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

• Published in: Wei sheng wu hsüeh pao Vol. 52; no. 3; p. 345
• Main Authors: Tang, Wei, Li, Yahua, Liu, Lu, Zhang, Junxia, Xian, Hongquan
• Format: Journal Article
• Language: Chinese
• Published: China 04.03.2012
• Subjects: Chitinases - chemistry; Chitinases - genetics; Chitinases - isolation & purification; Chitinases - metabolism; Cloning, Molecular; Enzyme Stability; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Pichia - genetics; Pichia - metabolism; Temperature; Trichoderma - chemistry; Trichoderma - enzymology
• ISSN: 0001-6209

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). 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. 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