Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs
Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs
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DOI：
10.1271/bbb.69.1711
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发表时间：
2005-09-01
影响因子：
1.6
通讯作者：
Watanabe, H
中科院分区：
文献类型：
--
作者：
Ni, JF;Takehara, M;Watanabe, H
在纤维素酶基因中，动物的纤维素酶基因以难以过表达著称。通过对4种白蚁（Reticulitermes speratus、Nasutitermes takasagoensis、Coptotermes formosanus和acinaciformis）的内切- β -1,4-葡聚糖酶基因进行家族重组，构建了同源性为78.5-96%的嵌合文库。用刚果红平板法结合96孔微酶法对构建的文库进行筛选，获得了CMCase活性增强的克隆。突变基因在大肠杆菌细胞内以活性形式过表达。携带突变基因的大肠杆菌可溶性组分的内切- β -1,4-葡聚糖酶（CMCase）活性比野生型高20-30倍。该突变体酶具有较高的抗CMC活性和与天然酶相似的特性。
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.