Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases.     

Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei

During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K _m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 g polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues. Correspondence to: D. Jendrossek     

Biochemical evidence that phaZ gene encodes a specific intracellular medium chain length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442: characterization of a paradigmatic enzyme

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra- or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a corealpha/beta hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/exo-mcl-PHA depolymerases.     

Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater.

Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.     

Relationship between succinate transport and production of extracellular poly(3-hydroxybutyrate) depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6. 8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2- accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase

A approximately 35 kDa protein that has been described to be secreted by Paucimonas lemoignei during growth on succinate and to inhibit hydrolysis of denatured (crystalline) poly(3-hydroxybutyrate) (dPHB) by extracellular PHB depolymerases of P. lemoignei (PHB depolymerase inhibitor (PDI)) was purified and characterized. Purified PDI (M(r), 36 199 +/- 45 Da) inhibited hydrolysis of dPHB by two selected purified PHB depolymerases (PhaZ2 and PhaZ5) but did not inhibit the hydrolysis of water-soluble substrates such as p-nitrophenylbutyrate by PhaZ5 and PhaZ2. PDI revealed a high binding affinity to dPHB although it was not able to hydrolyze the crystalline polymer. However, purified PDI had a high hydrolytic activity if native (amorphous) PHB (nPHB) was used as a substrate. N-terminal sequencing of PDI revealed that it was identical to recently described extracellular PHB depolymerase PhaZ7 which is specific for nPHB and which cannot hydrolyze dPHB. To confirm that the inhibition of hydrolysis of dPHB by PhaZ7 is an indirect surface competition effect at high depolymerase concentration, the activity of PHB depolymerases PhaZ2 and PhaZ5 in the presence of different amounts of protein mixtures was determined. The components of NB or LB medium inhibited hydrolysis of the polymer in a concentration-dependent manner but had no effect on the hydrolysis of p-nitrophenylbutyrate by PHB depolymerases. In combination with PHB depolymerases PhaZ2 and PhaZ5 the protein PhaZ7 ("PDI") enables the bacteria to hydrolyze dPHB and nPHB simultaneously.     

Biochemical observations on medium-chain-length polyhydroxyalkanoate biosynthesis and accumulation in Pseudomonas mendocina

Certain Pseudomonads are capable of accumulating high levels of medium-chain-length polyhydroxyalkanates (PHAmcl) when grown with carbohydrates as the main carbon source. 3-OH acyl components of PHAmcl are derived from fatty acid synthase (FAS) and these components are accessed by action of 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase (transacylase). However, little is known with regard to the time courses of 3-OH acyl component occurrence and of transacylase activity during PHAmcl induction. Also, little is known with regard to the coupling mechanism between FAS and PHAmcl synthesis or whether the FAS pathway itself is specialized in PHAmcl-producing cells. Our results with regard to the time course of formation of 3-OH acids, 3-OH acyl-ACPs, and PHAmcl are consistent with the view that transacylase provides the key link between FAS and PHAmcl synthase. They also suggest that FAS specialization is not a feature of the mechanism. Further, we observed the formation of a 3-OH 10:0 homopolymer early in the induction phase followed by later formation of a mixed polymer containing 3-OH 8:0 and 3-OH 12:0 in addition to 3-OH 10:0. Early occurrence of 3-OH 10:0-CoA transacylase activity was coincident with homopolymer formation.     

Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system.

E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product, which is a regulatory protein required for expression of the A. eutrophus aco genes, and the A. eutrophus acoX gene product, which has an unknown function, respectively. The aco genes of C. magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.     

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis

Purification of poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75) from Paucimonas lemoignei is complicated because the bacterium produces several isoenzymes which are difficult to separate from each other. The phaZ5 gene of P. lemoignei encoding extracellular poly(3-hydroxybutyrate) depolymerase A was functionally expressed from the constitutive P43 promoter of pWB980 in a multiple protease-negative mutant of Bacillus subtilis (strain WB800) and secreted to the culture medium. The depolymerase (apparent M(r), 42 kDa; 1.9 mg purified protein per liter culture) was purified from cell-free culture fluid to homogenity by applying only one chromatography step in comparison to at least two necessary steps if poly(3-hydroxybutyrate) depolymerases are purified from P. lemoignei. The recombinant depolymerase lacked any carbohydrate content in contrast to the glycosylated depolymerase of the wild-type. Glycosylation was not essential for activity but enhanced the thermal stability of the enzyme at high temperature. Overexpression of poly(3-hydroxybutyrate) depolymerase in B. subtilis is more efficient than in Escherichia coli.     

Biochemical and molecular characterization of Staphylococcus simulans lipase.

Staphylococcus simulans strain secretes a non-induced lipase in the culture medium. Staphylococcus simulans lipase (SSL), purified to homogeneity, is a tetrameric protein (160 kDa) corresponding to the association of four lipase molecules. The 30 N-terminal amino acid residues were sequenced. This sequence is identical to the one of Staphylococcus aureus PS54 lipase (SAL PS54) and exhibits a high degree of homology with Staphylococcus aureus NCTC8530 lipase (SAL NCTC8530), Staphylococcus hyicus lipase (SHL) and Staphylococcus epidermis RP62A lipase (SEL RP62A) sequences. But the cloning and sequencing of the part of the gene encoding the mature lipase show some differences from SAL PS54 sequence, which suggest that it is a new sequence. The lipase activity was maximal at pH 8.5 and 37 degrees C. SSL is able to hydrolyze triacylglycerols without chain length specificity. A specific activity of about 1000 U/mg was measured on tributyrin or triolein as substrate at 37 degrees C and at pH 8.5 in the presence of 3 mM CaCl(2). In contrast to other staphylococcal lipases previously characterized, Ca(2+) is not required to express the activity of SSL. SSL was found to be stable between pH 4 and pH 9. The enzyme is inactivated after a few minutes when incubated at 60 degrees C. Using tripropionin as substrate, SSL does not present the interfacial activation phenomenon. In contrast to many lipases, SSL is able to hydrolyze its substrate in the presence of bile salts or amphiphilic proteins.     

Biochemical and molecular characterization of the insecticidal fragment of CryV.

Two C-terminal deletion constructs were made to study the effect of such deletions on the biological activity of the CryV protein of Bacillus thuringiensis subsp. kurstaki. The results of feeding on neonatal larvae of Ostrinia nubilalis (European corn borer [ECB]) indicated that the 50% lethal dose of the full-length CryV protein was 3.34 micrograms/g of diet (95% fiducial limits, 2.53 to 4.32 micrograms/g of diet). Removal of 71 amino acids (aa) from the C terminus had little effect on toxicity, whereas deletion of 184 aa abolished the insecticidal activity of the CryV protein completely. Truncations of the full-length CryV protein were also generated with trypsin and the midgut protease of ECB. The proteolytically treated products were characterized by determining their N-terminal amino acid sequences. The CryV protein was found to be cleaved by both proteases through a two-step process. Initially an intermediary form was generated which contained aa 45 of full-length CryV as its N-terminal end. The C-terminal end of this peptide was not experimentally determined. However, analysis of the deduced amino acid sequence of CryV indicated that the C-terminal end of the intermediary form is likely either aa 655 or 659. Further N-terminal processing of the intermediary form resulted in a protease-resistant core form. The core included aa 156 to aa 655 or 659. While the intermediary form retained 100% of the ECB larval toxicity, the core form exhibited only approximately 22% of the toxicity of the full-length protein.     

Biochemical and molecular characterization of gliadins

Gliadins account for about 40-50% of the total proteins in wheat seeds and play an important role on the nutritional and processing quality of flour. Usually, gliadins could be divided into alpha- (alpha/beta-), gamma- and omega-groups, whereas the low-molecular-weigh (LMW) gliadins were novel seed storage proteins. The low-molecular-weight glutenin subunits (LMW-GSs) were also designated as gliadins in a few literatures. The genes encoding gliadins were mainly located on the short arms of group 6 and group 1 chromosomes, and not evenly distributed. Repetitive sequences covered most of un-coding regions, which attributed greatly to the evolution of wheat genome. Primary structure of each gliadin has been divided into several domains, and the long repetitive domains consisted of peptide motifs. Conserved cysteine residues mainly formed intramolecular disulphide bonds. The rare potential intermolecular disulphide bonds and the long repetitive domains played an important role in the wheat flour quality. There was a general idea that gliadin genes, even prolamin genes, have a common origin and subsequent divergence lead to the gene polymorphism. The gamma-gliadins have been considered to be the most ancient of the wheat prolamin family. Several elements in the 5'-flanking (e.g. CAAT and TATA box) and the 3'-flanking sequences had been detected, which had been shown necessary for the proper expression of gliadins.     

Biochemical and molecular characterization of gliadins

Gliadins account for about 40–50% of the total proteins in wheat seeds and play an important role in the nutritional and processing quality of flour. Usually, gliadins can be divided into α-(α/β), γ-, and ω-groups, whereas the low-molecular-weight (LMW) gliadins are novel seed storage proteins. The low-molecular-weight glutenin subunits (LMW-GSs) are also designated as gliadins in a few publications. The genes encoding gliadins are mainly located on the short arms of group 6 and group 1 chromosomes, and not evenly distributed. Repetitive sequences cover most of the uncoding regions, which attributed greatly to the evolution of wheat genome. The primary structure of each gliadin is divided into several domains, and the long repetitive domains consist of peptide motifs. Conserved cysteine residues mainly form intramolecular disulfide bonds. The rare potential intermolecular disulfide bonds and the long repetitive domains play an important role in the quality of wheat flour. There is a general idea that gliadin genes, even prolamin genes, have a common origin and subsequent divergence leads to gene polymorphism. The γ-gliadins are considered to be the most ancient of the wheat prolamin family. Several elements in the 5′-flanking (e.g., CAAT and TATA box) and the 3′-flanking sequences have been detected, which has been shown to be necessary for the proper expression of gliadins. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 796–807. The text was submitted by the authors in English.     

Identification and characterization of two polyhydroxyalkanoate biosynthesis loci in Pseudomonas sp. strain 3Y2

A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha _Ps-1 and pha _Ps-2) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha _Ps-2 locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1 _Ps-2-phaC1 _Ps-2-phaZ _Ps-2-phaC2 _Ps-2-phaD _Ps-2. The pha _Ps-1 locus that contains phaC1 _Ps-1-phaZ _Ps-1 appears to have arisen by a duplication event that placed it downstream of a gene (ORF1 _Ps-1), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 _Ps-1 and phaC1 _Ps-2 were investigated by heterologous expression in Wautersia eutropha PHB⁻4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1 _Ps-1 to confer PHA synthesis, however, appeared reduced compared to that of PhaC1 _Ps-2, since cells harboring PhaC1 _Ps-1 accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1 _Ps-2. Primary sequence analysis revealed that PhaC1 _Ps-1 had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1 _Ps-2). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 _Ps-1 produces a protein product in the native strain, it is likely that PhaC1 _Ps-1 may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.     

Biochemical,molecular and anti-tumor characterization of L-methionine gamma lyase produced by local Pseudomonas sp. in Egypt

A soil inhabiting Pseudomonas sp. has been examined for producing L- methionine gamma-lyase enzyme. The identity of the tested bacteria was verified by VITEK2, and MALDI-TOF analysis in addition to molecular confirmation by 16S rDNA sequence and submitted in Genbank under accession number ON993898.1. Production of the targeted enzyme was done using a commercial medium including L-methionine, as the main substrate. This obtained enzyme was precipitated using acetone (1:1v/v) followed by purification with Sephadex G100 and sepharose columns. The specific activity of the purified enzyme (105.8 µmol/ mg/min) increased by 1.89 folds after the purification steps. The peptide fingerprint of the native MGL was verified from the proteomics analysis, with identical conserved active site domains with database-deposited MGLs. The molecular mass of the pure MGL denatured subunit was (>40 kDa) and that of the native enzyme was (>150 kDa) ensuring their homotetrameric identity. The purified enzyme showed absorption spectra at 280 nm and 420 nm for the apo-MGL and PLP coenzyme, respectively. Amino acids suicide analogues analysis by DTNB, hydroxylamine, iodoacetate, MBTH, mercaptoethanol and guanidine thiocyanate reduced the relative activity of purified MGL. From the kinetic properties, the catalytic effectiveness (K_cat/k_m) of Pseudomonas sp. MGL was 10.8 mM ^-1 S^-1 for methionine and 5.51 mM ^-1 S^-1 for cysteine, respectively. The purified MGL showed highly significant antiproliferative activity towards the liver carcinoma cell line (HEPG-2) and breast carcinoma cell line (MCF-7) with half inhibitory concentration values (IC₅₀) 7.23 U/ml and 21.14 U/ml, respectively. No obvious signs of toxicity on liver and kidney functions in the examined animal models were observed.     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

Biochemical and chemotaxonomic characterization of Pseudomonas stutzeri genomovars

A total of 48 strains representing the seven Pseudomonas stutzeri genomovars (DNA/DNA homology groups) were studied for cellular fatty acid composition, physiological characteristics and protein profiles. All strains were found to be homogeneous with respect to their fatty acid patterns. Numerical analysis of physiological properties demonstrated a considerable phenotypic heterogeneity within the genomovars. Characterization of the individual genomic groups on the basis of biochemical tests was not possible. In a numerical study of cellular protein patterns, two main clusters were obtained, one representing genomovars 1, 6 and 7 characterized by a high G + C content (mean value > 64 mol%), the other representing genomovars 2, 3, 4 and 5 with a low G + G content (< 64 mol%). The standardized cellular protein patterns have potential for differentiation of the genomic groupings within the species Ps. stutzeri as currently circumscribed.     

Poly(3-Hydroxyvalerate) Depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA_SCL) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596–607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA_SCL depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M_r], 43,610 Da) resembles precursors of other extracellular PHA_SCL depolymerases (28 to 50% identical amino acids). The mature protein (M_r, 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S₁₃₆, D₂₁₁, and H₂₆₉ similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA_SCL depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA_SCL depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA_SCL depolymerases.