Heterologous fusion gene expression and characterization of a novel carbohydrate binding module (Cbm36) to laccase (Lcc2).

Carbohydrate binding modules (CBMs) are commonly known to have the potential to increase binding of carbohydrate-active enzymes to target polysaccharides, thus the catalytic module can easily contact the substrate via CBM. Laccase is one of the most important enzymes used for delignification step in paper industry. This enzyme improves the product's quality, industrial process, and environmentally sound technologies. In this study, we have constructed the fusion gene encoding a novel CBM36 from β-Xylosidase B (GbtXyl43B) of Geobacillus thermoleovorans IT-08 and laccase (LCC2) of Pleurotus salmoneostramineus using overlapping polymerase chain reaction (PCR), and inserted into expression vectors pET-32a and pYHM1 for Escherichia coli BL21 codon plus and Saccharomyces cerevisiae BJ1824, respectively. The fusion protein, Cbm36-Lcc2, was successfully expressed both in E. coli and S. ceerevisiae and optimum pH and temperature were determined to be 5.0 and 60 °C, and 6.0 and 60 °C, respectively, with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate; ABTS) as the substrate. The thermostability and pH stability indicated that Cbm36-Lcc2 expressed in S. cerevisiae BJ1824 was more stable than in E.coli BL21 codon plus. The laccase activity for Cbm36-Lcc2 was two to three times higher than that for Lcc2 itself. This study developed the new insight that fusion of novel Cbm36 into Lcc2 could improve the thermostability of Lcc2 in its chimeric laccase form of Cbm36-Lcc2 compared to Lcc2 itself. • Laccase is one of enzymatically innovations for delignification processes that are more efficient and eco-friendly. • The novel CBM36 gene was successfully fused to LCC2 using an overlapping PCR, and could be expressed in heterologous host. • New insight displayed that the chimeric laccase could improve its thermostability and activity than that for Lcc2 itself. [ABSTRACT FROM AUTHOR]