## 4 Discussion
These data indicate that the PhaC1\({}_{16}\) enzyme was novel and the amino acid sequences in the usually conserved regions of PHA synthase were unique and some changes are identical to known PhaC1 mutations. [PERSON] et al. [57] reported three mutations (Ala295 Val, Ser482 Gly, and Leu484 Val) in the mutagenized versus wild-type PHA synthase of _P. putida_ Gpo1, which were responsible for addition of up to 60% 3 HB in co-polymers produced by _P. putida_ Gpo1. Comparison of the amino acids in positions 295, 482, and 484, which were identified as key amino acids for substrate specificity, revealed amino acids in PhaC1\({}_{16}\) that were different from both the _P. putida_ LS46 PHA synthase (Leu295 Ala, Thr482 Ser, and Val484 Ile) and the _P. putida_ Gpo1 PHA synthase. Three amino acids in the PHA synthase of _Pseudomonas_ sp. 61-3, which, when mutated (Ser325, Cys/Ser477, and Lys/Gln481 Leu), could add a greater mol % of 3 HB into copolymers. The PhaC1\({}_{16}\) enzyme had amino acids in these positions that were different than those in the _P. putida_ LS46 PhaC1 enzyme (Ser325 Cys and Gln481 Leu) [40; 56]. The amino acids in these two positions were also different from those encoded by the _Pseudomonas_ sp. 61-3 PHA synthase. This may be the reason for the greater incorporation of 3 HB in co-polymer produced by _P. putida_ LS46123. Mutations of Cys296 to Ser296 and His453 to Gln453 result in greater incorporation of (R)-3-hydroxyhexanoyl-CoA and (R)-3-hydroxydodecanoyl-CoA into MCL-PHAs, but not SCL-PHA monomers like (R)-3-hydroxybutylryl-CoA [51]. Another important difference in PhaC1\({}_{16}\) was Leu484 to Val484, which may result in increased 3-hydroxybutyrate content in PHAs. Likewise, transfer of plasmid with single mutations in PhaC1 of _Pseudomonas_ sp. 61-3 Q481M and S482G enhanced the (R)-3-hydroxyhexanoate monomer composition in the PHA accumulation by a _P. putida_ GPp104 (PHA\({}^{-}\)) [56]. Therefore, the amino acid sequence of PhaC1\({}_{16}\) is novel; it did not match any of the wild-type or mutated PhaC1 sequences. This could be the possible reason for the unique monomer composition of the polymer produced by _P. putida_ LS46123. The monomer composition of PHA polymers greatly influences their physical and thermal properties [8; 9]. Unlike the polymers produced by _P. putida_ LS46, polymers produced by _P. putida_ LS46123 on different substrates were semisolid. Therefore some of the physical property tests used for solid polymers, like [PERSON]'s modulus and stress strain tests, were not performed. Integration of the different peaks generated by \({}^{1}\)HNMR analysis demonstrated that polymers synthesized by _P. putida_ LS46123 are co-polymers of 3 HB and 3 HA. This was further confirmed by FT-IR analysis, which showed the stretching of the carbonyl group. The polymers synthesized by _P. putida_ LS46123 contained 40%-50% of 3 HB. _P. putida_ LS46123 recombinant accumulated higher 3 HB in PHAs than a recombinant of _P. putida_ KT2440, indicating the role of the host and some other necessary genesin the fatty acid metabolism pathways [31]. Generally, polymers with high 3 HB content have high melting temperature and toughness. The production of such polymers requires wide substrate specificity from the PHA synthase enzyme. _A. caviae, Pseudomonas_ sp. 61-3, and _P. stutzeri_ PHA synthases have been shown to have wide substrate specificity and were used to produce co-polymers of SCL- and MCL-monomers [57; 58]. The PhaC1\({}_{16}\) PHA synthase also displayed wide substrate specificity and could add both SCL- and MCL-monomers to the PHA polymers synthesized. However, unlike optimized PHA synthase of _P. stutzeri_, the PhaC1\({}_{16}\) enzyme added a greater proportion of 3 HHx and 3 HO subunits than 3 HD subunits, indicating that this PHA synthase had broad substrate specificity [59]. PHA copolymers with different monomer composition and contents are known to have different physical and thermal properties [19; 60; 61].