produce chiral amines, that can be found in more than 40% of pharmaceuticals. However, like all machines, enzymes can sometimes be finicky, struggling with efficiency and durability, which has been the main obstacle so far to ditching harmful chemical processes. Particularly, transaminases struggle with stability and solubility. In simple terms, an enzyme needs to dissolve well in a solution (solubility) and maintain its structure and activity over time (stability) to be effective. If an enzyme precipitates out of solution or loses its function quickly, it becomes less useful, driving up costs and reducing efficiency in industrial processes. For that reason, a lot of research is devoted in improving the enzymes produced in model microorganisms. Here, we tried to improve the stability of a transaminase isolated from the bacterium Burkholderia multivorans CDG1. This transaminase is particularly interesting for industrial use as it can maintain activity at very high temperatures and can be applied for a wide range of molecules. Firstly, we tried to improve stability by the addition of