Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system

An acidophilic beta-mannanase-encoding gene (Auman5A) from Aspergillus usamii YL-01-78 was amplified and inserted into pPIC9K and pPICZ alpha A vectors. The resulting recombinant vector, pPIC9K-Auman5A, was transformed into Pichia pastoris GS115. One strain having the highest recombinant beta-mannanase activity of 54.6 U/ml, labeled GSKM4-8, was chosen from the first-batch P. pastoris transformants. Then, the pPICZ alpha A-Auman5A was transformed into GSKM4-8 again. From the second-batch transformants, one strain (GSKZ alpha M4-2) with the highest beta-mannanase activity of 78.1 U/ml was obtained, and used to optimize expression conditions. As GSKZ alpha M4-2 was induced under the optimized conditions (initial pH value 6.5, induction period 120 h, methanol concentration 1.5 %, and induction temperature 32 A degrees C), beta-mannanase activity reached 162.8 U/ml. Protein and carbohydrate assays showed that the beta-mannanase, a glycoprotein with an apparent molecular weight of 49.8 kDa and a carbohydrate content of 21.3 %, was extracellularly expressed. It displayed the maximum activity at pH 3.0 and 70 A degrees C, and was stable at a pH range of 3.0-7.0 and at 60 A degrees C. Its activity was not significantly affected by metal ions tested and EDTA, but inhibited by Ag+ and Hg2+. Its most favorable substrate was locust bean gum, followed by konjac flour and guar gum. The K (m) and V (max) towards locust bean gum were 1.36 mg/ml and 415.8 U/mg, respectively. These results suggested that the beta-mannanase can be expressed with higher level and possesses superior enzymatic properties, making it a good candidate in industrial processes.