Extracellular PHB Depolymerase Research Articles

Purification and characterization of extracellular PHB depolymerase enzyme from Aeromonas caviae Kuk1-(34) and their biodegradation studies with polymer films.

PHB depolymerase enzymes are able to breakdown the PHB polymers and thereby get significant economic value in the bioplastics industry and for bioremediation as well. This study shows the purification of novel extracellular PHB depolymerase enzyme from Aeromonas caviae Kuk1-(34) using dialysis followed by gel filtration and HPLC. The purification fold and yield after HPLC were 45.92 and 27.04%, respectively. HPLC data showed a single peak with a retention time of 1.937min. GC-MS analysis reveals the presence of three compounds, of which 1-Dodecanol was found to be most significant with 54.48% area and 8.623-min retention time (RT). The molecular weight of the purified enzyme was obtained as 35kDa with Km and apparent Vmax values of 0.769mg/mL and 1.89 U/mL, respectively. The enzyme was moderately active at an optimum temperature of 35 °C and at pH 8.0. The stability was detected at pH 7.0-9.0 and 35-45 °C. Complete activity loss was observed with EDTA, SDS, Tween-20 at 5 mM and with 0.1% Triton X 100. A biodegradation study of commercially available biodegradable polymer films was carried out in a liquid medium and in soil separately with pure microbial culture and with purified enzyme for 7, 14, 28, and 49 consecutive days. In a liquid medium, with a pure strain of Aeromonas caviae Kuk1-(34), the maximum degradation (89%) was achieved on the PHB film, while no changes were observed with other polymer films. With purified enzyme in the soil, 71% degradation of the PHB film was noticed, and it was only 18% in the liquid medium. All such weight analysis were confirmed by SEM images where several holes, pits, grooves, crest, and surface roughness are clearly observed. Our results demonstrated the potential utility of Aeromonas caviae Kuk1-(34) as a source of extracellular PHB depolymerase capable of degrading PHB under a wide range of natural/ lab conditions.

A novel, thermotolerant, extracellular PHB depolymerase producer Paenibacillus alvei PHB28 for bioremediation of biodegradable plastics

Abstract Background Poly-β-hydroxybutyrate (PHB) is the most important and versatile class of biodegradable polymers used successfully in the medical, agricultural and industrial field. Idea is to find the novel isolate for degradation of biodegradable plastics that can enhance the bioremediation. Materials and methods Thirty-one PHB and PHB depolymerase enzyme producing isolates out of 80 mesophilic bacteria from Lucknow region were further screened for PHB degradation capability by secreting extracellular PHB depolymerase enzyme in minimal salt media supplemented with PHB (0.15%). Various biodegradable plastic films were tested by soil burial method for weight loss determination. Result 37.3% weight loss has been observed in PHB films when buried under the soil for 45 days in the presence of a novel PHB degrader identified as Paenibacillus alvei PHB28 by 16S rRNA sequencing (GenBank accession number KX886342). These Gram-negative, spore-forming, thermotolerant bacteria produce maximum PHB depolymerase (5.03 U/mL) at 45°C, pH 8.0, with 0.15% substrate concentration when incubated for 96 h with starch (0.1%) and yeast extract (0.01%) as an additional nutrient supplements. Conclusion To the best of our knowledge this is the first report of PHB depolymerase production by P. alvei PHB28 which may contribute successfully to combat plastic pollution and to sustain the green environment.

Efficient production of (R)-3-hydroxybutyric acid by Pseudomonas sp. DS1001a and its extracellular poly(3-hydroxybutyrate) depolymerase

We demonstrate a novel strategy to produce (R)-3-hydroxybutyric acid [(R)-3-HB] using Pseudomonas sp. DS1001a or its extracellular poly (3-hydroxybutyrate) (PHB) depolymerase. While approximately 77.8% PHB was degraded by Pseudomonas sp. DS1001a after 12h of cultivation, only 1.555g/l of 3-HB monomer was produced because of assimilation by the strain. About 8.58g/l (R)-3-HB was obtained after 8h of incubation using extracellular PHB depolymerase from Pseudomonas sp. DS1001a. The optimal temperature and pH of PHB depolymerase were 50°C and 8, respectively. CaCO3 increased (R)-3-HB yields to 23.97g/l by stabilizing the pH of the reaction system. High yields of the (R)-3-HB product and the low cost of the medium and CaCO3 indicate the feasibility of bioconversion of low-value PHB waste or industrial leftovers into high-value (R)-3-HB using extracellular PHB depolymerase.

Nanocomposites of poly(3‐hydroxybutyrate)/organomodified montmorillonite: Effect of the nanofiller on the polymer's biodegradation

ABSTRACTPoly(3‐hydroxybutyrate) (PHB) is a biopolymer that can be degraded by extracellular PHB depolymerase. This enzyme is secreted by various microorganisms, but bacterial PHB depolymerases are the most widely studied. The biodegradability rate depends on various factors. By controlling them, the biodegradability rate can change and be customized, and thus, the applications of the polymer can increase and become more diverse. In this work, the role of organomodified montmorillonite (OMMT) on PHB biodegradation was investigated. Using the melt‐mixing method, nanocomposites of PHB and OMMT as the nanofiller were prepared. The enzyme was isolated from the fungus Penicillium pinophilum and the enzymatic degradation was studied for both pure polymer and its nanocomposites. It was found that, after 25 days of enzymatic degradation, the mass loss was very low, while the polymer's average molecular weight as measured by gel permeation chromatography was significantly reduced (more than 50%). Additional peaks corresponding to PHB oligomers (from pentamers to nonamers) appeared after biodegradation. This behavior was observed for pure PHB and the hybrid materials. Scanning electron microscopy imaging of the biodegraded surfaces and analysis of these images showed that the higher amount of nanoclay (10 wt %) resulted in larger biodegraded area of the specimens. The results presented here demonstrate that the presence of the nanoclays enhances the biodegradation rate of pure PHB polymer and provide quantitative data for the biodegradation of PHB/organoclay hybrid materials. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41656.

A Simple Route for Purifying Extracellular Poly(3-hydroxybutyrate)-depolymerase from Penicillium pinophilum.

This work proposes the purification of an active and efficient enzyme, extracellular poly(3-hydroxybutyrate) (PHB)-depolymerase, suitable for industrial applications. This is achieved by the application of an easy, fast, and cheap route, skipping the chromatography step. Chromatography with one or two columns is a common step in the purification procedure, which however renders the isolation of the enzyme a time consuming and an expensive process. A strain of the fungus Penicillium pinophilum (ATCC 9644) is used for the isolation of extracellular PHB-depolymerase. The molecular weight of the purified enzyme is about 35 kDa and is estimated by gel electrophoresis (SDS-PAGE, 12% polyacrylamide). The enzymatic activity of the isolated enzyme is determined to be 3.56-fold similar to that found by other researchers that have used chromatography for the isolation. The as-isolated enzyme disintegrates the poly(3-hydroxybutyrate) (PHB) films successfully, as it is demonstrated by the biodegradation test results provided here.

Properties and enzymatic degradation of poly(acrylic acid) grafted polyhydroxyalkanoate films by plasma-initiated polymerization

Using plasma-initiated polymerization, poly(acrylic acid) (PAA) was grafted onto the film surfaces of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate] (P(3HB)), and two kinds of poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] (P(3HB-co-4HB)) with 4.7 mol% or 9.4 mol% of 4HB content. The graft ratio of PAA on the film surface increased drastically with acrylic acid concentration over 8%. Attenuated total reflection Fourier transform infrared spectroscopy demonstrated that the PAA grafted on the film surface was at least 70 nm thick. Mechanical and surface properties of PHA films grafted by PAA (PAA–PHA) were analyzed by tensile test, contact angle test, and scanning electron microscopy. While mechanical properties before and after plasma treatment remained unchanged, surface properties changed from hydrophobic to hydrophilic. Enzymatic degradation of PAA–PHA films was performed using an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The rate of enzymatic degradation was inhibited by PAA graft. However, the rate of enzymatic degradability was accelerated by exposing ungrafted polymer to the enzyme solution, when a PAA-grafted film was divided into several pieces.

Degradation of Poly (3-Hydroxybutyrate) (PHB) by Bacillus gelatini isolated from Vietnam

Poly (3-hydroxybutyrate) (PHB) is a natural polymer produced by many bacteria as a means to store carbon and energy. PHB have attracted commercial biotechnological interest because of their biodegradability and biocompatibility. Several aerobic and anaerobic PHB-degrading microorganisms have been isolated from soil, activated and aerobic sludge, seawater and lake water. The percentage of PHB-degrading microorganisms in the environment was estimated to be 0.5 - 9.6% of the total colonies. Majority of the PHB-degrading microorganisms were isolated at ambient or mesophilic temperatures and a few of them were able to degrade PHB at higher temperature. The composting at high temperature is one of the most promising technologies for recycling biodegradable plastics and the thermophilic microorganisms that could degrade polymers play an important role in the composting process. Thus, microorganisms that are capable of degrading various kinds of polyesters at high temperatures are of interest. In this study, we have isolated 11 thermophilic PHB-degrading bacteria strains. Among them strain B2 was able to use PHB as a carbon source for growth at high temperature (50oC). This bacteria grew at temperatures between 37 - 60oC, at NaCl concentration between 0.5 - 3% and at pH between 5 - 8. This strain was able to used sucrose, D-glucose, fructose, mannose... but not used D (+) glucosamin. This strain produced some extracellular enzymes such as cellulase, protease, catalase but not amylase.16S rDNA sequence analysis of strain B2 indicated that this novel isolation was related phylogenetically of Bacillus genus. Base on phenotypic, chemotaxonomic and phylogenetic results for strain B2, we describe a novel species named Bacillus gelatini. After 30 days of cultivation in the media with the concentration of PHB in the range of 1-2 g/l, B. gelatini degraded more than 50% total amount of PHB added. Strain B. gelatini can degrade not only PHB but also PLA, PCL at the rate of more than 20% after 20 days cultivation. For further studies, the purification of an extracellular PHB depolymerase from B. gelatini is necessary.

Identification and Characterization of a Novel Class of Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus sp. Strain NRRL B-14911

The catalytic, linker, and denatured poly(3-hydroxybutyrate) (dPHB)-binding domains of bacterial extracellular PHB depolymerases (PhaZs) are classified into several different types. We now report a novel class of extracellular PHB depolymerase from Bacillus sp. strain NRRL B-14911. Its catalytic domain belongs to type 1, whereas its putative linker region neither possesses the sequence features of the three known types of linker domains nor exhibits significant amino acid sequence similarity to them. Instead, this putative linker region can be divided into two distinct linker domains of novel types: LD1 and LD2. LD1 shows significant amino acid sequence similarity to certain regions of a large group of PHB depolymerase-unrelated proteins. LD2 and its homologs are present in a small group of PhaZs. The remaining C-terminal portion of this PhaZ can be further divided into two distinct domains: SBD1 and SBD2. Each domain showed strong binding to dPHB, and there is no significant sequence similarity between them. Each domain neither possesses the sequence features of the two known types of dPHB-binding domains nor shows significant amino acid sequence similarity to them. These unique features indicate the presence of two novel and distinct types of dPHB-binding domains. Homologs of these novel domains also are present in the extracellular PhaZ of Bacillus megaterium and the putative extracellular PhaZs of Bacillus pseudofirmus and Bacillus sp. strain SG-1. The Bacillus sp. NRRL B-14911 PhaZ appears to be a representative of a novel class of extracellular PHB depolymerases.

Y443F mutation in the substrate-binding domain of extracellular PHB depolymerase enhances its PHB adsorption and disruption abilities

Extracellular poly[( R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ RpiT1) from Ralstonia pickettii T1 adsorbs to the PHB surface via its substrate-binding domain (SBD) and cleaves the PHB chain using its catalytic domain. Our previous study (Biomacromolecules 2010; 11: 113–119) has suggested that the hydrophobic interaction between the amino acid residues at positions 441, 443, and 445 in the SBD and the PHB surface plays a crucial role in facilitating the association phase of the enzyme adsorption process. In the present study, in order to improve PhaZ RpiT1 for effective PHB degradation, we targeted Tyr at position 443 for substitution with a more highly hydrophobic amino acid residue because its hydrophobicity shows medium to high degree compared to those of general naturally occurring amino acid residues. We designed a mutant enzyme with an amino acid substitution at this position, taking the following factors into consideration: (1) to achieve higher hydrophobicity than the original residue, (2) to retain the β-sheet structure, and (3) to change as little as possible the volume of the amino acid residue after the substitution. As a result, the substitution of Tyr443 with Phe (Y443F) was considered to be appropriate. The purified Y443F enzyme showed identical CD spectrum and hydrolysis activity for a water-soluble substrate with the wild type, indicating that the mutation had no influence on the structure and the ester bond cleavage activity. In contrast, the Y443F enzyme had higher PHB degradation activity than the wild type. Kinetic analysis of PHB degradation suggests that this amino acid substitution promoted not only the adsorption of the mutant enzyme to PHB, but also the disruption of the PHB surface to enhance the hydrolysis of the PHB polymer chain.

A novel poly(3-hydroxybutyrate)-degradingStreptoverticillium kashmirenseAF1 isolated from soil and purification of PHB-depolymerase

A new bacterial strain, capable of degrading poly(3-hydroxybutyrate) (PHB) was isolated from soil. This organism, identified as Streptoverticillium kashmirense AF1, secreted PHB depolymerases both on solid as well as in liquid mineral salt medium containing poly(3-hydroxybutyrate) as sole carbon source. The optimum production of PHB depolymerase was observed at pH 8 and 7, at 45 degrees C, 1% substrate concentration and in the presence of lactose as an additional carbon source. The extracellular PHB depolymerase was purified by gel permeation chromatography using Sephadex G-75. The Streptoverticillium kashmirense AF1 produced two types of PHB depolymerases having molecular weights of about 37 and 45 kDa as determined by SDS-PAGE. The difference in dry cell mass and amount of CO2 evolved in the test and control calculated gravimetrically through Sturm test indicated the degradative capabilities of Streptoverticillium kashmirense AF1.

Mechanical properties and enzymatic degradation of poly[( R)-3-hydroxybutyrate] fibers stretched after isothermal crystallization near Tg

Poly[( R)-3-hydroxybutyrate] (P(3HB)) fibers with high tensile strength were prepared by stretching the fibers after isothermal crystallization near the glass transition temperature. Two samples with different molecular weights ( M w = 0.7 × 10 6 and 4.3 × 10 6) were used to investigate the effect on tensile strength. Increasing the time for isothermal crystallization of P(3HB) fibers resulted in a decrease in the maximum draw ratio. But, the tensile strength of P(3HB) fibers increased remarkably when the isothermal crystallization time was prolonged to more than 24 h. The tensile strength of low-molecular-weight drawn fibers was higher than that of high-molecular-weight fibers. Therefore, it can be concluded that this procedure does not increase the tensile strength of the high-molecular-weight drawn fibers. This is because, in this drawing method, small crystal nuclei grow initially during the isothermal crystallization process. Then, the molecular chains between the small crystal nuclei that acted as the entanglement points are oriented by stretching. In the case of the high-molecular-weight fibers, because the molecular length between the entanglement points of the small crystal nuclei is too long, the molecular chains are not sufficiently oriented by the stretching process. However, in the case of the low-molecular-weight fibers, the molecular length is suitable for generating the extended chains. Based on the result of X-ray analysis of P(3HB) fibers stretched after isothermal crystallization, fibers have the oriented α-form crystal with 2 1 helix conformation and β-form with planar zigzag conformation. The enzymatic degradation of the stretched P(3HB) fibers was performed by using an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The enzymatic erosion rate of β-form was faster than that of α-form in the P(3HB) fibers stretched after isothermal crystallization.

Fermentative production of (R)-(−)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase ofBacillus megateriumN-18-25-9

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.

An extracellular poly (3-hydroxybutyrate) depolymerase from Penicillium sp. DS9713a-01

Penicillium sp. DS9713a-01 was obtained by ultraviolet (u.v.) light mutagenesis from the Penicillium sp. DS9713a which can degrade poly (3-hydroxybutyrate) (PHB). The enzymatic activity of DS9713a-01 was 97% higher than that of the wild-type strain. The DS9713a-01 mutant could completely degrade PHB films in 5 days; however, the wild-type strain achieved only 61% at the same time. The extracellular PHB depolymerase was purified from the culture medium containing PHB as the sole carbon source by filtration, ammonium sulfate precipitation and chromatography on Sepharose CL-6B. The molecular weight of the PHB depolymerase was about 15.1kDa determined by SDS-polyacrylamide gel electrophoresis. The optimum activity of the PHB depolymerase was observed at pH 8.6 and 50 °C. The enzyme was stable at temperatures below 37 °C and in the pH range from 8.0 to 9.2. The activity of PHB depolymerase could be activated or inhibited by some metal ions. The apparent Km value was 0.164 mg ml−1. Mass spectrometric analysis of the water-soluble products after enzymatic degradation revealed that the primary product was the monomer, 3-hydroxybutyric acid.

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

Synthesis and characterization of biodegradable triblock copolymers based on bacterial poly[(R)‐3‐hydroxybutyrate] by atom transfer radical polymerization

AbstractBiodegradable poly(tert‐butyl acrylate)–poly[(R)‐3‐hydroxybutyrate]–poly (tert‐butyl acrylate) triblock copolymers based on bacterial poly[(R)‐3‐hydroxybutyrate] (PHB) were synthesized by atom transfer radical polymerization. The chain architectures of the triblock copolymers were confirmed by 1H NMR and 13C NMR spectra. Gel permeation chromatography analysis was used to estimate the molecular weight characteristics and lengths of the PHB and poly(tert‐butyl acrylate) blocks of the copolymers. The thermal properties of the copolymers were studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA showed that the triblock copolymers underwent stepwise thermal degradation and had better thermal stability than their respective homopolymers, whereas DSC analyses showed that a microphase‐separation structure was formed only in the triblock copolymers with the longer PHB block. As a similar result, from wide‐angle X‐ray diffraction experimentation, the crystalline phase of PHB could not be seen evidently in the triblock copolymers with the shorter PHB block. The enzymatic hydrolysis of the copolymer films was carried at 37 °C and pH 7.4 in a potassium phosphate buffer with an extracellular PHB depolymerase from Penicillum sp. The biodegradability of the triblock copolymers increased with an increase in the PHB block content. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4857–4869, 2005

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: Application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.

The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases.

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.

Thermotolerant poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characterization of the PHB depolymerase of Schlegelella sp. KB1a.

Eighteen gram-negative thermotolerant poly(3-hydroxybutyrate) (PHB)-degrading bacterial isolates ( T(max) approximately 60 degrees C) were obtained from compost. Isolates produced clearing zones on opaque PHB agar, indicating the presence of extracellular PHB depolymerases. Comparison of physiological characteristics and determination of 16S rRNA gene sequences of four selected isolates revealed a close relatedness of three isolates (SA8, SA1, and KA1) to each other and to Schlegelella thermodepolymerans and Caenibacterium thermophilum. The fourth strain, isolate KB1a, showed reduced similarities to the above-mentioned isolates and species and might represent a new species of Schlegelella. Evidence is provided that S. thermodepolymerans and C. thermophilum are only one species. The PHB depolymerase gene, phaZ, of isolate KB1a was cloned and functionally expressed in Escherichia coli. Purified PHB depolymerase was most active around pH 10 and 76 degrees C. The DNA-deduced amino acid sequence of the mature protein (49.4 kDa) shared significant homologies to other extracellular PHB depolymerases with a domain substructure: catalytic domain type 2-linker domain fibronectin type 3-substrate-binding domain type 1. A catalytic triad consisting of S(20), D(104), and H(138) and a pentapeptide sequence (GLS(20)AG) characteristic for PHB depolymerases (PHB depolymerase box, GLSXG) and for other serine hydrolases (lipase box, GXSXG) were identified.

Characterization of a thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2

Poly-3-hydroxybutyrate (P3HB) degradation capabilities of a novel bacterium, Anoxybacillus gonensis G2, were investigated. Both changes on film surfaces of the solution-cast films monitored by scanning electron microscopy (SEM) and weight loss up to 24% after 72 h exposure to A. gonensis G2 cultures indicated secretion of an active esterase responsible for the degradation of P3HB films. Kinetic parameters, V max and K m for the esterase activity of crude enzyme from A. gonensis G2 in the presence of p-nitrophenylbutyrate as substrate were observed as 50 U/L and 0.125 mM, respectively, in 50 mM phosphate buffer, pH 7.5 at 60 °C. The stimulation of the activity by Ca 2+ is an evidence for the requirement of Ca 2+ as a cofactor for the enzyme activity which is a characteristic for lipases/esterases. Inhibition of the esterase activity by metal chelating agents such as ethylenediamine tetraacetate, azide and cyanide has also supported the requirement of a metal ion for the activity. The thermal and pH stability profiles for the enzyme showed that the thermophilic bacterium A. gonensis G2 secretes an extracellular thermoalkalophilic PHB depolymerase active at 60 °C, and stable at this temperature for 120 min at pH 7.5 and for 24 h at pH 7.5–9.5 range at 4 °C by retaining over 75% of its initial activities.