Zhang Ruifu, Fan Zhiliang, Kasuga Takao
Biological and Agricultural Engineering Department, One Shields Avenue, Davis, CA 95616, USA.
Protein Expr Purif. 2011 Jan;75(1):63-9. doi: 10.1016/j.pep.2010.08.003. Epub 2010 Aug 13.
A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a His(6)-tag (rNC-CDH1) was successfully expressed and secreted. rNC-CDH1 was produced at the level of 652 IU/L after 2 days of cultivation in the induction medium. The His(6)-tagged rNC-CDH1 was purified through a one-step Ni-NTA affinity column under non-denaturing conditions. The purified rNC-CDH1 has a CDH activity of 745 1IU/L (0.89 mg protein/mL), with a specific CDH activity of 8.37 IU/mg. The purity of the enzyme was examined by SDS-PAGE, and a single band corresponding to a molecular weight of about 120 kDa was observed. Activity staining confirmed the CDH activity of the protein band. The purified rNC-CDH1 has maximum CDH activity at pH 4.5, and a rather broad temperature optimum of 25-70°C. Kinetic analysis showed cellobiose and cellooligosaccharides are the best substrates for rNC-CDH1. The K(m) value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. k(cat) remains relatively constant when the chain length changes.
已克隆来自粗糙脉孢菌FGSC 2489菌株的编码纤维二糖脱氢酶（CDH）的基因，并在AOX1甲醇诱导型启动子的控制下在异源宿主毕赤酵母中表达。成功表达并分泌了没有天然信号序列且与His(6)-标签融合的重组CDH（rNC-CDH1）。在诱导培养基中培养2天后，rNC-CDH1的产量为652 IU/L。His(6)-标签化的rNC-CDH1在非变性条件下通过一步Ni-NTA亲和柱纯化。纯化后的rNC-CDH1的CDH活性为745 IU/L（0.89 mg蛋白质/mL），比CDH活性为8.37 IU/mg。通过SDS-PAGE检测酶的纯度，观察到一条对应分子量约为120 kDa的单一条带。活性染色证实了蛋白条带的CDH活性。纯化后的rNC-CDH1在pH 4.5时具有最大CDH活性，最适温度范围较宽，为25-70°C。动力学分析表明，纤维二糖和低聚纤维糖是rNC-CDH1的最佳底物。rNC-CDH1对低聚纤维糖的K(m)值随着葡萄糖基单元的延长而增加。当链长变化时，k(cat)保持相对恒定。
