Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens

• Main Authors: Ming-Chyuan Hong, 洪明全
• Other Authors: Ming-Chung Chang
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/88171068460611357883

碩士 === 國立成功大學 === 生物化學研究所 === 82 === A gram-negative bacterium, Acinetobacter radioresistens, possesses lipase activity even under high alkaline conditions (pH11), has been isolated and identified in our laboratory. Be- cause alkaline lipase is useful for industrial practices, in- creasing its yield in industrial production is desirable. To achieve this goal, we started to clone the alkaline lipase gene from A. radioresistens and to analyze the genetic information by means of molecular biology. An A. radioresistens genomic library was constructed in Escherichia coli. Thirteen colonies exhibited lipase activity on the tributyrin-ampicillin agar plate were iso- lated from the genomic library. One of the postive clones, desig- nated "pTB2" contained 4.7 kb DNA fragment of A. radioresistens, showed the strongest alkaine lipase activity. This 4.7 kb DNA fragment was subcloned into pUC19 and pBCSK(+/-). The results of subcloning of various restriction fragments indicated that the alkaline lipase gene was located on the 1.6 kb SmaI-SphI region. Using exonuclease III unidirectional deletion method, twelve pro- gressive deletion clones were isolated, the nucleotide sequence of the deletion clones were determined in both orientation. An open reading frame of 1260 nucleotides which encoded a protein of 420 amino acid residues with a predicted molecular weight of 45 kD was identified. The deduced amino acid sequence contains a se- quence, Val-Val-Gly-His-Ser-Gln-Gly-Gly-His, which matches exclu- sively to the consensus sequences of lipase, Gly-X1-Ser- X2-Gly. The result of SDS-polyacylamide gel electrophoresis analysis re -vealed that the molecular weight of the alkaline lipase was approximately 45 kD, which were corresponding with the size derived from the nucleotide sequence and the minicell analysis.