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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED STRATEGIES FOR ADVANCE MANAGEMENT OF FRUIT, NUT, AND OAK TREE DISEASES

Location: Crops Pathology and Genetics Research

Title: Expression of Cellobiose Dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization

Authors
item Zhang, Ruifu -
item Zhiliang, Fan -
item Kasuga, Takao

Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 10, 2010
Publication Date: N/A

Interpretive Summary: Cellobiose dehydrogenases (CDH) play important roles in cellulose and lignin degradation. We cloned a gene encoding for CDH from a filamentous model fungus Neurospora crassa. We then expressed the gene in a yeast Pichia pastoris, purified the enzyme, and characterized several enzymatic properties.

Technical Abstract: 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 his6-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 His6-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 7,451 IU/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 Km value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. kcat remains relatively constant when the chain length changes.

Last Modified: 9/21/2014
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