Bridges to Stability: Engineering Disulfide Bonds Towards Enhanced Lipase Biodiesel Synthesis

Abstract

Computational design of disulfide bonds was performed for lipase from Geobacillus stearothermophilus T6 (LipT6) for enhanced methanol stability and improved biodiesel production. Thirteen double mutants comprising new cysteine pairs were screened and evaluated for their stability in 70 % methanol. Superior stability was found with variant E251C/G332C (M13) having a 5.5‐fold higher hydrolysis activity and enhanced unfolding temperature (Tm) of +7.9 °C in methanol compared with wild‐type. Moreover, M13 converted nearly 80 % waste chicken oil to biodiesel, representing a 2.4‐fold improvement relative to the WT. Structural studies using X‐ray crystallography confirmed the existence of the engineered disulfide bonds shedding light on the link between the bond location and backbone architecture with its stabilization impact. Rational integration of disulfide bonds is suggested to be a feasible method to promote elevated stability in organic solvents for various industrial applications such as biodiesel synthesis.

Improved thermostability of creatinase from Alcaligenes Faecalis through non-biased phylogenetic consensus-guided mutagenesis

“…Over the years, many different approaches have been used to modify the stability of proteins. Classic protein stability design strategies are usually based on disulfide bonds design [7], optimization of protein surface charges [8], B-factor design [9], Proline effect design [10]. Recently, design tools rely on the energy function or the machine-learning algorithm have been developed to predict changes in protein stability, such as CC/PBSA [11], I-Mutant 2.0 [12], Fold-X [13], MUpro [14], PopMUSIC [15], Rosetta [16].…”

Advances in Rational Protein Engineering toward Functional Architectures and Their Applications in Food Science

“…After sequence mutagenesis, 13 double mutants comprising new cysteine pairs were evaluated, finally obtaining variant E251C/G332C with superior stability and improved hydrolysis activity. 34 Currently, successful cases have been reported in the range of improving protein stability and folding, 34 altering specificity and binding affinity, 35 implanting novel enzymatic activities, and preparing high-ordered assemblies. 36,37 The de novo design process usually starts with an unknown sequence and backbone structure and finally requires the determination of the protein sequence that will fold into a certain structure with novel functionality (Figure 2D).…”

Towards one‐pot consolidated bioprocessing of cellulose to biodiesel: lipase‐catalyzed transesterification at cellulose‐coated oil‐in‐water emulsions as micro‐reactors

“…But the main barriers of enzyme‐catalyzed processes for their industrial implementation are high cost of enzymes, performance (loss of activity) and recycling problems 11,15 . Several groups have studied methods to improve enzymatic production of biodiesel by transesterification including enzyme immobilization, enzyme engineering, enzyme pre‐ and post‐treatments and microwave heating 16‐25 …”