Biodegradation of polyhydroxyalkanoic acids

Stimulated by the commercial availability of bacteriologically produced polyesters such as poly[(R)-3-hydroxybutyric acid], and encouraged by the discovery of new constituents of polyhydroxyalkanoic acids (PHA), a considerable body of knowledge on the metabolism of PHA in microorganisms has accumulated. The objective of this essay is to give an overview on the biodegradation of PHA. The following topics are discussed: (i) general considerations of PHA degradation, (ii) methods for identification and isolation of PHA-degrading microorganisms, (iii) characterization of PHA-degrading microorganisms, (iv) biochemical properties of PHA depolymerases, (v) mechanisms of PHA hydrolysis, (vi) regulation of PHA depolymerase synthesis, (vii) molecular biology of PHA depolymerases, (viii) influence of the physicochemical properties of PHA on its biodegradability, (ix) degradation of polyesters related to PHA, (x) biotechnological aspects of PHA and PHA depolymerases. Received: 28 May 1996 / Received revision: 5 August 1996 / Accepted: 12 August 1996     

Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35

A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2% N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45 degrees C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity when p-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.     

A second polycaprolactone depolymerase from Fusarium, a lipase distinct from cutinase

Polycaprolactone (PCL), a synthetic polyester with applications in biodegradable plastics, is degraded by a variety of microorganisms, including fungal phytopathogens. These pathogens secrete cutinase, which hydrolyzes cutin, the polyester structural component of plant cuticle, releasing ω-hydroxy fatty acids that induce cutinase synthesis. Our laboratory previously reported that growth of Fusarium solani on PCL requires cutinase, which is active as a PCL depolymerase and induced by the products of its action on PCL. A mutant strain of F. solani in which the cutinase gene is deleted was unable to grow on PCL and did not secrete PCL depolymerase activity in the media tested. It is now shown that this mutant produces a PCL depolymerase in media containing lipase inducers. Wild-type strains also produce this second PCL depolymerase, which is induced by Tween 80 and tributyrin, but not by PCL or cutin. The second depolymerase shows interfacial activation, indicating that it is a lipase. PCL may thus be a substrate but not an inducer of depolymerases that degrade it, and screening microorganisms on medium with PCL as the sole source of carbon and energy may fail to reveal strains with active PCL depolymerases, because of the absence of an inducer. Surprisingly, Tween 80 induces both cutinase and lipase activities in wild-type F. solani. Received: 31 March 1998 / Received revision: 27 July 1998 / Accepted: 8 August 1998     

Biotechnology and microbiology of coal degradation

For several years it has been known that fungi and bacteria can attack and even liquefy low rank coals. This review covers the progress in coal biotechnology and microbiology, mainly during the last decade, from describing the first effects to elucidating the mechanisms used by the microorganisms. More than one mechanism is responsible for microbial coal degradation/liquefaction: oxidative enzymes (peroxidases, laccases), hydrolytic enzymes (esterases), alkaline metabolites and natural chelators. Due to the heterogeneous structure of coal, which is described in one section, and for economic reasons the review focuses on the enzymatic depolymerization of brown coal. Approaches which seem not so promising are discussed (anaerobic, reductive pathways, chemical pretreatment). Finally the possible applications and products in this field are summarized, as lignite with a worldwide production of about 940 million tons a year will continue to play an important economic role in the future. Received: 19 October 1998 / Received revision: 16 December 1998 / Accepted: 21 December 1998     

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agar-plate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 °C respectively. Received: 2 February 1998 / Received revision: 20 April 1998 / Accepted: 27 April 1998     

A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria as inclusion bodies, and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. In this paper, Fourier-transform infrared spectroscopy (FT-IR) was used to carry out rapid qualitative analysis of PHA in intact bacterial cells. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mclHA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, showed their strong characteristic band at 1728 cm⁻¹, 1740 cm⁻¹ or 1732 cm⁻¹ respectively. Other accompanying bands near 1280 cm⁻¹ and 1165 cm⁻¹ helped identify the types of PHA. The intensity of the methylene band near 2925 cm⁻¹ provided additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm⁻¹, 1744 cm⁻¹ or 1739 cm⁻¹, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. The FT-IR results were further confirmed by gas chromatography analysis. Received: 14 October 1998 / Received revision: 29 December 1998 / Accepted: 30 December 1998     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

Degradation of alicyclic molecules by Rhodococcus ruber CD4

The present work describes investigations on the bacterial degradation of the alicyclic molecule cyclododecane. It represents a structure where the initial degradative steps have to be similar to a “subterminal” attack as there is no “terminal” part of the molecule. We were able to show that the gram-positive bacterium Rhodococcus ruber CD4 DSM 44394 oxidizes cyclododecane to the corresponding alcohol and ketone, the latter being subject to ring fission by a Baeyer-Villiger oxygenase. This key enzyme is an NADPH- and O₂-dependent flavoprotein with a substrate specificity for bigger rings. The further metabolism of the resulting lactone gives rise to an ω-hydroxyalkanoic acid that is susceptible to common β-oxidation. Due to its alicyclic character and its ring size, cyclododecane is comparable to aliphatic bridge components that are an important element in the coal texture. They contribute to the three-dimensional coal structure and thus could serve as a valuable target for the oxidative abilities of R. ruber CD4 to reduce the molecular mass of coal. Received: 2 July 1998 / Received revision: 27 October 1998 / Accepted: 30 October 1998     

Poly(hydroxyalkanoate) biosynthesis from triglyceride substrates

The biosynthesis of poly(hydroxyalkanoates) (PHA) by Pseudomonas resinovorans from triglyceride substrates was investigated. Each triglyceride, whether animal fat or vegetable oil, supported cellular growth to relatively high average cell yields (3.3 ± 0.2 g/l). PHA yields ranged from 1.1 g/l to 2.1 g/l, representing approximately 45% of the bacterial cell dry weight. The repeat-unit composition of the polymers was determined by gas chromatography (GC) and GC/mass spectrometry of the β-hydroxyalkanoate methyl esters from the hydrolyzed polymers. With the exception of PHA from soybean oil (PHA-soy), each polyester was composed of β-hydroxyacyl moieties with chain lengths ranging from C₄ to C₁₄, with C₈ and C₁₀ being the predominant species. PHA-soy contained an additional fraction (2%) of C₁₆ monomers. The alkyl side-chains of the PHA contained varying degrees of unsaturation. PHA from coconut oil was composed entirely of saturated side-chains, whereas PHA-soy contained 4.2 mol% olefinic groups in its side-chains. The increase in the degree of side-chain unsaturation caused decreased melting temperatures, enthalpies of fusion, and glass transition temperatures. The molar masses of the polymers were relatively constant and ranged from 6.5 × 10⁴ to 10.1 × 10⁴ g/mol. Received: 2 September 1997 / Received revision: 21 November 1997 / Accepted: 2 January 1998     

Characterisation of a starch-hydrolysing enzyme of Aspergillus niger

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 °C and glucose, maltose and maltodextrins at 70 °C as primary products, suggested significant applications for the enzyme in starch-processing industries. Received: 29 October 1998 / Received revision: 11 January 1999 / Accepted: 19 January 1999