Dissecting the evolvability landscape of the CalB active site toward aromatic substrates

Abstract

A key event in the directed evolution of enzymes is the systematic use of mutagenesis and selection, a process that can give rise to mutant libraries containing millions of protein variants. To this day, the functional analysis and identification of active variants among such high numbers of mutational possibilities is not a trivial task. Here, we describe a combinatorial semi-rational approach to partly overcome this challenge and help design smaller and smarter mutant libraries. By adapting a liquid medium transesterification assay in organic solvent conditions with a combination of virtual docking, iterative saturation mutagenesis, and residue interaction network (RIN) analysis, we engineered lipase B from P. antarctica (CalB) to improve enzyme recognition and activity against the bulky aromatic substrates and flavoring agents methyl cinnamate and methyl salicylate. Substrate-imprinted docking was used to target active-site positions involved in enzyme-substrate and enzyme-product complexes, in addition to identifying ‘hot spots’ most likely to yield active variants. This iterative semi-rational design strategy allowed selection of CalB variants exhibiting increased activity in just two rounds of site-saturation mutagenesis. Beneficial replacements were observed by screening only 0.308% of the theoretical library size, illustrating how semi-rational approaches with targeted diversity can quickly facilitate the discovery of improved activity variants relevant to a number of biotechnological applications.

Combined Theoretical and Experimental Study to Unravel the Differences in Promiscuous Amidase Activity of Two Nonhomologous Enzymes

“…Other studies have reported that Trp104 is essential and forms a hydrophobic pocket in CALB, 58 − 60 while residues such as Ile189, Val190, and Thr138 have also been proven to be crucial in the kinetic performance of CALB. 60 − 62 …”

“…In a directed evolution study by Arnold and collaborators, despite many of the tested mutations related to the surface, the mutation of Met358 was found to improve the kinetic activity in organic solvents. Other studies have reported that Trp104 is essential and forms a hydrophobic pocket in CALB, − while residues such as Ile189, Val190, and Thr138 have also been proven to be crucial in the kinetic performance of CALB. − …”

Application of freeze-dried Yarrowia lipolytica biomass in the synthesis of lipophilic antioxidants

“…Molecular modeling was used, and based on this computer-aided investigation, researchers claimed that at least one methylene bridge between the carboxylic group and aromatic ring is necessary for the reactions to proceed. Assumedly, this has to do with the molecule's flexibility, and thus, the possibility of it adapting to the hydrophobic cavity (Santos et al 2019 ). Moreover, unsaturated phenolic acids (cinnamic, p -coumaric, and caffeic), as well as α-substituted phenolic acids are not suitable substrates for CALB esterification (Otto et al 2000 ).…”

Enzymatic synthesis of novel aromatic‐aliphatic polyesters with increased hydroxyl group density

“…This problem is currently addressed by protein engineering studies, which should contribute to synthesis optimization in the future. [52] For the purpose of a potential economic applicability, diacids available on large scale have to be used instead of activated diester. Their low solubility in a number of diols and high melting points already add a general challenge to the enzymatic polymerization process.…”

“…This problem is currently addressed by protein engineering studies, which should contribute to synthesis optimization in the future. [ 52 ]…”