Lipase catalyzes the hydrolysis of long chain fatty acid ester to glycerol and fatty acids. The lipase gene was transferred from an unidentified bacterial species to Escherichia coli JM83 by a molecular cloning using pUC19 as a vector.
The lipase from an unidentified bacterial species was characterized. It was maximally active at pH 8 to 9, and its optimum temperature was 40°C.
The chromosomal DNA was isolated from an unidentified bacterial species and partially digested with Sau3AI. The pUC19 vector was digested with BamHI and dephosphorylated with Calf intestinal alkaline phosphatase. The digested chromosomal DNA fragments and linearized pUC19 vector were ligated, and the resulting recombinant plasmids were transformed to E. coli JM83. The first screening was carried out on the agar plate containing tributyrin to esterase or lipase activities. The second screening was performed on the agar plate containing olive oil and rhodamine-B to select the lipase activity. The recombinant plasmid isolated from the clone exhibiting lipase activity was designated as pUL9. pUL9 was linearized and digested with exonuclease Bal31 to reduce the size of the insert DNA and self ligated. The resulting recombinant plasmid was designated as pUL920. The nucleotide sequence of pUL920 was analyzed.
리파아제는 triglyceride 의 긴 사슬을 가수분해하여 glycerol, 지방산, partial glyceride 를 생성하는 효소로, 본 실험에서는 산탄식 방법으로 미확인 균주로부터 E. coli JM83으로 리파아제 유전자를 클로닝하였다. 이 균주의 리파아제가 활성을 나타내는 최적 pH 는 8.0 - 9.0 이고 최적온도는 섭씨 40도였다.
리파아제 유전자의 클로닝에 있어서 두가지 스크리닝 방법이 이용되었고 이 방법들에서 활성을 나타내는 균주는 하나였다. 이 유전자는 pUL9 으로 명명되었고 그것의 크기는 1.6Kb 였다. 이들을 Bal31 으로 처리하여 pUL19에 서브클로닝 하였다. 이 유전자는 pUL920으로 명명되었고 pUL9을 갖는 균주보다 더 큰 활성을 나타내었다.
이 유전자 내에는 하나의 Xhol, 두개의 Sau3Al, 그리고 두개의 HaeIII 부위를 갖고 있다. 따라서 이들을 염기서열 결정에 이용하여 부분적으로 염기서열을 분석하였다.
