Development of a magnetically separable co-immobilized laccase and versatile peroxidase system for the conversion of lignocellulosic biomass to vanillin

Lignin obtained from renewable biomass is a potential feedstock for the synthesis of various value-added chemicals through efficient biocatalytic routes. The ligninolytic enzymes-assisted depolymerization of lignin to vanillin constitutes the most commercially attractive and promising approach in green chemistry as vanillin constitutes the second most prevalent flavoring agent. Thus, in the present work, immobilized laccase and versatile peroxidase, and further, a co-immobilized laccase and versatile peroxidase system on magnetic silica microspheres (MSMS) were developed to generate a robust biocatalytic system that mediates the depolymerization of lignin obtained from <i>Casuarina equisetifolia</i> biomass. The depolymerization of lignin by free and immobilized laccase showed a vanillin yield of 24.8% and 23%, respectively, at pH 4.0 in 6 h at 30°C against a vanillin yield of 20% and 21.7% with the free and immobilized versatile peroxidase, respectively, at pH 5.0°C and 50°C. Comparatively, the system with the co-immobilized laccase and versatile peroxidase exhibited a 1-fold and 1.2-fold higher vanillin yield than the free and immobilized laccase system, respectively. On comparing with the versatile peroxidase system, the co-immobilized biocatalytic system displayed 1.3-fold and 1.2-fold increased vanillin yield than the free and immobilized versatile peroxidase system, respectively, at a pH of 6.0 in 6 h at 30°C with an enzyme concentration of 1 U/ml. The reusability studies of the co-immobilized biocatalytic system exhibited that both the enzymes retained up to 40% of its activity till sixth cycle. <i>Implications</i>: The waste biomass of <i>Casuarina equisetifolia</i> is widely available around the coastal regions of India which does not have any agricultural or industrial applications. The present work exploits the lignocellulosic content of the <i>Casuarina</i> biomass to extract the lignin, which provides a renewable alternative for the production of the commercially high-valued compound, vanillin. This work also integrates a co-immobilized biocatalytic process comprising of laccase and versatile peroxidase leading to an environmentally benign enzymatic process for the depolymerization of lignin to vanillin.

从可再生生物质中获取的木质素，是通过高效生物催化途径合成多种高附加值化学品的潜在原料。以木质素降解酶辅助解聚木质素制备香兰素的路径，是绿色化学领域最具商业吸引力与应用前景的策略——这是由于香兰素是全球第二大常用调味剂。因此，本研究开发了固定化漆酶、固定化多功能过氧化物酶，以及二者在磁性二氧化硅微球（magnetic silica microspheres, MSMS）上的共固定化体系，构建了一套稳定的生物催化系统，用于解聚取自木麻黄（<i>Casuarina equisetifolia</i>）生物质的木质素。在30℃、pH 4.0条件下反应6h时，游离漆酶与固定化漆酶催化的木质素解聚反应香兰素得率分别为24.8%与23%；而游离多功能过氧化物酶与固定化多功能过氧化物酶在pH 5.0、50℃条件下的香兰素得率则分别为20%与21.7%。相较而言，共固定化漆酶与多功能过氧化物酶的体系，其香兰素得率分别比游离漆酶体系、固定化漆酶体系高出1倍与1.2倍。在30℃、pH 6.0、酶浓度1 U/mL条件下反应6h时，该共固定化生物催化体系的香兰素得率，相较于游离多功能过氧化物酶体系与固定化多功能过氧化物酶体系，分别提升1.3倍与1.2倍。对该共固定化生物催化体系的重复使用性能研究表明，两种酶在第6个循环时仍可保留最高40%的催化活性。<i>研究启示</i>：印度沿海地区广泛分布木麻黄（<i>Casuarina equisetifolia</i>）废弃生物质，这类生物质目前无任何农业或工业应用价值。本研究利用木麻黄生物质的木质纤维素组分提取木质素，为商业化高附加值化合物香兰素的生产提供了一条可再生替代路径。本研究还集成了包含漆酶与多功能过氧化物酶的共固定化生物催化工艺，构建了一种环境友好的酶法工艺，用于将木质素解聚制备香兰素。