Synthesis And Characterization Of Copolyesters P(2HB-co-3HB) And P(3MP-co-3HB) In Vitro

Polyhydroxyalkanoates?PHAs?is a kind of polymer polyester synthesized by microorganism.It has thermoplastic properties similar to traditional petroleum plastics,and has good biocompatibility and biodegradability.As a new environmentfriendly green material instead of traditional petroleum plastics,it has attracted wide attention.At present,the high production cost,single structure and poor processing performance of PHAs restrict the application of PHAs in packaging and other fields.In order to solve the problem of PHAs monomer structure,2-hydroxybutyrate?2HB?and 3-mercapto propionic acid?3MP?were used as an alternative substrate for 3-hydroxybutyrate?3HB?.The controllable synthesis of P?2HB-co-3HB?and P?3MPco-3HB?was achieved based on the self designed in vitro synthesis pathway.The structures and properties of the synthesized copolyesters were analyzed by means of ¹H-NMR,GPC,DSC and TGC.First,the three enzymes needed in the synthetic pathway were prepared by means of in vitro recombination and overexpression.Their activity?Acetyl-CoA synthetase?ACS?is derived from Pelotomaculum thermopropionicum JCM10971.CoA transferase?PCT?is derived from Clostridium propionicum JCM1430.PHA synthase?PhaC?is derived from Ralstonia eutropha.?were determined.The results showed that the concentrations and activities of the three enzymes obtained by the mass expression were:ACS?2.9 mg/mL;0.174 U/mg??PCT?15 mg/mL;4.302 U/mg??PhaC?5.3 mg/mL;4.49 U/mg?.Secondly,different proportions of 2HB and 3HB were used as substrates.In the presence of CoA and acetic acid,three enzymes were added to prepare P?2HB-co-3HB?in vitro synthesis reaction.¹H-NMR results showed that when the mole ratio of 2HB/3HB was0/100,25/75 and 50/50,the synthetic yield of the copolymer was 1.52 g/L?0.94 g/L?0.60g/L.The results of GPC of incorporation ratio of 2HB in P?2HB-co-3HB?was 30 mol%showed that the molecular weight of the synthesized PHAs decreased slightly with the increase of 2HB components,and the weight average molecular weight decreased from89.2 KDa to 70.4 KDa.Further analysis of the thermal properties of the copolymer P?30mol%2HB-co-3HB?showed that the glass transition temperature?T_g?,crystallization temperature?T_c?and melting temperature?T_m?of the polyester were respectively-18.7?,36.5?and 80.4?,and the decomposition temperature T_d?10%?was 237.2?.It shows that compared with both homopolyester PHB and copolyester P?9 mol%2HB-co-3HB?,the content of 30 mol%2HB in copolyester greatly changes the thermal properties of homopolyester PHB.Finally,we used 3MP and 3HB as substrates in the in vitro reaction system and constructed the copolymerization of P?3MP-co-3HB?in vitro.The structure and properties of the complexes were also analyzed.The results showed that when the molar ratio of3MP/3HB was 0/100,25/75 and 50/50,the yield of PHAs was 2.0 g/L,0.717 g/L and 0.26g/L,respectively.The ¹H-NMR results show that the molar ratios of 3MP in the copolyester P?3MP-co-3HB?are 60 mol%and 80 mol%respectively in the reactions of 3MP/3HB?25/75?and 3MP/3HB?50/50?.The GPC results showed that the molecular weight of the synthesized copolyester increased greatly with the increase of 3MP molar fraction,and the weight average molecular weight increased from 89.2 KDa to P?60 mol%2HB-co-3HB?97.5 KD and P?80 mol%2HB-co-3HB?102.8 KDa.Further analysis of the thermal properties of the copolyester P?3MP-co-3HB?containing 60 mol%and 80 mol%3MP showed that the glass transition temperature?T_g?,crystallization temperature?T_c?,melting temperature?T_m?and decomposition temperature?T_d?of P?60 mol%2HB-co-3HB?were-2.3?,47.7?,134.9?and 271.4?;P?80 mol%2HB-co-3HB?were 43.4?,97.7?,112.5?and 239.7?,respectively.Compared with the homopolyester PHB,copolyester,with the increase of the molar ratio of 3MP,T_m has an obvious decreasing trend,while T_d has an obvious upward trend.These results show that the addition of 3MP in the copolyester greatly changes the structure and thermal properties of homopolyester PHB.In this study,copolyesters P?2HB-co-3HB?and P?3MP-co-3HB?containing 2HB and3MP were successfully synthesized by monophasic reaction system in vitro,which provided a new method for the study of PHAs modification.In addition,this in vitro synthesis pathway provides a new idea for the synthesis of special PHAs in vivo.