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

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

摘要:

摘要:【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17.93U/mL，蛋白表达量为6.19g/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。

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．