Cloning, Sequencing, and Expression of the Gene Encoding a Multidomain Endo-β-1,4-Xylanase from Paenibacillus curdlanolyticus B-6, and Characterization of the Recombinant Enzyme
Cloning, Sequencing, and Expression of the Gene Encoding a Multidomain Endo-β-1,4-Xylanase from Paenibacillus curdlanolyticus B-6, and Characterization of the Recombinant Enzyme
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DOI：
10.4014/jmb.0804.293
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发表时间：
2009-03-01
影响因子：
2.8
通讯作者：
Ratanakhanokchai, Khanok
中科院分区：
文献类型：
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作者：
Waeonukul, Rattiya;Pason, Patthra;Ratanakhanokchai, Khanok
编码木聚糖酶Xyn 10A的解凝乳类芽孢杆菌B-6 xyn 10A基因的核苷酸序列由3，828个核苷酸组成，编码具有1，276个氨基酸的蛋白质，预测分子量为142，726 Da。三个家族22碳水化合物结合模块（CBM）、家族10糖基水解酶（木聚糖酶）催化结构域、家族9 CBM、富含甘氨酸的区域和三个表面层同源（SLH）结构域。通过在纤维素上的单一步骤亲和纯化从重组大肠杆菌纯化Xyn 10A。它能有效地水解农业废弃物和纯不溶性木聚糖，尤其是低取代不溶性木聚糖。水解产物为一系列短链低聚木糖，表明纯化的酶是内切β-1，4-木聚糖酶。Xyn 10A与各种不溶性多糖结合，包括Avicel、α-纤维素、不溶性桦木和燕麦斯佩耳特木聚糖、几丁质和淀粉，以及解凝乳酶原杆菌B-6的细胞壁片段，表明CBM和SLH结构域在Xyn 10A中完全起作用。从Xyn 10A中去除CBM强烈降低了植物细胞壁水解的能力。这些结果表明，Xyn 10A的CBMs在植物细胞壁的水解中起着重要作用。
The nucleotide sequence of the Paenibacillus curdlanolyticus B-6 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular mass of 142,726 Da. Sequence analysis indicated that Xyn10A is a multidomain enzyme comprising nine domains in the following order: three family 22 carbohydrate-binding modules (CBMs), a family 10 catalytic domain of glycosyl hydrolases (xylanase), a family 9 CBM, a glycine-rich region, and three surface layer homology (SLH) domains. Xyn10A was purified from a recombinant Escherichia coli by a single step of affinity purification on cellulose. It could effectively hydrolyze agricultural wastes and pure insoluble xylans, especially low substituted insoluble xylan. The hydrolysis products were a series of short-chain xylooligosaccharides, indicating that the purified enzyme was an endo-beta-1,4-xylanase. Xyn10A bound to various insoluble polysaccharides including Avicel, alpha-cellulose, insoluble birchwood and oat spelt xylans, chitin, and starches, and the cell wall fragments of P. curdlanolyticus B-6, indicating that both the CBM and the SLH domains are fully functioning in the Xyn10A. Removal of the CBMs from Xyn10A strongly reduced the ability of plant cell wall hydrolysis. These results suggested that the CBMs of Xyn10A play an important role in the hydrolysis of plant cell walls.