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Title: Isolation, cloning and expression of NAD+ glycohydrolase from neurospora crassa
Authors: TAN KER SIN
Keywords: NAD+ glycohydrolase, neurospora crassa, pichia pastoris
Issue Date: 17-Mar-2008
Citation: TAN KER SIN (2008-03-17). Isolation, cloning and expression of NAD+ glycohydrolase from neurospora crassa. ScholarBank@NUS Repository.
Abstract: NAD+ glycohydrolase (NADase) from Neurospora crassa is a glycoprotein that catalyzes the hydrolysis of NAD+ to ADP-ribose and nicotinamide. It is used as one of the reagents in the cycling assay which functions to remove endogenous NAD+. Conidia were found to have higher NADase activities than mycelia. Conidial NADase is different from mycelial NADase in terms of their optimum pH, Km and carbohydrate moiety. Conidial NADase has a Km of 280 uM while the Km of mycelial NADase is 500 uM. Optimum pH for conidial NADase is pH 7. The mycelia NADase is active over a wide range of pH. N-linked deglycosylation reduced the size of the protein from 42 kDa to 32 kDa which suggested that the carbohydrate contributes 20% of the molecular mass. The native form of the protein is predominantly a dimer of 75 kDa without interdisulfide bond. Conidial NADase was purified using affinity columns, either cibacron blue 3GA agarose or blue sepharose CL-6B. The sequence of NADase was revealed and identified by mass spectrometry analysis. The DNA sequence was cloned into intracellularly expression vector, pPICZB and secretion expression vector, pPICZalphaA. The recombinant protein was expressed in the methylotropic yeast, Pichia pastoris. The extracellularly expressed protein has higher molecular weight than intracellularly expressed protein due to glycosylation. The native recombinant protein is a dimer or trimer bonded together by interdisulfide bond. The enzyme activity was confirmed by in-gel substrate staining and fluorimetric NADase assay. The recombinant proteins were applied in the cycling assay for NAD+. It has been shown that the recombinant proteins are effective in removing NAD+.
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