Yong Hwan Kim

Improvement of catalytic performance of lignin peroxidase for the enhanced degradation of lignocellulose biomass based on the imbedded electron-relay in long-range electron transfer route

BackgroundAlthough lignin peroxidase is claimed as a key enzyme in enzyme-catalyzed lignin degradation, in vitro enzymatic degradation of lignin was not easily observed in lab-scale experiments. It implies that other factors may hinder the enzymatic degradation of lignin. Irreversible interaction between phenolic compound and lignin peroxidase was hypothesized when active enzyme could not be recovered after the reaction with degradation product (guaiacol) of lignin phenolic dimer.ResultsIn the study of lignin peroxidase isozyme H8 from white-rot fungi Phanerochaete chrysosporium (LiPH8), W251 site was revealed to make the covalent coupling with one moiety of monolignolic radical (guaiacol radical) by LC-MS/MS analysis. Hypothetical electron-relay containing W251 residue was newly suggested based on the observation of repressed radical coupling and remarkably lower electron transfer rate for W215A mutant. Furthermore, the retardation of the suicidal radical coupling between the W251 residue and the monolignolic radical was attempted by supplementing the acidic microenvironment around the W251 residue to engineer radical-robust LiPH8. Among many mutants, mutant A242D showed exceptional catalytic performances by yielding 21.1- and 4.9-fold higher increases of kcat and kcat/KM values, respectively, in the oxidation of non-phenolic model lignin dimer.ConclusionsA mechanism-based suicide inhibition of LiPH8 by phenolic compounds was firstly revealed and investigated in this work. Radical-robust LiPH8 was also successfully engineered by manipulating the transient radical state of radical-susceptible electron-relay. Radical-robust LiPH8 will play an essential role in degradation of lignin, which will be consequently linked with improved production of sugars from lignocellulose biomass.

Enzymes for Environment

Strict regulations on the disposal of wastes to the environment require improvement of waste treatment processes. In recent years, extensive research on biological processes has been conducted to enable industrial, agricultural, municipal, and commercial facilities to reduce their harmful impacts on the environment. Biological processes such as activated sludge process are the most economical method when treating broad range of compounds in aqueous solution.

Enzymes in Non-conventional Media

Traditionally enzyme reactions have been in many cases performed in aqueous buffer systems. Since the environments inside the cells are rather hydrophilic in some part and hydrophobic in other part of the cells, enzyme reactions can be also performed in hydrophobic condition. Historically, organic solvent was used for steroid bioconversion. Since solubilities of substrate and product steroids are very low in water, they can be solubilized using organic solvent system.

Thermodynamics and Stability

There are many advantages of enzymes such as substrate specificity, mild reaction conditions. However, there are also disadvantages such as high cost and instability of enzymes which give limitation for commercial applications.

Production of Enzymes

The first step in enzyme production is the selection of the enzyme source. Enzymes can be derived from microorganisms through fermentation processes, as well as plant and animal sources. Table 3.1 presents industrially important enzymes and their sources.

Biosynthesis of Enzymes

The central carbon atom covalently bonded by amino, carboxyl, and R group in the structure is called the alpha carbon (Cα). The side chain R group varies in chemical composition, size, and interaction with water as reflected in their polarity. There are 20 standard amino acids used as common building blocks for peptides and proteins.

Engineering Tools for Enzymes

Since new millennia, application of industrial enzymes in manufacturing process including pharmaceuticals, fine chemicals, bio-based chemicals remarkably gained much attention. There is a fundamental limitation to apply industrial enzymes since enzymes have not been evolved to meet the requirements as biocatalysts.

Enzyme Reaction Kinetics

Since enzyme reaction, in many cases, follows first-order kinetics at low substrate concentration, and zero-order kinetics at high substrate concentration, simple enzyme reaction mechanism was suggested. During the course of enzyme, enzymes form a complex with the substrate. The mechanism is called as Michaelis–Menten kinetics for one-substrate reaction.

Enzymes for Chemicals and Polymers

Isolated enzymes have been used as highly specific catalyst in organic chemicals synthesis. However, industrial significance of enzyme reactions was especially emphasized in the 1970s with the production of high fructose corn syrup (HFCS) . Recombinant DNA technology also enables the efficient production of enzymes, making them cheaply available for use.

Regeneration of Cofactors

Enzymes such as oxidoreductases and transferases are able to catalyze industrially useful reactions. However, these enzymes are often cofactor dependent. Cofactors are relatively low molecular weight compounds that are required for the enzymatic reactions.