Pseudomonas lemoignei has five poly(hydroxyalkanoic acid) (PHA) depolymerase genes: A comparative study of bacterial and eukaryotic PHA depolymerases

Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas lemoignei: poly(3-hydroxybutyrate) (PHB), depolymerase A (M _r , 55,000), and PHB depolymerase B (M _r , 67,000) were specific for PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) as substrates. The third depolymerase additionally hydrolyzed poly(3-hydroxyvalerate) (PHV) at high rates (PHV depolymerase;M _r , 54,000). The N-terminal amino acid sequences of the three purified proteins, of a fourth partially purified depolymerase (PHB depolymerase C), and of the PHB depolymerases ofComamonas sp. were determined. Four PHA depolymerase genes ofP. lemoignei (phaZ1,phaZ2,phaZ3, andphaZ4) have been cloned inEscherichia coli, and the nucleotide sequence ofphaZ1 has been determined recently (D. Jendrossek, B. Müller, and H. G. Schlegel,Eur. J. Biochem. 218, 701–710, 1993). In this study the nucleotide sequences ofphaZ2 andphaZ3 were determined.PhaZ1,phaZ2, andphaZ4 were identified to encode PHB depolymerase C, PHB depolymerase B, and PHV depolymerase, respectively.PhaZ3 coded for a novel PHB depolymerase ofP. lemoignei, named PHB depolymerase D. None of the four genes harbored the PHB depolymerase A gene, which is predicted to be encoded by a fifth depolymerase gene ofP. lemoignei (phaZ5) and which has not been cloned yet. The deduced amino acid sequences ofphaZ1–phaZ3 revealed high homologies to each other (68–72%) and medium homologies to the PHB depolymerase gene ofAlcaligenes faecalis T₁ (25–34%). Typical leader peptide amino acid sequences, lipase consensus sequences (Gly-Xaa-Ser-Xaa-Gly), and unusually high proportions of threonine near the C terminus were found in PhaZ1, PhaZ2, and PhaZ3. Considering the biochemical data of the purified proteins and the amino acid sequences, PHA depolymerases ofP. lemoignei are most probably serine hydrolases containing a catalytical triad of Asp, His, and Ser similar to that of lipases. A comparison of biochemical and genetic data of various eubacterial and one eukaryotic PHA depolymerases is provided also.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

Degradation of poly(3-hydroxybutyrate), PHB,by bacteria and purification of a novel PHB depolymerase fromComamonas sp.

Bacteria capable of growing on poly(3-hydroxybutyrate), PHB, as the sole source of carbon and energy were isolated from various soils, lake water, activated sludge, and air. Although all bacteria utilized a wide variety of monomeric substrates for growth, most of the strains were restricted to degrade PHB and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Five strains were also able to decompose a homopolymer of 3-hydroxyvalerate, PHV. Poly(3-hydroxyoctanoate), PHO, was not degraded by any of the isolates. One strain, which was identified asComamonas sp., was selected, and the extracellular depolymerase of this strain was purified from the medium by ammonium sulfate precipitation and by chromatography on DEAE-Sephacel and Butyl-Sepharose 4B. The purified PHB depolymerase was not a glycoprotein. The relative molecular masses of the native enzyme and of the subunits were 45,000 or 44,000, respectively. The purified enzyme hydrolyzed PHB, P(3HB-co-3HV), and—at a very low rate—also PHV. Polyhydroxyalkanoates, PHA, with six or more carbon atoms per monomer or characteristic substrates for lipases were not hydrolyzed. In contrast to the PHB depolymerases ofPseudomonas lemoignei andAlcaligenes faecalis T₁, which are sensitive toward phenylmethylsulfonyl fluoride (PMSF) and which hydrolyze PHB mainly to the dimeric and trimeric esters of 3-hydroxybutyrate, the depolymerase ofComamonas sp. was insensitive toward PMSF and hydrolyzed PHB to monomeric 3-hydroxybutyrate indicating a different mechanism of PHB hydrolysis. Furthermore, the pH optimum of the reaction catalyzed by the depolymerase ofComamonas sp. was in the alkaline range at 9.4.     

Characterization and enzymatic degradation of a cross-linked bacterial polyester

The bacterial polyester, poly(-hydroxybutyrate-co--hydroxyvalerate) (PHB/V), was cross-linked with 1, 5, 7, 10, 20, and 30 wt% benzoyl peroxide by thermal decomposition reactions. Solvent extractions were carried out to determine the cross-linked fractions of the films. The sol/gel data were used to estimate cross-link densities. Films of PHB/V cross-linked with 10% benzoyl peroxide were placed in contact with purified depolymerase A secreted byP. lemoignei. These samples exhibited weight loss rates which were half that of un-cross-linked PHB/V, but the network was degraded completely by the enzyme. The results of this study suggest that anendo-type enzymatic degradation may occur, in addition to theexo-type activity, which is normally presumed to occur with theP. lemoignei depolymerase system.     

Enzymatic Degradation and Aminolysis of Microbial Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Single Crystals

Solution-grown single crystals of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] were hydrolyzed by polyhydroxybutyrate (PHB) depolymerase from Ralstonia pickettii T1. Enzymatic degradation proceeded from the edges of lamellar crystals, yielding serrated contour and small crystal fragments. Gel permeation chromatography analysis revealed that the molecular weights of the crystals decreased during enzymatic degradation, suggesting that the enzymatic hydrolysis of chain-folding regions at the crystal surfaces occurred in addition to the enzymatic degradation at crystal laterals or edges. After P(3HB-co-4HB) single crystals were aminolysed in 20% aqueous methylamine solution to remove the folded-chain regions and enzymatic degradation by lipase from Rhizopus oryzae to remove 4HB components at crystal surfaces of single crystal aminolyzed, it was found that a small amount (up to ca. 2 mol%) of 4HB component can be incorporated into the P(3HB) mother crystal lattice irrespective of the 4HB content.     

Properties of a Poly(3-hydroxybutyrate) Depolymerase from Penicillium funiculosum

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a lipase box sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.     

Biochemical and Genetic Characterization of an Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Acidovorax Sp. Strain TP4

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X₁-Ser-X₂-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that ²⁰Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.     

Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

The extracellular poly(-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of 40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent K _m value of purified enzyme for PHB was 119 g/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.     

Characterization of a poly(3-hydroxybutyrate) depolymerase fromaureobacterium saperdae: Active site and kinetics of hydrolysis studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase was purified fromAureobacterium saperdae cultural medium by using hydrophobic interaction chromatography. The isolated enzyme was composed of a single polypeptide chain with a molecular mass of 42.7 kDa as determined by SDS-PAGE and by native gel filtration on TSK-HW-55S. The enzyme was not a glycoprotein. Its optimum activity occurred at pH 8.0 and it showed a broad pH stability, ranging from pH 3 to pH 11.N-Bromosuccinamide and 2-hydroxy-5-nitrobenzyl bromide completely inactivated the enzyme, suggesting the involvement of tryptophan residues at the active site of the protein. The enzyme was very sensitive to diisopropyl fluorophosphate and diazo-dl-norleucine methyl ester, showing the importance of serine and carboxyl groups. The modification of cysteine residues byp-hydroxy mercuricbenzoate did not cause a loss of activity, whereas dithiothreitol rapidly inactivated the enzyme, revealing the presence of disulfide bonds.A saperdae depolymerase acted on the surface layer of PHB films and the degradation proceeded by surface erosion releasing monomers and dimers of 3-hydroxybutric acid. The degradation of PHB films byA. saperdae depolymerase was partially inhibited in the presence of excess amounts of enzyme. This phenomenon, already observed by Mukaiet al. with poly(hydroxyalkanoates) depolymerases fromAlcaligenes faecalis, Pseudomonas pickettii, andComamonas testosteroni, was analyzed according to the kinetic model proposed by these authors. The experimental data evidenced a general agreement with the kinetic model, although higher initial degradation rates were found withA. saperdae depolymerase.     

Thermal Properties and Biodegradability Studies of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

For investigating the relationship between thermal properties and biodegradability of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), several films of PHBV containing different polyhydroxyvalerate (HV) fractions were subjected to degradation in different conditions for up to 49 days. Differential scanning calorimetry (DSC), thermogravimetry (TG), specimen weight loss and scanning electron microscopy (SEM) were performed to characterize the thermal properties and enzymatic biodegradability of PHBV. The experimental results suggest that the degradation rates of PHBV films increase with decreasing crystallinity; the degradability of PHBV occurring from the surface is very significant under enzymatic hydrolysis; the crystallinity of PHBV decreased with the increase of HV fraction in PHBV; and no decrease in molecular weight was observed in the partially-degraded polymer.     

Biodegradability of degradable plastics exposed to anaerobic digested sludge and simulated landfill conditions

The biodegradabilities of various plastics by anaerobic digested sludge were measured and compared with the biodegradabilities under simulated landfill conditions. Bacterial poly(3-hydroxy-butyrate-co-3-hydroxyvalerate) (PHB/HV; 92/8, w/w), a natural aliphatic polyester, degraded nearly to completion within 20 days of cultivation by anaerobic digested sludge, while synthetic aliphatic polyesters such as poly-lactic acid, poly(butylene succinate), and poly (butylene succinate-co-ethylene succinate) did not degrade at all in 100 days. Cellophane, which was used as a control material, exhibited a similar degradation behavior to PHB/HV. Under simulated landfill conditions, PHB/HV degraded quite well within 6 months. Synthetic aliphatic polyesters also showed significant weight losses through 1 year of cultivation. The acidic environment inside simulators generated by the degradation of biodegradable food wastes which comprised 34 % of municipal solid waste seems to cause the weight loss of synthetic aliphatic polyesters.     

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.     

Utilization of Broken Rice for the Production of Poly(3-hydroxybutyrate)

The feasibility of utilizing non edible rice (broken rice) for production of fine materials such as poly(3-hydroxybutyrate) (PHB) was considered as one of the alternative ways of keeping the environment clean for sustainable development. Thus, production of PHB from broken rice by simultaneous saccharification and fermentation (SSF) was investigated. During the SSF process, the rice (15% w/v) material was hydrolyzed to glucose, which was utilized by Cupriavidus necator for growth and production of PHB. The PHB content reached 38% at 58 h fermentation. The PHB had weight average molar mass (Mw) and polydipersity index of 3.82 × 10⁵ (g/mol) and 4.15, respectively. Differential calorimetric scan of the PHB showed a melting temperature (Tm) of 176 °C. Given that the PHB was a homopolymer (which consisted of (R)-3-hydroxybutyric acid monomers), it was thought that broken rice could be a raw material for production of both PHB and (R)-3-hydroxybutyric acid. This SSF process would not only help in the utilization of broken rice or non edible rice, but would also serve as a model for utilization of other raw materials that contain starch for production of PHB.     

Bacterial Poly(Hydroxyalkanoate) Polymer Production from the Biodiesel Co-product Stream

A co-product stream from soy-based biodiesel production (CSBP) containing glycerol, fatty acid soaps, and residual fatty acid methyl esters (FAME) was utilized as a fermentation feedstock for the bacterial synthesis of poly(3-hydroxybutyrate) (PHB) and medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers. Pseudomonas oleovorans NRRL B-14682 and P. corrugata 388 grew and synthesized PHB and mcl-PHA, respectively, when cultivated in up to 5% (w/v) CSBP. In shake flask culture, P. oleovorans grew to 1.3 ± 0.1 g/L (PHA cellular productivity = 13–27% of the bacterial cell dry weight; CDW) regardless of the initial CSBP concentration, whereas P. corrugata reached maximum cell yields of 2.1 g/L at 1% CSBP, which tapered off to 1.7 g/L as the CSBP media concentration was increased to 5% (maximum PHA cellular productivity = 42% of the CDW at 3% CSBP). While P. oleovorans synthesized PHB from CSBP, P. corrugata produced mcl-PHA consisting primarily of 3-hydroxyoctanoic acid (C_8:0; 39 ± 2 mol%), 3-hydroxydecanoic acid (C_10:0; 26 ± 2 mol%) and 3-hydroxytetradecadienoic acid (C_14:2; 15 ± 1 mol%). The molar mass (M_n) of the PHB polymer decreased by 53% as the initial CSBP culture concentration was increased from 1% to 5% (w/v). In contrast, the M_n of the mcl-PHA polymer produced by P. corrugata remained constant over the range of CSBP concentrations used.     

Confirmation of colonization of degrading bacterium strain SC-17 on poly(3-hydroxybutyrate) cast film

To clarify the mechanism of microbial degradation owing to colonization ofPseudomonas sp. strain SC-17 on a poly(3-hydroxybutyrate) (PHB) cast film surface, morphological and spectroscopic analyses of the degraded film were investigated and colonization kinetics on the films is discussed. By spectroscopic analysis of unique hemispherical degradation marks, cells of strain SC-17 adhering to the marks' surface were confirmed. To account for the hemispherical hole formation and their linear enlargement with culture time, a three-dimensional colony growth model toward the interior of the film was developed. The model explained the hemispherical hole formation well. It was concluded that the hemispherical holes resulted from the colonization of strain SC-17 on the film surfaces. It was further determined that the microbial degradation by strain SC-17 is initiated from small pits formed on the PHB film surface.     

Production and Purification of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Degrading Enzyme from Streptomyces sp. AF-111

A poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading bacterial strain designated as AF-111 was isolated from sewage sludge sample. The bacterium was identified by 16S rRNA gene sequencing. The results revealed that strain AF-111 showed 99 % similarity with Streptomyces althioticus strain NRRL B-3981 and designated as Streptomyces sp. strain AF-111. An extracellular PHBV depolymerase enzyme was produced under optimized conditions and purified through ammonium sulphate fractionation and column chromatography. The enzyme was purified to homogeneity, indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and molecular weight was found to be approximately 51 kDa. Effect of temperature, pH, metal ions and inhibitors on the PHBV depolymerase activity was determined. The enzyme was stable at wide range of temperature (35–55 °C) and pH (6–8). PHBV depolymerase was stable in the presence of different metal ions except iron and zinc which had inhibitory effect on depolymerase activity. Both ethylenediamine teteracetic acid and phenylmethyl sulphonyl fluoride strongly inhibited enzyme activity which indicates that this enzyme belongs to the serine hydrolase family like other polyhydroxyalkanoate depolymerases. The results show that a depolymerase from strain AF-111 can effectively degrade PHBV, therefore, it can be applied in the process of biochemical monomer recycling.     

Fungal Degradation of the Bioplastic PHB (Poly-3-hydroxy- butyric acid)

PHB (poly-3-hydroxybutyric acid) is a thermoplastic polyester synthesized by Ralstonia eutropha and other bacteria as a form of intracellular carbon and energy storage and accumulated as inclusions in the cytoplasm of these bacteria. The degradation of PHB by fungi from samples collected from various environments was studied. PHB depolymerization was tested in vials containing a PHB-containing medium which were inoculated with isolates from the samples. The degradation activity was detected by the formation of a clear zone below and around the fungal colony. In total, 105 fungi were isolated from 15 natural habitats and 8 lichens, among which 41 strains showed PHB degradation. Most of these were deuteromycetes (fungi imperfecti) resembling species of Penicillium and Aspergillus and were isolated mostly from soils, compost, hay, and lichens. Soil-containing environments were the habitats from which the largest number of fungal PHB degraders were found. Other organisms involved in PHB degradation were observed. A total number of 31 bacterial strains out of 67 isolates showed clear zones on assay medium. Protozoa, possible PHB degraders, were also found in several samples such as pond, soil, hay, horse dung, and lichen. Lichen, a fungi and algae symbiosis, was an unexpected sample from which fungal and bacterial PHB degraders were isolated.     

Molecular structure of extracellular poly(3-hydroxybutyrate) depolymerase fromAlcaligenes faecalis T1

The amino acid sequence of a peptide containing an active serine was examined with poly(3-hydroxybutyrate) (PHB) depolymerase ofAlcaligenes faecalis T1. The sequence Cys-Asn-Ala-Trp-Ala-Gly-Ser-Asn-Ala-Gly-Lys was obtained. This amino acid sequence around the active serine does not fit any reported sequence of other esterases and proteases. On the other hand, a segment of the amino acid sequence of PHB depolymerase ofA. faecalis was homologous to the type III sequence of fibronectin. Similar sequences have been reported in some type of bacterial chitinase and cellulases, and PHB depolymerase seems to have an overall similarity to these bacterial extracellular hydrolases.     

Effects of higher-order structure of poly(3-hydroxybutyrate) on its biodegradation. II. Effects of crystal structure on microbial degradation

As one of a series of studies concerning the relationship between the higher-order structure and the biodegradability of a biodegradable plastic, the effects of the crystal structure of the plastic on microbial degradation were investigated. Bacterial poly(d-(–)-3-hydroxybutyrate) (PHB) films which had a wide range of crystallinity were prepared by the melt-quenching method. Results of the microbial degradation indicated that the development of crystallinity evidently depressed the microbial degradability. From scanning electron microscopy (SEM) observations, it is suggested that the microbial degradation proceeded in at least two manners. One was preferential degradation of the amorphous region leaving the crystalline lamellae intact, which was considered to be a homogeneous enzymatic degradation over the surface. The other was nonpreferential spherical degradation on the surface. The SEMs indicate that the spherical holes were the result of colonization by degrading bacteria. The holes varied in size and number with the change of crystal structure. Therefore, it is considered that the crystal structure of PHB also influenced the physiological behavior of the degrading bacteria on the PHB surface.     

Effect of Lignosulfonate on the Thermal and Morphological Behavior of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

The effect of lignosulfonate on poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV, was studied by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The PHBV/lignosulfonate samples were prepared by melt mixing in an internal mixer. SEM showed that PHBV/lignosulfonate samples present a cracked surface that is more intense in mixtures with high lignosulfonate proportions. According to DSC, melting and glass transition temperatures of the PHBV matrix decrease with lignosulfonate addition. The same effect was observed for melting enthalpies (ΔH_m), which indicates a decrease of crystallinity. TGA showed that thermal stability of PHBV/lignosulfonate samples was shifted to lower temperatures, which indicates the existence of an interaction between the thermal decomposition processes of PHBV and lignosulfonate.     

Degradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Some Soil Aspergillus spp.

Systematic screening of 45 soil fungi for degradation polyhydroxyalkanoic acids (PHAs) has led to the selection of 6 potent Aspergillus isolates belonging to A. flavus, A. oryzae, A. parasiticus, and A. racemosus. Degradation of PHAs as determined by tube assay method revealed that these Aspergillus spp. were more efficient in degrading poly(3-hydroxybutyrate) [P(3HB)] compared to copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB-co-16% 3HV). Moreover, the extent of degradation in mineral base medium was much better than those in complex organic medium. For all the Aspergillus spp. tested, maximum degradation was recorded at a temperature of 37°C with significant inhibition of growth. The optimum pH range for degradation was 6.5–7.0 with degradation being maximum at pH 6.8. The extent of polymer degradation increased with increase in substrate concentration, the optimum concentration for most of the cultures being 0.4% and 0.2% (w/v) for P(3HB) and P(3HB-co-16%3HV) respectively. Supplementation of the degradation medium with additional carbon sources exerted significant inhibitory effect on both P(3HB) and P(3HB-co-16%3HV) degradation.