Cloning and Functional Expression of an Acidophilic β-Mannanase Gene (Anman5A) from Aspergillus niger LW-1 in Pichia pastoris

A cDNA fragment of the AnmanSA, a gene that encodes an acidophilic beta-mannanase of Aspergillus niger LW-1 (abbreviated as AnManSA), was cloned and functionally expressed in Pichia pastoris. Homology alignment of amino acid sequences verified that the AnManSA belongs to the glycoside hydrolase (GH) family 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) assay demonstrated that the recombinant AnManSA (reAnMan5A), a N-glycosylated protein with an apparent molecular weight of 52.0 kDa, was secreted into the medium. The highest reAnManSA activity expressed by one P. pastoris tr.ansformant, labeled as GSAnMan4-12, reached 29.0 units/mL. The purified reAnManSA displayed the highest activity at pH 3.5 and 70 degrees C. It was stable at a pH range of 3.0-7.0 and at a temperature of 60 degrees C or below. Its activity was not signifidantly 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.