Enzymatic catalyzed hydrolysis in water structured media

BURGOS I; CLOP EM; SANCHEZ JM; NOLAN VM; PERILLO MA; CLOP PD

Conferencia; 5th International Conference on Catalysis & Chemical Engineering; 2021

Macromolecular crowded (MC) environments exhibit large surface areas incontact with the solvent. This can affect the water availability as a solvent and,in hydrolytic reactions, its availability as a reagentwhen water takes part of the reaction rate limiting step. This occurs in the β-Galactosidase(β-Gal) catalyzed hydrolysis of ortho-nitrophenyl-β-D-galactopyranoside(ONPG), but not of its para- isomer (PNPG). Here we studied these reactions bothin solutions containing a high molecular mass polymer (poliethyleneglycol, PEG6000)and with β-Gal confined in the nanopores of a sol-gel silicate matrix. In bothconditions, [1H]NMR showed several water populations, those with thesmallest T1 grew up as a function of PEG concentration and with the aging-timedependent polymerization level of silicate matrices (gels pore size decreasedwith aging), reflecting the increasing solvent immobilization. In bothconditions, spectroscopic analysis (fluorescence, CD, FTIR) showed that, comparedto dilute solutions, β-Gal exhibited a more open conformation, higher thermalstability and slower unfolding kinetics, reflecting that the hydrophobic effectwas also affected. In MC conditions, rate vs [substrate] data revealed biphasicand monophasic kinetics for the hydrolysis of ONPG and PNPG, respectively. Inthe case of ONPG, one of the components became constant while the otherexhibited a MC-dependent kcat. In conjunction, kinetic datareflected complex combinations of MC -induced effects on enzyme structure,water structure and protein diffusion (demonstrated byanalytical-ultracentrifugation). The analysis performed in this work, providesa perspective with potential application to all hydrolases in MC environmentsand in conditions of technological interest5th International Conference on Catalysis & Chemical Engineering. 22-26 February 2021. CCE 2021 Virtual. https://catalysis.unit