期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 75, 期 1, 页码 63-69出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2010.08.003
关键词
Cellobiose dehydrogenase; Neurospora crassa; Pichia pastoris; Heterologous expression; Characterization
类别
资金
- University of California Davis
- California Energy Commission
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 7451 IU/L (0 89 mg protein/mL) with a specific CDH activity of 837 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 degrees 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 (C) 2010 Elsevier Inc All rights reserved
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