Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad 38040, Pakistan.
Mol Biol Rep. 2011 Jun;38(5):3227-33. doi: 10.1007/s11033-010-9996-2. Epub 2010 Mar 9.
We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.
我们研究了嗜热毛壳菌木聚糖酶 11A 基因在巴斯德毕赤酵母中的异源表达，并对纯化基因产物的热稳定性进行了表征。为此，我们在巴斯德毕赤酵母 GS115 中通过 AOX1 启动子控制下克隆了嗜热毛壳菌的木聚糖酶 11A 基因。经过 96 小时的生长，带有内含子的重组木聚糖酶（xyn698：带有内含子的基因）的最大胞外活性为 15.6 U/ml，而没有内含子的重组木聚糖酶（xyn669）的最大胞外活性为 1.26 U/ml。基因产物明显被纯化到均相水平。纯重组木聚糖酶活性的最适温度为 70°C，酶在 80°C 孵育 10 分钟后保留其 40.57%的活性。它在底物水解过程中表现出较低的活化能、焓、吉布斯自由能、熵和木聚糖结合能的需求，这表明它具有热稳定性。pH 依赖性催化表明，它在 pH 5.5-8.5 的范围内相当稳定。这表明该基因在巴斯德毕赤酵母中成功进行了加工，并且保持了热稳定性，可能有资格将其潜在用于造纸和纸浆以及动物饲料应用。
