Hydroxyalkanoate monomers

In Table 1, the recombinant bacteria harboring the heterologous PHA biosynthesis genes and synthesized PHAs with novel hydroxyalkanoate monomer have been summarized. 

Lenz, R.W., Farcet, C., Dijkstra, P.J., Goodwin, S., and Zhang, S.M. (1999) Extracellular polymerization of 3-hydroxyalkanoate monomers with the polymerase of Alcaligenes eutrophus. Int.J. Biol. Macromol., 25, 55-60. 

PHAs are polyesters composed of several units of hydroxyalkanoate monomers linked to each other through ester linkages. Figure 12.1 represents PHA s general formula where n is the number of monomer units in each polymer chain, which varies between 100 and 30,000, Ri and R2 the side chain that includes alkyl groups with 1-13 carbons and x in the main chain which ranges from 1 to 4. 

PHAs can be either thermoplastic or elastomeric materials with variable mechanical, thermal stability and durability properties. They are water insoluble and impermeable to oxygen [14]. Due to the stereospecificity of the PHA synthase, all the hydroxyalkanoate monomers incorporated in the polymer are in the R(—) configuration, resulting in an optically pure polymer [15]. Additionally, they are biodegradable hence, they can be degraded and metabolized by microbes and by enzymes within the human body biocompatible, they do not generate toxic byproducts and some of them are piezoelectric, a property known to stimulate cell growth [4]. 

Polyhydroxyalkanoate(s) (PHA) are a group of natural biopolymers which are synthesised by a wide variety of microbial genera. PHA are biodegradable and biocompatible thermoplastics consisting of a repeated chain of various hydroxyalkanoate monomers. 

Xi JZ, Wu Q, Yan YB, Zhang ZM, Yu HP, MK Cheung, Zhang RQ and Chen GQ, Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317. Antonie van Leeuwenhoek, 2000, 78 43 9. 

Aliphatic polyesters based on monomers other than a-hydroxyalkanoic acids have also been developed and evaluated as drug delivery matrices. These include the polyhydroxybutyrate and polyhydroxy valerate homo- and copolymers developed by Imperial Chemical Industries (ICI) from a fermentation process and the polycaprolactones extensively studied by Pitt and Schindler (14,15). The homopolymers in these series of aliphatic polyesters are hydrophobic and crystalline in structure. Because of these properties, these polyesters normally have long degradation times in vivo of 1-2 years. However, the use of copolymers and in the case of polycaprolactone even polymer blends have led to materials with useful degradation times as a result of changes in the crystallinity and hydrophobicity of these polymers. An even larger family of polymers based upon hydroxyaliphatic acids has recently been prepared by bacteria fermentation processes, and it is anticipated that some of these materials may be evaluated for drug delivery as soon as they become commercially available. 

The polymerase is stereospecific. It accepts only the D-(-)-stereoisomer which is generally formed by the NADPH-linked reductase. With respect to chain length of the activated fatty acids the specificity of the polymerase varies in different organisms. It links not only C4 3-acyl moieties but also C5 compounds when forming the polyester molecule [26]. It also polymerizes 3-hy-droxy-, 4-hydroxy-, and 5-hydroxyalkanoates from C3 to C5 monomers, but not C6 or higher (e.g., in R. eutropha) [27-31]. In pseudomonads, in contrast, it links C6 to C14 3-hydroxyalkanoyl-CoA [32]. 

Fig. 2A-F. Hydroxy acid monomers of PHAs with varied polymer backbones A standard 3-hydroxyalkanoic acid monomer B 2-methyl-3-hydroxyalkanoic acid monomer C 2,2-di-methyl-3-hydroxyalkanoic acid monomer D 4-hydroxyalkanoic acid monomer E 5-hydroxy-alkanoic acid monomer F 6-hydroxyalkanoic acid monomer. (R indicates alkyl residue or H)...

An alternative to the extraction of intact PHA polymer is the isolation of PHA monomers, oligomers, or various derivatives such as esters [74]. PH As are composed of stereo-chemically pure P-3-hydroxyacids, and therefore can be used as a source of optically pure organic substrates for the chemical and pharmaceutical industry [79]. In this protocol, the defatted cake containing PHA polymer would be chemically treated to obtain the PHA derivatives. For example, transesterification of the meal with methanol would give rise to methyl esters of 3-hydroxyalkanoic acids. The PHA derivatives would then be separated from the meal with appropriate solvents. One potential disadvantage of this method is the potential alteration of the quality of the residual meal if the harsh chemical treatments required for the production of PHA derivatives lead to protein or amino acid breakdown. 

PHA is produced in Alcaligenes eutrophus from acetyl CoA in three steps and the last step is the chain growth polymerization of hydroxyalkanoate CoA esters catalyzed by PHA polymerase (synthase), yielding PHA of high molecular weight. Kinetics and mechanism of the polymerization of hydroxyalkanoyl CoA monomers with this bacterial polymerase have been investigated. 

Since the chemical structure and monomer composition of a specific polymer are the most important factors in determining the polymer s physical and material properties, a short recapitulation of typical representatives of microbially synthesized poly(hydroxyalkanoates) is presented in this section. A more detailed overview on this issue is available from References [19-21], but is not within our scope here. The monomer composition of PHAs depends on the nature of the carbon source and the microorganisms used. This way, numerous monomers have been introduced into PH A chains [3-9]. PHAs have been divided roughly into two classes [19]. 

The ring-expansion carbonylation of epoxides is the most widely studied field in the epoxide carbonylation chemistry since the product lactones are highly attractive targets particularly, /1-lactones are useful compounds due to their versatility in organic synthesis [ 14,15] as well as their utilization as monomers to produce poly(3-hydroxyalkanoate)s, naturally occurring biodegradable polyesters [16-19]. 

Another interesting example of lactones are the p-hydroxyalkanoates, whose ROP affords poly(p-hydroxyalkanoate)s (PHAs), a class of aliphatic polyesters naturally produced by bacteria (Fig. 3) [12, 13]. Poly(3-(R)-hydroxybutyrate) (PHB) is a typical example. PHB is a stiff thermoplastic material with relatively poor impact strength, but the incorporation of other monomers can improve the mechanical properties. 

The mechanism for bacterial synthesis of PHA is not the simple dehydration reaction between alcohol and carboxyl groups. It is more complicated and involves the coenzyme A thioester derivative of the hydroxyalkanoic acid monomer (produced from the organic feedstock available to the bacteria) [Kamachi et al., 2001], Growth involves an acyl transfer reaction catalyzed by the enzyme PHA synthase (also called a polymerase) [Blei and Odian,... 

The reaction mechanism for the polymerization of a hydroxyalkanoic acid (Eqs. 2-243 through 2-246) is a chain polymerization, often called an activated monomer polymerization. The active site of lipase is its serine a-amino acid unit, which contains a hydroxyl group. The acyl carbon of the hydroxyalkanoic acid undergoes nucleophilic attack by the hydroxyl group of serine to form lipase-activated monomer (Eq. 2-243). Initiation consists of reaction... 

Huijberts, G. N. M., Eggink, G., de Waard, P, Huisman, G. W., and Witholt, B. 1992. Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers. Appl. Environ. Microbiol., 58, 536-544. 

PHAs are mainly composed of R-(-)-3-hydroxyalkanoic acid monomers. These can be broadly subdivided into two groups ... 

While the first efforts [11, 12] of the biotechnological generation of aliphatic homopolyesters and random copolyesters have been restricted essentially on monomers from 3-hydroxybutyric acid (3HB) and 3-hydroxypentanic acid (3HV), newer investigations concentrate on mraiomers with branches in the range of medium chain length, the so-called thermoplastic elastomers [13—15]. Steinbiichel et al. [3] catalogued more than 100 hydroxyalkanoic acids as craistituents of biosynthetic PHAs. 

Pseudomonas putida KT2442 cultivated on glucose accumulates poly(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers. Apjd. Environ Microbiol,... 

Poly(jS-hydroxyalkanoates) (PHAs) are a class of poly(esters) which are synthesised by many bacteria as energy reserve and carbon source when exposed to an excess of carbon under unbalanced growth conditions [82]. The produced PHAs can account for up to 80% of the bacterium s dry weight. This has led to the biotechnological production of PHAs. Depending on the substrates provided to the bacteria, different monomers can be produced which results in different PHA (co)polymers [83]. 

In addition to the research being done on the incorporation of a larger monomer variety in /3-hydroxyalkanoates, a mutant form of the PHA synthase enzyme was observed to enable the polymerisation of 2-hydroxyacids and the incorporation of lactic acid in the polymer backbone [80]. The properties of these materials were found to be distinctly different from those of the PHAs synthesised starting from 3-hydroxyacids. 

---