[Characterization and production optimization of a chitinase (Tachil) from Trichoderma asperellum in recombinant Pichia pastoris expression system].

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. Tachil had a broad range of temperature and pH adaption with the optimal reaction temperature at 50 degrees C and pH 5.5. It was stable at the temperature below 50 degrees C, 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 beta-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 6.19 g/L.
CONCLUSION: The recombinant strain GS-tachi1-K showed high expression of tachi1 and the protein secreted by GS-tachi1-K had high chitinase activity. It will provide theoretical basis for further research and application in this chitinase.