title: Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system.
abstract: Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4°C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaC(Re) achieved 1.76 U mg⁻¹, 29.05, 16.23 and 95.32%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37°C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro.
authors.affiliation: Bio-refinery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, 135 Yuan-Tung Road, Chung-li, Taoyuan 320, Taiwan. lanchiwei@saturn.yzu.edu.tw
authors.forename: John Chi-Wei
authors.initials: JC
authors.lastname: Lan
authors.forename: Chun-Yi
authors.initials: CY
authors.lastname: Yeh
authors.forename: Chih-Chi
authors.initials: CC
authors.lastname: Wang
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Yang
authors.forename: Ho-Shing
authors.initials: HS
authors.lastname: Wu
mesh_terms: D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005591:Chemical Fractionation / Q000379:methods*; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014867:Water
chemical_list: D008670:Metals; D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D014867:Water; D011092:Polyethylene Glycols; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase
keywords: Aqueous two-phase systems; PHA synthase; Polyhydroxyalkanoates; Recombinant Escherichia coli; Recovery