Extracellular PHB Depolymerase Research Articles

Visualization of Enzymatic Degradation of Poly[(R)-3-hydroxybutyrate] Single Crystals by an Extracellular PHB Depolymerase

Single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with different molecular weights (Mn = 2500 and Mn = 48 000) were prepared in a mixture of chloroform and ethanol. The enzymatic de...

Heterogeneous kinetics of the enzymatic degradation of poly(β-hydroxyalkanoates)

The kinetics of the enzymatic degradation reactions of the naturally produced polyesters poly(β-hydroxybutyrate) (PHB) and poly(β-hydroxybutyrate- co-β-hydroxyvalerate) (PHBV) by appropriate depolymerases were studied. Specifically, the interaction between the extracellular PHB depolymerase of Pseudomonas lemoignei and both PHB powder and solution-cast films of PHBV was investigated. Kinetic analysis of titristatically and turbidimetrically determined rates of degradation has revealed that the observed degradation behaviour was inconsistent with classical Michaelis-Menten enzymatic kinetics. Depending upon the relative concentrations of enzyme and substrate, each exhibited a range of kinetic dependencies from zero order to first order. At high relative concentrations of enzyme, the reaction rate became inversely proportional to enzyme concentration. A new kinetic treatment was derived which predicted the observed variations in kinetic dependence on both enzyme and substrate, and it successfully described previously published kinetic data for this and other PHB depolymerases. The degradation of PHB by the PHB depolymerase of Aspergillus fumigatus M2A was investigated for comparison. While the specific rate constants of this enzyme differed from that of P. lemoignei, the general kinetic model was still applicable.

Effects of Solid-State Structures on the Enzymatic Degradability of Bacterial Poly(hydroxyalkanoic acids)

The effects of solid-state structures on the rate of enzymatic erosion for melt-crystallized films of bacterial poly(hydroxyalkanoic acids) (PHAs) have been studied at 37 °C and pH 7.4 in the aqueous solution of an extracellular PHB depolymerase from Alcaligenes faecalis. In this study, the following PHA homopolymer and copolymers were used; poly(3-hydroxybutyric acid) (P(3HB)), poly(3-hydroxybutyric acid-co-7 mol % 3-hydroxyvaleric acid), poly(3-hydroxybutyric acid-co-22 mol % 3-hydroxyvaleric acid), poly(3-hydroxybutyric acid-co-10 mol % 4-hydroxybutyric acid), poly(3-hydroxybutyric acid-co-4 mol % 3-hydroxyhexanoic acid), and poly(3-hydroxybutyric acid-co-11 mol % 3-hydroxyhexanoic acid). All PHA films were crystallized isothermally at temperatures of 60−140 °C for different periods from the melt. The crystallinity of the films of P(3HB) homopolymer ranged from 53 to 80%, while those of PHA copolymers ranged from 39 to 71%, determined by X-ray crystallography and depending on the crystallization condit...

Adsorption kinetics of bacterial PHB depolymerase on the surface of polyhydroxyalkanoate films

The kinetics of adsorption and hydrolysis by an extracellular PHB depolymerase from Alcaligenes faecalis were studied at 37°C on the surface of five types of polyhydroxyalkanoate (PHA) films. The films of poly[(R)-3-hydroxybutyrate](P(3HB)), poly(3-hydroxypropionate)(P(3HP)), and poly(4- hydroxybutyrate)(P(4HB)) were hydrolyzed by the enzyme, while the films of poly[(S)-2-hydroxypropionate)(P(2HP)) and poly(6-hydroxyhexanoate)(P(6HH)) were not eroded. The PHB depolymerase with binding and catalytic domains adsorbed on the surface of all PHA films used, and the adsorption kinetics were found to obey the Langmuir isotherm. The cross-area per one molecule of enzyme binding to the surface of PHA film was estimated to be 17 ± 8 (nm 2/molecule). It has been concluded that the binding domain of enzyme is non-specific for the binding to the surface of PHA films, while the active site in a catalytic domain is specific for the hydrolysis of PHA molecules

Effect of tacticity on enzymatic degradability of poly(β‐hydroxybutyrate)

AbstractFilms of synthetic poly(β‐hydroxybutyrate)s (PHB)s ranging from highly isotactic to moderately syndiotactic were cast from each of several polymer fractions isolated from ring‐opening polymerizations of racemic β‐butyrolactone and were subjected to degradation by the extracellular PHB depolymerases of the bacterium Pseudomonas lemoignei and the fungus Aspergillus fumigatus M2A. Films of bacterially produced PHB were also degraded for comparison. Gravimetric determinations of enzymatic degradation were measured relative to control experiments in which no depolymerase was added. Each sample was exposed to the same concentration of enzyme, as determined by the activity of the enzyme solutions towards bacterial PHB powder, and the degradation behavior varied strongly with the tacticity of the samples. Weight loss was greatest for the bacterial polymer, with films being completely degraded by either enzyme in 48 h. The degradation of the synthetic samples by both enzymes followed similar trends, although the fungal depolymerase effected less rapid degradation for all synthetic samples than did the bacterial enzyme. Of the synthetic PHB samples, those of intermediate tacticity (55–60% isotactic diads) exhibited the greatest rate of weight loss, while those most syndiotactic in nature (34–45% isotactic) degraded the most slowly. The observed rates are interpreted in terms of isotactic contents, tacticity sequence distributions, and sample crystallinities.

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases produced by Agrobacterium sp. K-03

A poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Agrobacterium sp. strain K-03, isolated from soil, secreted two PHB depolymerases into the culture fluid when it was cultivated on PHB, 3-hydroxybutyrate (3HB), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 43 mol% 3-hydroxyvalerate (P(3HB-co-43%3HV)) as the sole source of carbon. The two extracellular PHB depolymerases, designated E1 and E2, were purified from the culture fluid using DEAE-Toyopearl 650M column chromatography followed by gel filtration in Sephadex G-75 column chromatography. E1 and E2 are basic proteins having molecular weights of 46 and 44 kDa, and isoelectric points of 9.0 and 8.9, respectively. The optimal pHs of E1 and E2 for degradation of PHB are 8.1 and 7.9, respectively, and both of them have optimal PHB degradation temperatures of 45°C. Their enzymatic activities were considerably inhibited by dithiothreitol, phenylmethanesulfonyl fluoride, and Tween 20. The K m values of E1 and E2 for PHB were 17.8 μg/ml and 70.5 μg/ml, respectively. At pH 8.0 and 30°C, both enzymes completely degraded PHB, and they degraded 94% of P(3HB-co-43%3HV). The methyl ester of 3HB dimer was degraded by each enzyme to form the free dimer and the free monomer of 3HB.

Miscibility, Thermal Properties, and Enzymatic Degradability of Binary Blends of Poly[(R)-3-hydroxybutyric acid] with Poly(ε-caprolactone-co-lactide)

The miscibility, morphology, and biodegradability of binary blends of poly[(R)-3-hydroxybutyric acid] (P[(R)-3HB]) (Mn = 300 000) with poly(ε-caprolactone-co-lactide) (P(CL-co-LA)) (Mn = 1500−40 000) have been studied by means of differential scanning calorimetry (DSC), optical microscopy, scanning electron microscopy (SEM), enzymatic hydrolysis, and biodegradation in river water. Copolymers of ε-caprolactone (CL) and (R,S)-lactide (LA) with a wide range of compositions were prepared by ring-opening copolymerization of ε-caprolactone with (R,S)-lactide in the presence of aluminum triisopropoxide as an initiator. The P(CL-co-LA) samples were found to have a random sequence distribution of monomer units by 13C NMR analysis. The P(CL-co-LA) films were hydrolyzed by a lipase from Rhizopus delemar, and the rates of enzymatic hydrolysis were higher than that of PCL homopolymer. DSC analysis revealed that the solid-state structure of P[(R)-3HB]/P(CL-co-LA) blends was strongly dependent on the copolymer composition of the P(CL-co-LA) component. The miscible blends of P[(R)-3HB] were prepared with amorphous P(CL-co-LA) ranging from 30 to 100 mol % LA. The spherulites of P[(R)-3HB] in the miscible P[(R)-3HB]/P(CL-co-LA) blends were volume-filled, and the spherulitic growth rate decreased with an increase in the content of the P(CL-co-LA) component. The enzymatic hydrolysis of P[(R)-3HB]/P(CL-co-LA) blend films was carried out at 37 °C and pH 7.4 in a potassium phosphate buffer with an extracellular PHB depolymerase from Alcaligenes faecalis. The rates of enzymatic hydrolysis on the films of P[(R)-3HB]/P(CL-co-LA) blends decreased with an increase in the P(CL-co-LA) content.

Enzymatic Degradability of Poly(β-Hydroxybutyrate) as a Function of Tacticity

Abstract The enzymatic degradation of films of synthetic poly[(R,S)-β-hydroxybutyrate], PHB, of various tacticities was investigated by weight loss measurement. Extracellular PHB depolymerases of the bacterium Pseudomonas lemoignei and the fungus Aspergillus fumigatus M2A were used. In both enzyme systems the nearly atactic samples showed the greatest weight loss; this maximum occurred at a slightly lower percent isotacticity in the P. lemoignei system than in the A. fumigatus. The maximum degradation rate for the P. lemoignei system was double that of the A. fumigatus; natural PHB degraded an order of magnitude more rapidly in both enzyme systems. As isotacticity increased, synthetic PHB showed decreased degradation while syndiotactic samples did not appreciably degrade. Results are interpreted in terms of both crystallinity and stereochemistry; the A. fumigatus system is more affected than the P. lemoignei by the presence of the S repeat units. Total weight loss data suggest that the enzymes are capable...

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.

Enzymatic degradation of poly[( R)-3-hydroxybutyrate] by Comamonas testosteroni ATSU of soil bacterium

A bacterium capable of degrading poly(3-hydroxybutyrate) (P(3HB)) was isolated from the soil and identified as Comamonas testosteroni. The strain C.testosteroni ATSU excreted an extracellular PHB depolymerase and grew on P(3HB) as a sole carbon source. C.testosteroni also grew on 3-hydroxybutyrate P(3HB), malate, and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (P(3HB- co-3HV)). The secretion of PHB depolymerase was induced in the presence of P(3HB) and P(3HB- co-3HV). The PHB depolymerase was purified to electrophoretic homogeneity from the culture medium by hydrophobic column chromatography, preparative isoelectric focusing and gel filtration, and its molecular weight was determined as 49 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The optimum activity of degrading P(3HB) by the depolymerase was observed at pH 8·5 and 70°C. The enzymatic hydrolysis of P(3HB) film produced water-soluble products composed of the monomer and dimer of 3-hydroxybutyric acid. The rate of production of the monomer and dimer increased to a maximum value with the concentration of PHB depolymerases, followed by a gradual decrease. The properties of PHB depolymerase from C.testosteroni ATSU were compared with those of PHB depolymerase from Alcaligenes faecalis TI, Pseudomonas pickettii YM-b and Comamonas testosteroni YM1004.

Synthesis of a block copolymer of poly(3-hydroxybutyrate) and poly(ethylene glycol) and its application to biodegradable polymer blends

A block copolymer {P[(R,S)-HB-b-EG]} of atactic poly[(R,S)-3-hydroxybutyrate] {P[(R,S)-HB]} and poly(ethylene glycol) (PEG) was prepared by the ring-opening polymerization of β-butyrolactone in the presence of a macroinitiator (PEG/ZnEt2/H2O) which had been produced by the reaction of α,ω-dihydroxy PEG ( $$\overline M $$ n=3000) with ZnEt2/H2O (1/0.6) catalyst. The block copolymer ( $$\overline M $$ n=10,500, $$\overline M $$ w/ $$\overline M $$ n=1.2) was an A-B-A triblock copolymer comprising atactic P[(R,S)-HB] (A) and PEG (B) segments. The miscibility, physical properties, and biodegradability of binary blends of microbial poly[(R)-3-hydroxybutyrate] {P[(R)-HB]} with the block copolymer P[(R,S)-HB-b-EG] has been studied. The glass-transition temperature (T g) data showed that the P[(R)-HB]/P[(R,S)-HB-b-EG] blend was miscible in the amorphous state. The P[(R)-HB] film became flexible and tough by means of blending with P[(R,S)-HB-b-EG] block copolymer. The enzymatic degradation of blend films was carried out at 37°C and pH 7.4 in a 0.1M phosphate solution of an extracellular PHB depolymerase fromAlcaligenes faecalis. The enzymatic degradation took place solely on the surface of the blend films.

Enzymatic degradation of binary blends of microbial poly(3-hydroxybutyrate) with enzymatically active polymers

The miscibility and biodegradability of binary blends of microbial poly(3-hydroxybutyrate) (PHB) with poly(β-propiolactone) (PPL), poly(ethylene adipate) (PEA) and microbial poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHB-HV, HB 24 mol% and HV 76 mol%) have been studied by analysis of differential scanning calorimetry (DSC), scanning electron micrography (SEM) and enzymatic degradation. The glass transition temperature ( T g) data indicated that the blends of PHB/PPL, PHB/PEA and PHB/PHB-HV were immiscible in the amorphous state. The enzymatic degradation of the PHB-based blend films was carried out at 37°C and pH 7·4 in an aqueous solution of an extracellular PHB depolymerase from Alcaligenes faecalis T1. The films of PPL, PEA and PHB-HV polymers, as well as the films of PHB, were degraded by the PHB depolymerase. The rates of enzymatic degradation of the PHB/PPL, PHB/PEA and PHB/PHB-HV blend films were higher than the rate of each polymer component film. In the SEMs of the surface and cross-section of a PHB/PPL (26:74, w/w) film, large holes, 10–30 μm in diameter, were detected not only on the surface but also inside the film. The acceleration of enzymatic degradation of the blend films may be caused by an increase in the surface area of film active to the PHB depolymerase.

Enzymatic degradation of poly(3-hydroxybutyrate)-based blends: poly(3-hydroxybutyrate)/poly(ethylene oxide) blend

The compatibility and biodegradability of a blend of poly(3-hydroxybutyrate) (PHB) and poly(ethylene oxide) (PEO) were studied by analysis of differential scanning calorimetry and enzymatic degradation. The PHB/PEO blend exhibited a single glass-transition temperature ( T g) and the T g values depended upon the blend composition, which indicates that the blend is compatible in the amorphous state. The enzymatic degradation profiles of the blend films were studied at 37°C and pH 7·4 in an aqueous solution of an extracellular PHB depolymerase from Alcaligenes faecalis T1. The rate of enzymatic degradation of the blend film was faster than that of PHB film. Scanning electron micrographs of the blend films after degradation showed that many holes were formed in the films by the elution of the PEO component into an aqueous buffer. It has been concluded that the acceleration of enzymatic degradation of the PHB/PEO blend films is caused by the permeation of PHB depolymerase into the films through the holes.

Enzymatic degradation and morphologies of binary blends of microbial poly(3-hydroxy butyrate) with poly(ε-caprolactone), poly(1,4-butylene adipate and poly(vinyl acetate)

The miscibility, morphology and biodegradability of binary blends of poly(3-hydroxy butyrate) (PHB) with poly(ε-caprolactone) (PCL), poly(1,4-butylene adipate) (PBA) and poly(vinyl acetate) (PVAc) have been studied by analysis of differential scanning calorimetry, mechanical properties, scanning electron micrography (SEM) and enzymatic degradation. The glass-transition temperature ( T g) data indicated that the PHB/PCL and PHB/PBA blends were immiscible in the amorphous state, while the PHV/PVAc blend was miscible. On the basis of the mechanical properties and SEM of the immiscible blends, it was suggested that the PHB/PCL blend has a macro-phase separated structure and the PHB/PBA blend has a modulated structure with a micro-phase separation. The enzymatic degradation of the PHB-based blend films was carried out at 37°C and pH 7·4 in an aqueous solution of an extracellular PHB depolymerase from Alcaligenes faecalis T1. The profiles of enzymatic degradation of PHB-based blends were strongly dependent upon the polymer component blended with PHB. In the case of PHB/PCL blend film, a complicated dependence of PCL weight fraction on the rate of enzymatic degradation was observed. In contrast, the weight loss of PHB/PBA or PHB/PVAc blend film by the PHB depolymerase decreased monotonously with increase in the weight fraction of PBA or PVAc. The enzymatic degradation data have been discussed in connection with the morphologies of PHB-based blend films.

Enzymatic degradation of microbial poly(3‐hydroxybutyrate) films

AbstractThe effects of crystallinity and spherulite size on the enzymatic degradation of microbial poly(3‐hydroxybutyrate) (PHB) films have been studied at 37°C and pH 7,4 in aqueous solutions of an extracellular PHB depolymerase from Alcaligenes faecalis T1. The rate of enzymatic degradation of PHB films decreases with an increase in crystallinity, but it is little influenced by the size of PHB spherulites. It was suggested that the PHB depolymerase firstly hydrolyzes the PHB chains in the amorphous state on the surface of the films and subsequently erodes the PHB chains in the crystalline state.

Ilyobacter delafieldii sp. nov., a metabolically restricted anaerobic bacterium fermenting PHB

Enrichments from an estuarine sediment with crotonate as substrate resulted in the isolation of a motile, gram-negative, obligately anaerobic rod with pointed ends, designated strain 10cr1. The organism was asporogenous, did not reduce sulfur, sulfate, thiosulfate, nitrate, oxygen or fumarate, and had a mol %G+C ratio of 29. Strain 10cr1 was able to ferment crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly-β-hydroxybutyric acid (PHB). Acetate, propionate, butyrate, CO2 and H2 were the fermentation products. When grown on PHB there was accumulation of 3-hydroxybutyrate once growth had ceased, indicating degradation of PHB to the monomer. The 3-hydroxybutyrate formed during growth of the culture was fermented to acetate, butyrate and H2. Experimental evidence suggested the production of an extracellular PHB depolymerase. The cells were not attached to the PHB granules. This is the first isolation of an anaerobic bacterium capable of degrading exogenous PHB. This strain is described as a new species, Ilyobacter delafieldii sp. nov., and strain 10cr1 (=DSM 5704) is designated as the type (and at present, only) strain.