重组毕赤酵母表达棘孢木霉几丁质酶Tachi1的酶学性质研究及表达条件优化

【目的】对转棘孢木霉几丁质酶基因tachi1的毕赤酵母工程菌GS-tachi1-K进行诱导表达,研究重组几丁质酶Tachi1的酶学性质,优化表达条件。【方法】对GS-tachi1-K进行甲醇诱导培养,纯化目的蛋白Tachi1进行几丁质酶酶学性质的研究;通过单因素和正交试验对GS-tachi1-K菌株产几丁质酶Tachi1表达条件进行优化。【结果】GS-tachi1-K表达的几丁质酶Tachi1表观分子量约为44 kDa,酶反应最适的温度和pH分别为50℃和5.5,具有较宽的温度、pH适用范围;50℃以下保持较高的酶活力,在碱性条件下稳定性较差;受Ag+、Hg2+、Cu2+、Fe2+和高浓度的SDS及β-巯基乙醇强烈抑制。该菌株的最佳表达条件为:pH为6.5,甲醇诱导浓度为0.5%,起始细胞浓度为OD600=2,甲醇诱导时间为180 h;几丁质酶Tachi1活力可达17.93 U/mL,蛋白表达量为6.19 g/L。【结论】成功实现了棘孢木霉新几丁质酶基因tachi1的毕赤酵母高效分泌表达,工程菌GS-tachi1-K具有高表达量和表达产物酶活性高两个特点,明确了几丁质酶Tachi1的酶学性质和最佳诱导表达条件,为该几丁质酶及其基因的深入研究和开发利用奠定了基础。

Characterization and production optimization of a chitinase (Tachi1) 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.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 6.19 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.