Studying The Role of Aprotic and Protic Solvents on Extremophilic Laccase From Thermus thermophilus for Solvent-Mediated One-Pot Biocatalysis.

In present times, globally, solvent-mediated biocatalysis is gaining a pivotal role in sustainable biotechnological solutions for organic synthesis. The versatile green catalyst, laccase, a multicopper oxidoreductase, has gained significant importance in aromatic bioconversion in solvent systems. Laccases portray varied behavioral patterns and functionality in solvents. With this interest, the influence of aprotic polar organic solvents and a benchmark out-of-the-ordinary protic solvent, an ionic liquid, i.e., 1-Ethyl-3-methylimidazolium acetate (EmimOAc), on extremophilic laccase from Thermus thermophilus, TtL, was elucidated. In this study, a signal peptide engineering strategy was adopted for improved enzyme expression. The first 28 amino acids corresponding to the signal peptide from an evolutionarily close E. coli' s laccase, copper efflux oxidase, tagged as 'sp', were incorporated with TtL by replacing TtL's signal peptide (first 23 amino acids), hence, spTtL was attained. Furthermore, micro-anaerobic condition was followed to reduce inclusions, and spTtL was purified. Subsequently, the spTtL's thermal unfolding and its activity against EmimOAc, DMSO, and acetone were studied as a prime highlight. At higher organic solvents, the thermal unfolding of spTtL directly exhibited constraints in spTtL' s melting point, showing reduced thermal stability. Interestingly, at higher EmimOAc (10-20% V/V), spTtL' s stability was infinitesimally affected; however, only 40% activity was retained in 50% V/V EmimOAc. In EmimOAc, inhibition kinetics of spTtL indicated a mixed competitive and non-competitive inhibition. Conclusively, this work emphasizes thermal unfolding understanding of spTtL in acetone, DMSO, and EmimOAc, which serves as a prerequisite for aiming/tailoring solvent media for one-pot laccase-based catalysis.