Cloning and Expression of Chitin Deacetylase from Rhizopus oryzae

• Main Authors: LIN, CHING-YI, 林靜宜
• Other Authors: CHUNG, YUN-CHIN
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/x96x87

碩士 === 靜宜大學 === 食品營養學系 === 105 === Chitin deacetylase (CDA) is an enzyme, which catalyses the hydrolysis of N-acetamido group of N-acetyl-D-glucosamine (GlcNAc) residues in chitin and converts chitin to chitosan. Chitosan shows many bioactivities, such as antibacterial, anti-tumor, immunomodulatory and inhibition of lipid absorption. In this study, chitin deacetylase DNA was ligated with a strong promoter in an expression vector to increase the activity and yield of the gene prodouct. Total RNA was extracted from mycelia of Rhizopus oryzae and the cDNA was prepared by reverse transcription. The target DNA was amplified by polymerase chain reaction and ligated with pGAPZαB expression vector. The recombinant expression plasmid was transformed into Pichia pastoris by electroporation. The transformants were selected by a specific medium, and the biochemical properties of the recombinant CDA were evaluated. DNA sequence of the obtained cda showed high homology with that of chitin deacetylase from Rhizopus circinans according to National Center for Biotechnology Information (NCBI). These two CDAs showed 3 bp different in their DNA sequences, but their amino acid sequences were identical. SDS-PAGE and western blot showed that recombinant protein was expressed as intracellular protein with molecular weight about 100 kDa, which was much greater than it was expected as 62.02 kDa. After digested by glycosidase, molecular weight of the recombinant CDA was decreased to about 70 kDa, which matched the molecular weight of combining CDA with the signal peptide of pGAPZαB.