A multidomain xylanase from a Bacillus sp with a region homologous to thermostabilizing domains of thermophilic enzymes
A multidomain xylanase from a Bacillus sp with a region homologous to thermostabilizing domains of thermophilic enzymes
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DOI：
10.1099/13500872-145-8-2163
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发表时间：
1999-08-01
期刊：
影响因子：
2.8
通讯作者：
Pastor, FIJ
中科院分区：
文献类型：
--
作者：
Blanco, A;Díaz, P;Pastor, FIJ
从芽孢杆菌BP-23中克隆了木聚糖酶C基因，并在大肠杆菌中进行了表达。测定了含有木聚糖酶基因的3538bpDNA片段的核苷酸序列，其开放阅读框为3258bp，编码一个120 567 Da的蛋白质。将推导出的木聚糖酶C的氨基酸序列与已知的β-葡聚糖酶序列进行比较，表明该酶是一个含有3个不同结构域的模块蛋白。该酶的中心区域是催化结构域，它与家族10木聚糖酶高度同源，与家族IX纤维素结合结构域同源的区域位于木聚糖酶C的C末端区域，而该酶的N末端区域与几种嗜热酶中的热稳定结构域同源，木聚糖酶C显示出类似于中温微生物酶的活性图谱。最大活性在45摄氏度，酶只在55摄氏度或更低的温度下才稳定。桦木木聚糖酶的主要产物是木四糖、木三糖、木二糖和木糖，而该酶对长度增加的低聚木糖的活性增加，表明该酶为内切木聚糖酶，构建了缺失与热稳定结构域同源区域的木聚糖酶C。截短酶的最适活性温度低于全长酶，35℃而不是45℃，热稳定性降低，导致在55℃孵育2小时后酶完全失活。
The gene xynC encoding xylanase C from Bacillus sp, BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120 567 Da, A comparison of the deduced amino acid sequence of xylanase C with known beta-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology to family 10 xylanases, A domain homologous to family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal region of the enzyme shows homology to thermostabilizing domains found in several thermophilic enzymes, Xylanase C showed an activity profile similar to that of enzymes from mesophilic microorganisms. Maximum activity was found at 45 degrees C, and the enzyme was only stable at 55 degrees C or lower temperatures. Xylotetraose, xylotriose, xylobiose and xylose were the main products from birchwood xylan hydrolysis, whilst the enzyme showed increasing activity on xylo-oligosaccharides of increasing length, indicating that the cloned enzyme is an endoxylanase, A deletion derivative of xylanase C, lacking the region homologous to thermostabilizing domains, was constructed. The truncated enzyme showed a lower optimum temperature for activity than the full-length enzyme, 35 degrees C instead of 45 degrees C, and a reduced thermal stability that resulted in a complete inactivation of the enzyme after 2 h incubation at 55 degrees C.