Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2005-09, Vol.69 (9), p.1711-1720
• Main Authors: Ni, J.(National Inst. of Agrobiological Sciences, Tsukuba, Ibaraki (Japan)), Takehara, M, Watanabe, H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; animal cellulase; Animals; Biological and medical sciences; CELLULASE; Cellulase - biosynthesis; Cellulase - genetics; CELULASA; Computational Biology; Coptotermes; Coptotermes formosanus; DNA Shuffling; DNA, Complementary - biosynthesis; endo-β-1,4-D-glucanase; ESCHERICHIA COLI; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; ISOPTERA; Isoptera - enzymology; Isoptera - genetics; Molecular Sequence Data; MUTANT; MUTANTES; MUTANTS; Mutation; Nasutitermes; overexpression; Reticulitermes speratus; Sequence Alignment; termite; Up-Regulation
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.69.1711

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.