Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs.
Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-beta-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-beta-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.
Ni
Jinfeng
J
National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Takehara
Motomi
M
Watanabe
Hirofumi
H
D000595:Amino Acid Sequence; D000818:Animals; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics*; D019295:Computational Biology; D032621:DNA Shuffling; D018076:DNA, Complementary / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D020049:Isoptera / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D015854:Up-Regulation
D018076:DNA, Complementary; D002480:Cellulase