[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.


CONCLUSIONS
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.
Agricultural Applied Microbiology Laboratory, College of Life Science, Qingdao Agricultural University, Qingdao 266109, China. tianti_121@163.com
Wei
W
Tang
Yahua
Y
Li
Lu
L
Liu
Junxia
J
Zhang
Hongquan
H
Xian
D002688:Chitinases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology*
D005656:Fungal Proteins; D002688:Chitinases