Valorization of Agricultural Lignocellulosic Biomass through Application of Thermoalkaliphilic Laccase from Caldalkalibacillus thermarum Strain TA2.A1

Abstract

Lignin is a complex aromatic heteropolymer with a three-dimensional structure composed of phenylpropanoid aryl-C3 units (p-coumaryl, coniferyl, and sinapyl alcohols) via a variety of linkages such as β-O-4 aryl ether, β-β, β-5, 5-5, and 5-O-4 bond. This structural complexity renders lignin valuable for the production high-value chemical synthons and simultaneously recalcitrant for the utilization of the carbohydrate polymers in lignocellulosic biomass. Laccase is considered as a “green” catalyst due to its catalytic cycle: the reduction of one molecule of oxygen and the concomitant oxidation of four substrates molecules with the production of two molecules of water as only by-product. Laccase is able to perform single-electron oxidation of various phenolic compounds to the corresponding radical species which can undergo further hydration, oxidation, polymerization, or depolymerization reactions and therefore it is widely used as a biocatalyst in diverse industrial applications. The present study is focused on characterization of a novel thermoalkaliphilic laccase from Caldalkalibacillus thermarum strain TA2.A1 and the enzymatic treatment on lignocellulosic biomass to investigate its potential use in the valorization of lignin. The following experiments were performed to proceed with this study. Firstly, a novel thermoalkaliphilic laccase, CtLac, was cloned and expressed in E. coli and the enzymatic properties were characterized. Secondly, the potential application of CtLac in the depolymerization of lignin with the production of various value-added chemicals was explored using various sources of lignocellulosic biomass and lignin. Thirdly, the effect of CtLac-pretreatment on saccharification of various lignocellulosic biomass was investigated with an observation of surface morphological analysis of CtLac-pretreated biomass. Lastly, the combination of random mutagenesis and site-directed mutagenesis was performed to improve enzymatic activity of CtLac. A novel laccase CtLac from C. thermarum strain TA2.A1 was cloned and successfully expressed in E. coli and it showed versatile properties such as high thermostability and resistance to the various surfactants, organic solvents, and halides. Besides, CtLac catalyzed the oxidative dimerization of a dimeric lignin model compound, GGGE. Moreover, CtLac showed the efficient depolymerization of the various lignocellulosic biomass and lignin used in this study, with the enhanced production of vanillin and p-hydroxybenzaldehyde as major benzaldehyde chemicals. CtLac-pretreatment led to the highest yield of reducing sugar in rice straw samples with severe surface morphological changes. The optimal conditions for CtLac-pretreatment were determined as temperature 70℃, pH 8.0, and 12 h of reaction time. From the mutagenesis study, it was revealed that Valine at the position of 243 might be one of important amino acids contributing to the catalytic efficiency of CtLac in the lack of protein structure. In summary, the findings in the present study suggest that CtLac has a high potential for the application in the valorization of agricultural lignocellulosic biomass, considering its versatile properties.