Abstract
An acidophilic β-mannanase-encoding gene (Auman5A) from Aspergillus usamii YL-01-78 was amplified and inserted into pPIC9K and pPICZαA vectors. The resulting recombinant vector, pPIC9K-Auman5A, was transformed into Pichia pastoris GS115. One strain having the highest recombinant β-mannanase activity of 54.6 U/ml, labeled GSKM4-8, was chosen from the first-batch P. pastoris transformants. Then, the pPICZαA-Auman5A was transformed into GSKM4-8 again. From the second-batch transformants, one strain (GSKZαM4-2) with the highest β-mannanase activity of 78.1 U/ml was obtained, and used to optimize expression conditions. As GSKZαM4-2 was induced under the optimized conditions (initial pH value 6.5, induction period 120 h, methanol concentration 1.5 %, and induction temperature 32 °C), β-mannanase activity reached 162.8 U/ml. Protein and carbohydrate assays showed that the β-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 °C, and was stable at a pH range of 3.0–7.0 and at 60 °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 β-mannanase can be expressed with higher level and possesses superior enzymatic properties, making it a good candidate in industrial processes.
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Acknowledgments
This work was financially supported by the National Nature Science Foundation of China (No. 31271811), Doctoral Research Funds of Jiangnan University (No. JUDCF11011) and Postgraduate Innovation Training Project of Jiangsu (No. CXZZ11_0480). We are grateful to Prof. **anzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.
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C.D. Tang and J. Guo, the two first authors, contributed equally to this work.
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Tang, CD., Guo, J., Li, JF. et al. Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system. Ann Microbiol 64, 561–569 (2014). https://doi.org/10.1007/s13213-013-0689-7
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DOI: https://doi.org/10.1007/s13213-013-0689-7