Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens
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...
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ndltd-TW-082NCKU01070012015-10-13T15:33:33Z http://ndltd.ncl.edu.tw/handle/88171068460611357883 Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens Acinetobacterradioresistens耐鹼性脂質■基因之選殖及其蛋白質之表現 Ming-Chyuan Hong 洪明全 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. Ming-Chung Chang 張敏政 1994 學位論文 ; thesis 78 zh-TW |
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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.
|
author2 |
Ming-Chung Chang |
author_facet |
Ming-Chung Chang Ming-Chyuan Hong 洪明全 |
author |
Ming-Chyuan Hong 洪明全 |
spellingShingle |
Ming-Chyuan Hong 洪明全 Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens |
author_sort |
Ming-Chyuan Hong |
title |
Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens |
title_short |
Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens |
title_full |
Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens |
title_fullStr |
Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens |
title_full_unstemmed |
Molecular Analysis and Expression of the Alkaline Lipase Gene of Acinetobacter radioresistens |
title_sort |
molecular analysis and expression of the alkaline lipase gene of acinetobacter radioresistens |
publishDate |
1994 |
url |
http://ndltd.ncl.edu.tw/handle/88171068460611357883 |
work_keys_str_mv |
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