Polyurethane Degradation by Pseudomonas

Three Pseuomonads have been isolated for their ability to utilize a polyester PU as the sole carbon and energy source. Interestingly, three species of bacteria produce

different PUase activities that are inhibited by serine hydrolase inhibitors. These data suggest that either esterase and/or protease activities are involved in the degradation of Impranil (Fig. 14.4).

Growth of Comamonas acidovorans on colloidal polyester-polyurethane resulted in the growth parameters for Ks and imax of 0.3 mg ml-1 and 0.7 doublings h-1, respectively (Allen et al. 1999). A 42 kDa PUase enzyme displaying esterase/protease activity has been purified and characterized (Allen et al. 1999). Nakajima-Kambe et al. (1995, 1997) reported a strain of C. acidovorans that could utilize solid polyester PU as the sole carbon and nitrogen source. These authors indicated the role of an extracellular membrane bound esterase activity in PU degradation. Purification of the membrane bound esterase revealed a thermally labile protein having a 62 kDa molecular mass (Akutsu et al. 1998). C. acidovorans strain TB-35 was isolated from the soil samples for its ability to degrade polyester PU (Nakajima-Kambe et al. 1995). Solid cubes of polyester PU were synthesized with various polyester segments. The cubes were completely degraded after 7 days incubation when they were supplied as the sole carbon source and degraded 48% when they were the sole carbon and nitrogen source. Analysis of the breakdown products of the PU revealed that the main metabolites were derived from the polyester segment of the polymer. Gas chromatographic analysis revealed the metabolites produced were diethylene glycol, trimethylolpropane, and dimethy-ladipic acid. In agreement with these findings, Gautam et al. (2007) examined the biodegradation of polyester-polyurethane foam by P. chlororaphis ATCC 55729. Concentrations of ammonia and diethylene glycol increased over time with an increase of bacterial growth and a decrease in PU mass. A possible biodegradative pathway of PU is shown schematically (Fig. 14.5). Further analysis of strain i 1

Esterase j CH3

-O_C— N — R—N — C— O— C— C — O — C —C —O—C —C—C — C — C—C— O—C — C — c—O— Adipic Acid

Di-isocyanate

Diethylene Glycol

Diethylene Glycol

Adipic Acid

Trimethylol Propane

Dimethyl Adipic Acid Trimethylol Propane

Fig. 14.5 Theoretical degradative pathway of polyester-polyurethane by esterase activity of Pseudomonas

H2COH

TB-35 revealed that the degradation products from the polyester PU were produced by an esterase activity (Nakajima-Kambe et al. 1997). Strain TB-35 possesses two esterase enzymes, a soluble, extracellular and one membrane-bound. The membrane-bound enzyme was found to catalyze the majority of the polyester PU degradation. The membrane-bound PUase enzyme was purified and characterized (Akutsu et al. 1998). The protein has a molecular mass of 62 kDa, heat stable up to 65°C and is inhibited by PMSF. The structural gene, pudA, for the PU esterase was cloned in Escherichia coli. Upon nucleotide sequencing of the open reading frame (ORF), the predicted amino acid sequence contained a Gly-X -Ser-X-Gly motif characteristic of serine hydrolases. The highest degree of homology was detected with the Torpedo californica acetylcholinesterase (T ACh E), possessing the Ser-His-Glu catalytic triad, with the glutamate residue replacing the usual aspartate residue. Similarity in the number and positions of cysteine and salt bonds was very apparent between PudA and T AchE, as were also identities of sequences and their positions in the a-helix and b-strand regions between the two. In the neighborhood of the glutamate residue of the Ser199-His433-Glu324 catalytic domain of PudA, there were three hydrophobic domains, one of which constituted the surface-binding domain, which occurred in the C-terminus of most bacterial poly(hydroxyalkanoate)(PHA) depolymerases.

Growth of Pseudomonas fluorescens on PU resulted in values of 0.9 mg ml—1 and 1.6 doublings h—1 for Ks and imax, respectively (Howard and Blake 1999). Two PUase enzymes have been purified and characterized from this bacterial isolate, a 29 kDa protease (Howard and Blake 1999) and a 48 kDa esterase (Vega et al. 1999). In addition, to the enzymology of the Puases, the gene encoding a 48 kDa protein has been cloned and expressed in E. coli (Vega et al. 1999). The gene encoding PulA has been sequenced (Genbank, Accession AF144089). The deduced amino acid sequence has 461 amino acid residues and a molecular mass of 49 kDa. The PulA amino acid sequence showed high identity with Group I lipases (58-75%).

Growth of Pseudomonas chlororaphis on polyurethane resulted in values of 0.9 mg ml-1 and 1.3 doublings h-1 for Ks and imax, respectively (Ruiz et al. 1999a). Two PUase enzymes have been purified and characterized, a 65 kDa esterase/protease and a 31 kDa esterase (Ruiz et al. 1999b. A third PUase enzyme, 60 kDa esterase, has been partially purified and characterized (Ruiz et al. 1999a). Two genes encoding PUase activity from P. chlororaphis have been cloned in E. coli (Stern and Howard 2000; Howard et al. 2001). Both genes can be expressed in E. coli. However, the PueA enzyme is secreted in the recombinant E. coli and displays a beta-zone of clearing on polyurethane agar plates while PueB is not secreted in the recombinant E. coli and displays an alpha-zone of clearing on polyurethane agar plates. In addition, PueB has been noted to display esterase activity towards q-nitrophenylacetate, q-nitrophenylpropionate, q-nitrophenylbu-tyrate, q-nitrophenylcaproate, and q-nitrophenylcaprylate while PueA has been reported to display esterase activity only towards q-nitrophenylacetate and q-nitrophenylpropionate.

Upon cloning PueA (Stern and Howard 2000) and PueB (Howard et al. 2001) from P. chlororaphis in Escherichia coli, the recombinant proteins were noted to have a high homology to Group I lipases. This family of lipases and other serine hydrolases, are characterized by an active serine residue that forms a catalytic triad in which an aspartate or glutamate and a histidine also participate (Jaeger et al. 1994; Persson et al. 1989; Winkler et al. 1990). Sequence analysis of the two-polyurethanase genes revealed that both encoded proteins contain serine hydro-lase-like active site residues (G-H-S-L-G) and a C-terminal nonapeptide tandem called repeat in toxin (RTX), (G-G-X-G-X-D-X-X-X) repeated three times. Group I lipases lack an N-terminal signal peptide but instead contain a C-terminal secretion signal. The secretion of these enzymes occurs in one step through a three-component, ATP-binding cassette (ABC) transporter, Type I secretion system (Arpigny and Jaeger 1999). Proteins secreted by Type I systems typically exhibit two features: (1) an extreme C-terminal hydrophobic secretion signal located within the last 60 amino acids that is not cleaved as part of the secretion process and (2) roll structure stabilized by glycine-rich RTX motifs. The RTX repeats form a Ca2+ roll. These ions co-ordinated between adjacent coils of the motifs are thought to be important for proper presentation of the secretion signal to the secretion machinery, but their exact role is controversial.

Comparison between the amino acid and nucleotide sequences of these two genes revealed that they share 42 and 59% identity, respectfully (Table 14.2). Parsimony analysis of the predicted amino acid sequences for PueA, PueB, PudA, and PulA polyurethanase enzymes and similar lipase enzymes was also performed (Fig. 14.6). Interestingly the PUase enzymes do not form a single cluster, but appear to be distributed among multiple lineages (Howard et al. 2001). These analyses suggest that the PUase enzymes so far studied have evolved from lipases, and are not derived from a single source.

Howard et al. (2007) identified a gene cluster resembling a binding-protein-dependent ABC transport system in Pseudomonas chlororaphis in connection with PueA and PueB (Fig. 14.7). The identified ABC transport system

Table 14.2 Identity comparison of PueB and other serine hydrolases

Protein

Length

%Identity

Strain

Accession

(aa/nt)

(aa/nt)a

number

PueB

567/1704

100/100

Pseudomonas chlororaphis

EF175556

PueA

617/1801

42/59

Pseudomonas chlororaphis

EF175556

PulA

451/1353

24/41

Pseudomonas fluorescens

AF144089

PudA

548/1644

11/31

Comamonas acidovorans

AB009606

TliA

476/1428

26/40

Pseudomonas fluorescens B52

AF083061

LipA

613/1789

36/53

Serratia marcescens SM6

BAA02519

Lipase

617/1801

39/55

Pseudomonas sp. MIS38

BAA84997

LipApf33

476/1428

27/41

Pseudomonas fluorescens 33

BAA36468

Lipase

449/1338

25/39

Pseudomonas fluorescens SIK W1

JQ1227

a Amino acid and nucleotide identities were determined with Bioedit version 4.8.8 program a Amino acid and nucleotide identities were determined with Bioedit version 4.8.8 program

Fig. 14.6 Single most parsimonious tree inferred from the phylogenetic analysis of polyurethanases and lipases. The numbers above the branches depict total character support/ bootstrap support for each branch and node. Branch lengths reflect number of changes estimated along each branch

Fig. 14.6 Single most parsimonious tree inferred from the phylogenetic analysis of polyurethanases and lipases. The numbers above the branches depict total character support/ bootstrap support for each branch and node. Branch lengths reflect number of changes estimated along each branch

consists of three components: an ATPase-binding protein (ABC), an integral membrane protein (MFP), and an outer membrane protein (OMP). The ABC pathway has been shown to mediate translocation of an alkaline protease in Pseudomonas aeruginosa (Doung et al. 1994). Also, the ABC pathway has been shown to be involved in secretion of a lipase from Serratia marcescens

Pseudomonas fluorescens (GeneBank Accession # AF083061)

|ABC Protei^^ [Membrane Fusion Protei^^> | Outer Membrane [Thermostable Lipase*^

1736 bp 1736 bp 1445 bp 1430 bp

Pseudomonas fluorescens (GeneBank Accession # B015053)

ABC Protein Membrane Fusion Protein Outer Membrane Protein PspA PspB Thermostable Lipase

1751 bp 1334 bp 1334 bp 2957 bp 3119 bp 1430 bp

Pseudomonas chlororaphis (EF175556)

|ABC Protei^^ |Membrane Fusion Pretei^^- | Outer Membrane PspA Psp^^^j PspA PspB ^

1781 bp 1320 bp 1362 bp 1698 bp 2978 bp 3125 bp 1851 bp

Fig. 14.7 Comparison of the gene clusters from two strains of Pseudomonas fluorescens and the PUase gene cluster from Pseudomonas chlororaphis. The ABC Reporter Protein, Membrane Fusion Protein and Outer Membrane Protein are involved in Type I translocation of the extracellular protein. The PspA and PspB proteins are serine protease homologues

(Akatsuka et al. 1995), which is located separately from the lipase gene on the chromosome and secretes protease, lipase and S-layer proteins (Kawai et al. 1998). A gene cluster (accession number AF083061) was identified for an ABC transporter specific for a lipase in Pseudomonas fluorescens SIK W1 (Ahn et al. 1999) and a similar gene cluster (accession number AB015053) was identified in Pseudomonas fluorescens 33 for a lipase gene and two serine proteases (Kawai et al. 1999). Interestingly, when the two ABC exporter gene clusters of Pseudomonas fluorescens are compared to the ABC exporter gene cluster of the one found in Pseudomonas chlororaphis, a unique gene arrangement is observed (Fig. 14.7). It appears that the novel gene arrangement observed is a combination of the two P. fluorescens gene clusters, and may have resulted through either a rearrangement or an insert ional event between the two ABC gene clusters observed in P. fluorescens.

Further investigation of the gene cluster involved growth studies to compare the effects of a PueA deficient strain and a PueB deficient strain with the wild type strain in polyurethane utilization (Table 14.3). Pseudomonas chlororaphis wild type and its PueA derivatives when grown on 1% Impranil DLNTM YES medium exhibited a lag phase growth for the first 3 h and then was followed by logarithmic growth for 6 h. The wild type reached a cell density of 2.31 x 108 ± 0.87. The PueA mutant, P. chlororaphis pueA::Kanr, had an 80% decrease in cell number (4.66 x 107 ± 0.13), whereas both the complements, P. chlororaphis pueA::Kanr pPueA-1 and P. chlororaphis pPueA-1 had an increase in cell densities, 2.86 x 108 ± 0.09 (25% increase) and 3.85 x 108 ± 0.98 (65% increase), respectively. The results obtained from the cell densities of each strain were reflected in the growth kinetic studies. Values for Ks and imax for polyurethane utilization were elucidated by varying the Impranil concentration from 0.18 to

Table 14.3 Growth kinetic analysis of P. chlororaphis and its derivatives using polyurethane as the sole carbon source

Strain 1max Doubling time Ks Cell density

Table 14.3 Growth kinetic analysis of P. chlororaphis and its derivatives using polyurethane as the sole carbon source

Strain 1max Doubling time Ks Cell density

P. chlororaphis (wild type)

1.32

31.5

0.800

2.31

X

108

±

0.87

P. chlororaphis pueA::Kanr

1.09

38.2

0.917

4.66

x

107

±

0.13

P. chlororaphis pueA::Kanr

1.41

29.5

0.710

2.86

X

108

±

0.09

(pPueA-1)

P. chlororaphis (pPueA-1)

1.54

27.0

0.649

3.85

X

108

±

0.98

P. chlororaphis pueB::Kanr

1.19

34.9

0.893

2.35

X

108

±

0.148

P. chlororaphis pueB::Kanr

1.37

30.4

0.735

3.59

X

108

±

0.187

(pPueB-1)

P. chlororaphis (pPueB-1)

1.41

29.5

0.781

3.99

X

108

±

0.813

The concentrations of Impranil DLNTM used were: 9.0, 6.0, 3.0, 1.5, 0.75, 0.54, 0.375, and 0.18 mg ml-1 . Each concentration was prepared in triplicate

The concentrations of Impranil DLNTM used were: 9.0, 6.0, 3.0, 1.5, 0.75, 0.54, 0.375, and 0.18 mg ml-1 . Each concentration was prepared in triplicate

9.0 mg ml-1. P. chlororaphis wild type exhibited a imax of 1.32 whereas, the PueA insert ional mutant, P. chlororaphis pueA::Kanr, exhibited a imax of 1.09. It would be hypothesized that a deletion of the pueA gene would result in a decrease in growth rate. However, a large decrease in growth obtained from the insert ional mutant may indicate that PueA plays a major role as compared to PueB in polyurethane degradation by P. chlororaphis. When multiple copies of pueA gene were introduced into either the wild type, P. chlororaphis pPueA-1, a imax value of 1.54, or the mutant, P. chlororaphis pueB::Kanr, pPueA-1, a imax value of 1.41, was obtained. An increase in the growth rate seems plausible since more PueA produced from the added plasmid would reflect more polyurethane degraded, resulting in an increase in the amount of nutrients available to the cells.

The PueB mutant, P. chlororaphis pueB::Kanr, had a 18% decrease in cell number (2.35 x 108 ± 0.148) whereas, both the complement, P. chlororaphis pueB::Kanr pPueB-1 and P. chlororaphis pPueB-1 had an increase in cell densities, 3.59 x 108 ± 0.187 and 3.99 x 108 ± 0.813, respectively. The results obtained from the cell densities of each strain were reflected in the growth kinetic studies. Values for Ks and imax for polyurethane utilization were elucidated by varying the Impranil concentration from 0.18 to 9.0 mg ml-1. P. chlororaphis wild type exhibited a imax of 1.31. When multiple copies of the pueB gene were introduced into the wild type, P. chlororaphis pPueB-1, a imax value of 1.41 was obtained which was similar to the complement, P. chlororaphis pueB::Kanr pPueB-1, imax value of 1.37. An increase in growth rate seems plausible since more PueB produced would reflect more polyurethane degraded resulting in an increase in the amount of nutrients available to the cells. However, these values are small and may indicate that PueB plays a minor role as compared to PueA in polyurethane degradation by P. chlororaphis. The insertion mutant, P. chlororaphis pueB::Kanr, displayed a imax value of 1.19. Again, it would be hypothesized that the deletion of the pueB gene would result in a decrease in growth rate.

However, this small variation compared to the wild type suggests that degradation of polyurethane by P. chlororaphis may be more dependent on PueA.

14.5.3 Binding of Polyurethane by Polyurethanase Enzymes

Enzyme molecules can easily come in contact with water-soluble substrates thus allowing the enzymatic reaction to proceed rapidly. However, the enzyme molecules are thought to have an extremely inefficient contract with insoluble substrates (e.g. PU). In order to overcome this obstacle, enzymes that degrade insoluble substrates posses some characteristic that allows them to adhere onto the surface of the insoluble substrate (Van Tilbeurgh et al. 1986; Fukui et al. 1988; Hansen 1992).

The observations made by Akutsu et al. (1998) for the polyurethanase PudA indicate that this enzyme degrades PU in a two-step reaction: hydrophobic adsorption onto the PU surface followed by the hydrolysis of the ester bonds of PU. The PU esterase was considered to have a hydrophobic-PU-surface binding domain (SBD) and a catalytic domain. The SBD was shown to be essential for PU degradation. The structure observed in PudA has also been reported in PHA depoly-merase, which degrades PHA. PHA is insoluble polyester synthesized as a food reserve in bacteria. In PHA depolymerase enzymes, the hydrophobic SBD has been determined by amino acid sequence analysis and its various physicochemical and biological properties (Fukui et al. 1988; Shinomiya et al. 1997). Another class of enzymes that contain a SBD is cellulases. Several cellulase enzymes have been observed to contain three main structural elements: the hydrolytic domain, a flexible hinge region, and a C-terminus tail region involved in substrate binding (Knowles et al. 1987; Bayer et al. 1985; Langsford et al. 1987).

So far, only two types of PUase enzymes have been isolated and characterized: a cell associated, membrane bound PU-esterase (Akutsu et al. 1998) and soluble, extracellular PU-esterases (Ruiz et al. 1999b; Allen et al. 1999; Vega et al. 1999). The two types of PUases seem to have separate roles in PU degradation. The membrane bound PU-esterase would allow cell-mediated contact with the insoluble PU substrate while, the cell-free extracellular PU-esterases would bind to the surface of the PU substrate and subsequent hydrolysis. Both enzyme actions would be advantageous for the PU-degrading bacteria. The adherence of the bacteria cell to the PU substrate via the PUase would allow for the hydrolysis of the substrate to soluble metabolites which would then be metabolised by the cell. This mechanism of PU degradation would decrease competition between the PU-degrading cell with other cells and also allow for more adequate access to the metabolites. The soluble, extracellular PU-esterase would, in turn, hydrolyze the polymer into smaller units allowing for metabolism of soluble products and easier access for enzymes to the partially degraded polymer.

Studies addressing binding of PUase to soluble PU have been also performed. The equilibrium binding of Impranil DLN (polyester-polyurethane) to purified PueA from Pseudomonas chlororaphis was studied by kinetic exclusion assays

Fig. 14.8 Equilibrium binding of Impranil DLN to PueA. The concentration of occupied polyurethane binding sites present in different reaction mixtures of PueA and soluble Impranil DLN were determined by kinetic exclusion assays on a flow fluorimeter as described in the text. Each determination was expressed as a fraction of the total PueA in solution and plotted versus the concentration of free soluble polyurethane. Each datum represents the average of at least two determinations. The parameters for the curve drawn through the data were determined by nonlinear regression analysis using a one-site homogeneous binding model

Fig. 14.8 Equilibrium binding of Impranil DLN to PueA. The concentration of occupied polyurethane binding sites present in different reaction mixtures of PueA and soluble Impranil DLN were determined by kinetic exclusion assays on a flow fluorimeter as described in the text. Each determination was expressed as a fraction of the total PueA in solution and plotted versus the concentration of free soluble polyurethane. Each datum represents the average of at least two determinations. The parameters for the curve drawn through the data were determined by nonlinear regression analysis using a one-site homogeneous binding model conducted on a KinExA flow fluorimeter. Briefly, the KinExA comprises an immunoassay instrument that exploits an immobilized form of the polyurethane substrate to separate and quantify the fraction of unoccupied binding sites that remain in solution reaction mixtures of PueA and soluble polyurethane. In this case, the immobilized polyurethane was Bayhydrol 110 adsorption coated onto polystyrene beads, while the soluble polyurethane was Impranil DLN. The results of these binding studies are summarized in Fig. 14.6. Kinetic exclusion assays conducted with 6.6 ig ml-1 PueA in the absence of soluble polyurethane produced fluorescence signals of greater than 2.2 V with mvolt noise. In the presence of increasing concentrations of soluble Impranil DLN, the fluorescence signal attributed to PueA with unoccupied binding sites decreased to an extrapolated constant value at an infinitely high concentration of the soluble polyurethane that represented nonspecific binding to the beads. The fraction of soluble PueA that contained unoccupied polyurethane binding sites was calculated as the ratio of the difference between the fluorescence signal observed in the absence of Impranil DLN minus that observed in its presence, divided by the difference in fluorescence signals between zero and an infinitely highly high concentration of the soluble polyurethane.

The binding data in Fig. 14.8 were fit to a one-site homogeneous binding model with an apparent equilibrium dissociation constant of 220 ± 30 mg ml-1 Impranil DLN. Since both the soluble Impranil DLN and the immobilized Bayhydrol 110 are hydrolysable substrates for the active PueA enzyme, care was taken to perform individual measurements in such a manner as to minimize the time of exposure of the polyurethane substrates to the active PueA. Thus the PueA-Impranil DLN mixtures were assayed within two minutes of mixing, while the PueA captured on the immobilized Bayhydrol was exposed to the fluorescent labeling reagents and

Fig. 14.9 Electron micrographs of embedded Bayhydrol 110TM polyurethane particles. a Electron micrograph of polyurethane particles taken at a magnification of x 15,000. b Electron micrographs of Immunogold-labeled PueA (1:5,000,000,000 dilution of 0.83 mg ml-1 PueA) bound to embedded polyurethane particle at x15,000 magnification

Fig. 14.9 Electron micrographs of embedded Bayhydrol 110TM polyurethane particles. a Electron micrograph of polyurethane particles taken at a magnification of x 15,000. b Electron micrographs of Immunogold-labeled PueA (1:5,000,000,000 dilution of 0.83 mg ml-1 PueA) bound to embedded polyurethane particle at x15,000 magnification wash buffer within 4 min of the initial exposure of the hydrolase to the immobilized substrate. Control experiments demonstrated that much longer exposure times (at least 3-fold longer) were required before a time-dependent deterioration in individual fluorescence signals could be detected.

Electron micrographs were used in conjunction with the analysis of binding via the KinExA 3000, Kinetic Exclusion Assay unit. Grids were analyzed at high magnification and electron micrographs were produced from sections incubated in 1:5,000,000 PU and 1:5,000,000,000 PueA (Fig. 14.9). The TEM analysis of PueA, showed PueA to have a high affinity for the polyurethane substrate. Binding was found to be so extensive, that only the most dilute concentrations of PueA could be used to allow for visualization of areas with individual immunogold labeling.

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