Browse > Article

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);
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 Klingbeil, B., R.M. Kroppenstedt, and D. Jendrossek. 1996. Taxonomic identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. FEMS Microbiol. Lett. 142, 215-221   DOI
2 An effective family shuffling method using single-stranded DNA /
[ Kikuchi,M.;K.Ohnishi;S.Harayama ] / Gene   DOI
3 Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates /
[ Anderson,A.J.;E.A.Dawes ] / Microbiol. Rev.
4 In vivo evolution of the Aeromonas punctata polyhydroxyalkanoate(PHA) synthase: isolation and characterization of modified PHA synthases with enhanced activity /
[ Amara,A.A.;A.Steinbuchel;B.H.A.Rehm ] / Appl. Microbiol. Biotechnol.   DOI   ScienceOn
5 Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1 /
[ Nojiri,M.;T.Saito ] / J. Bacteriol   DOI
6 Display of poly(3-hydroxybutyrate) depolymerase on the surface of Escherichia coli /
[ Park,S.H. ] / MS thesis, Chungnam National University
7 DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution /
[ Stemmer,W.P. ] / Proc. Natl. Acads Sci.   DOI   ScienceOn
8 /
[ Sambrook,J.;E.F.Fritsch;T.Maniatis ] / Molecular cloning: a laboratory manual(2nd ed.)
9 Biosynthesis of polyhydroxyalkanoate copolyester containing cyclohexyl groups by Pseudomonas oleovorans /
[ Kim,D.Y.;S.B.Jung;G.G.Choi;Y.B.Kim;Y.H.Rhee ] / Int. J. Biol. Macromol.   DOI   ScienceOn
10 Hydrolysis of native poly(hydroxybutyrate) granules(PHB), crystalline PHB, and artificial amorphous PHB tranules by intracellular and extracellular depolymerases /
[ Merrick,J.M.;R.Steger;D.Dombroski ] / Int. J. Biol. Macromol.   DOI
11 Production of PHA depolymerase A from Paucimonas lemoignei in Bacillus subtilis /
[ Braaz,R.;S.L.Wong;D.Jendrossek ] / FEMS Microbiol. Lett.   DOI   ScienceOn
12 Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein /
[ Jung,H.C.;J.H.Park;S.H.Park;J.M.Lebeault;J.G.Pan ] / Enzyme Microb. Technol.   DOI   ScienceOn
13 Structure and enzymatic degradation of poly(3-hydroxybutyrate) copolymer single crystals with an extracellular PHB depolymerase from Alcaligenes faecalis T1 /
[ Iwata,T.;Y.Doi;S.Nakayama;H.Sasatsuki;S.Teramachi ] / Int. J. Biol. Macromol.   DOI   ScienceOn
14 Molecular characterization of the poly(3-hydroxybutyrate)(PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model /
[ Rehm,B.H.;R.V.Antonio;P.Spiekermann;A.A.Amara;A.Steinbuchel ] / Biochim. Biophys. Acta.   DOI   ScienceOn
15 Taxonomic identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerases gene /
[ Klingbeil,B.;R.M.Kroppenstedt;D.Jendrossek ] / FEMS Microbiol. Lett.   DOI
16 Analysis of mutational effects of a polyhydroxybutyrate(PHB) polymerase on bacterial PHB accumulation using an in vivo assay system /
[ Taguchi,S.;A.Maehara;K.Takase;M.Nakahara;H.Nakamura;Y.Doi ] / FEMS Microbiol. Lett.   DOI   ScienceOn
17 Determination of the active sites serine of the poly(3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei(PhaZ5) and of Alcaligenes faecalis /
[ Shinohe,T.;M.Nojiri;T.Saito;T.Stanislawski;D.Jendrossek ] / FEMS Microbiol. Lett.   DOI   ScienceOn
18 Characterization of an extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas alcaligenes LB-19 /
[ Kim,D.Y.;J.S.Nam;Y.H.Rhee ] / Biomacromol.   DOI   ScienceOn
19 Effect of levulinic acid on the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstonia eutropha KHB-8862 /
[ Chung,S.H.;G.G.Choi;H.W.Kim;Y.H.Rhee ] / J. Microbiol.   과학기술학회마을
20 Enhancement of the thermostability and hydrolytic activity of xylanase by random gene shuffling /
[ Shibuya,H.;S.Kaneko;K.Hayashi ] / J. Biochem.   DOI   ScienceOn
21 Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes /
[ Suenaga,H.;M.Mitsuoka;Y.Ura;T.Watanabe;K.Furukawa ] / J. Bacteriol.   DOI   ScienceOn
22 Biodegradation of microbial and synthetic polyesters by fungi /
[ Kim,D.Y.;Y.H.Rhee ] / Appl. Microbiol. Biotechnol.   DOI
23 Analysis of shuffled gene libraries /
[ Joern,J.M.;P.Meinhold;F.H.Arnold ] / J. Mol. Biol.   DOI   ScienceOn
24 Nonhydrolytic fragmentation of a poly[(R)-3-hydroxybutyrate] single crystal revealed by use of a mutant of polyhydroxybutyrate depolymerase /
[ Murase,T.;Y.Suzuki;Y.Doi;T.Iwata ] / Biomacromol.   DOI   ScienceOn