Heterologous Overexpression of a Mutant Termite Cellulase Gene in Escherichia coli by DNA Shuffling of Four Orthologous Parental cDNAs

Abstract

Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-β-1,4-glucanase genes from four different termite species (Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus, and Coptotermes acinaciformis) sharing 78.5–96% homology in amino acid sequence. The constructed library was screened by Congo red plate assay combined with 96-well micro-enzyme assay, and clones showing enhanced CMCase activities were obtained. The mutated genes were overexpressed in Escherichia coli intracellularly as an active form. The endo-β-1,4-glucanase (CMCase) activity in soluble fractions of E. coli harboring the mutant genes was 20–30 fold higher than that of wild-type genes. The mutant enzyme showed high activity against CMC and properties similar to those of the native enzymes.

• animal cellulase
• endo-β-1,4-D-glucanase
• termite
• DNA shuffling
• overexpression