The exo-β-1, 4-glucanase gene (exoglucanase gene) previously cloned from Clostridium thermocellum in our laboratory was tried to transfer from the Escherichia coli host cell to Bacillus subtilis cell to produce the enzyme extracellularly. The unnecessary parts of both N-terminal and C-terminal regions of the exoglucanase gene contained in pCE64 were removed to increase expression efficiency. The constructed plasmid was designated as pCX33 in which a 3.3 kilobase fragment containing the exoglucanase gene fused to the lacZ gene is contained. After confirming expression of the exoglucanase gene in E. coli, the exoglucanase gene in pCX33 was retransferred to a Bacillus expression vector pJH31 to make a new plasmid pJX33. Bacillus subtilis transformed with pJX33 was grown in LB medium. The Bacillus subtilis transformant produced 719 mU/ml of exoglucanase which was completely secreted into the culture medium.
Cloning되어있는 Clostridium thermocellum의 exo-β-1, 4-glucanase 유전자를, 그 효소가 세포 밖으로 생성되어 나오도록 숙주인 Eschrichia coli 에서 Bacllus subtilis로 옮기려고 시도했다.
발현 효율을 높이기 위하여 pCE64에 들어있는 exoglucanase 유전자의 불필요한 N-말단과 C-말단 부위를 제거했다. 재조합된 plasmid인 pCX33은 lacZ 와 fusion된 exoglucanase 유전자를 포함하는 3.3kb의 DNA 조각을 갖는다. E.coli에서 exoglucanase 유전자의 발현을 확인한 후, pCX33의 exoglucanase를 Bacillus expressin vector인 pJH31로 다시 옮겨서 새로운 plasmid인 pJX33을 만들었다. pJX33으로 형질전환된 Bacillus subtilis를 LB 배지에서 키웠다. 이 Bacillus transformant는 exoglucanase를 완전히 배지로 분비했고, 이때 생성된 효소의 활성도는 719mU/ml(culture broth)였다.