| Summary: | 博士 === 國立成功大學 === 生命科學系碩博士班 === 97 === The blue fluorescent protein gene (bfpvv) and extracellular lipase gene (lipA) with its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned, sequenced, and characterized respectively.
1. The blue fluorescent protein (BFPVV) gene, bfpvv, from Vibrio vulnificus CKM-1 was cloned and sequenced. Expression of the bfpvv gene in Escherichia coli XL1B could be visualized through the illumination with UV source. Nucleotide sequence analysis predicted a single open reading frame of 717 bp encoding a 239-amino-acid polypeptide with a calculated molecular mass of 25.8 kDa. The BFPVV protein has been produced in E. coli XL1B or E. coli BL21. The nucleotide sequence of bfpvv gene and its deduced amino acid sequence showed significant homology to those of the short-chain dehydrogenase / reductase (SDR) family proteins from various organisms. Some functionally important residues in SDR were strictly conserved in BFPVV, such as an active site-Tyr145, a catalytic site-Lys149 and a common GlyXXXGlyXGly pattern in the N-terminal part of the molecule. By changing three amino acid residues, Tyr145, Lys149, and Gly9 to Phe, Ile, and Val, respectively, it was found that G9V mutant did not generate blue fluorescence, while the mutants Y145F and K149I have 126% and 68.5% fluorescence compared with the wild-type BFPVV.
2. The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced. Nucleotide sequence analysis and alignments of amino acid sequences suggest that LipA is a member of family I.1 lipase and that LipB is a lipase activator of LipA. The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB. In the hydrolysis of p-nitrophenyl esters using the reactivated LipA, optimum chain lengths for the acyl moiety on the substrate were C12 to C14.
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