Heterologous Overexpression of a Mutant Termite Cellulase Gene in Escherichia coli by DNA Shuffling of Four Orthologous Parental cDNAs
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概要
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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.
- 社団法人 日本農芸化学会の論文
著者
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WATANABE Hirofumi
National Institute of Agrobiological Sciences
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NI Jinfeng
National Institute of Agrobiological Sciences
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TAKEHARA Motomi
National Institute of Agrobiological Sciences
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Ni Jinfeng
National Institute Of Agrobiological Sciences:state Key Laboratory Of Microbial Technology Shandong
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Watanabe Hirofumi
National Inst. Of Agrobiological Sciences
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- Purification and Molecular Cloning of Xylanases from the Wood-Feeding Termite, Coptotermes formosanus Shiraki
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