Process Optimization for Concentration and Stabilization of Recombinant Endoxylanase Expressed in Bacillus subtilis

Bacillus subtilis에서 발현된 재조합 Endoxylanase 농축과 안정화 공정의 최적화

A strong constitutive PJH promoter from Bacillus sp. was applied to overexpress the endoxylanase gene (639 bp) in Bacillus subtilis. The expression plasmid, pJHKJ4, was designed to contain the $P_{JH}$ promoter and open reading frame of endoxylanase including its own promoter. The plasmid was introduced into B. subtilis DB431 and the resulting transformant was grown on LB glucose medium. At the end of cultivation, the endoxylanase activity in the culture supernatant reached about 140 DIm!. The enzyme in the supernatant was concentrated by ultrafiltration (MW cut-off 10 kDa and 30 kDa) and ammonium sulfate precipitation. For the concentration of the enzyme, ultrafiltration was more efficient than 70% ammonium sulfate precipitation. The stabilization of concentrated enzyme solution at $50^{\circ}C$ was examined with various stabilizers such as NaCI, glycerol, polyethylene glycol, sorbitol, and $CaCI_2$. The most effective stabilizers were found to be NaCI and $CaCI_2$.

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.