Expression of raw starch-digesting amylase gene feom Cytophaga sp. in Bacillus subtilis

• Main Authors: Hui-Chen Hung, 洪慧貞
• Other Authors: Chii-Ling Jeang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/45426318384522609351

博士 === 國立中興大學 === 食品科學系 === 88 === In our previous study, the raw-starch digesting amylase gene from a soil bacterium, Cytophaga sp., was selected and constructed in plasmid, pAcUW21, and then transformed into the host cells, Escherichia coli. The recombinant plasmid was designated as pARMH10. E. coli harboring the pARMH10 expressed the gene product, raw-starch digesting amylase (RSDA) intracellular. For simplifying the followed-up purification procedures, we subclone the rsda gene into Bacillus subtilis which often secret the foreign proteins. There were two shuttle recombinant plasmids constructed in this work. A DNA fragment of 3.2 kb in length was deleted from pARMH10. The shortened product was ligated with pWP18, a plasmid containing replication region of B. subtilis. The reconstructed plasmid was designated as pAWR, and transformed into B. subtilis. Besides, a PCR product of 2.7 kb including the rsda structural gene and 5’-upstream region obtained by using pARMH10 plasmid as template was ligated with pHY300PLK, a shuttle vector. The recombinant plasmid was designated as pHR and transformed into B. subtilis. The transformants, B. subtilis (pAWR) and B. subtilis (pHR), produce RSDA extra-cellular. Primer extension experiments indicated that the transcription start site lies 34 bp upstream of the ATG start codon, and allowed the identification of —35 (TTGACT) and —10 (TATAGT) promoter-like sequences. Higher amount of RSDA was produced by B. subtilis carrying pAWR grown on 2YT medium than B. subtilis carrying pHR. Fourty-eight and ninety-five percentage of recombinant plasmids was retained as B. subtilis (pAWR) and B. subtilis (pHR), respectively, grown on 2YT medium for 60 hr without adding tetracycline. B. subtilis RSDA was purified through three steps, the purification folds was about 54 and activity recovery was 33% . The N-termnial amino acid sequence of B. subtilis RSDA determinated was AATNGTMMQYFE WYVPN, which is consistent with that of Cytophaga sp. The enzyme biochemical properties produced from different hosts shows that RSDA produced by Cytophaga sp. and B. subtilis are the same ,however, E.coli (pAWR) are different . From above results, we proposed the system could be employed in the production of large amount of enzyme for starch industrial processed application.