Cloning and functional expression of an acidophilic β-mannanase gene (Anman5A) from Aspergillus niger LW-1 in Pichia pastoris.
A cDNA fragment of the Anman5A, a gene that encodes an acidophilic β-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-polyacrylamide gel electrophoresis (SDS-PAGE) 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 °C. It was stable at a pH range of 3.0-7.0 and at a temperature of 60 °C or below. Its activity was not significantly affected by an array of metal ions and ethylenediaminetetraacetic acid (EDTA). The K(m) and V(max) of the reAnMan5A, toward locust bean gum, were 1.10 mg/mL and 266.7 units/mg, respectively.
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China.
Jian-Fang
JF
Li
Shun-Ge
SG
Zhao
Cun-Duo
CD
Tang
Jun-Qing
JQ
Wang
Min-Chen
MC
Wu
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D005656:Fungal Proteins; D044902:beta-Mannosidase