Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization |
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| Authors: | Ruifu Zhang Zhiliang Fan Takao Kasuga |
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| Institution: | 1. Department of Chemistry, University of Rochester, 14611 Rochester, NY, USA;2. CMC Diabetes Formulation Development, Novo Nordisk, Novo Alle Bagsvaerd, 2880, Denmark;3. Protein Engineering, Novozymes A/S, Bagsvaerd 2880, Denmark;4. Department of Organic Chemistry, Center for Physical Sciences and Technology, Sauletekio av. 3, LT-10257 Vilnius, Lithuania;5. Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, 20506 Malmö, Sweden;1. Department of Chemistry, Institute for Advanced Studies in Basic Sciences, P.O. Box 45195-1159, Gava Zang, Zanjan, Iran;2. Department of Analytical Chemistry/Biochemistry and Structural Biology, Lund University, P.O. Box 124, 22100 Lund, Sweden;3. Food Biotechnology Laboratory, Department of Food Sciences and Technology, BOKU – University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria;4. Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71454, Iran |
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| 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 7451 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. |
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