Characterization and production optimization of a chitinase (Tachi1) from Trichoderma asperellum in recombinant Pichia Pastoris expression system

Abstract:

Abstract:［Objective］We characterized a chitinase (Tachi1) from Trichoderma asperellum and optimized its production conditions，by methanol induction of the recombinant strain Pichia pastoris GS-tachi1-K transformed with the gene tachi1 (GenBank accession: GU457411)．［Methods］ We purified Tachi1 from the fermentation broth to analyze enzymatic properties after it was secreted by GS-tachi1-K．The production conditions of GS-tachi1-K were optimized by single-factor experiment and orthogonal experiment．［Results］The molecular weight of Tachi1 was about 44 kDa．Tachi1 had a broad range of temperature and pH adaption with the optimal reaction temperature at 50℃ and pH 5. 5. It was stable at the temperature below 50℃，yet less stable under alkaline conditions．Its activity was significantly reduced by 0.05 mol/L of Ag+，Hg2+，Cu2+，Fe2+，1% of Sodium dodecyl sulfate (SDS) and 10 mmol/L of β-mercaptoethanol．The optimum conditions obtained were: initial cell density with an OD600 equal to 2，0.5% of methanol，pH 6.5，induction time 180 h． Under the optimized condition，the activity of Tachi1 reached 17.93 U/mL and the expression of tachi1 was 6.19g/L．［Conclusion］The recombinant strain GS-tachi1-K showed high expression of tachi1 and the protein secreted by GStachi1-K had high chitinase activity．It will provide theoretical basis for further research and application in this chitinase．

Key words:Keywords: Pichia pastoris，Trichoderma asperellum，chitinase，enzymatic properties，production conditions