Effects Of Deep Eutectic Solvents On The Catalytic Efficiency And Stability Of ω-Transaminase And Uridine-diphosphate Glycosyltransferase

The efficient synthesis of chiral amines at a large scale to meet the high demand from the pharmaceutical industries which require them as precursors for the biosynthesis of chiral drugs is challenging as a result of poor stability and low catalytic efficiency of the ω-transaminases(ω-TAs)conventional buffers.Plant uridine diphosphate glycosyltransferases(UGTs)are other enzymes which are essential for the biosynthesis of precursors with pharmaceutical value.Currently,an outmanoeuvring potent alternative to conventional organic solvents have emerged,with exquisite characteristics desirable to the environment and biocatalysis.These types of solvents are named deep eutectic solvents(DESs)and are formed from self-association of a hydrogen bond acceptor(HBA)and a hydrogen bond donor(HBD)at a certain stoichiometric molar ratio after thermal heating.On the basis of the promising prognoses,we explored the potency of various DESs in the stability and activity of two enzymes which are potent in the pharmaceutical industry.The main objective of this study was to evaluate the effects of deep eutectic solvents on the stability and catalytic efficiency ofω-transaminase variants from Aspergillus terreus and UGTs form Sorbus aucuparia(S.aucuparia).A combination of HBA and various HBDs were used to synthesise various DESs which showed different activities on the variants.The ω-TAs which displayed higher activity in the aqueous solutions of the DESs were selected for further downstream applications.Subsequently,the optimisation of the reaction parameters of the two selected ω-TA variants in a binary and a ternary based DES was done using response surface method(RSM).The enzyme kinetics studies and thermal stability assays were conducted,inorder to ascertain the effects of the DESs on these enzyme parameters.The most potent DES which enhanced the activity,thermal stability and storage stability of the ω-TA variant was a ternary alcoholbased ternary DES(ChCl:EG:PG).Furthermore,a comprehensive interaction mechanism between the most potent DES and ω-TA was illustrated by comparing the molecular docking simulation studies and experimental data.The two UGTs from S.aucuparia(SaUGTs)demonstrated regioselectivity towards the glycosylation of the three biphenyls from S.aucuparia in conventional buffer solution.The product yield was low and the UGTs were unstable,such that the optimum reaction conditions for UGT activity were not obtained.Since the SaUGTs displayed activity on the biphenyls,we considered it necessary to optimise the glycosylation of the biphenyls for efficient yield of the biphenyl glycosides in deep eutectic solvents.However,when glycosylation was performed in the synthesised DESs,neither the pure DES nor the aqueous solutions of the DESs enhanced the activity of the SaUGTs.The DESs which were tested did not improve the stability of the SaUGTs relative to the conventional buffer,hence,other downstream applications in deep eutectic solvents were not evaluated.The DESs which were evaluated in this study were able to solubilise the substrates,thereby replacing the use of the organic solvent DMSO which can impart some negative effects on both the stability of the enzyme and the environment.This study illustrated the potency of DESs as a sustainable,cost effective and environmentally friendly solvent engineering strategy to enhance the stability and catalytic efficiency of ω-TAs.