| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The Pharenochaete chrysosporium cdh gene (U46081.1) for mature enzyme was synthesized and cloned in pUC57 plasmid by GenScript, Piscataway, NJ, USA. The PCR reaction (94 °C for 4 min, 1 cycle; 94 °C 1 min/50 °C 1 min/72 °C for 2.45 min, 30 cycles; 72 °C for 10 min, 1 cycle) contained Pfu DNA polymerase (0.04 u/µL), dNTP mix (0.2 mM), forward primer (0.5 µM)(5′-ATGCTAGCCAGAGTGCCTCACAGTTTACC-3′), reverse primer (0.5 µM) (5′- ATGGATCCTCAAGGACCTCCCGCAAG-3′), template CDH-pUC57 (30 ng). The purified fragments were restriction digested with NheI and BamHI enzyme and ligated in the pCTCON2 vector. The ligated gene was used to transform Echerichia coli DH5α. Gene length was checked with DNA electrophoresis and EBY100 yeast cells were transformed with CDH-pCTCON2 construct. | Get A Quote |
Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the a... More
