Protein Engineering of an Artificial P450BM3 Peroxygenase System Enables Highly Selective O-Demethylation of Lignin Monomers

Abstract

The O-demethylation of lignin monomers, which has drawn substantial attention recently, is critical for the formation of phenols from aromatic ethers. The P450BM3 peroxygenase system was recently found to enable the O-demethylation of different aromatic ethers with the assistance of dual-functional small molecules (DFSM), but these prepared mutants only have either moderate O-demethylation activity or moderate selectivity, which hinders their further application. In this study, we improve the system by introducing different amino acids into the active site of P450BM3, and these amino acids with different side chains impacted the catalytic ability of enzymes due to their differences in size, polarity, and hydrophobicity. Among the prepared mutants, the combination of V78A/F87A/T268I/A264G and Im-C6-Phe efficiently catalyzed the O-demethylation of guaiacol (TON = 839) with 100% selectivity. Compared with NADPH-dependent systems, we offer an economical and practical bioconversion avenue.

Solid-State Fermentation withStreptomycesas an Ecofriendly Route to Tune Lignin Properties and Its Use as a Binder in Adhesive Formulation

“…The great production of xylanase by both strains corroborates the important role of hemicellulolytic enzymes on grass lignocellulose degradation, as previously described . It is also important to take into account that these enzymes are being used for countless biotechnological applications such as bio-bleaching of wood pulps, enzymatic treatment of animal feeds, production of bulk chemicals, direct modification of biopolymers, fermentable sugars production as well as food additives, and ingredients in laundry detergents, among others. ,,− From this point of view, the obtaining of enzymes with proven industrial interest on residual substrates provides an added value to the whole process.…”

Hijacking Chemical Reactions of P450 Enzymes for Altered Chemical Reactions and Asymmetric Synthesis

“…As the name indicates, DFSM has two groups: an anchoring group, usually hydrophobic in nature, is connected to a basic group, which acts as a general acid-base catalyst to activate H 2 O 2 through an appropriate length of a linker (Figure 7B). Decoy and DFSM strategies have been extensively used to alter the substrate specificity and chemical reaction of both NADPH-and H 2 O 2 -dependent P450s, suggesting that these strategies, complemented by protein engineering technology and whole-cell catalysis, would help to generate a combination of toolboxes for developing practical P450 biocatalysts for the synthesis of novel and commercial chiral chemical products [7,8,[87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103] (Figure 7). Elsewhere, there are review articles that have extensively discussed decoy and DFSM strategies, as applied to different P450s to tune or switch the chemical reactions and to accept the non-natural substrates [6,7,96].…”