极端嗜热厌氧菌株NA10多结构域纤维素酶的表征

Katsuhide Miyake, Yuichi Machida, Kouji Hattori, Shinji Iijima
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引用次数: 2

摘要

测定了极端嗜热厌氧菌NA10 β-葡聚糖酶基因的核苷酸序列。该开放阅读框长度超过3000 bp,编码一个分子量为113 kDa的多肽。该蛋白的氨基酸序列与Caldocellum saccharticum的双功能纤维素酶CelB具有高度的同源性。根据与CelB的同源性,NA10 β-葡聚糖酶似乎包含三个结构域:n端,中心和c端结构域。其中,N端和c端结构域可能是催化结构域,中心结构域可能是纤维素结合结构域。这些结构域通过富含脯氨酸和苏氨酸的区段(PT box)相互连接。gst融合的c端结构域显示CMCase和MUCase活性,而gst融合的n端结构域活性非常弱。酶谱分析表明,含有β-葡聚糖酶基因的重组大肠杆菌产生的蛋白分子量约为113 kDa,与DNA序列推断的结果吻合较好。然而,Western blot分析表明,该全长蛋白的数量非常少。此外,还检测到几个具有CMCase活性的小蛋白。Northern blot分析表明,似乎存在假定的内部转录起始位点。小分子质量物种的产生似乎是由于基因起始位点的替代转录和翻译,或部分蛋白质水解。
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Characterization of a multidomain cellulase from an extremely thermophilic anaerobe strain NA10

The nucleotide sequence of a β-glucanase gene from an extremely thermophilic anaerobe NA10 was determined. The open reading frame extended over 3000 bp and encoded a polypeptide with a molecular mass of 113 kDa. The deduced amino acid sequence of this protein exhibited high homology to a bifunctional cellulase CelB of Caldocellum saccharolyticum. Based on the homology to CelB, the NA10 β-glucanase appears to comprise three domains: N-terminal, central, and C-terminal domains. Among these, N- and C-terminal domains apper to be catalytic domains, and the central domain to be a cellulose binding domain. These domains were joined with each other by proline and threonine rich segments (PT box). The GST-fused C-terminal domain showed CMCase and MUCase activities, but the activities of the GST-fused N-terminal domain were very weak. Zymogram analysis revealed that recombinant Escherichia coli containing the β-glucanase gene produced a protein with a molecular mass of approximately 113 kDa, which was in good agreement with that deduced from the DNA sequence. However, Western blot analysis indicated that the amount of this full length protein was very small. Several smaller abundant proteins which exhibited CMCase activity were also detected. Northern blot analysis indicated that there appear to be putative internal transcriptional initiation sites. Generation of small molecular mass species appear to be due to alternative transcription and translation from the initiation sites within the gene, or partial proteolysis.

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