Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K _m and V _max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.     

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.     

Covalent and metal-chelate immobilization of a modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid

The stereospecific L-2-haloacid dehalogenase DehCI from Pseudomonas CBS3 was tagged with a peptide tail containing six histidines and overexpressed in Escherichia coli. The His-tagged protein was purified after a single-step affinity chromatography on Zn(2+)-chelating sepharose. The activity of the modified protein was tested after immobilization on Zn(2+)-chelating sepharose and on covalently bound acrylic polymer. Both immobilization systems were used for the transformation of racemic 2-chloropropionic acid into D-lactate and D-chloropropionic acid. Although immobilization on chelating sepharose produced a limited increase in stability, covalent immobilization on acrylic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C or pH 11, whereas no activity could be detected under these conditions in the soluble or chelate-immobilized enzyme. Both forms of immobilization extended the enzyme effective storage periods, and after 10 cycles of reutilization, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively.     

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.     

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.     

Biochemical and molecular characterisation of the 2,3-dichloro-1-propanol dehalogenase and stereospecific haloalkanoic dehalogenases from a versatile <Emphasis Type="Italic">Agrobacterium</Emphasis> sp.

We previously reported the presence of both haloalcohol and haloalkanoate dehalogenase activity in the Agrobacterium sp. strain NHG3. The versatile nature of the organism led us to further characterise the genetic basis of these dehalogenation activities. Cloning and sequencing of the haloalcohol dehalogenase and subsequent analysis suggested that it was part of a highly conserved catabolic gene cluster. Characterisation of the haloalkanoate dehalogenase enzyme revealed the presence of two stereospecific enzymes with a narrow substrate range which acted on d -2-chloropropionic and I-2-chloropropionoic acid, respectively. Cloning and sequencing indicated that the two genes were separated by 87 bp of non-coding DNA and were preceded by a putative transporter gene 66 bp upstream of the d-specific enzyme.     

Molecular Characterization of Monochloroacetate-Degrading Arthrobacter sp. Strain D2 Isolated from Universiti Teknologi Malaysia Agricultural Area

Arthrobacter sp. strains D2 and D3 and Labrys sp. strain D1 capable of degrading 20 mM monochloroacetic acid (MCA) were isolated from soil contaminated with herbicides and pesticides. All three isolates were able to grow on MCA as the sole source of carbon and energy with concomitant chloride ion release in the growth medium (19 mM). Strains D2 and D3 (cells doubling time 7 ± 0.3 h) grew four times faster than D1 (26 ± 0.1 h). Strain D2 was then further investigated and could also grow in 10 mM of monobromoacetic acid (MBA), 2,2-dichloropropionic acid (2,2DCP), d,l-2-chloropropionic acid (D,L2CP), l-2-chloropropionic acid (L-2CP), d-2-chloropropionic acid (D-2CP), and glycolate as the sole sources of carbon and energy. Dehalogenase gene amplification using group I primers revealed a 410-bp polymerase chain reaction (PCR) product, but there was none using group II primers. The partial amino acid sequence analysis of group I DehD2 dehalogenase showed at least 32% identity to the corresponding regions of DehE, DhlIV, DehI, and D,L-DEX, with key amino acid residues Ser188, Ala187, and Asp189. These amino acid residues were involved in substrate binding and catalysis and were conserved in the partial amino acid sequence.     

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.     

Biodegradation and metabolic pathway of β-chlorinated aliphatic acid in Bacillus sp. CGMCC no. 4196

In this study, a bacterial Bacillus sp. CGMCC no. 4196 was isolated from mud. This strain exhibited the ability to degrade high concentration of 3-chloropropionate (3-CPA, 120 mM) or 3-chlorobutyrate (30 mM), but not chloroacetate or 2-chloropropionate (2-CPA). The growing cells, resting cells, and cell-free extracts from this bacterium had the capability of 3-CPA degradation. The results indicated that the optimum biocatalyst for 3-CPA biodegradation was the resting cells. The 3-CPA biodegradation pathway was further studied through the metabolites and critical enzymes analysis by HPLC, LC-MS, and colorimetric method. The results demonstrated that the metabolites of 3-CPA were 3-hydroxypropionic acid (3-HP) and malonic acid semialdehyde, and the critical enzymes were 3-CPA dehalogenase and 3-HP dehydroxygenase. Thus, the mechanism of the dehalogenase-catalyzed reaction was inferred as hydrolytic dehalogenation which was coenzyme A-independent and oxygen-independent. Finally, the pathway of β-chlorinated aliphatic acid biodegradation could be concluded as follows: the β-chlorinated acid is first hydrolytically dehalogenated to the β-hydroxyl aliphatic acid, and the hydroxyl aliphatic acid is oxidized to β-carbonyl aliphatic acid by β-hydroxy aliphatic acid dehydroxygenase. It is the first report that 3-HP was produced from 3-CPA by β-chlorinated aliphatic acid dehalogenase.     

Crystallization and preliminary X-ray crystallographic studies of L-2-haloacid dehalogenase from Pseudomonas sp. YL

The dimeric L -2-haloacid dehalogenase from Pseudomonas sp. YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n-propanol. The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 Å, b = 62.78 Angst; c = 50.84 Å, and β = 122.4°, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 Å resolution.     

Immobilisation of the D-2-haloacid dehalogenase fromPseudomonas putida strain AJ1/23

A variety of procedures were used to immobilise D-2-haloacid dehalogenase. Natural polymer supports were insufficiently robust to withstand degradation by high concentrations of 2-chloropropionate. The best results were obtained with enzyme covalently attached to controlled-pore glass via a diazo linkage. The immobilisation procedure was optimised with respect to enzyme loading, pH, temperature and the presence of substrate during attachment. Immobilisation significantly modified the kinetics of the enzyme, in particular improving its temperature stability and ability to withstand mildly alkaline conditions where it is most active. The performance of the immobilised preparation in batch and plug-flow bioreactors was assessed. Biocatalyst half-life in plug-flow reactors was better than in batch bioreactors whereas effectiveness factors, although concentration dependent in the batch reactor, were similar at least with 200 mM D,L-2-CPA as substrate.     

Distribution of Dehalococcoidia in the Anaerobic Deep Water of a Remote Meromictic Crater Lake and Detection of Dehalococcoidia-Derived Reductive Dehalogenase Homologous Genes

Here we describe the natural occurrence of bacteria of the class Dehalococcoidia (DEH) and their diversity at different depths in anoxic waters of a remote meromictic lake (Lake Pavin) using 16S rRNA gene amplicon sequencing and quantitative PCR. Detected DEH are phylogenetically diverse and the majority of 16S rRNA sequences have less than 91% similarity to previously isolated DEH 16S rRNA sequences. To predict the metabolic potential of detected DEH subgroups and to assess if they encode genes to transform halogenated compounds, we enriched DEH-affiliated genomic DNA by using a specific-gene capture method and probes against DEH-derived 16S rRNA genes, reductive dehalogenase genes and known insertion sequences. Two reductive dehalogenase homologous sequences were identified from DEH-enriched genomic DNA, and marker genes in the direct vicinity confirm that gene fragments were derived from DEH. The low sequence similarity with known reductive dehalogenase genes suggests yet-unknown catabolic potential in the anoxic zone of Lake Pavin.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

X-ray structure and mechanism of ZgHAD,a l-2-haloacid dehalogenase from the marine Flavobacterium Zobellia galactanivorans

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l -2-haloacid dehalogenase (l -2-HAD) from the marine Bacteroidetes Zobellia galactanivorans Dsij^T (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l -2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l -2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l -2-HAD.     

Cloning and expression of a haloacid dehalogenase fromPseudomonas sp. strain 19 S

A dehalogenase gene specifying the utilization of a variety of haloacids byPseudomonas sp. Strain 19S has been cloned and expressed inE. coli. Our cloning strategy employed specific amplification of a fragment homologous toPseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digestedPseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified inE. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.Abbreviations 2-CPA 2-chloropropionate - MCA monochloro acetate - IPTG isopropyl-1-thio--D-galactoside - NBT nitroblue tetrazolium salt - PCR polymerase chain reaction - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside - X-phosphate 5-bromo-4-chloro-3-indolyl phosphate     

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.     

A mechanistic analysis of enzymatic degradation of organohalogen compounds

Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the α-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH(2)-dependent manner.     

An insight into the distribution,genetic diversity,and mycotoxin production of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in soils of pistachio orchards

In the present study, 193 Aspergillus strains were isolated from a total of 100 soil samples of pistachio orchards, which all of them were identified as Aspergillus flavus as the most abundant species of Aspergillus section Flavi existing in the environment. Approximately 59%, 81%, and 61% of the isolates were capable of producing aflatoxins (AFs), cyclopiazonic acid (CPA), and sclerotia, respectively. The isolates were classified into four chemotypes (I to IV) based on the ability to produce AFs and CPA. The resulting dendrogram of random amplified polymorphic DNA (RAPD) analysis of 24 selected A. flavus isolates demonstrated the formation of two separate clusters. Cluster 1 contained both aflatoxigenic and non-aflatoxigenic isolates (17 isolates), whereas cluster 2 comprised only aflatoxigenic isolates (7 isolates). All the isolates of cluster 2 produced significantly higher levels of AFs than those of cluster 1 and the isolates that produced both AFB₁ and AFB₂ were found only in cluster 2. RAPD genotyping allowed the differentiation of A. flavus from Aspergillus parasiticus as a closely related species within section Flavi. The present study has provided for the first time the relevant information on distribution and genetic diversity of different A. flavus populations from nontoxigenic to highly toxigenic enable to produce hazardous amounts of AFB₁ and CPA in soils of pistachio orchards. These fungi, either toxigenic or not-toxigenic, should be considered as potential threats for agriculture and public health.     

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the α-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH₂-dependent manner.     

2-卤代酸脱卤酶研究进展

2-卤代酸脱卤酶(EC 3.8.1.X)催化2-卤代酸脱卤水解形成相应的2-羟基酸。该类酶不仅能够降解环境中的卤代污染物,而且具有宽广底物谱和高效手性拆分特性,因而在环保和手性中间体的绿色合成中具有广阔应用前景。目前已经对多种2-卤代酸脱卤酶进行生化特性表征,并对酶分子三维结构及催化机制进行了深入研究。文中从2-卤代酸脱卤酶的来源、蛋白质结构与催化反应机制、催化特性及应用方面等研究取得的新进展进行综述,并展望了2-卤代酸脱卤酶的进一步研究方向。