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Modifying Surface Charges of a Thermophilic Laccase Toward Improving Activity and Stability in Ionic Liquid.

ABSTRACT: The multicopper oxidase enzyme laccase holds great potential to be used for biological lignin valorization alongside a biocompatible ionic liquid (IL). However, the IL concentrations required for biomass pretreatment severely inhibit laccase activity. Due to their ability to function in extreme conditions, many thermophilic enzymes have found use in industrial applications. The thermophilic fungal laccase from Myceliophthora thermophila was found to retain high levels of activity in the IL [C₂C₁Im][EtSO₄], making it a desirable biocatalyst to be used for lignin valorization. In contrast to [C₂C₁Im][EtSO₄], the biocompatibility of [C₂C₁Im][OAC] with the laccase was markedly lower. Severe inhibition of laccase activity was observed in 15% [C₂C₁Im][OAc]. In this study, the enzyme surface charges were modified via acetylation, succinylation, cationization, or neutralization. However, these modifications did not show significant improvement in laccase activity or stability in [C₂C₁Im][OAc]. Docking simulations show that the IL docks close to the T1 catalytic copper, likely interfering with substrate binding. Although additional docking locations for [OAc]^- are observed after making enzyme modifications, it does not appear that these locations play a role in the inhibition of enzyme activity. The results of this study could guide future enzyme engineering efforts by showing that the inhibition mechanism of [C₂C₁Im][OAc] toward M. thermophila laccase is likely not dependent upon the IL interacting with the enzyme surface.

SUBMITTER: Stevens JC

PROVIDER: S-EPMC9213733 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The development of advanced biofuels from lignocellulosic biomass will require the use of both efficient pretreatment methods and new biomass-deconstructing enzyme cocktails to generate sugars from lignocellulosic substrates. Certain ionic liquids (ILs) have emerged as a promising class of compounds for biomass pretreatment and have been demonstrated to reduce the recalcitrance of biomass for enzymatic hydrolysis. However, current commercial cellulase cocktails are strongly inhibited by most of the ILs that are effective biomass pretreatment solvents. Fortunately, recent research has shown that IL-tolerant cocktails can be formulated and are functional on lignocellulosic biomass. This study sought to expand the list of known IL-tolerant cellulases to further enable IL-tolerant cocktail development by developing a combined in vitro/in vivo screening pipeline for metagenome-derived genes. RESULTS:Thirty-seven predicted cellulases derived from a thermophilic switchgrass-adapted microbial community were screened in this study. Eighteen of the twenty-one enzymes that expressed well in E. coli were active in the presence of the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) concentrations of at least 10% (v/v), with several retaining activity in the presence of 40% (v/v), which is currently the highest reported tolerance to [C2mim][OAc] for any cellulase. In addition, the optimum temperatures of the enzymes ranged from 45 to 95°C and the pH optimum ranged from 5.5 to 7.5, indicating these enzymes can be used to construct cellulase cocktails that function under a broad range of temperature, pH and IL concentrations. CONCLUSIONS:This study characterized in detail twenty-one cellulose-degrading enzymes derived from a thermophilic microbial community and found that 70% of them were [C2mim][OAc]-tolerant. A comparison of optimum temperature and [C2mim][OAc]-tolerance demonstrates that a positive correlation exists between these properties for those enzymes with a optimum temperature >70°C, further strengthening the link between thermotolerance and IL-tolerance for lignocelluolytic glycoside hydrolases.

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Modifying Surface Charges of a Thermophilic Laccase Toward Improving Activity and Stability in Ionic Liquid.

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