Grépinet O, Chebrou M C, Béguin P
Département des Biotechnologies, Institut Pasteur, Paris, France.
J Bacteriol. 1988 Oct;170(10):4582-8. doi: 10.1128/jb.170.10.4582-4588.1988.
The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined. The putative xynZ gene was 2,511 base pairs long and encoded a polypeptide of 837 amino acids. A region of 60 amino acids containing a duplicated segment of 24 amino acids was found between residues 429 and 488 of xylanase Z. This region was strongly similar to the conserved domain found at the carboxy-terminal ends of C. thermocellum endoglucanases A, B, and D. Deletions removing up to 508 codons from the 5' end of the gene did not affect the activity of the encoded polypeptide, showing that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis. Expression of xylanase activity in Escherichia coli was increased up to 220-fold by fusing fragments containing the 3' end of the gene with the start of lacZ present in pUC19. An internal translational initiation site which was efficiently recognized in E. coli was tentatively identified 470 codons downstream from the actual start codon.
测定了编码嗜热栖热放线菌胞外木聚糖酶Z的xynZ基因的核苷酸序列。推测的xynZ基因长2511个碱基对，编码一个含837个氨基酸的多肽。在木聚糖酶Z的429至488位氨基酸残基之间发现了一个包含一段24个氨基酸重复片段的60个氨基酸的区域。该区域与嗜热栖热放线菌内切葡聚糖酶A、B和D羧基末端发现的保守结构域高度相似。从基因5'端缺失多达508个密码子并不影响所编码多肽的活性，表明活性位点位于蛋白质的C端一半，且保守区域不参与催化。通过将含有基因3'端的片段与pUC19中lacZ的起始部分融合，大肠杆菌中木聚糖酶活性的表达提高了220倍。在实际起始密码子下游470个密码子处初步鉴定出一个在大肠杆菌中能被有效识别的内部翻译起始位点。
