The 46th Academic Congress of Taiwan Association for Food Science and Technology

Secretory expression of β-agarase form Paenibacillus agarexedens in Bacillus subtilis

HJ Lin, YS Chou, WC Huang, SL Hsuan, ZW Chen, JH Lin, JP Wang

Agarases are hydrolytic enzymes that degrade agar into oligosaccharides. According to the cleave pattern, agarases are classified into α-agarase (EC 3.2.1.158) and β-agarase (EC 3.2.1.81). Paenibacillus agarexedens, a bacterium isolated from meadow soil, was found to have agarase activity. The gene encoding a novel β-agarase has been cloned and expressed in Escherichia coli in our previous study. However, the purification of recombinant β-agarase from E. coli is time-consuming and complicated. The objective of this study is to express β-agarase extracellularly in Bacillus subtilis for developing simplified downstream processes of recombinant β-agarase. In addition, the optimized medium composition for the production of β-agarase in B. subtilis was also determined by using response surface methodology. The gene encoding β-agarase was inserted into a B. subtilis expression vector. Then, the resulting plasmid was transformed into B. subtilis. The results of SDS-PAGE and western blot showed that the β-agarase was successfully secreted by B. subtilis. The β-agarase activity of B. subtilis cultured with optimized medium is 55-fold higher than that of cultured with LB broth. Downstream processes for high recovery of β-agarase from supernatant of B. subtilis will be further determined.