A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active ß-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

New alkali tolerant ß-galactosidase from Paracoccus marcusii KGP - A promising biocatalyst for the synthesis of oligosaccharides derived from lactulose (OsLu), the new generation prebiotics.

The enzyme ß-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose (OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular ß-galactosidase produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular weight as determined by SDS PAGE and Q-TOF-LC/MS. ß-Galactosidase was highly active at 50 °C and pH 6-8. The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH range of 5-9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5 M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on enzyme activity were also studied. The kinetic parameters KM and Vmax of ß-galactosidase were 1 mM and 8.56 µmoles/ml/min and 72.72 mM and 11.81 µmoles/ml/min on using oNPG and lactose as substrates. P. marcusii ß-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L lactulose at 40 °C. Thus, in this study we identified a new ß-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides.

Gene switch for l-glucose-induced biopharmaceutical production in mammalian cells.

In this study, we designed and built a gene switch that employs metabolically inert l-glucose to regulate transgene expression in mammalian cells via d-idonate-mediated control of the bacterial regulator LgnR. To this end, we engineered a metabolic cascade in mammalian cells to produce the inducer molecule d-idonate from its precursor l-glucose by ectopically expressing the Paracoccus species 43P-derived catabolic enzymes LgdA, LgnH, and LgnI. To obtain ON- and OFF-switches, we fused LgnR to the human transcriptional silencer domain Krüppel associated box (KRAB) and the viral trans-activator domain VP16, respectively. Thus, these artificial transcription factors KRAB-LgnR or VP16-LgnR modulated cognate promoters containing LgnR-specific binding sites in a d-idonate-dependent manner as a direct result of l-glucose metabolism. In a proof-of-concept experiment, we show that the switches can control production of the model biopharmaceutical rituximab in both transiently and stably transfected HEK-293T cells, as well as CHO-K1 cells. Rituximab production reached 5.9 µg/ml in stably transfected HEK-293T cells and 3.3 µg/ml in stably transfected CHO-K1 cells.

Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid.

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C10-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s-1 mM-1 to 119 s-1 mM-1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L-1 day-1 and 160 g L-1 day-1, respectively.

A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2 ß2 or (α2 ß2 )2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

Single amino acid mutation altered substrate specificity for L-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans.

scyllo-inositol dehydrogenase, isolated from Paracoccus laeviglucosivorans (Pl-sIDH), exhibits a broad substrate specificity: it oxidizes scyllo- and myo-inositols as well as L-glucose, converting L-glucose to L-glucono-1,5-lactone. Based on the crystal structures previously reported, Arg178 residue, located at the entry port of the catalytic site, seemed to be important for accepting substrates. Here, we report the role of Arg178 by using an alanine-substituted mutant for kinetic analysis as well as to determine the crystal structures. The wild-type Pl-sIDH exhibits the activity for scyllo-inositol most preferably followed by myo-inositol and L-glucose. On the contrary, the R178A mutant abolished the activities for both inositols, but remained active for L-glucose to the same extent as its wild-type. Based on the crystal structures of the mutant, the side chain of Asp191 flipped out of the substrate binding site. Therefore, Arg178 is important in positioning Asp191 correctly to exert its catalytic activities.Abbreviations: IDH: inositol dehydrogenase; LB: Luria-Bertani; kcat: catalyst rate constant; Km: Michaelis constant; NAD: nicotinamide dinucleotide; NADH: nicotinamide dinucleotide reduced form; PDB; Protein Data Bank; PDB entry: 6KTJ, 6KTK, 6KTL.

A novel chlorpyrifos hydrolase CPD from Paracoccus sp. TRP: molecular cloning, characterization and catalytic mechanism

Background: Biodegradation is a reliable approach for efficiently eliminating persistent pollutants such as chlorpyrifos. Despite many bacteria or fungi isolated from contaminated environment and capable of degrading chlorpyrifos, limited enzymes responsible for its degradation have been identified, let alone the catalytic mechanism of the enzymes. Results: In present study, the gene cpd encoding a chlorpyrifos hydrolase was cloned by analysis of genomic sequence of Paracoccus sp. TRP. Phylogenetic analysis and BLAST indicated that CPD was a novel member of organophosphate hydrolases. The purified CPD enzyme, with conserved catalytic triad (Ser155-Asp251-His281) and motif Gly-Asp-Ser-Ala-Gly, was significantly inhibited by PMSF, a serine modifier. Molecular docking between CPD and chlorpyrifos showed that Ser155 was adjacent to chlorpyrifos, which indicated that Ser155 may be the active amino acid involved in chlorpyrifos degradation. This speculation was confirmed by site-directed mutagenesis of Ser155Ala accounting for the decreased activity of CPD towards chlorpyrifos. According to the key role of Ser155 in chlorpyrifos degradation and molecular docking conformation, the nucleophilic catalytic mechanism for chlorpyrifos degradation by CPD was proposed. Conclusion: The novel enzyme CPD was capable of hydrolyze chlorpyrifos and Ser155 played key role during degradation of chlorpyrifos.

Characterization of haloalkalophilic organic solvent tolerant protease for chitin extraction from shrimp shell waste.

Halophilic organic solvent tolerant protease (HOSP) producing Paracoccus saliphilus APCMST-CS5 was isolated from the marine sediment samples and identified through 16S rRNA sequence analysis. P. saliphilus APCMST-CS5 registered maximum HOSP production of 1,321.70U/ml in the medium contained the most significant parameters such as shrimp shell powder (SSP), CaCl2, NaCl, and sardinella powder (SP), obtained through Placket-Burman and Response Surface Methods. HOSP was further purified to 22.68 fold purity with 29.71 U/mg specific activity and its molecular weight was 39kDa. The HOSP was stable at 60°C, 9.0 pH, 3.0M NaCl concentration and it also showed maximum activity at other tested parameters. Interestingly the purified HOSP showed better antibiofilm ability against tested pathogens. Also, the HOSP effectively deproteinized (85.64%) shrimp shell chitin which in turn maximum and exhibited higher antioxidant activity. The commercial and experimental shrimp shell chitin showed similar peak pattern in FTIR and 13C CP/MAS NMR spectral analysis.

Exploration of the 1-deoxy-d-xylulose 5-phosphate synthases suitable for the creation of a robust isoprenoid biosynthesis system.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for production of two precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). Previously, we attempted the overexpression of endogenous DXS in Synechocystis sp. PCC6803, and revealed that although the mRNA level was 4-fold higher, the DXS protein level was only 1.5-fold higher compared with those of the original strain, suggesting the lability of endogenous DXS protein. Therefore, for the creation of a robust isoprenoid synthesis system, it is necessary to build a novel MEP pathway by combining stable enzymes. In this study, we expressed 11 dxs genes from 9 organisms in Escherichia coli and analyzed their protein solubility. Furthermore, we purified the recombinant DXSes and evaluated their specific activities and protease tolerance, thermostability, and feedback inhibition tolerance. Among DXSes we examined in this study, the highest protein solubility was observed in Paracoccus aminophilus DXS (PaDXS). The DXS with the highest activity was one from Rhodobacter capsulatus (RcDXSA). The highest protease tolerance, thermostability, and tolerance of feedback inhibition were found in Bacillus subtilis DXS (BsDXS), RcDXSA, PaDXS, BsDXS, respectively. These DXSes can be potentially used for the design of robust isoprenoid synthesis system.

An endolytic mutanase from novel strain Paracoccus mutanolyticus: its application potential in dentistry.

Mutanase, α-(1→3)-glucanase, belonging to the family glucanohydrolase, catalyses mutan [α-(1→3)-glucan] synthesized by cariogenic streptococci and hence has potential in caries prophylaxis. A novel bacterial strain with potential to produce higher mutanase (glucanohydrolase) activity was isolated from soils contaminated with cellulosic waste. One of the isolated strains, RSP-02, was subjected to biochemical and 16S rRNA molecular analysis, and we noticed that it belongs to the genus Paracoccus. The mutanase production (800- 1200 U l-1) in this strain was growth associated and substrate induced, and the activity was comparable with the strains reported earlier. The enzyme displayed a molecular mass of 138 kDa by native PAGE studies, showed endolytic activity and produced nigerose as end product. In vitro studies revealed production of 140±2.82 µg of glucose equivalents in 30 min from the biofilm formed on glass surface indicating its potentiality in dentistry. To the best of our knowledge, this is the first report on the production of mutanase by Paracoccus sp.; hence, this isolated bacterial strain is designated as Paracoccus mutanolyticus RSP-02.

A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2-3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.

The isolation and characterization of the novel chlorothalonil-degrading strain Paracoccus sp. XF-3 and the cloning of the chd gene.

Chlorothalonil (CTN) is one of the most widely used fungicides and is often detected in the environment. Here, we report the isolation and characterization of a novel CTN-degrading bacterial strain XF-3 from long-term CTN-contaminated sites and identify it as a strain of the Paracoccus sp. The isolate could utilise CTN as the sole source of carbon and energy for growth. The optimal pH and temperature for degradation by XF-3 were 7.0 and 30°C, respectively. The CTN degradation gene was cloned by PCR. Although the results of a BLAST sequence search indicated that this gene has a 99% similarity with chd (a gene encoding the CTN hydrolytic dehalogenase), its hydrolytic efficiency for CTN was slightly greater than the chd from strain CTN-3. This is the first report of this gene from the genus Paracoccus. Therefore, there is a practical significance and a potential value of the isolated novel strain, XF-3.

Bis-Fe(IV): nature's sniper for long-range oxidation.

Iron-dependent enzymes are prevalent in nature and participate in a wide range of biological redox activities. Frequently, high-valence iron intermediates are involved in the catalytic events of iron-dependent enzymes, especially when the activation of peroxide or molecular oxygen is involved. Building on the fundamental framework of iron-oxygen chemistry, these reactive intermediates constantly attract significant attention from the enzymology community. During the past few decades, tremendous efforts from a number of laboratories have been dedicated to the capture and characterization of these intermediates to improve mechanistic understandings. In 2008, an unprecedented bis-Fe(IV) intermediate was reported in a c-type diheme enzyme, MauG, which is involved in the maturation of a tryptophan tryptophylquinone cofactor of methylamine dehydrogenase. This intermediate, although chemically equivalent to well-characterized high-valence iron intermediates, such as compound I, compound ES, and intermediate Q in methane monooxygenase, as well as the hypothetical Fe(V) species in Rieske non-heme oxygenases, is orders of magnitude more stable than these other high-valence species in the absence of its primary substrate. It has recently been discovered that the bis-Fe(IV) intermediate exhibits a unique near-IR absorption feature which has been attributed to a novel charge-resonance phenomenon. This review compares the properties of MauG with structurally related enzymes, summarizes the current knowledge of this new high-valence iron intermediate, including its chemical origin and structural basis, explores the formation and consequences of charge resonance, and recounts the long-range catalytic mechanism in which bis-Fe(IV) participates. Biological strategies for storing oxidizing equivalents with iron ions are also discussed.

Overexpression and characterization of lycopene cyclase (CrtY) from marine bacterium Paracoccus haeundaensis.

Lycopene cyclase converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into beta-carotene rings via the monocyclic gamma-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The Km values for lycopene and NADPH were 3.5 microM and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

Canthaxanthin production with modified Mucor circinelloides strains.

Canthaxanthin is a natural diketo derivative of ß-carotene primarily used by the food and feed industries. Mucor circinelloides is a ß-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the ß-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 µg/g (dry mass) of canthaxanthin.

An L-glucose catabolic pathway in Paracoccus species 43P.

BACKGROUND: L-Glucose, the enantiomer of D-glucose, was believed not to be utilized by any organisms. RESULTS: An L-glucose-utilizing bacterium was isolated, and its L-glucose catabolic pathway was identified genetically and enzymatically. CONCLUSION: L-Glucose was utilized via a novel pathway to pyruvate and D-glyceraldehyde 3-phosphate. SIGNIFICANCE: This might lead to an understanding of homochirality in sugar metabolism. An L-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing L-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD(+)-dependent L-glucose dehydrogenase was detected as having sole activity toward L-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. L-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward L-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize L-gluconate to glycolytic intermediates, D-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to L-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for L-glucose catabolism.

Methyl-beta-cyclodextrin enhanced biodegradation of polycyclic aromatic hydrocarbons and associated microbial activity in contaminated soil.

The contamination of soils by polycyclic aromatic hydrocarbons (PAHs) is a widespread environmental problem and the remediation of PAHs from these areas has been a major concern. The effectiveness of many in situ bioremediation systems may be constrained by low contaminant bioavailability due to limited aqueous solubility or a large magnitude of sorption. The objective of this research was to evaluate the effect of methyl-beta-cyclodextrin (MCD) on bioaugmentation by Paracoccus sp. strain HPD-2 of an aged PAH-contaminated soil. When 10% (W/W) MCD amendment was combined with bioaugmentation by the PAH-degrading bacterium Paracoccus sp. strain HPD-2, the percentage degradation of total PAHs was significantly enhanced up to 34.8%. Higher counts of culturable PAH-degrading bacteria and higher soil dehydrogenase and soil polyphenol oxidase activities were observed in 10% (W/W) MCD-assisted bioaugmentation soil. This MCD-assisted bioaugmentation strategy showed significant increases (p < 0.05) in the average well color development (AWCD) obtained by the BIOLOG Eco plate assay, Shannon-Weaver index (H) and Simpson index (lambda) compared with the controls, implying that this strategy at least partially restored the microbiological functioning of the PAH-contaminated soil. The results suggest that MCD-aided bioaugmentation by Paracoccus sp. strain HPD-2 may be a promising practical bioremediation strategy for aged PAH-contaminated soils.

Integration of a bacterial ß-carotene ketolase gene into the Mucor circinelloides genome by the Agrobacterium tumefaciens-mediated transformation method.

Plasmids introduced in Mucor circinelloides (and most transformable Mucorales) tend to replicate autonomously, and hardly ever integrate in the genome. This is critical if we want to express exogenous genes, because plasmids are easily lost during vegetative growth, and the ratio of plasmid molecules/nuclei is invariably low. Linearized molecules of DNA have been used to get their genomic integration but the transformation efficiency drops extremely. We have developed and highly optimized an efficient Agrobacterium-mediated transformation system for M. circinelloides to facilitate the integration of transforming DNA in the genome of the recipient strain that could also be used for other Mucorales.

Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides.

The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with K(m) and k(cat) values of 29.5 µM and 49.2 s(-1), respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.

Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities.

A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0-10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k (cat) and K (m) were 2.8 s(-1) and 158 µM, respectively, and the catalytic efficiency value (k (cat)/K (m)) was 0.018 µM(-1) s(-1). In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.