Expression of raw starch-digesting amylase gene from Cytophaga sp. in Escherichia coli

• Main Authors: Chen, Li - Shien, 陳立賢
• Other Authors: Chii-Ling Jeang
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/25512646311538035108

碩士 === 國立中興大學 === 食品科學系 === 86 === AbstractThe importance of the enzyme saccharification of raw starch has been well recognized from the viewpoints of energy saving and effective biomass utilization. Previously, we isolated a bacterium - Cytophaga sp. from soil, that had a great potential for production of raw starch digesting amylase ( RSDA ). In this study, the rsda gene of Cytophaga sp. was cloned in phage particles using Lambda ZAP II. A kb DNA fragment of Cytophaga sp. chromsomal DNA digested with EcoR I was hybridized with the DIG-labeled PCR probe derived from the N- and an internal N- terminal amino acid sequence of RSDA. Through a series of subcloning for locating the rsda-coding region, the DNA insert was reduced to 3.2 kb and was ligated with pAcUW21. We designated the recombinant plasmid pARMH10. The expressed RSDA with a molecular weight of approximately 60 kDa, had the property to hydrolyze raw starch as does the RSDA purified from Cytophaga sp.. The sequence of 3.2 kb inserted DNA from subclone was obtained upon ABI BigDye Terminator Cycle Sequencing. The rsda gene consists of 1,455 bp encoding a protein of 485 amino acid residues, which have a characteristic signal peptide of 19 amino acid residues upstream of its N- terminus. A typical promoter sequence and Shine-Dalgarno ( S. D. ) region were located upstream of the initiation codon ATG.