Abstract

The 7.1 Mdal Xbaf fragment of Bacillus pasteurii ATCC 11859 containing gene for urease was inserted into the Xbal site of bifunctional plasmid pGR71, and its urease gene was cloned and expressed in E. coil RRI. But the cloned gene was not expressed in Bacillus subtilis BR151 in consequence of deletion of inserted DNA fragment. The recombinant plasmid thus formed was named pGU66. The restriction map of the plasmid pGU66 was determined, and the size of the plasmid was estimated to be 12.6 Mdal by double digestion of restriction enzymes of the plasmid. The urease of the cloned strain was accumulated in periplasmic space and very similiar to that of donor strains in their enzymatic properties.

미생물중 urease생성능이 아주 강한 B. pasteurii의 Hind III partial digest 된 chromosomal DNA를 E. coli-B. subtilis bifunctional plasmid vector pGR 71으로 E. coli RR1 균주에 cloning 하므로써 그 urease gene을 expression시킬 수 있었다. 그러나 B. subtilis에서는 insertion DNA fragment의 deletion으로 expression되지 않았다. Cloning된 E.coli RR1 균주로부터 분리 정제한 urease gene함유 Plasmid(pGU66)의 restriction map을 작성하여 본 결과 7.1 Mdal의 insertion fragment가 삽입된 12.6Mdal의 plasmid에 Hind III, Bgl II, Xba I, Sal I등 몇 개의 cleavage site 위치를 찾을 수 있었다. Cloning된 E. coli의 urease는 periplasmic space에 많은 비율로 축적되며, 그 효소학적 성질은 donor인 B.pasteurii의 그것과 매우 유사하였다.

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