Engineering lignocellulolytic enzymes for industrial applications: Engineering of laccase and endoglucanase

Abstract

Energy consumption, especially electricity demand, is continually rising, while non-renewable fossil energy sources such as petroleum, natural gas, and coal are depleting. Fossil energy production also causes greenhouse gas emissions, which accelerate climate change. Efforts are being made to reduce reliance on these energy sources by increasing wind, water, and geothermal energy production. However, additional measures are still needed to fill the resulting energy gap. This shortfall can be addressed by biofuels, which can be produced from biomass. The world's most common biomass is lignocellulose, which consists of wood, forest residues, peat, straw, and agricultural biomass.

Lignocellulose has a complex molecular structure, making it difficult to break down. After pretreatment, it is broken into its main components—cellulose, hemicellulose, and lignin—which are further broken down into molecular fractions or fully soluble molecules. These can then be processed to produce bio-based chemicals and products. This process is called lignocellulose valorization, where the biomass is catalytically "upgraded" into new raw materials or products. Waste streams from existing industrial processes using lignocellulose are also being repurposed more efficiently. For example, lignin waste from paper mills, previously burned for energy, is now used as raw material for new products.

Enzymes are proteins that act as biological catalysts in chemical reactions. Using enzymes in industry can reduce energy and chemical consumption, making processes more sustainable, economically profitable, and environmentally friendly. Industrial processes are large-scale, so enzyme production must be as efficient as possible to keep production costs and industrial use affordable. Additionally, industrial processes require remarkably stable and resilient enzymes, especially under extreme heat and pH conditions. Enzyme engineering can improve production yield and enzyme properties to meet industrial needs.

This work focused on developing two bacterial enzymes - laccase (from Bacillus wacoensis) and endoglucanase (from Spirochaeta thermophila) - and their further utilisation in industrial processes. Public databases were searched for gene sequences of enzymes that were expected to have the desired catalytic activity. These sequences were synthesised, cloned, and produced using the standard industrial microbe Escherichia coli for the first time. The enzymes were characterised to determine their specific activities and thermal and pH stability under different conditions. The laccase enzyme was stable in alkaline, high-pH conditions and retained its activity, but its soluble yield in E. coli was very low. The goal of enzyme engineering was to increase the solubility of laccase and further improve its activity. As a result of the engineering, both the yield and activity were significantly improved, allowing the enzyme to be utilised in lignin depolymerisation at high pH and to separate small molecular lignin fractions of different sizes. Thanks to the successful development process, the enzyme's industrial use became feasible, and it remains unique for its laccase properties in lignin depolymerisation.

The second enzyme studied was endoglucanase, which had a good production yield in E. coli but was not stable enough at high temperatures for use in many industrial processes. Its engineering aimed to improve thermal stability and further increase activity. As a result of the engineering, its specific activity was significantly increased, and its substrate specificity was altered. However, no improvement in thermal stability was achieved. Endoglucanase is currently used as part of an enzyme cocktail for cellulosic fibre modification processes, but its engineered properties still need to be improved to replace existing alternative enzymes sustainably.