The objective of this study is to develop an efficient strategy for directed evolution, termed
"Corner Engineering", to establish a mutant library and screen for beneficial mutants. Differing
from traditional directed evolution, this study first employs computational prediction of protein
secondary structure to identify potential beneficial sites, followed by the design of degenerate
primers for site-saturation mutagenesis. This approach allows for the creation of a more precise
mutant library. Through two rounds of screening, beneficial mutants exhibiting increased resistance
in low eutectic solvents are identified, ultimately achieving enhanced enzymatic biocatalytic
transformations in such solvents.
Deep Eutectic Solvents
Configure choline chloride and acetamide, choline chloride and ethylene glycol, and
tetrabutylphosphonium bromide and ethylene glycol as subsequent screening solutions. Perform
robustness validation using the wild-type enzyme to ensure the stability of the established
screening system.
BSLA and Bs2Est
Bacillus subtilis lipase A （BSLA） belongs to the lipase subfamily I-4, with its mature form
consisting of 181 amino acids. BSLA is one of the smallest lipases, with a molecular weight of only
19 kDa. It lacks the lid structure present in most lipases and is considered one of the smallest α/β
fold hydrolases. It has a broad substrate range and excellent enzymatic properties, holding
significant potential for industrial applications. Therefore, this enzyme was selected as a model
enzyme to explore the Corner Engineering strategy.
Bacillus subtilis esterase (Bs2Est), a pivotal enzyme in enzymatic depolymerization,
efficiently hydrolyzes saccharide products into TPA. This enzyme exhibits multiple turn secondary
structures and has a molecular weight of approximately 54 kDa. In this study, we have chosen this
enzyme to further validate the effectiveness of the strategy.
Corner Engineering
Predicted Sites: The enzyme's secondary structure was predicted using EMBI-EBI.
Design Primers: The two Degenerate codons SRC (S= C, G; R= A, G) and RAA (R= A, G) were mixed in a
ratio of 1:1 to cover the charged amino acid (H, R, D, E, and K).
Site Saturated Mutagenesis: For site-saturated mutagenesis (SSM), the template plasmid was stepwise
mutated by PCR according to the QuikChange mutagenesis method. The PCR product was digested with
DpnI (20 U, 37 °C, overnight) and transformed into Escherichia coli BL21(DE3).
Determination of Beneficial Mutants: A crude enzyme solution (5 μL) was added to 95 μL DES/buffer in
each well. After 2h of incubation at room temperature, the release of pNPB was recorded by measuring
A410 nm on a microtiter plate reader (Biotek Synergy HI, USA) over 8 min.
