engleski

Activity and operational stability of halohydrin dehalogenases in aqueous-organic biphasic systems

Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that show exceptional selectivity, activity, and versatility regarding accepting nucleophiles in epoxide ring-opening reactions.[1] HHDHs can be employed in the synthesis of important and versatile pharmaceutical building block (S)-2-(4- fluorophenyl)-3-hydroxypropanenitrile through kinetic resolution of rac-2-(4- fluorophenyl)oxirane.[1, 2] The major limitations of this reaction are poor substrate solubility and its susceptibility to hydrolytic decomposition. Introduction of organic phase in the aqueous reaction media can have a positive effect both on the solubility and hydrolytic stability of epoxide, leading to higher volume productivity. However, possible side effects of the organic phase introduction are enzyme activity and operational stability decrease, hence they need to be experimentally investigated and quantified. In this work, we determined the activity of wild-type HheC from Agrobacterium radiobacter in the presence of a wide solvent/buffer ratio range. As a result, correlation between solvent logP values and concentration of half-inactivation (C50) was observed, whereby C50 represents concentration of solvent that irreversibly reduces enzyme initial activity by half.[3]In addition to affecting the initial enzyme activity, the organic solvent may also affect the enzyme activity during continuous use. Hence, enzyme stability in selected solvents and at different ratios during time was monitored. Besides HheC, we examined the co-solvent activity and stability of thermostable variant ISM-4 obtained with iterative saturation mutagenesis, [4] as resistance to high temperatures is often accompanied with enhanced co-solvent activity and stability.[5] Finally, based on the experiments performed on a model system, we investigated the co-solvent operational stability of ISM-4 during (S)-2-(4-fluorophenyl)-3-hydroxypropanenitrile synthesis. We continually monitored the enzyme activity during reaction and estimated the values of enzyme deactivation rate constant (kd, min-1). References [1] Z. Findrik Blažević ; N. Milčić ; M. Sudar ; M. Majerić Elenkov ; Adv. Synth. Catal. 2021, 363 (2), 388-410. [2] I. Dokli ; N. Milčić ; P. Marin ; M. Svetec Miklenić ; M. Sudar ; L. Tang ; Z. Findrik Blažević ; M. Majerić Elenkov ; Catal. Commun. 2021, 152, 106285. [3] V. Stepankova ; S. Bidmanova ; T. Koudelkova ; Z. Prokop ; R. Chaloupkova ; J. Damborsky ; Adv. Synth. Catal. 2013, 3 (12), 2823-2836. [4] Z. Wu ; W. Deng ; Y. Tong ; Q. Liao ; D. Xin ; H. Yu ; J. Feng ; L. Tang ; App. Microbiol. Biotechnol. 2017, 101, 3201–3211. [5] H. Arabnejad ; M. Dal Lago ; P. A. Jekel ; R. J. Floor ; A-M. W. H. Thunnissen ; A. C. T. van Scheltinga ; H. J. Wijma ; D. B. Janssen ; Protein. Eng. Des. Sel. 2017, 30 (3), 173-187.

halohydrin dehalogenase ; aqueous-organic system ; operational stability

nije evidentirano