Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China.
Enzyme Microb Technol. 2011 Jul 10;49(2):223-8. doi: 10.1016/j.enzmictec.2011.04.005. Epub 2011 Apr 15.
The glycoside hydrolase β-1,3-glucomannanase is an enzyme that specifically breaks the β-1,3 glycosidic bond of the glucomannan, the main cell wall constituent of some yeasts. In this work, a codon optimized DNA sequence of the MAN5C gene from Penicillium lilacinum ATCC 36010 was expressed in the yeast Pichia pastoris under the control of AOX1 promoter. The recombinant protein plMAN5C was purified from the shake flask culture and the stirred-tank bioreactor culture in yields of 30.0mg/l and 224.0mg/l, respectively. The purified protein had a specific activity of 14.6 U/mg at 37 °C, pH 4.5. Biochemical analysis showed that the optimal temperature and pH for plMAN5C were 50 °C and 4.5, respectively. The recombinant plMAN5C was efficient in lysis of the cell wall of the red yeast Rhodosporidium toruloides to form protoplast. Our work provided an effective system for heterogeneous production of β-1,3-glucomannanase, which should facilitate a more convenient application of this enzyme in biotechnology and other related areas.
β-1,3-葡聚糖酶是一种糖苷水解酶，能够特异性地切断葡聚糖的β-1,3 糖苷键，葡聚糖是某些酵母细胞壁的主要成分。在这项工作中，我们通过优化密码子，在毕赤酵母中表达了里氏木霉 ATCC 36010 来源的 MAN5C 基因，该基因受 AOX1 启动子的调控。重组蛋白 plMAN5C 可从摇瓶培养和搅拌罐生物反应器培养中分别以 30.0mg/L 和 224.0mg/L 的产量进行纯化。该纯化蛋白在 37°C、pH4.5 时的比活为 14.6U/mg。生化分析表明，plMAN5C 的最适温度和 pH 值分别为 50°C 和 4.5。重组 plMAN5C 能有效裂解红色酵母罗伦隐球酵母的细胞壁，形成原生质体。我们的工作为β-1,3-葡聚糖酶的异源生产提供了一个有效的系统，这将有助于该酶在生物技术和其他相关领域更方便地应用。
