• 한국미생물·생명공학회지
• /
• 제32권4호
• /
• pp.322-327
• /
• 2004

구성적 promoter인 $P_{JH}$ promoter 하류에 연결된 Bacillus sp. 유래의 endoxylanase를 B. subtilis에서 과발현 하였다. 발현된 plasmid, pJHKJ4는 자체 promoter($P_{B}$)와 Bacillus의 강력한 promoter($P_{JH}$)가 tandem으로 위치하여 transcription되었다. B.subtilis DB431에 형질전환된 균주를 glucose가 함유된 LB 배지에서 배양 후 총활성 및 분비효율은 140 U/ml 으로 나타났다. 배양상등액은 ultrafiltration(MW cut off 10 kDa and 30 kDa)과 염석법으로 농축하였다. 효소의 농축에서 ultrafiltration이 70% ammonium sulfate precipitation 보다 효과적이었다. $50^{\circ}C$ 에서 농축된 효소액의 안정화는 NaCl, glycerol, sorbitol, and $CaCl_{2}$ 등의 다양한 안정제를 농도별로 첨가하여 안정제의 효과를 검토한 결과 가장 효과적인 안정제는 NaCl과 $CaCl_{2}$ 이었다.

• 생명과학회지
• /
• 제10권2호
• /
• pp.125-130
• /
• 2000

To overproduce endoxylanase from a recombinant Bacillus subtilis harboring the pJHKJ4 plasmid, the effects of carbon and nitrogen sources on the cell growth and expression level of endoxylanase were investigated in the flask cultures. Among the various carbon and nitrogen sources tested, glucose and maltose as carbon source and yeast extract as nitrogen source were found to be the most effective for the cell growth and the endoxylanase expression. When the concentration of glucose was increased from 0.5% to 5%, the highest activity of extracellular endoxylanse, 166 unit/$m\ell$, was observed at 2% glucose. In case of maltose, the endoxylanase was stably produced at the level of 180 unit/$m\ell$, regardless of the concentration of maltose. The higher the concentration of yeast extract, the greater cell growth and endoxylanase expression were obtained. However, the highest endoxylanase activity per unit cell mass was observed with 1% yeast extract. With the optimized medium (2% glucose, 1% yeast extract, etc), about 630 unit/$m\ell$ of endoxylanse was expressed through the batch fermentation in a fermentor, which expression level corresponded to about 0.7 g-endoxylanase protein /$\ell$. It was also found that the plasmid was stably maintained above 70% level, and more than 90% of endoxylanase activity was detected in the extracellular medium.