Sunna Anwar, Gibbs Moreland D, Bergquist Peter L
Department of Biological Sciences, Macquarie University, North Ryde, Sydney, NSW 2109, Australia1.
Department of Molecular Medicine, University of Auckland Medical School, Private Bag 92019, Auckland, New Zealand2.
Microbiology (Reading). 2000 Nov;146 ( Pt 11):2947-2955. doi: 10.1099/00221287-146-11-2947.
The nucleotide sequence of the complete xynA gene, encoding a novel multidomain xylanase XynA of 'Caldibacillus cellulovorans', was determined by genomic-walking PCR. The putative XynA comprises an N-terminal domain (D1), recently identified as a xylan-binding domain (XBD), homologous to non-catalytic thermostabilizing domains from other xylanases. D1 is followed by a xylanase catalytic domain (D2) homologous to family 10 glycosyl hydrolases. Downstream of this domain two cellulose-binding domains (CBD), D3 and D4, were found linked via proline-threonine (PT)-rich peptides. Both CBDs showed sequence similarity to family IIIb CBDs. Upstream of xynA an incomplete open reading frame was identified, encoding a putative C-terminal CBD homologous to family IIIb CBDs. Two expression plasmids encoding the N-terminal XBD plus the catalytic domain (XynAd1/2) and the xylanase catalytic domain alone (XynAd2) were constructed and the biochemical properties of the recombinant enzymes compared. The absence of the XBD resulted in a decrease in thermostability of the catalytic domain from 70 degrees C (XynAd1/2) to 60 degrees C (XynAd2). Substrate-specificity experiments and analysis of the main products released from xylan hydrolysis indicate that both recombinant enzymes act as endo-1, 4-beta-xylanases, but differ in their ability to cleave small xylooligosaccharides.
通过基因组步移PCR确定了编码嗜热纤维芽孢杆菌（Caldibacillus cellulovorans）新型多结构域木聚糖酶XynA的完整xynA基因的核苷酸序列。推测的XynA包含一个N端结构域（D1），最近被鉴定为木聚糖结合结构域（XBD），与其他木聚糖酶的非催化热稳定结构域同源。D1之后是一个与10家族糖基水解酶同源的木聚糖酶催化结构域（D2）。在该结构域的下游，发现两个纤维素结合结构域（CBD），D3和D4，通过富含脯氨酸-苏氨酸（PT）的肽连接。两个CBD都与IIIb家族CBD具有序列相似性。在xynA的上游鉴定出一个不完整的开放阅读框，编码一个与IIIb家族CBD同源的推定C端CBD。构建了两个表达质粒，分别编码N端XBD加催化结构域（XynAd1/2）和单独的木聚糖酶催化结构域（XynAd2），并比较了重组酶的生化特性。XBD的缺失导致催化结构域的热稳定性从70℃（XynAd1/2）降至60℃（XynAd2）。底物特异性实验和木聚糖水解释放的主要产物分析表明，两种重组酶均作为内切-1,4-β-木聚糖酶起作用，但在切割小木寡糖的能力上有所不同。
