Substrate Report for: Polybutylene-succinate-co-adipate

Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZCma , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORFCma (a putative periplasmic substrate binding protein that is within the same operon of phaZCma ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZCma has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORFCma can potentially be a universal element for enhancing the secretion of recombinant protein.

Biodegradable plastics (BPs) have attracted much attention since more than a decade because they can easily be degraded by microorganisms in the environment. The development of aliphatic-aromatic co-polyesters has combined excellent mechanical properties with biodegradability and an ideal replacement for the conventional nondegradable thermoplastics. The microorganisms degrading these polyesters are widely distributed in various environments. Although various aliphatic, aromatic, and aliphatic-aromatic co-polyester-degrading microorganisms and their enzymes have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. In this review, we have reported some new microorganisms and their enzymes which could degrade various aliphatic, aromatic, as well as aliphatic-aromatic co-polyesters like poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), poly(epsilon-caprolactone) (PCL), poly(ethylene succinate) (PES), poly(L-lactic acid) (PLA), poly(3-hydroxybutyrate) and poly(3-hydoxybutyrate-co-3-hydroxyvalterate) (PHB/PHBV), poly(ethylene terephthalate) (PET), poly(butylene terephthalate) (PBT), poly(butylene adipate-co-terephthalate (PBAT), poly(butylene succinate-co-terephthalate) (PBST), and poly(butylene succinate/terephthalate/isophthalate)-co-(lactate) (PBSTIL). The mechanism of degradation of aliphatic as well as aliphatic-aromatic co-polyesters has also been discussed. The degradation ability of microorganisms against various polyesters might be useful for the treatment and recycling of biodegradable wastes or bioremediation of the polyester-contaminated environments.

Poly(tetramethylene succinate-co-tetramethylene adipate) (PBSA) and poly(tetramethylenesuccinate) (PBS) were hydrolyzed experimentally into water-soluble oligomers and monomers by Chromobacterium extracellular lipase. The oligomers were identified by high-performance liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance, which indicated that a total of 28 oligomer species were liberated from PBSA, and that 13 of them were identical to the hydrolysates from PBS. Moreover, 20 of the species were polyester-based compounds of monomer units, and the other 8 species were small amounts of diurethane compounds. Bis(hydroxybutyl) succinate (BSB) and bis(hydroxybutyl) hexamethylene dicarbamate (BHB) were the typical oligomers and were chemically synthesized. Biodegradability of BSB and BHB was examined for 28 d in the activated sludge, and analysis of the results of this study indicated that the final conversion rate of constituent carbon to carbon dioxide was estimated at 80 mol% for BSB and 10 mol% for BHB. The remaining amount of carbon in the undegraded BHB was 20 mol%. In the presence of BSB, the biodegradability of BHB was increased by about 1.5 times. The suggestion was made that BSB induced a growth of microorganisms and helped BHB degradation. This is consistent with the observation that the biodegradation of BHB in native soil for 60 d reached > 60%.

Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZCma , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORFCma (a putative periplasmic substrate binding protein that is within the same operon of phaZCma ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZCma has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORFCma can potentially be a universal element for enhancing the secretion of recombinant protein.

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. In the fungus Aspergillus oryzae, the hydrophobin RolA and the polyesterase CutL1 are co-expressed when the sole available carbon source is the biodegradable polyester polybutylene succinate-co-adipate (PBSA). RolA promotes the degradation of PBSA by attaching to the particle surface, changing its structure and interacting with CutL1 to concentrate CutL1 on the PBSA surface. We previously reported that positively charged residues in RolA and negatively charged residues in CutL1 are cooperatively involved in the ionic interaction between RolA and CutL1. We also reported that hydrophobin RodA of the model fungus Aspergillus nidulans, which was obtained via an A. oryzae expression system, interacted via ionic interactions with CutL1. In the present study, phylogenetic and alignment analyses revealed that the N-terminal regions of several RolA orthologs contained positively charged residues and that the corresponding negatively charged residues on the surface of CutL1 that were essential for the RolA-CutL1 interaction were highly conserved in several CutL1 orthologs. A PBSA microparticle degradation assay, a pull-down assay using a dispersion of Teflon particles, and a kinetic analysis using a quartz crystal microbalance revealed that recombinant A. nidulans RodA interacted via ionic interactions with two recombinant A. nidulans cutinases. Together, these results imply that ionic interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.

Aspergillus oryzae hydrophobin RolA adheres to the biodegradable polyester polybutylene succinate-co-adipate (PBSA) and promotes PBSA degradation by interacting with A. oryzae polyesterase CutL1 and recruiting it to the PBSA surface. In our previous studies, we found that positively charged amino acid residues (H32, K34) of RolA and negatively charged residues (E31, D142, D171) of CutL1 are important for the cooperative ionic interaction between RolA and CutL1, but some other charged residues in the triple mutant CutL1-E31S/D142S/D171S are also involved. In the present study, on the basis of the 3D-structure of CutL1, we hypothesized that D30 is also involved in the CutL1-RolA interaction. We substituted D30 with serine and performed kinetic analysis of the interaction between wild-type RolA and the single mutant CutL1-D30S or quadruple mutant CutL1-D30S/E31S/D142S/D171S by using quartz crystal microbalance. Our results indicate that D30 is a novel residue involved in the ionic interaction between RolA and CutL1.

Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA-CutL1 interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (KD = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (KD = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.

Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca2+ ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca2+ changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65-75 degrees C and pH 6.5-8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5-9 and at temperatures up to 65 degrees C for 24 h with 40 % activity remaining after incubation for 1 h at 70 degrees C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 degrees C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca2+ activation, and stabilization.

Biodegradable plastics (BPs) have attracted much attention since more than a decade because they can easily be degraded by microorganisms in the environment. The development of aliphatic-aromatic co-polyesters has combined excellent mechanical properties with biodegradability and an ideal replacement for the conventional nondegradable thermoplastics. The microorganisms degrading these polyesters are widely distributed in various environments. Although various aliphatic, aromatic, and aliphatic-aromatic co-polyester-degrading microorganisms and their enzymes have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. In this review, we have reported some new microorganisms and their enzymes which could degrade various aliphatic, aromatic, as well as aliphatic-aromatic co-polyesters like poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), poly(epsilon-caprolactone) (PCL), poly(ethylene succinate) (PES), poly(L-lactic acid) (PLA), poly(3-hydroxybutyrate) and poly(3-hydoxybutyrate-co-3-hydroxyvalterate) (PHB/PHBV), poly(ethylene terephthalate) (PET), poly(butylene terephthalate) (PBT), poly(butylene adipate-co-terephthalate (PBAT), poly(butylene succinate-co-terephthalate) (PBST), and poly(butylene succinate/terephthalate/isophthalate)-co-(lactate) (PBSTIL). The mechanism of degradation of aliphatic as well as aliphatic-aromatic co-polyesters has also been discussed. The degradation ability of microorganisms against various polyesters might be useful for the treatment and recycling of biodegradable wastes or bioremediation of the polyester-contaminated environments.

Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable plastics (BPs) such as agricultural mulch films composed of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA). An enzyme named PaE was isolated and the gene encoding PaE was cloned from the strain by functional complementation in Saccharomyces cerevisiae. The deduced amino acid sequence of PaE contains 198 amino acids with a predicted molecular weight of 20,362.41. High identity was observed between this sequence and that of cutinase-like enzymes (CLEs) (61-68%); therefore, the gene encoding PaE was named PaCLE1. The specific activity of PaE against emulsified PBSA was 54.8+/-6.3 U/mg. In addition to emulsified BPs, PaE degraded solid films of PBS, PBSA, poly(epsilon-caprolactone), and poly(lactic acid).

wo bacteria, Burkholderia cepacia PBSA-1 and Pseudomonas aeruginosa PBSA-2, capable of degrading poly(butylene succinate-co-butylene adipate) (PBSA) were isolated from activated sludge soil and cultivating soil in Korea by using the enrichment culture and the clear zone test at 27 C and 37 C. In this study, two bacteria showed a very high activity for PBSA degradation so that 78% of PBSA with an initial concentration of 0.01% was mineralized into CO2 during 40 days of the modified Sturm test. To analyze the PBSA-degrading enzyme encoding gene, lip A, PCR, cloning and sequencing were performed on the PBSA-degrading strains. The lip A appeared at about 1.5 kb. The amino acid sequences possessed the lipase box, Gly-His-Ser-Gln-Gly and sequences of the two strains received their accession number of EF189714 and EF189715, respectively, from GenBank. The lip A showed 87.9% homology between those of B. cepacia PBSA-1 and P. aeruginosa PBSA-2. Also they showed 83.2-86.9% and 85.9-89.7% homology compared with the Pseudomonas lipase subfamily, respectively. However, the genes of B. cepacia PBSA-1 and P. aeruginosa PBSA-2 exhibited no more than 22.9-26.9% and 21.3-23.5% of homology, respectively, when compared with the previously reported bioplastics-degrading enzyme encoding genes. Consequently, the PBSA-degrading enzyme lipase gene, lip A, was presumed to be a characteristic gene distinct from other bioplastics-degrading enzyme encoding genes.

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.

Poly(tetramethylene succinate-co-tetramethylene adipate) (PBSA) and poly(tetramethylenesuccinate) (PBS) were hydrolyzed experimentally into water-soluble oligomers and monomers by Chromobacterium extracellular lipase. The oligomers were identified by high-performance liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance, which indicated that a total of 28 oligomer species were liberated from PBSA, and that 13 of them were identical to the hydrolysates from PBS. Moreover, 20 of the species were polyester-based compounds of monomer units, and the other 8 species were small amounts of diurethane compounds. Bis(hydroxybutyl) succinate (BSB) and bis(hydroxybutyl) hexamethylene dicarbamate (BHB) were the typical oligomers and were chemically synthesized. Biodegradability of BSB and BHB was examined for 28 d in the activated sludge, and analysis of the results of this study indicated that the final conversion rate of constituent carbon to carbon dioxide was estimated at 80 mol% for BSB and 10 mol% for BHB. The remaining amount of carbon in the undegraded BHB was 20 mol%. In the presence of BSB, the biodegradability of BHB was increased by about 1.5 times. The suggestion was made that BSB induced a growth of microorganisms and helped BHB degradation. This is consistent with the observation that the biodegradation of BHB in native soil for 60 d reached > 60%.