Hydrolysis of polyesters by serine proteases

The substrate specificity of -chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(-hydroxybutyrate) (PHB), poly(ethylene succinate) (PES), poly(ethylene adipate) (PEA), poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBS/A), poly[oligo(tetramethylene succinate)-co-(tetramethylane carbonate)] (PBS/C), and poly(-caprolactone) (PCL). -Chymotrypsin could degrade PLA and PEA with a lower activity on PBS/A. Proteinase K and subtilisin degraded almost all substrates other than PHB. Trypsin and elastase had similar substrate specificities to -chymotrypsin.     

Isolation and Characterization of Polyester-Based Plastics-Degrading Bacteria from Compost Soils

Four potential polyester-degrading bacterial strains were isolated from compost soils in Thailand. These bacteria exhibited strong degradation activity for polyester biodegradable plastics, such as polylactic acid (PLA), polycaprolactone (PCL), poly-(butylene succinate) (PBS) and polybutylene succinate-co-adipate (PBSA) as substrates. The strains, classified according to phenotypic characteristics and 16S rDNA sequence, belonging to the genera Actinomadura, Streptomyces and Laceyella, demonstrated the best polyester- degrading activities. All strains utilized polyesters as a carbon source, and yeast extract with ammonium sulphate was utilized as a nitrogen source for enzyme production. Optimization for polyester-degrading enzyme production by Actinomadura sp. S14, Actinomadura sp. TF1, Streptomyces sp. APL3 and Laceyella sp. TP4 revealed the highest polyester-degrading activity in culture broth when 1% (w/v) PCL (18 U/mL), 0.5% (w/v) PLA (22.3 U/mL), 1% (w/v) PBS (19.4 U/mL) and 0.5% (w/v) PBSA (6.3 U/mL) were used as carbon sources, respectively. All strains exhibited the highest depolymerase activities between pH 6.0–8.0 and temperature 40–60°C. Partial nucleotides of the polyester depolymerase gene from strain S14, TF1 and APL3 were studied. We determined the amino acids making up the depolymerase enzymes had a highly conserved pentapeptide catalytic triad (Gly-His-Ser-Met-Gly), which has been shown to be part of the esterase-lipase superfamily (serine hydrolase).     

Microbial degradation of aliphatic and aliphatic-aromatic co-polyesters

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(ε-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.     

Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

Study on the enzymatic degradation of PBS and its alcohol acid modified copolymer

Enzymatic hydrolytic degradation of polybutylene succinate (PBS), poly(polybutylenesuccinate-co-1,4-cyclohexane dimethanol) (PBS/CHDM) and poly(polybutylene succinate-co-diglycolic acid) (PBS/DGA) in mixed solvent of tetrahydrofuran (THF) and toluene was examined. Lipase was used as catalyst to degrade polymers with molecular weight of more than 100,000, and the molecular weight of products ranged from hundreds to thousands. Thermal decomposition temperatures of all products were below 250°C. The degradation products of both PBS/CHDM and PBS/DGA showed two melting points at about 85 and 99°C. Mass spectrometry (MS) was employed to obtain the molecular weight of oligomers extracted from the products, which proved to be low-polyesters with the molecular weight of less 1,000. The butanediol (BDO) monomer was found in PBS/CHDM degradation product for the first time.     

Purification,characterization, and cloning of the gene for a biodegradable plastic-degrading enzyme from Paraphoma-related fungal strain B47-9

Paraphoma-related fungal strain B47-9 secreted a biodegradable plastic (BP)-degrading enzyme which amounted to 68 % (w/w) of the total secreted proteins in a culture medium containing emulsified poly(butylene succinate-co-adipate) (PBSA) as sole carbon source. The gene for this enzyme was found to be composed of an open reading frame consisting of 681 nucleotides encoding 227 amino acids and two introns. Southern blot analysis showed that this gene exists as a single copy. The deduced amino acid sequence suggested that this enzyme belongs to the cutinase (E.C.3.1.1.74) family; thus, it was named P araphoma-related fungus cutinase-like enzyme (PCLE). It degraded various types of BP films, such as poly(butylene succinate), PBSA, poly(butylene adipate-co-terephthalate), poly(ε-caprolactone), and poly(dl-lactic acid). It has a molecular mass of 19.7 kDa, and an optimum pH and temperature for degradation of emulsified PBSA of 7.2 and 45 °C, respectively. Ca²⁺ ion at a concentration of about 1.0 mM markedly enhanced the degradation of emulsified PBSA.     

Degradation of polycaprolactone at 50 °C by a thermotolerant Aspergillus sp.

A thermotolerant Aspergillus sp. strain ST-01 degrading poly(-caprolactone) films was isolated. The polyester was degraded and assimilated giving 36 mg of cell from 100 mg sample and 10 mg yeast extract after 6 days at 50 °C. The degradation products were identified as succinic acid, butyric acid, valeric acid, and caproic acid. The isolate also degraded more than 90% film samples of polyhydroxybutyrate (PHB) and poly(tetramethylene succinate-co-tetramethylene adipate) at 50 °C.     

Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization

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(ε-caprolactone), and poly(lactic acid).     

Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics

Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.     

Degradation of poly(tetramethylene succinate) by thermophilic actinomycetes

Various thermophilic actinomycetes were screened for their ability to degrade a high melting point, aliphatic polyester, poly(tetramethylene succinate) (PTMS), at 50 °C. By using the clear zone method, Microbispora rosea, Excellospora japonica and E. viridilutea were found to have PTMS-degrading activity. In a liquid culture with 100 mg PTMS film, M. rosea subsp. aerata IFO 14046 degraded about 50 mg film sample after 8 days. Degradation at the amorphous regions of the PTMS film was observed by scanning electron microscopy. This strain was also able to completely degrade poly(-caprolactone).     

Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

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.This revised version was published online in February 2005 with corrections to Table 1.     

Blends of aliphatic polyesters. VI. Lipase-catalyzed hydrolysis and visualized phase structure of biodegradable blends from poly(epsilon-caprolactone) and poly(L-lactide)

Phase-separated biodegradable polymer blends were prepared from poly(epsilon-caprolactone) (PCL) and poly(L-lactide) (PLLA), and Rhizopus arrhizus lipase-catalyzed hydrolysis and phase structure of the blend films were investigated. Gravimetry revealed that the lipase-catalyzed hydrolysis of PCL in PCL- and PLLA-rich phases is disturbed by the presence of PLLA. Polarimetry confirmed the occurrence of a predominant hydrolysis of PCL and subsequent removal of the hydrolyzed water-soluble PCL oligomers in the blend films. Gravimetry and gel permeation chromatography of the non-blended PLLA film indicated that R. arrhizus lipase has no catalytic effect on the hydrolysis of PLLA. The phase structure of the blend films could be visualized by selective enzymatic removal of one component and subsequent scanning electron microscopic observation.     

Depolymerisation and biodegradation of a synthetic tanning agent by activated sludges, the bacteria Arthrobacter globiformis and Comamonas testosteroni, and the fungus Cunninghamella polymorpha

Degradation of a synthetic tanning agent CNSF (a condensation product of 2-naphthalenesulfonic acid (2-NSA) and formaldehyde) by four activated sludges, two previously characterised bacterial strains, Arthrobacter sp. 2AC and Comamonas sp. 4BC, and the fungus Cunninghamella polymorpha, was studied in batch culture at 25°C by determining the changes in the concentrations of CNSF and its component monomers and oligomers (n2–n11). The loss of individual oligomers was correlated with the length of the NSA-CH₂ chain. Approximately 25% of the total CNSF was degraded (i.e. mineralised) by the microbes contained in the four activated sludges and by the two bacterial isolates but with different lag phases and at different overall rates. The decline in CNSF concentration was due almost entirely to the biodegradation of the monomers (34.3% of CNSF) and, in particular, 2-NSA (27% of CNSF). There was no change in the n2–n11 components. The growth of C. polymorpha, on the other hand, arose from extracellular depolymerisation of CNSF oligomers and the biodegradation of the lower molecular mass products. Between 38% and 42% of total CNSF was degraded by C. polymorpha at 25°C. The order of oligomer degradation was inversely related to degree of polymerisation. Eighty percent and 90% of the n4 and n5 and 100% oligomers n6–n11 were degraded after 120h. At a higher temperature (37°C) oligomers n4–n11 were degraded completely after 120h. A combination of biodegradation (75%) and sorption to fungal biomass (25%) accounted for the measured loss of all oligomers from the solution phase. The CNSF degradation rates and the volume of fungal biomass produced (and therefore the extent of biosorption) were dependent on the presence of a second carbon source (both optimum at glucose 5g/l). This is the first report that identifies and distinguishes between depolymerisation, sorption and biodegradation processes in the removal of CNSF and its component oligomers. The use of combinations of the depolymerising fungus C.polymorpha, and the monomer-degrading bacteria, Arthrobacter sp. 2AC and Comamonas sp. 4BC, have potential for wastewater treatment.     

Asp30 of Aspergillus oryzae cutinase CutL1 is involved in the ionic interaction with fungal hydrophobin RolA

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.     

Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis

Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca²⁺ or Mg²⁺ at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).     

Improved activity of a lipase by vacuum drying on to a hydrophobic microporous support

Summary Lipase from Rhizopus arrhizus was immobilized by physical adsorption on hydrophobic microporous polypropylene supports. The immobilized enzyme catalyst was employed for the hydrolysis of palm kernel olein in the presence of n-hexane. The initial rate of lipolysis for vacuum dried immobilized lipase is nearly double that of air dried. The initial rate of lipolysis declines with increase of drying time. Immobilized lipase clearly reveals a relatively high initial rate after 30 days of storage at 4 °C. Stability of the immobilized lipase in buffer could be enhanced up to three-fold that of the free lipase.     

Involvement of hydrophobic amino acid residues in C7–C8 loop of Aspergillus oryzae hydrophobin RolA in hydrophobic interaction between RolA and a polyester

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. The Cys3–Cys4 and Cys7–Cys8 loops of hydrophobins are thought to form hydrophobic segments involved in adsorption of hydrophobins on hydrophobic surfaces. When the fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate-co-adipate (PBSA), A. oryzae produces hydrophobin RolA, which attaches to PBSA. Here, we analyzed the kinetics of RolA adsorption on PBSA by using a PBSA pull-down assay and a quartz crystal microbalance (QCM) with PBSA-coated electrodes. We constructed RolA mutants in which hydrophobic amino acids in the two loops were replaced with serine, and we examined the kinetics of mutant adsorption on PBSA. QCM analysis revealed that mutants with replacements in the Cys7–Cys8 loop had lower affinity than wild-type RolA for PBSA, suggesting that this loop is involved in RolA adsorption on PBSA.     

Limiting concentrations of activated mononucleotides necessary for poly(C)-directed elongation of oligoguanylates

Summary Selected imidazolide-activated nucleotides have been subjected to hydrolysis under conditions similar to those that favor their template-directed oligomerization. Rate constants of hydrolysis of the P–N bond in guanosine 5-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5-monophosphate imidazolide (ImpG), k_h, have been determined in the presence/absence of magnesium ion as a function of temperature and polycytidylate [poly(C)] concentration. Using the rate constant of hydrolysis of 2-MeImpG and the rate constant of elongation, i.e., the reaction of an oligoguanylate with 2-MeImpG in the presence of poly(C) acting as template, the limiting concentration of 2-MeImpG necessary for oligonucleotide elongation to compete with hydrolysis can be calculated. The limiting concentration is defined as the initial concentration of monomer that results in its equal consumption by hydrolysis and by elongation. These limiting concentrations of 2-MeImpG are found to be 1.7 mM at 37°C and 0.36 mM at 1°C. Boundary conditions in the form of limiting concentration of activated nucleotide may be used to evaluate a prebiotic model for chemical synthesis of biopolymers. For instance, the limiting concentration of monomer can be used as a basis of comparison among catalytic, but nonenzymatic, RNA-type systems.We also determined the rate constant of dimerization of 2-MeImpG, k₂=0.45±0.06 M^–1 h^–1 in the absence of poly(C), and 0.45±0.06k₂0.97±0.13 M^–1 h^–1 in its presence at 37°C and pH 7.95. This dimerization, as well as the trimerization of 2-MeImpG, which represent the first steps in the oligomerization reaction, are markedly slower than the elongation of longer oligoguanylates, (pG) _n n>6. This means that in the presence of low concentrations of 2-MeImpG (1.7 mM) the system directs the elongation of longer oligomers more efficiently than the formation of short oligomers such as dimers and trimers. These results will be discussed as a possible example of chemical selection in template-directed reactions of nucleotides.     

Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei

During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K _m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 g polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues. Correspondence to: D. Jendrossek