Cloning and Functional Expression of an Acidophilic 尾-Mannanase Gene (Anman5A) from Aspergillus niger LW-1 in Pichia pastoris
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- 作者：Jian-Fang Li ; Shun-Ge Zhao ; Cun-Duo Tang ; Jun-Qing Wang ; Min-Chen Wu
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2012
- 出版时间：January 25, 2012
- 年：2012
- 卷：60
- 期：3
- 页码：765-773
- 全文大小：584K
- 年卷期：v.60,no.3(January 25, 2012)
- ISSN：1520-5118
文摘
A cDNA fragment of the Anman5A, a gene that encodes an acidophilic 尾-mannanase of Aspergillus niger LW-1 (abbreviated as AnMan5A), was cloned and functionally expressed in Pichia pastoris. Homology alignment of amino acid sequences verified that the AnMan5A belongs to the glycoside hydrolase (GH) family 5. Sodium dodecyl sulfate鈥損olyacrylamide gel electrophoresis (SDS鈥揚AGE) assay demonstrated that the recombinant AnMan5A (reAnMan5A), a N-glycosylated protein with an apparent molecular weight of 52.0 kDa, was secreted into the medium. The highest reAnMan5A activity expressed by one P. pastoris transformant, labeled as GSAnMan4-12, reached 29.0 units/mL. The purified reAnMan5A displayed the highest activity at pH 3.5 and 70 掳C. It was stable at a pH range of 3.0鈥?.0 and at a temperature of 60 掳C or below. Its activity was not significantly affected by an array of metal ions and ethylenediaminetetraacetic acid (EDTA). The Km and Vmax of the reAnMan5A, toward locust bean gum, were 1.10 mg/mL and 266.7 units/mg, respectively.