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Enhancement of PHB depolymerase Activity from Alcaligenes faecalis T1 by DNA Shuffling  

신동성 (충남대학교 자연과학대학 미생물학과)
이영하 (충남대학교 자연과학대학 미생물학과)
남진식 (충남대학교 자연과학대학 미생물학과)
Publication Information
Korean Journal of Microbiology / v.39, no.2, 2003 , pp. 76-82 More about this Journal
Abstract
To prepare evolved PHB depolymerase with increased activity for PHB or P(3HB-co-3HV) compared to the activity of the original PHB depolymerase from Alcaligenes faecalis T1, random mutation of the cloned PHB depolymerase gene was performed by using a DNA shuffling method. A library of mutated PHB depolymerase genes from A. faecalis T1 was fused to the ice nucleation protein (INP) gene from Pseudomonas syringae in pJHCl 1 and approximately 7,000 transformants were isolated. Using M9 minimal medium containing PHB or P(3HB-co-3HV) as the carbon source, mutants showing alteration in PHB depolymerase activity were selected from the transformants. The PHB depolymease activity of the transformants was confirmed by the formation of halo around colony and the turbidity decrease tests using culture supermatants. The catalytic activity of PHB depolymerase of the best mutant II-4 for PHB or P(3HB-co-13 mol% 3HV) was approximately 1.8-fold and 3.2-fold, respectively, higher than that of the original PHB depolymerase. DNA sequence analysis revealed that three amino acid residues (Ala209Val, Leu258Phe, and Asp263Thr) were substituted in II-4. From the mutational analysis, it was presumed that the substitution of amino acids near catalytic triad to more hydrophobic amino acids enhance the catalytic activity of PHB depolymerase from A. faecalis T1.
Keywords
Alcaligenes faecalis T1; DNA shuffling; PHB depolymerase; poly(3-hydroxyalkanoate);
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