A gene encoding a novel extremely thermostable 1,4-β-xylanase isolated directly from an environmental DNA sample
A gene encoding a novel extremely thermostable 1,4-β-xylanase isolated directly from an environmental DNA sample
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DOI：
10.1007/s00792-002-0296-1
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发表时间：
2003-02-01
期刊：
影响因子：
2.9
通讯作者：
Bergquist, PL
中科院分区：
文献类型：
--
作者：
Sunna, A;Bergquist, PL
使用细菌或芽孢杆菌特异性rDNA引物，通过PCR从热池环境DNA样品中扩增小亚基（SSU）rRNA基因（rDNA）。通过限制性片段长度多态性（RFLP）分析鉴定了独特的rDNA类型，并确定了代表性序列。利用家族10糖苷水解酶共有引物对热池环境样品中木聚糖酶基因进行PCR扩增，研究了热池环境样品中木聚糖酶基因的存在情况和多样性。获得了三种不同木聚糖酶的部分序列，并使用基因组步移PCR（GWPCR）结合巢式引物对获得了独特的1，741-bp核苷酸序列。对该序列的分析鉴定了由xynA开放阅读框编码的推定的XynA蛋白。该单模块新型木聚糖酶与家族10糖苷水解酶具有序列相似性。纯化的重组酶XynA在E.大肠杆菌在100 ℃和pH6.0时具有最佳活性，在90 ℃时具有极强的热稳定性。该酶对不同种类的木聚糖和低聚木糖具有高度的专一性。
Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100degreesC and pH 6.0, and was extremely thermostable at 90degreesC. The enzyme showed high specificity toward different xylans and xylooligosaccharides.