Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.     

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.     

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Gluconate and Glucose by Recombinant Aeromonas hydrophila and Pseudomonas putida

Aeromonas hydrophila 4AK4 and Pseudomonas putida GPp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) using gluconate and glucose rather than fatty acids. A truncated tesA gene, encoding cytosolic thioesterase I of Escherichia coli which catalyzes the conversion of acyl-ACP into free fatty acids, was introduced into A. hydrophila 4AK4. When grown in gluconate, the recombinant A. hydrophila 4AK4 synthesized 10% (w/w) PHBHHx containing 14% (mol/mol) 3-hydroxyhexanoate. If additional PHBHHx synthesis genes, phaPCJ, were over-expressed with the truncated tesA in A. hydrophila 4AK4, the PHBHHx content increased to 15% (w/w) and contained 19% (mol/mol) 3-hydroxyhexanoate. Recombinant P. putida GPp104 harboring phaC encoding PHBHHx synthase of A. hydrophila, phaB encoding acetoacetyl-CoA reductase of Wautersia eutropha and phaG encoding 3-hydroxyacyl-ACP-CoA transferase of P. putida, synthesized 19% (w/w) PHBHHx containing 5% (mol/mol) 3-hydroxyhexanoate from glucose. The results suggest that the engineered pathways were applicable to synthesize PHBHHx from unrelated carbon sources such as gluconate and glucose.     

Enzymes of arginine utilization and their formation in Aeromonas formicans NCIB 9232

In Aeromonas formicans two inducible catabolic pathways of L-arginine have been characterized. The arginine decarboxylase is induced by arginine which also induces the three enzymes of the arginine deiminase pathway but only in stress conditions such as a shift from aerobic growth conditions to very low oxygen tension. Addition of glucose to medium containing arginine leads to repression of the enzymes involved in the arginine deiminase pathway while exogenous cAMP prevents that repression of enzyme synthesis by glucose. This suggests that the induction of arginine deiminase pathway is regulated by carbon catabolite repression and the energetic state of the cell.     

Diversity of nodule-endophytic agrobacteria-like strains associated with different grain legumes in Tunisia

This study represents the first report describing the genetic diversity of nodule-endophytic agrobacteria isolated from diverse legumes and their phylogenetic relationships with the valid species of agrobacteria, as well as the non-recognized genomospecies of the former Agrobacterium tumefaciens (Rhizobium radiobacter). The genetic diversity of a collection of 18 non-nodulating agrobacteria-like strains, previously isolated from root nodules of Vicia faba, Cicer arietinum and Phaseolus vulgaris from different geographical regions of Tunisia, was studied by REP-PCR and PCR-RFLP of the 16S-23S rDNA IGS, as well as by sequence analysis of the 16S rDNA and the housekeeping genes recA and atpD. The aim of the work was to study the genetic diversity of the different isolates and to check for any host-specificity. The results from the different techniques were congruent and suggested a specific interaction for P. vulgaris, whereas no specific endophytic interaction was observed for V. faba and C. arietinum. The phylogenetic analysis clearly indicated that some isolates were affiliated to R. radiobacter or to its non-recognized genomic species (genomovars G2, G4 and G9). However, the other isolates probably constitute new species within Rhizobium (Agrobacterium) and Shinella.     

C<Subscript>1</Subscript> compounds as auxiliary substrate for engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Toxic effects of toluene on the growth of activated sludge bacteria

Two bacteria were isolated from the activated sludge sample of a wastewater treatment plant in Dublin by enrichment culture technique with toluene as the sole source of carbon and energy. They were identified as Aeromonas caviae (To-4) and Pseudomonas putida (To-5). The growth of these bacteria depended on the manner in which toluene was supplied. In general, growth was better when toluene was supplied in the vapour phase. When toluene was added directly to the growth medium it was found to be toxic to the organisms but the toxic effect could be alleviated in the presence of other carbon sources and by the acclimation of the cells. The growth of To-4 on toluene has never been previously reported.     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

The mechanism of action of hexahydro-1,3,5-triethyl-s-triazine

Summary The mechanism of antimicrobial action of hexahydro-1,3,5-triethyl-s-triazine (HHTT) was studied using the HHTT-resistant isolate,Pseudomonas putida 3-T-15², its HHTT-sensitive, novobiocin-cured derivative,P. putida 3-T-15² 11:21,P. putida ATCC 12633,Pseudomonas aeruginosa PA01 andEscherichia coli J53 (RP4). HHTT was oxidized byP. putida 3-T-15², while respiration ofP. putida 3-T-15² 11:21 was inhibited by HHTT. Chemical assays showed that HHTT released formaldehyde.P. putida 3-T-15² was highly resistant to formaldehyde, whileP. putida 3-T-15² 11:21 was highly sensitive to formaldehyde. Both HHTT and formaldehyde acted similarly to inhibit proline uptake in bacterial cells and to inhibit the synthesis of the inducible enzymes, -galactosidase and glucose-6-phosphate dehydrogenase. HHTT did not have uncoupler-like activity.P. putida 3-T-15² used either HHTT or ethylamine, a component of HHTT, as a nitrogen source for growth, but neither HHTT, ethylamine or formaldehyde served as a carbon and energy source for growth. We concluded that a major mechanism of antimicrobial action of HHTT was through its degradation product, formaldehyde.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Leaching of zinc from an industrial filter dust withPenicillium,Pseudomonas andCorynebacterium: Citric acid is the leaching agent rather than amino acids

Summary Heterotrophic microorganisms are able to solubilize metals via excreted metabolites-most often di- or tricarboxylic acids but also amino acids. With amino acids Cu, Zn, Au, Ni, U, Hg and Sb have been solubilized from metal oxides, metal sulfides or elementary metals. In this work it was investigated if excreted amino acids play a role in the leaching of zinc from a zinc oxide containing industrial filter dust. Two bacteria-Pseudomonas putida andCorynebacterium glutamicum-and a fungus-Penicillium simplicissimum were used.P. putida andP. Simplicissimum have already been used to solubilize zinc oxide, whereasC. glutamicum was used because of its known ability to excrete amino acids. Amino acids in culture fluids were analyzed via derivatization with phenyl isothiocyanate, separation on a RP-18 column and UV-detection. All three microorganisms solubilized zinc from the filter dust and excreted much more citric acid than amino acids. Thus citric acid rather than amino acids was regarded to be the leaching agent. Of the two bacteriaP. putida was more resistant towards the heavy metalcontaining filter dust.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

Inactivation of type I polyhydroxyalkanoate synthase in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> resulted in discovery of another potential PHA synthase

Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC _mcl) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC _mcl gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.     

Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of the dilution rate on the utilisation of a mixture of glucose and methanol

The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h^-1 (H. polymorpha) and 0.26h^-1, respectively (Kloeckera sp. 2201) which significantly exceeded _max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C₁ Methanol - C₆ glucose; D dilution rate (h^-1) - D _c critical dilution rate (h^-1) - q _s specific, rate of substrate consumption (g substrate [g cell dry weight]^-1 h^-1) - q _CO2 and q _O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]^-1 h^-1) - RQ respiration quotient (q _CO2 q _O2 ¹ ) - s ₀(C₁) and s ₀(C₆) are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - s residual substrate concentration in the culture liquid (g l^-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl^-1) - Y _X/C6 growth yield on glucose (g cell dry weight [g substrate]^-1     

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Glyphosate degradation byAgrobacterium radiobacter isolated from activated sludge

Summary Two species of bacteria capable of growth onN-phosphonomethylglycine (glyphosate) were isolated from a bench scale sequencing batch reactor degrading a waste stream containing glyphosate. The enrichment and isolation medium contained defined salts and glyphosate as the sole carbon and energy source. Glyphosate was stoichiometrically degraded to aminomethylphosphonic acid (AMPA). The bacteria have been identified asAgrobacterium radiobacter andAchromobacter Group V D.     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.