Characterisation of an l-Haloacid Dehalogenase from the Marine Psychrophile Psychromonas ingrahamii with Potential Industrial Application

The recombinant l-haloacid dehalogenase from the marine bacterium Psychromonas ingrahamii has been cloned and over-expressed in Escherichia coli. It shows activity towards monobromoacetic (100 %), monochloroacetic acid (62 %), S-chloropropionic acid (42 %), S-bromopropionic acid (31 %), dichloroacetic acid (28 %) and 2-chlorobutyric acid (10 %), respectively. The l-haloacid dehalogenase has highest activity towards substrates with shorter carbon chain lengths (≤C3), without preference towards a chlorine or bromine at the α-carbon position. Despite being isolated from a psychrophilic bacterium, the enzyme has mesophilic properties with an optimal temperature for activity of 45 °C. It retains above 70 % of its activity after being incubated at 65 °C for 90 min before being assayed at 25 °C. The enzyme is relatively stable in organic solvents as demonstrated by activity and thermal shift analysis. The V _max and K _m were calculated to be 0.6 μM min^?1 mg^?1 and 1.36 mM with monobromoacetic acid, respectively. This solvent-resistant and stable l-haloacid dehalogenase from P. ingrahamii has potential to be used as a biocatalyst in industrial processes.     

Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.     

Phylogenetic diversity and characterization of 2-haloacid degrading bacteria from the marine sponge <Emphasis Type="Italic">Hymeniacidon perlevis</Emphasis>

A total of 139 2-haloacid degrading bacteria strains were isolated from the marine sponge Hymeniacidon perlevis using a modified enrichment medium and a pH indicator method. After screening on indicator agar and 2-chloropropionic acid (2-CPA) liquid medium, 11 isolates with high degrading activities were characterized and initially identified. Seven of the 11 isolates were able to degrade 2-CPA at 8% salt, and four isolates (DEH 66, DEH 99, DEH125 and DEH138) degraded 2-CPA at 15% salt. Eight of the 11 isolates utilized all four types of organohalogen compounds used in this study. The DEH99 and DEH138 isolates exhibited the best enantioselectivity towards (S)-2-chloropropionic acid (S-CPA) and (R)-2-chloropropionic acid (R-CPA), respectively. The dehalogenase activities of DEH84 against racemic CPA, DEH99 against S-CPA, DEH138 against R-CPA and DEH130 against racemic CPA were 0.16U/mg, 0.06U/mg, 0.12U/mg and 0.19U/mg, respectively. Based on 16S rRNA sequence analysis, the 11 isolates were clustered into the Rhodobacteraceae family of α-proteobacteria and the Pseudomonadaceae family of γ-proteobacteria. To our knowledge, this is the first report detailing the isolation of organisms of Pseudomonas stuzeri sp. and the Rhodobacteraceae family with 2-haloacid dehalogenase activity from marine sponges.     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Partial purification, stereospecificity and stoichiometry of three dehalogenases from a Rhizobium species

Abstract A fast-growing species of Rhizobium that utilized 2,2-dichloropropionate (2,2DCP) and d,l -2-chloropropionate ( dl -2CP) as sole sources of carbon and energy was shown to contain three inducible dehalogenases. These enzymes differed in their substrate specificities: dehalogenase II degraded 2,2DCP, d - and l -2CP, monochloroacetate (MCA) and dichloroacetate (DCA) whilst dehalogenase I showed activity only towards l -2CP and DCA. Dehalogenase III liberated halide from d -2CP and MCA. This is the first report of a dehalogenase acting solely on the d -isomer of a haloalkanoate. All three dehalogenases inverted the isomeric configuration during dehalogenation, forming d (−) and l (+) lactate from l - and d -2CP, respectively.     

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.

DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.     

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.     

Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.     

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia,phylum Chloroflexi

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated ‘DEH-J10'' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO₂ or oxidation of organics completely to CO₂. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.     

Characterisation of Arthrobacter sp. S1 that can degrade α and β-haloalkanoic acids isolated from contaminated soil

A bacterium identified as Arthrobacter sp. S1 by 16S rRNA was isolated from contaminated soil. This is the first reported study that Arthrobacter could utilize both α-halocarboxylix acid (αHA) [2,2-dichloropropionic acid (2,2-DCP) and D,L-2-chloropropionic acid (D,L-2-CP)] and β-halocarboxylix acid (βHA) [3-chloropropionic acid (3CP)] as sole source of carbon with cell doubling times of 5?±?0.2, 7?±?0.1, and 10?±?0.1 h, respectively. More than 85 % chloride ion released was detected in the growth medium suggesting the substrates used were utilized. To identify the presence of dehalogenase gene in the microorganism, a molecular tool that included the use of oligonucleotide primers specific to microorganisms that can grow in halogenated compounds was adapted. A partial putative dehalogenase gene was determined by direct sequencing of the PCR-amplified genomic DNA of the bacterium. A comparative analysis of the deduced amino acid sequence data revealed that the amino acid sequence has a low identity of less than 15 % to both group I and group II dehalogenases, suggesting that the current putative dehalogenase amino acid sequence was completely distinct from both α-haloacids and β-haloacids dehalogenases. This investigation is useful in studying the microbial populations in order to monitor the presence of specific dehalogenase genes and to provide a better understanding of the microbial populations that are present in soil or in water systems treating halogenated compounds.     

Identification of 4-Deoxy-L-Etychro-Hexoseulose Uronic Acid Reductases in an Alginolytic Bacterium <Emphasis Type="Italic">Vibrio splendidus</Emphasis> and their Uses for L-Lactate Production in an <Emphasis Type="Italic">Escherichia coli</Emphasis> Cell-Free System

4-Deoxy-L-erythro-hexoseulose uronic acid (DEH) reductase is a key enzyme in alginate utilizing metabolism, but the number of characterized DEH reductase is quite limited. In this study, novel two DEH reductases, VsRed-1 and VsRed-2, were identified in marine bacterium Vibrio splendidus, and the recombinant enzymes were expressed in an Escherichia coli system and purified by Ni-NTA chromatography. The optimal pH and temperature of the recombinant VsRed-1 and VsRed-2 were pH 7.5, 30 °C, and pH 7.0, 35 °C, respectively. The specific activities of VsRed-1 (776 U/mg for NADH) and VsRed-2 (176 U/mg for NADPH) were the highest among the DEH reductases reported so far. We also demonstrated that DEH could be converted to L-lactate with a yield of 76.7 and 81.9% in E. coli cell-free system containing VsRed-1 and VsRed-2 enzymes, respectively, indicating that two DEH reductases can be employed for production of biofuels and bio-chemicals from brown macroalgae biomass.     

Purification and characterization of a fibrino(geno)lytic protease from cultured natural isolate of a cyanobacterium, Anabaena fertilissima

An isolate of the cyanobacterium Anabaena from paddy fields was cultured and identified as Anabaena fertilissima based on morphometric features and 16S rRNA gene sequence matching. Cell extracts prepared using bead beater hydrolyzed casein. The caseinolytic protease with native molecular mass of 49 kDa was purified using ammonium sulfate fractionation, hydrophobic, affinity and ion-exchange chromatography, and gel filtration. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified protease was resolved in 17-kDa homologue of microcompartment protein and 27-kDa fragment of unknown protein. The enzyme in native state was digested with gelatin and fibrin in substrate gels producing bands corresponding to ca. 49 kDa. Moreover, a plasmin-specific substrate d-Val-Leu-Lys p-nitroanilide was also hydrolyzed with apparent K _m?=?0.18 mM and V _max?=?4.9?×?10^?7?M?s^?1; while Ca²⁺ stimulated, phenylmethanesulfonyl fluoride, leupeptin, and chelators completely abolished the amidolytic activity. The enzyme exhibited pH and temperature stability over a wide range. Upon incubation with fibrinogen, the Aα- and Bβ-chains preferentially cleaved, though the products thus resolved on SDS-PAGE moved at masses different from those of thrombin- and plasmin hydrolysates, and unlike thrombin, cross-linking of fibrinopeptides was not observed. In the plate assays, fibrinolysis was revealed at comparable strengths to that of plasmin, and the dissolute so obtained upon SDS-PAGE lacked bands corresponding to γ-dimer. Consequently, the degraded D-Dimer peptides appeared. The cyanobacterial protease displayed several unique properties not found in microbial and snake venom fibrinolytic enzymes.     

Purification and characterization of D-2-haloacid dehalogenase from Pseudomonas putida strain AJ1/23

A D-2-haloacid dehalogenase was isolated and purified to homogeneity from Pseudomonas putida strain AJ1/23. The enzyme catalysed the stereospecific dehalogenation of the D-isomer of 2-chloropropionate. Using a new ion-chromatograph assay, the enzyme was found to catalyse the dehalogenation of short-chain 2-halocarboxylic acids. Maximum enzyme activity occurred at pH 9.5 and 50 degrees C and the enzyme was insensitive to most -SH reagents. The enzyme has an Mr of about 135,000 and appears to be composed of four subunits of identical Mr.     

Cu2+ Inhibits Photosystem II Activities but Enhances Photosystem I Quantum Yield of Microcystis aeruginosa

Responses of photosystem I and II activities of Microcystis aeruginosa to various concentrations of Cu²⁺ were simultaneously examined using a Dual-PAM-100 fluorometer. Cell growth and contents of chlorophyll a were significantly inhibited by Cu²⁺. Photosystem II activity [Y(II)] and electron transport [rETR_max(II)] were significantly altered by Cu²⁺. The quantum yield of photosystem II [Y(II)] decreased by 29 % at 100 μg L^?1 Cu²⁺ compared to control. On the contrary, photosystem I was stable under Cu²⁺ stress and showed an obvious increase of quantum yield [Y(I)] and electron transport [rETR_max(I)] due to activation of cyclic electron flow (CEF). Yield of cyclic electron flow [Y(CEF)] was enhanced by 17 % at 100 μg L^?1 Cu²⁺ compared to control. The contribution of linear electron flow to photosystem I [Y(II)/Y(I)] decreased with increasing Cu²⁺ concentration. Yield of cyclic electron flow [Y(CEF)] was negatively correlated with the maximal photosystem II photochemical efficiency (F _v/F _m). In summary, photosystem II was the major target sites of toxicity of Cu²⁺, while photosystem I activity was enhanced under Cu²⁺ stress.     

Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1

Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaH₂PO₄–Na₂HPO₄) with acetamide as the substrate and showed activity over the pH range of 4–8 and the temperature range of 4–95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and l-glutamine. Enzyme activity was induced by 1 mM Ca²⁺, 1 mM Al³⁺, and 1–10 mM Mg²⁺, but strongly inhibited by Zn²⁺, Cu²⁺, Ni²⁺, and Fe³⁺. The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5 % methanol nor 5 % isopropanol had any significant effect on AmiE activity (99 and 96 % retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78 % identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications.     

Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli

The goal of this work was to produce high levels of endoglucanase in Escherichia coli for its potential usage in different industrial applications. Endoglucanase gene was amplified from genomic DNA of Bacillus subtilis JS2004 by PCR. The isolated putative endoglucanase gene consisted of an open reading frame of 1,701 nucleotides and encoded a protein of 567 amino acids with a molecular mass of 63-kDa. The gene was cloned into pET-28a(+) and expressed in E. coli BL21 (DE3). Optimum temperature and pH of the recombinant endoglucanase were 50 °C and 9, respectively which makes it very attractive for using in bio-bleaching and pulp industry. It had a K _M of 1.76 μmol and V _max 0.20 μmol/min with carboxymethylcellulose as substrate. The activity of recombinant endoglucanse was enhanced by Mg²⁺, Ca²⁺, isopropanol and Tween 20 and inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ and SDS. The activity of this recombinant endoglucanase was significantly higher than wild type. Therefore, this recombinant enzyme has potential for many industrial applications involving biomass conversions, due to characteristic of broad pH and higher temperature stability.     

The role of antioxidants enzymes of E. coli ASU3, a tolerant strain to heavy metals toxicity, in combating oxidative stress of copper

The current study describes the isolation and characterization of E. coli from wastewater that collected from El-Malah canal in Assiut, Egypt. Twelve isolates were investigated for heavy metal resistance by which one of them showed multiple metal resistances. Furthermore, the bacterium was identified as E. coli ASU3 according to biochemical tests and then, preserved at Assuit University Mycological Centre with accession number AUMC B83. It exhibited high minimal inhibitory concentrations for metals and antibiotic resistance. The order of metals toxicity to the bacterium was Cr⁶⁺ > Cu²⁺ > Co²⁺ > Pb²⁺ > Ni²⁺ > Cr³⁺ > Cd²⁺ > Zn²⁺. Total protein content of E. coli ASU3 decreased with the increase of copper concentration. Under exposure of different concentrations of copper, the induction of antioxidant enzymes such as catalase, peroxidase and ascorbate peroxidase was increased and these antioxidant enzymes can contribute to combating oxidative stresses.     

Resolution and characterization of two soluble calcium-dependent protein kinases from silver beet leaves

Two soluble Ca²⁺-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca²⁺-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different K_m values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca²⁺ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca²⁺. A millimolar concentration of Mg²⁺ is required in addition to a micromolar concentration Ca²⁺ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C.     

Expression and characterization of a novel isocitrate dehydrogenase from Streptomyces diastaticus No. 7 strain M1033

Isocitrate dehydrogenase (IDH) is one of the key enzymes in tricarboxylic acid cycle, widely distributed in Archaea, Bacteria and Eukarya. Here, we report for the first time the cloning, expression and characterization of a monomeric NADP⁺-dependent IDH from Streptomyces diastaticus No. 7 strain M1033 (SdIDH). Molecular mass of SdIDH was about 80 kDa and showed high amino acid sequence identity with known monomeric IDHs. Maximal activity of SdIDH was observed at pH 8.0 (Mn²⁺) and 9.0 (Mg²⁺), and the optimal temperature was 40 °C (Mn²⁺) and 37 °C (Mg²⁺). Heat-inactivation studies showed that SdIDH remained about 50 % activity after 20 min of incubation at 47 °C. SdIDH displayed a 19,000 and 32,000-fold (k _cat/K _m) preference for NADP⁺ over NAD⁺ with Mn²⁺ and Mg²⁺, respectively. Our work implicate that SdIDH is a divalent metal ion-dependent monomeric IDH with remarkably high coenzyme preference for NADP⁺. This work may provide fundamental information for further investigation on the catalytic mechanism of monomeric IDH and give a clue to disclose the real cause of IDH monomerization.     

Biochemical characterization of NADP+-dependent isocitrate dehydrogenase from Microcystis aeruginosa PCC7806

Microcystis aeruginosa is the key symptom of water eutrophication and produces persistent microcystins. Our special attention was paid to the isocitrate dehydrogenase (IDH) of M. aeruginosa (MaIDH) because it plays important roles in energy and biosynthesis metabolisms and its catalytic product 2-oxoglutarate provides the carbon skeleton for ammonium assimilation and also constitutes a signaling molecule of nitrogen starvation in cyanobacteria. Sequence alignment showed that MaIDH shared significant sequence identity with IDHs from other cyanobacteria (>80 %) and other bacteria (>45 %). The subunit molecular weight of MaIDH was determined to be 52.6 kDa by filtration chromatography, suggesting MaIDH is a typical homodimer. The purified recombinant MaIDH was completely NADP⁺-dependent and no NAD⁺-linked activity was detectable. The K _m values for NADP⁺ were 32.24 and 71.71 μM with Mg²⁺ and Mn²⁺ as a sole divalent cation, and DL-isocitrate linked K _m values were 32.56 μM (Mg²⁺) and 124.3 μM (Mn²⁺), respectively. As compared with Mn²⁺, MaIDH showed about 2.5-times and 4-times higher affinities (1/K _m) to NADP⁺ and dl-isocitrate with Mg²⁺. The optimum activity of MaIDH was found at pH 7.5, and its optimum temperature was 45 °C (Mn²⁺) and 50 °C (Mg²⁺). Heat-inactivation studies showed that heat treatment for 20 min at 45 °C caused a 50 % loss of enzyme activity. MaIDH was completely divalent cation dependent as other typical dimeric IDHs and Mn²⁺ was its best activator. Our study is expected to give a better understanding of primary metabolic enzymes in M. aeruginosa. This would provide useful basic information for the research of controlling the blue-green algae blooms through biological techniques.