Application of recombinant gene technology for production of polyhydroxyalkanoic acids: Biosynthesis of poly(4-hydroxybutyric acid) homopolyester

Screening of a large number of bacteria revealed several strains, which utilize 1,4-butanediol and/or 4-hydroxybutyric acid (4HB) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA) containing 4HB as one constituent among others (mostly 3-hydroxybutyric acid). However, none of the wild-type strains investigated in this study was able to produce a homopolyester consisting solely of 4HB. Only several poly(3-hydroxybutyric acid)-leaky mutants ofAlcaligenes eutrophus strain JMP222 synthesized poly(4HB) homopolyester, which amounted to approximately 10% (w/w) of the cellular dry matter. If the PHA synthase structural gene ofA. eutrophus strain H16 was expressed in these mutants, the amount of poly(4HB) was increased to approximately 30% (w/w). The occurrence of poly(4HB) was demonstrated by gas chromatographic as well as¹H and¹³C nuclear magnetic resonance spectroscopic analysis.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

Biodegradability and Biodegradation of Polyesters

A variety of biodegradable plastics have been developed in order to obtain useful materials that do not cause harm to the environment. Among the biodegradable plastics, aliphatic polyesters such as: poly(3-hydroxybutyrate) (PHB), poly(ε-caprolactone) (PCL), poly(butylene succinate) (PBS), and poly(l-lactide) (PLA) have become the focus of interest because of their inherent biodegradability. However, before their widespread applications, comprehensive studies on the biodegradability and biodegradation mechanisms of these polyesters are necessary. Thus, this paper describes the degradation mechanisms and the effects of various factors on the degradation of polyesters. The distribution of polymer-degrading microorganisms in the environment, different microorganisms and enzymes involved in the degradation of various polyesters are also discussed.     

Enzymatic Degradation of Poly(l-Lactic Acid): Effects of UV Irradiation

Amorphous and crystallized poly(l-lactic acid) (PLLA-A and PLLA-C, respectively) films were prepared, and the proteinase K-catalyzed enzymatic degradation of UV-irradiated and non-irradiated PLLA-A and PLLA-C films was investigated for periods up to 10 h (PLLA-A) and 60 h (PLLA-C). The molecular weights of both the PLLA-A and PLLA-C films can be manipulated by altering the UV irradiation time. The enzymatic weight loss values of the UV-irradiated PLLA films were higher than or similar to those of the non-irradiated PLLA film, when compared with the specimens of same crystallinities. UV irradiation is expected to cause the PLLA films to undergo chain cleavage (a decrease in molecular weight) and the formation of C=C double bonds. It seems that the acceleration effects from decreased molecular weight on enzymatic degradation were higher than or balanced with the disturbance effects caused by the formation of C=C double bonds. After enzymatic degradation, a fibrous structure appeared on the spherulites of the UV-irradiated PLLA-C film. This structure may have arisen from chains containing or neighboring on the C=C double bonds, which were enzymatically undegraded and assembled on the film surface during enzymatic degradation. The results of this study strongly suggest that UV irradiation will significantly affect the biodegradation behavior of PLLA materials in the environment.     

Chemical Recovery of Useful Chemicals from Polyester (PET) Waste for Resource Conservation: A Survey of State of the Art

In this paper we present the main technologies developed over the last decades on chemical recycle of PET from a practical and economical point of view. We show details of plants used to carry out solvolitic reactions emphasising steps of purification, which are sometimes not considered when discussing chemical, recycle but which, in fact, are the key feasibility factors. The recycling or modification of PET to obtain monomers and other useful chemicals as intermediates and additives is carefully considered.     

Fast and Living Ring-Opening Polymerization of l-Lactide Initiated with In-situ–Generated Calcium Alkoxides

The ring-opening polymerization of l-lactide with calcium alkoxides generated in-situ from bis(tetrahydrofuran)calcium bis[bis(trimethylsilyl)amide] and 2-propanol are presented. The polymerization in THF at room temperature proceeds rapidly and in a living manner, giving poly(l-lactide)s of controlled molecular weight, low polydispersity, and tailored end-functionalities. Kinetic studies show the absence of an induction period and a pseudo-first order rate constant of 6.41 L mol^–1 min^–1, which is significantly higher than for related Y₅(-O)(O ⁱ Pr)_13– or aluminum alkoxide-initiated polymerizations. The initiation involves a two-step process: (1) alcoholysis of bis(tetrahydrofuran)calcium bis[bis(trimethylsilyl)amide] to give the corresponding calcium alkoxide and (2) ring-opening of l-lactide via acyl-oxygen cleavage and insertion into the calcium-alkoxide bond. In the presence of excess alcohol, fast and reversible exchange between free alcohol molecules and coordinated alkoxide ligands takes place. This allows tuning of the poly(l-lactide) molecular weight over a wide range.     

Biodegradability of a Selected Range of Polymers and Polymer Blends and Standard Methods for Assessment of Biodegradation

Synthetic polymers are important to the packaging industry but their use raises aesthetic and environmental concerns, particularly with regard to solid waste accumulation problems and the threat to wildlife. Some concerns are addressed by attention to problems associated with source reduction, incineration, recycling and landfill. Others are addressed by the development of new biodegradable polymers either alone or in blends. Materials used for biodegradable polymers include various forms of starch and products derived from it, biopolyesters and some synthetic polymers. Starch is rapidly metabolised and is an excellent base material for polymer blends or for infill of more environmentally inert polymers where it is metabolised to leave less residual polymer on biodegradation. This should help to improve the environmental impact of waste disposal. A number of standard methods have been developed to estimate the extent of biodegradability of polymers under various conditions and with a variety of organisms. They tend to be used mainly in the countries where they were developed but there is much overlap between the standards of different countries and wide scope for development of consistent and international standards.     

Accumulation of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) by mutants and recombinant strains ofAlcaligenes eutrophus

Alcaligenes eutrophus accumulated a terpolyester of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), and 4-hydroxyvaleric acid (4HV) during cultivation with 4HV as carbon and energy source under nitrogen starvation. The polyester accumulated by wild-type strains under these conditions contained 4HV at a molar fraction of approximately 5 mol% only. A catabolic pathway of 4HV was postulated, which included the activation of 4HV to 4HV-CoA and a conversion of 4HV-CoA to 3HV-CoA. Tn5::mob-induced mutants were isolated fromA. eutrophus HF39, which were affected in 4HV and/or valeric acid catabolism. Among 83 mutants were 27 4HV-negative or 4HV-leaky mutants; two mutants were identified which accumulated a terpolyester with a molar fraction of 10.1 to 22.7 mol% 4HV. In addition, a further increase in the molar fraction of 4HV in poly(3HB-co-3HV-co-4HV) and a two- to fourfold increase in the PHA synthase activity were monitored in these mutants or others and also in HF39, if the cells were complemented with the hybrid plasmid pHP1014::PP1, which contained the PHA biosynthesis genes ofA. eutrophus H16. Application of mutagenesis plus recombinant DNA techniques resulted in the accumulation of a terpolyester with up to 30 mol% 4HV and with approximately equimolar fractions of 3HB, 3HV, and 4HV.     

Natural and Artificial Functionalized Biopolyesters. II. Medium-Chain Length Polyhydroxyoctanoates from Pseudomonas Strains

Sixteen Pseudomonas strains have been tested with a view to developing medium-chain length polyhydroxyalkanoates. Four strains were selected and it is shown that their ability for producing three different polyesters with variable properties was dependent on the strains and substrates. Otherwise, Pseudomonas oleovorans was grown on a mixture of sodium octanoate and undecenoate salts at a 90/10 mol/mol ratio. The corresponding copolymer, bearing lateral double bonds, was chemically modified in the carboxy group. Finally, the ability to tailor-make functional bacterial polyesters aimed at temporary therapeutic applications is demonstrated.     

Natural and Artificial Functionalized Biopolyesters I. Chemoenzymatic Mediated Synthesis of Chiral Precursors

Design of optically active polymeric materials for temporary therapeutic and environmental applications requires the working-out of functionalized polymers with structures and properties adjusted to the considered applications. Biocompatibility of selected polymers is a required property in regard to the interactions between living organisms and macromolecular systems. It is therefore important to prepare highly optically active monomers and their corresponding hydrolyzable and biocompatible polymers. Bacterium Clostridium tetanomorphum is an useful source of enzymes for bioconversion and particularly in the chemoenzyrnatic route to optically active alkylmalolactonic acid esters and their optically pure stereoisomers. 3-Methylaspartase, involved in the glutamate fermentation pathway, is a very interesting enzyme, which can provide chiral precursors with high optically purity. This chemoenzymatic strategy can be used to prepare the four stereoisomers of 3-methylmalolaconic acid esters, by enzymatic resolution of natural and artificial stereoisomers of 3-methylaspartic acid. A series of 3-alkymalolactonic acid esters with alkyl equal to ethyl or isopropyl, have been also synthesized and transformed into corresponding optically active polyesters.