Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization.
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.
Zhang
Ruifu
R
Biological and Agricultural Engineering Department, One Shields Avenue, Davis, CA 95616, USA.
Fan
Zhiliang
Z
Kasuga
Takao
T
D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004795:Enzyme Stability; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase