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Expression, Purification and Enzymatic Activity of α-1,2 Mannosidase I Derived from Trichoderma reesei in Pichia pastoris

Expression, Purification and Enzymatic Activity of α-1,2 Mannosidase I Derived from Trichoderma reesei in Pichia pastoris

Siqiang Li1,2, Tiantian Wang1, Peng Sun1, Airong Gao1, Xin Gong1, Yuanhong Xu2, Baogen Wang2, Jun Wu1* and Bo Liu1*

1Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. 
2School of Biological and Food Processing Engineering, Huanghuai University, 76 Kaiyuan Road, Zhumadian 463000, China.
 
Siqiang Li and Tiantian Wang contributed equally to this article.
 
*      Corresponding author: junwu1969@163.com; liubo7095173@163.com

ABSTRACT

α-1,2 mannosidase I (MDS I) is a desired tool enzyme to modify oligosaccharides and their analogues in structurally homogeneous and defined forms in vitro. This study was aimed to explore the acquisition of an effective MDS I in vitro. For this purpose a Pichia pastoris strain GS115 harboring a recombinant MDS I derived from Trichoderma reesei was constructed via conventional molecular cloning methods, and expressed in a 5-liter fermentation tank. The target protein was purified in three-step purification and identified by peptide mass of fingerprint. The enzymatic activity and optimal reaction conditions of MDS I were detected using DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. We obtained MDS I with a purity exceeding 90% in gram scales, which was capable of digesting α-1,2 linked mannose residues in high selectivity. The highest enzymatic activity of MDS I occurred at a pH of 7.0 and a temperature of 42°C. Enzymatic activity of MDS I was also influenced by metal ions, which were increased to 22% and 17%, respectively, by Co2+ and Cu2+ (2 mmol/L each), while were inhibited to some extent by Ca2+, Mg2+, Mn2+ and Zn2+. This study has laid the foundation for the application of MDS I in future glycol-engineering research.

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Pakistan Journal of Zoology

October

Pakistan J. Zool., Vol. 56, Iss. 5, pp. 2001-2500

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