Gary T. Howard

Biodegradation of polyurethane: a review

Lack of degradability and the closing of landfill sites as well as growing water and land pollution problems have led to concern about plastics. Increasingly, raw materials such as crude oil are in short supply for the synthesis of plastics, and the recycling of waste plastics is becoming more important. As the importance of recycling increases, so do studies on elucidation of the biodegradability of polyurethanes. Polyurethanes are an important and versatile class of man-made polymers used in a wide variety of products in the medical, automotive and industrial fields. Polyurethane is a general term used for a class of polymers derived from the condensation of polyisocyanates and polyalcohols. Despite its xenobiotic origins, polyurethane has been found to be susceptible to biodegradation by naturally occurring microorganisms. Microbial degradation of polyurethanes is dependent on the many properties of the polymer such as molecular orientation, crystallinity, cross-linking and chemical groups present in the molecular chains which determine the accessibility to degrading-enzyme systems. Esterase activity (both membrane-bound and extracellular) has been noted in microbes which allow them to utilize polyurethane. Microbial degradation of polyester polyurethane is hypothosized to be mainly due to the hydrolysis of ester bonds by these esterase enzymes.

Growth of Pseudomonas fluorescens on a polyester–polyurethane and the purification and characterization of a polyurethanase–protease enzyme

A Pseudomonas fluorescens was found to degrade and utilize a polyester polyurethane as a sole carbon and energy source. Polyurethane utilization by P. fluorescens. followed simple Michaelis–Menten kinetics. The Ks and μmax values were 0.9 mg ml−1 and 1.61 doublings · h−1, respectively. The enzymes from P. fluorescens responsible for polyurethane degradation were found to be extracellular. Analysis of the polyurethane degrading proteins using non-denaturing polyacrylamide gel electrophoresis revealed one active protein band with an Rf value of 0.083. A polyurethane degrading enzyme was purified and displayed protease activity. This enzyme was inhibited by phenylmethylsulfonyl fluoride and had a molecular weight of 29,000 daltons.

Growth of Pseudomonas chlororaphis on apolyester–polyurethane and the purification andcharacterization of a polyurethanase–esterase enzyme

Abstract A Pseudomonas chlororaphis was found to degrade and utilize apolyester polyurethane as a sole carbon and energy source. Polyurethane utilization by P.chlororaphis followed simple Michaelis–Menten kinetics. The K s and μ max values were 0.802 mg·ml −1 and 1.316 doublings·h −1 , respectively. The enzymes from P. chlororaphis responsible for polyurethanedegradation were found to be extracellular. Analysis of the polyurethane degrading proteins, usingnon-denaturing polyacrylamide gel electrophoresis, revealed three active protein bands with R f values of 0.25, 0.417 and 0.917. A polyurethane degrading enzyme was purifiedand displayed esterase activity. This enzyme was inhibited by phenylmethylsulfonyl fluoride andhad a molecular weight of 27,000 daltons.

Growth of Bacillus subtilis on polyurethane and the purification and characterization of a polyurethanase-lipase enzyme

Abstract A soil microorganism capable of degrading polyurethane was isolated from a mesocosm study. This organism was identified as Bacillus subtilis through 16s rRNA sequencing. The ability of this organism to degrade polyurethane was characterized by the measurement of its growth kinetics and the purification and characterization of a polyurethane degrading enzyme. The growth kinetics for this microorganism was obtained on two substrates: Impranil DLNTM and tributyrin. The growth of B. subtilis on polyurethane at certain concentrations (1.5– 0.18 mg ml −1 ) and tributyrin at all concentrations followed simple Monod growth kinetics. Lineweaver–Burke analysis of the data was performed to yield a μmax value of 0.7626±0.04799 doublings h −1 and Ks value of 0.08559±0.03291 mg ml −1 for Impranil DLNTM and a μmax value of 0.46003±0.05407 doublings h −1 and Ks value of 0.04065±0.2336 μM for tributyrin. At higher concentrations of Impranil DLNTM (9.0– 3.0 mg ml −1 ) Monod kinetics were not observed due to the binding of polyurethane to the cell wall of the B. subtilis. The purified protein as determined by SDS-PAGE has a molecular weight of approximately 28 kDa . Substrate specificity was examined using p-nitrophenyl substrates with varying carbon lengths. The highest substrate specificity was observed for p-nitrophenyl-acetate with an activity of 45 U mg −1 . Additionally, the enzyme is heat labile and not inhibited by phenylmethylsulfonylfluoride (PMSF), adenylmethylsulfonylfluoride (AMSF), ethylenediamine-tetra acetic acid (EDTA), or Bromelain.

Cloning and expression in Escherichia coli of apolyurethane-degrading enzyme from Pseudomonasfluorescens

Abstract A polyester polyurethane (PU)-degrading enzyme, PU esterase, derived from Pseudomonas fluorescens, a bacterium that utilizes polyester PU as the sole carbon source,was purified to homogeneity as indicated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. This enzyme was a soluble, extracellular protein with a molecular mass of 48 kDa and was inhibited by phenylmethylsulfonylfluoride (PMSF). A genomic library of Ps.fluorescens was constructed using the Escherichia coli bacteriophage l vector lZAPII. A recombinant phage exhibiting activity against Impranil DLN was isolated. The geneencoding the polyurethanase (PUase) protein was subcloned into a plasmid expression vectorpT7-6 and expressed in E. coli. Upon expression, the PUase was secreted by the host,displayed esterase activity which was inhibited by PMSF, and in vivo 35S-methionine labeling of the gene product encoded by the open reading frame of the clone insertrevealed a single polypeptide with a molecular mass of 48 kDa.

Purification and characterization of twopolyurethanase enzymes from Pseudomonas chlororaphis

Two polyester polyurethane (PU)-degrading enzymes from Pseudomonaschlororaphis, a bacterium that utilizes polyester PU as the sole carbon and energy source,were purified to electrophoretic homogeneity as indicated by sodium dodecyl-polyacrylamide gelelectrophoresis (SDS–PAGE). Both enzymes are extracellular, soluble proteins with molecularweight of 63,000 Da and 31,000 Da. The 63,000 Da protein exhibits both esterase and proteaseactivities toward r-nitrophenylacetate and hide powder azure respectively. The enzyme has anoptimum pH of 8.5 for esterase activity and an optimum pH of 7.0 for protease activity. The31,000 Da protein exhibits esterase activity toward r-nitrophenylacetate, butyrate and propionate,and has an optimum pH of 8.5. In addition, the enzyme activities of both proteins are heat stableafter 10 min at 100°C and are inhibited 50% by the addition of 1 mMphenylmethylsulfonylfluoride indicating both are serine-hydrolases.

Purification and characterization of a solublepolyurethane degrading enzyme from Comamonasacidovorans

Abstract A soluble esterase involved in the biodegradation of polyester polyurethane (PU) waspurified to apparent electrophoretic homogeneity in high yield, ∼83%. The enzyme displayed asingle band on both non-denaturing (ND-) and sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) with an apparent molecular mass of 42 kDa. Using ρ -nitrophenylacetate as the substrate, the enzyme displayed steady-state kinetic parameters K m and V max of 51.5 mM and 180 U mg −1 respectively. Esteraseactivity was thermally stable and could be inhibited with phenylmethylsulfonylfluoride (PMSF)and soybean trypsin inhibitor (STI).

Adherence and growth of a Bacillus species on an insoluble polyester polyurethane

Abstract Laser Doppler velocimetry, electrical impedance, and static light scattering measurements were employed to quantify the growth of a Bacillus species at the expense of a heterogeneous, insoluble polyester polyurethane. Progress of the culture was arbitrarily divided into 3 phases: adherence of polyurethane to the bacteria, bacterial multiplication, and aggregation of the bacteria with the remaining, undegraded polymer. Observations with all 3 instruments indicated that multiple binding of the small colloidal polyurethane to the bacterium occurred over the course of 4 to 5 hours. During this extended lag period, the average volume of the bacterium–polyurethane complex increased to nearly three times that of the bacterium alone. The Bacillus cells then entered an exponential phase of growth with a doubling time of 55 minutes. Beyond 8 hours, the number of free bacteria in the culture declined concomitant with the appearance of large aggregrates composed of hundreds of cells and residual polyurethane. Further culture development ceased beyond 24 hours, leaving portions of the heterogeneous substrate unmetabolized. Phase contrast, scanning electron, and transmission electron microscopic observations provided qualitative corroboration of the significant events in the culture deduced from the instrumental analyses. These data demonstrate that physical measurements of colloidal suspensions may be used to study and quantify the complex interactions of bacteria with heterogeneous insoluble substrates.

Cloning, nucleotide sequencing and characterization of a polyurethanase gene (pueB) from Pseudomonas chlororaphis

Abstract A second gene (pueB, polyurethane esterase B) encoding an extracellular polyurethanase (PueB) was cloned from Pseudomonas chlororaphis into Escherichia coli. The recombinant polyurethanase showed esterase activity when assayed with various p-nitrophenyl substrates and lipase activity when assayed with triolein. Nucleotide sequencing of pueB showed an open reading frame of 1695 bp encoding a 60-kDa protein of 565 amino acid residues, with the serine hydrolase consensus sequence GXSXG and a C-terminal secretion signal (G-G-X-G-X-D-X-X-X). Unlike the PueA polyurethanase, PueB contains a putative N-terminal signal peptide. Comparison between the amino acid and nucleotide sequences of these two genes revealed that they share 42% and 59% identity respectfully. Parsimony analysis of the predicted amino acid sequences for the PueB, PueA, and other polyurethanase enzymes and similar lipase enzymes was performed. Interestingly the polyurethanase enzymes do not form a single cluster, but appear to be distributed among multiple lineages. These analyses suggest that the polyurethanase enzymes thus far studied have evolved from lipases, and are not derived from a single source.

Sensitive plate assay for screening and detection of bacterial polyurethanase activity

G.T. HOWARD, J. VICKNAIR AND R.I. MACKIE. 2001.