Engineering a Bacterial DyP-Type Peroxidase for Enhanced Oxidation of Lignin-Related Phenolics at Alkaline pH

Abstract

Dye-decolorizing peroxidases (DyPs) are a family of microbial heme-containing peroxidases that show important properties for lignocellulose biorefineries due to their ability to oxidize lignin-related compounds. Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by errorprone PCR of the ppDyP gene followed by high-throughput screening allow identification of the 6E10 variant showing a 100-fold enhanced catalytic efficiency (k cat /K m ) for 2,6dimethoxyphenol (DMP), similar to that exhibited by fungal lignin peroxidases (∼10 5 M −1 s −1 ). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin, and the lignin phenolic model dimer guaiacylglycerol-β-guaiacyl ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units in comparison to the wild type, showed resistance to hydrogen peroxide inactivation, and was produced at 2-fold higher yields. The acquired mutations in the course of the evolution affected three amino acid residues (E188K, A142V, and H125Y) situated at the surface of the enzyme, in the second shell of the heme cavity. Biochemical analysis of hit variants from the laboratory evolution, and single variants constructed using site-directed mutagenesis, unveiled the critical role of acquired mutations from the catalytic, stability, and structural viewpoints. We show that epistasis between A142V and E188K mutations is crucial to determine the substrate specificity of 6E10. Evidence suggests that ABTS and DMP oxidation occurs at the heme access channel. Details of the catalytic cycle of 6E10 were elucidated through transient kinetics, providing evidence for the formation of a reversible enzyme−hydrogen peroxide complex (Compound 0) barely detected in the majority of heme peroxidases studied to date.

Study on the cleavage of alkyl-O-aryl bonds by in situ generated hydroxyl radicals on an ORR cathode

“…Reagents with mild oxidation capability, such as Co, V, Mn oxides and their complexes catalyzed O 2 or organic oxidants (DDQ, TEMPO, ABTS and so on), have been used to depolymerize lignin. [11][12][13][14][15][16][17][18][19][20][21] However, the recovery of these catalysts and the high cost of organic oxidants hinder the large-scale application. [22][23][24] Reactive oxygen species (ROS), mainly H 2 O 2 , cOH, and cO 2 À , could attack the active site on the benzene ring, leading to the breaking of benzene ring from its side chain.…”

Multienzyme Immobilized Polymeric Membrane Reactor for the Transformation of a Lignin Model Compound

“…LC-MS analysis of the reaction products of GGE conversion revealed multiple oxidized products with m / z ranging from 251 [M + Na] + up to 979 [M + Na] + . Enzymatic conversion of GGE (MW 320) to multiple polymeric oxidized products with higher molecular weight has been reported by other researchers, as well [51]. It is noteworthy to point out that the generation of such oligomeric products is enzyme dependent.…”

CAZyome and Sugar Transportome Unveil Singular Aspects of the Lignocellulolytic Enzyme System of Penicillium echinulatum 2HH

“…Surprisingly, an encoding-gene of dye-decolorizing peroxidase (DyP) was found in P. echinulatum 2HH. This family of hemecontaining peroxidases is active on lignin-related compounds and contains important properties for lignocellulose biorefineries [30]. Apparently, this enzyme is not a particular adaptation of P. echinulatum 2HH, considering that the orthologs of this DyP peroxidase were found in P. brasilianum, P. subrubescens and P. chrysogenum, with the exception of P. oxalicum 114-2.…”