Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
Appl Biochem Biotechnol. 2014 Feb;172(4):2253-65. doi: 10.1007/s12010-013-0672-6. Epub 2013 Dec 19.
Bioconversion of cellulosic material into glucose needs cellulase enzymes. One of the most important organisms that produces cellulases is Trichoderma reesei, whose cellulose enzymes are probably the most widely used in the industry. However, these enzymes are not stable enough at high pH and temperatures. The optimized synthetic endoglucanase II gene with Pichia pastoris codon preferences was secretary expressed in P. pastoris. Recombinant enzyme characterization showed maximum activity at pH 4.8 and temperature 75 °C, and it demonstrated increasing thermal stability in high temperature. The enzyme maintained its activity in a wide pH range from 3.5 to 6.5. The optimization of fermentation medium was carried out in shaking flasks. Recombinant protein expression at optimum conditions (pH 7, temperature 25 °C, and 1 % methanol induction) for 72 h demonstrated 2,358.8 U/ml endoglucanase activity units. To our knowledge, this is the highest acidic thermophilic endoglucanase activity that is reported in crude intracellular medium in P. pastoris. We conclude that P. pastoris is an appropriate host for high-level expression of optimized endoglucanase gene with improved thermal stability.
纤维素物质转化为葡萄糖需要纤维素酶。产生纤维素酶的最重要的生物之一是里氏木霉，其纤维素酶可能是工业中应用最广泛的。然而，这些酶在高 pH 值和温度下不够稳定。经过优化的具有毕赤酵母密码子偏好的合成内切葡聚糖酶 II 基因在毕赤酵母中进行了分泌表达。重组酶的特性表明，其在 pH4.8 和 75°C 下具有最大活性，并且在高温下显示出增强的热稳定性。该酶在 pH3.5 到 6.5 的宽范围内保持活性。在摇瓶中对发酵培养基进行了优化。在最佳条件（pH7、温度 25°C 和 1%甲醇诱导）下进行 72 小时的重组蛋白表达，显示内切葡聚糖酶活性单位为 2358.8 U/ml。据我们所知，这是毕赤酵母粗细胞内培养基中报道的最高酸性嗜热内切葡聚糖酶活性。我们得出结论，毕赤酵母是表达优化后具有提高热稳定性的内切葡聚糖酶基因的合适宿主。
