Cloning, overexpression, and characterization of a novel organic solvent-tolerant lipase from Paenibacillus pasadenensis CS0611

We found a novel lipase gene in the Paenibacillus pasadenensis CS0611 strain.The lipase gene sequence was cloned into the pET-28a expression vector to construct a recombinant lipase protein containing 6×His tags at the C-and N-termini,respectively.High-level expression of the lipase in E.coli BL21(DE3)was obtained upon induction with IPTG at 20°C.The recombinant lipase activity was approximately 1631-fold higher than the wild type.His-tagged recombinant lipase was purified rapidly and efficiently by using Ni-charged affinity chromatography with 63.5%recovery and a purification factor of 10.78.The purified lipase was stable in a broad range of temperatures and pH values,with the optimal temperature and pH being 50°C and 7.0,respectively.Its activity was stimulated to different degrees in the presence of metal ions such as Ca2+,Mg2+,and some non-ionic surfactants.In addition,the purified lipase was activated by a series of water-miscible organic solvents such as some short carbon chain alcohols and was highly tolerant to some water-immiscible organic solvents.

Methanol tolerance upgrading of Proteus mirabilis lipase by machine learning‑assisted directed evolution

For many crucial industrial applications,enzyme-catalyzed processes take place in harsh organic solvent environments.However,it remains a challenging problem to improve enzyme stability in organic solvents.This study utilized the MLDE(machine learning-assisted directed evolution)protocol to improve the methanol tolerance of Proteus mirabilis lipase(PML).The machine learning(ML)models were trained based on 266 combinatorial mutants.Using top 3 in 22 regression models based on evaluation of tenfold cross-validation,the fitness landscape of the 8000 full-space combinatorial mutants was predicted.All mutants in the restricted library showed higher methanol tolerance,among which the methanol tolerance of G202N/K208G/G266S(NGS)was up to 13-fold compared with the wild-type.Molecular dynamics(MD)simulation showed that reconstructing of critical hydrogen bond network in the mutant region of NGS provides a more stable local structure.This compact structure may improve the methanol tolerance by preventing organic solvent molecules into the activity site and resisting structural destruction.This work provides a successful case of evolution guided by ML for higher organic solvent tolerance of enzyme,and may also be a reference for broad enzyme modifications.