| Summary: | 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.
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