Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris
Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris
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DOI：
10.1016/j.pep.2005.09.013
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发表时间：
2006-03-01
影响因子：
1.6
通讯作者：
Eurwilaichitr, L
中科院分区：
文献类型：
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作者：
Chantasingh, D;Pootanakit, K;Eurwilaichitr, L
从土曲霉BCC129中克隆了一个木聚糖酶基因，编码326个氨基酸，属于真菌糖基水解酶家族10。序列分析表明，该酶的前25个氨基酸为信号肽。因此，只有906bp的成熟木聚糖酶基因被克隆到酵母表达载体pPICZαA中，用于在巴斯德毕赤酵母中进行异源表达。甲醇诱导一天后，在SDS-PAGE凝胶上观察到一条约33 kDa的条带。用凝胶过滤柱层析对表达的酶进行纯化。以桦木木聚糖为底物，纯化的重组木聚糖酶在60℃、pH 5.0、K-m=4.8+/-0.07 mg/m l、V-max=757+/-14.54 m ol/min mg时酶活最高。此外，纯化的酶在40℃下孵育4h时，在4-10范围内表现出广泛的pH稳定性，在50℃下孵育30min时保持了90%的活力，具有中等的热稳定性，这使该酶在动物饲料和造纸工业中具有潜在的应用前景。(C)2005 Elsevier Inc.保留所有权利。
A full-length xylanase gene, encoding 326 amino acids belonging to the fungal glycosyl hydrolase family 10, from Aspergillus terreus BCC129 was cloned and sequenced. Sequence analysis suggested that the first 25 amino acids of this enzyme is the signal peptide. Therefore, only the mature xylanase gene of 906 bp was cloned into a yeast expression vector, pPICZ alpha A, for heterologous expression in Pichia pastoris. A band of approximately, 33kDa was observed on the SDS-PAGE gel after one day of methanol induction. The expressed enzyme was purified by gel filtration chromatography. The purified recombinant xylanase demonstrated optimal activity at 60 degrees C pH 5.0 and a K-m of 4.8 +/- 0.07 mg/ml and a V-max of 757 +/- 14.54 mu mol/min mg, using birchwood xylan as a substrate. Additionally, the purified enzyme demonstrated broad pH stability from 4 to 10 when incubated at 40 degrees C for 4 h. It also showed a moderate thermal stability since it retained 90% of its activity when incubated at 50 degrees C, 30 min, making this enzyme a potential use in the animal feed and paper and pulp industries. (c) 2005 Elsevier Inc. All rights reserved.