Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity

The extracellular poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis T1, which hydrolyzes both hydrophobic poly(3-hydroxybutyrate) and water-soluble oligomers of D(-)-3-hydroxybutyrate, lost its hydrolyzing activity toward the hydrophobic substrate on mile trypsin treatment, but retained its activity toward water-soluble oligomers. The molecular mass of the trypsin-treated enzyme was 44 kDa, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, which was 6 kDa smaller than that of the native enzyme (50 kDa). The trypsin-treated enzyme seemed to be less hydrophobic than the native one, because it was rather weakly adsorbed to a hydrophobic butyl-Toyopearl column compared with the native enzyme, and showed no ability to bind to poly(3-hydroxybutyrate), to which the native enzyme tightly bound. These results suggest that, in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers, but is necessary for the hydrolysis of hydrophobic substrates, and this hydrophobic site is removed from the enzyme by the action of trypsin.     

Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei

Extracellular poly(3-hydroxybutyrate) depolymerase was purified from the culture medium of Peudomonas lemoignei and separated into four isozymes (A1, A2, B1 and B2) by CM-Sepharose CL-6B chromatography. The molecular weights of A1 and A2 and those of B1 and B2 were estimated to be 54 000 and 58 000, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of A1, A2, B1 and B2 were found to be approximately pH 9.7, 10.0, 10.0 and 10.6, respectively, by isoelectric focusing. All four enzymes hydrolyzed poly(3-hydroxybutyrate) and oligomeric esters of D-(-)-3-hydroxybutyrate, but showed no activity toward the dimeric ester. Analysis of hydrolytic products of the oligomeric esters with A1 and B2 suggested that the enzymes hydrolyzed mainly the second and third ester bonds from the free hydroxy terminus at different frequencies, depending upon the chain length of the substrates.     

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.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Structural basis of poly(3-hydroxybutyrate) hydrolysis by PhaZ7 depolymerase from Paucimonas lemoignei

The crystal structure of poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 purified from Paucimonas lemoignei was determined at 1.90 Å resolution. The structure consists of a single domain with an α/β hydrolase fold in its core. The active site is analogous to that of serine esterases/lipases and is characterized by the presence of a catalytic triad comprising Ser136, Asp242, and His306. Comparison with other structures in the Protein Data Bank showed a high level of similarity with the Bacillus subtilis lipase LipA (RMSD, 1.55 Å). Structural comparison with Penicillium funiculosum PHB depolymerase, the only PHB depolymerase whose structure is already known, revealed significant differences, resulting in an RMSD of 2.80-3.58 Å. The two enzymes appear to utilize different types of solvent-exposed residues for biopolymer binding, with aliphatic and hydroxyl residues used in P. funiculosum PHB depolymerase and aromatic residues in PhaZ7. Moreover, the active site of P. funiculosum PHB depolymerase is accessible to the substrate in contrast to the active site of PhaZ7, which is buried. Hence, considerable conformational changes are required in PhaZ7 for the creation of a channel leading to the active site. Taken together, the structural data suggest that PhaZ7 and P. funiculosum PHB depolymerase have adopted different strategies for effective substrate binding in response to their diverse substrate specificity and the lack of a substrate-binding domain.     

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.     

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.     

Dynamic adsorption behavior of poly(3-hydroxybutyrate) depolymerase onto polyester surface investigated by QCM and AFM

Time-dependent adsorption behavior of poly(3-hydroxybutyrate) (PHB) depolymerase from Ralstonia pickettiiT1 on a polyester surface was studied by complementary techniques of quarts crystal microbalance (QCM) and atomic force microscopy (AFM). Amorphous poly(l-lactide) (PLLA) thin films were used as adsorption substrates. Effects of enzyme concentration on adsorption onto the PLLA surface were determined time-dependently by QCM. Adsorption of PHB depolymerase took place immediately after replacement of the buffer solutions with the enzyme solutions in the cell, followed by a gradual increase in the amount over 30 min. The amount of PHB depolymerase molecules adsorbed on the surface of amorphous PLLA thin films increased with an increase in the enzyme concentration. Time-dependent AFM observation of enzyme molecules was performed during the adsorption of PHB depolymerase. The phase response of the AFM signal revealed that the nature of the PLLA surface around the PHB depolymerase molecule was changed due to the adsorption function of the enzyme and that PHB depolymerase adsorbed onto the PLLA surface as a monolayer at a lower enzyme concentration. The number of PHB depolymerase molecules on the PLLA surface depended on the enzyme concentration and adsorption time. In addition, the height of the adsorbed enzyme was found to increase with time when the PLLA surface was crowded with the enzymes. In the case of higher enzyme concentrations, multilayered PHB depolymerases were observed on the PLLA thin film. These QCM and AFM results indicate that two-step adsorption of PHB depolymerase occurs on the amorphous PLLA thin film. First, adsorption of PHB depolymerase molecules takes place through the characteristic interaction between the binding domain of PHB depolymerase and the free surface of an amorphous PLLA thin film. As the adsorption proceeded, the surface region of the thin film was almost covered with the enzyme, which was accompanied by morphological changes. Second, the hydrophobic interactions among the enzymes in the adlayer and the solution become more dominant to stack as a second layer.     

Enzymatic hydrolysis of chemosynthesized atactic poly(3-hydroxybutyrate) by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4 and Ralstonia pickettii T1

The enzymatic degradability of chemosynthesized atactic poly([R,S]-3-hydroxybutyrate) [a-P(3HB)] by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pickettii T1 (PhaZ(ral)) and Acidovorax Sp. TP4 (PhaZ(aci)), defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were studied. The enzymatic degradation of a-P(3HB) by PhaZ(aci) depolymerase was confirmed from the results of weight loss and the scanning electron micrographs. The degradation products were characterized by one- and two-dimension (1)H NMR spectroscopy. It was found that a-P(3HB) could be degraded into monomer, dimer, and trimer by PhaZ(aci) depolymerase at temperatures ranging from 4 to 20 degrees C, while a-P(3HB) could hardly be hydrolyzed by PhaZ(ral) depolymerase in the same temperature range. These results suggested that the chemosynthesized a-P(3HB) could be degraded in the pure state by natural PHA depolymerase.     

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

Summary 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 K _m 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.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. 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). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. 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). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Structural basis for the substrate specificity of 3-hydroxybutyrate dehydrogenase

The substrate specificity of 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis with a non-native substrate, levulinic acid, was studied by analysis of the enzyme-substrate molecular interactions. The relation between structural and kinetic parameters was investigated considering the catalytic mechanism of the enzyme. The effects of key positive mutations (H144L, H144L/W187F) on the catalytic activity of the enzyme were studied by employing a surface analysis of its interatomic contacts between the enzyme and substrate atoms. The results revealed that the alteration of hydrogen bond network and rearrangement of the hydrophobic interactions between the active site and substrate molecule are the key structural basis for the change of the substrate specificity of 3-hydroxybutyrate dehydrogenase toward levulinic acid. With this approach, the structural basis for the substrate specificity of the enzyme could be elucidated in a quantitative manner.     

Structure and enzymatic degradation of poly(3-hydroxybutyrate) copolymer single crystals with an extracellular PHB depolymerase from Alcaligenes faecalis T1.

Lamellar single crystals of four random copolymers of (R)-3-hydroxybutyrate with different hydroxyalkanoates: poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyvalerate) (P(3HB-co-8%-3HV)), poly(3-hydroxybutyrate-co-10 mol%-4-hydroxybutyrate) (P(3HB-co-10%-4HB)), poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyhexanoate) (P(3HB-co-8%-3HH)) and poly(3-hydroxybutyrate-co-10 mol%-6-hydroxyhexanoate) (P(3HB-co-10%-6HH)), were grown from dilute solutions of chloroform and ethanol. All single crystals have lath-shaped morphology and the second monomer units seem to be excluded from the P(3HB) crystal, on the basis of the electron diffraction diagrams. The enzymatic degradation of P(3HB-co-8%-3HH) and P(3HB-co-10%-6HH) single crystals was investigated with an extracellular PHB depolymerase from Alcaligenes faecalis T1. Adsorption of an extracellular PHB depolymerase, examined using an immuno-gold labelling technique, demonstrated a homogeneous distribution of enzyme molecules with a low concentration on the crystal surfaces. Enzymatic degradation of single crystals progressed from the edges and ends of crystals to yield narrow cracks along their long axes and the small crystal fragments. Lamellar thicknesses of single crystals and molecular weights of copolymer chains remained unchanged during the enzymatic hydrolysis. The above results support the hypothesis that the hydrophobic adsorption of the enzyme contributes to increase the mobility of molecular chains of single crystals and generate the disordered chain-packing regions. The active-site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edges and ends with endo-exo enzymatic hydrolysis behaviour, termed processive degradation.     

Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: general characteristics and active site studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase has been isolated from Penicillium funiculosum cultural medium by a single hydrophobic column chromatography. The enzyme is a glycoprotein composed of a single polypeptide chain with a molecular mass of about 37,000 Da as analyzed by denatured sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by native gel filtration on Sephadex G-100. Its optimum activity occurs at pH 6.0. It has an isoelectric point of 5.8 and has a Km for PHB (average molecular weight = 45,000 Da) of 0.17 mg/ml. Various nonionic detergents competitively inhibit the enzyme with Ki values of 0.56 and 0.014% for Tween 80 and Triton X-100, respectively. The enzyme is extremely sensitive to diisopropyl fluorophosphate, mercuric ion, and dithiothreitol (DTT). However, sulfhydryl reagents have little or no effect on its activity. The inactivation by mercuric ion and DTT is reversible by mercaptoethanol and hydrogen peroxide, respectively. These data suggest that the enzyme may be a serine esterase and may contain an important disulfide bond. The enzyme is also inactivated by diazoacetyl and epoxide compounds at low pH, which can be prevented by PHB, indicating the presence of a critical carboxyl group at the active site. These characteristics of the enzyme are compared to other extracellular polymerases isolated from bacterial culture media.     

Assay of poly(3-hydroxybutyrate) depolymerase activity and product determination

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.     

Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis

Abstract Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S¹³⁸ ( P. lemoignei ) and S¹³⁹ ( A. faecalis ) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S¹⁹⁵ ( P. lemoignei ) and S¹⁹⁶ ( A. faecalis ), resulted in mutant proteinswith almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.