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

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.