The gene encoding a pre-endoxylanase of Bacillus sp. has been cloned and expressed in Escherichia coli and Bacillus subtilis. The pre-endoxylanase consisted of 28 amino acid-signal peptide and 213 amino acid endoxylanase. The signal peptide of 28-amino acid was processed both in E. coli and B. subtilis. The endoxylanase expressed in B. subtilis was identical to that expressed in E. coli. To increase the expression level of the endoxylanase in B. subtilis, a dimeric endoxylanase gene was constructed in a tandem unit using a gene amplification system. However, the dimeric endoxylanase gene cloned in rolling-cicle replicating plasmid spontaneously rearranged to a monomeric one in B. subtilis. To solve the plasmid instability in B. subtilis, the theta (Θ)-replicating plasmid was substituted for rolling-circle replicating one. The dimeric endoxylanase gene cloned in the theta (Θ)-replicating plasmid remained structurally stable in B. subtilis. Comparative analysis of endoxylanase expression revealed that the production of the endoxylanase in B. subtilis containing a dimeric endoxylanase gene was 50% greater than that of B. subtilis containing a monomeric one.
Pre-endoxylanase 유전자를 Escherichia coli와 Bacillus subtilis에서 발현 및 특성을 연구하였다. Pre-endoxylanase는 28개 amino acid의 signal peptide와 213개 amino acid의 endoxylanase로 구성되었다. E. coli와 B. subtilis에서 amino acid 28개의 signal peptide는 정확히 processing되었으며, B. subtilis에서 발현된 endoxylanase와 E. coli에서 발현된 endoxylanase는 특성이 같았다. B. subtilis에서 endoxylanase의 발현을 높이기 위해 dimeric endoxylanase gene을 construction 하였다. 그러나, rolling-cicle replicating plasmid에 cloning된 dimeric endoxylanase gene이 B. subtilis에서 monomeric endoxylanase gene으로 deletion 되었다. B. subtilis에서 이와 같은 plasmid instability를 해결하기 위해서 theta(Θ)-replicating plasmid로 plasmid를 대체하였다. 그 결과, theta(Θ)-replicating plasmid로 cloning된 dimeric endoxylanase gene은 B. subtilis에서 안정하게 유지되었다. Endoxylanase의 발현을 비교한 결과에 의하면, dimeric endoxylanase gene을 갖고 있는 B. subtilis monomer를 갖고 있는 B. subtilis 보다 약 50%의 증가를 나타내었다.
