Enzyme-catalyzed biodegradation of penicillin fermentation residues by ß-lactamase OtLac from Ochrobactrum tritici.

BACKGROUND: Biodegradation of antibiotics is a promising method for the large-scale removal of antibiotic residues in the environment. However, the enzyme that is involved in the biodegradation process is the key information to be revealed. RESULTS: In this study, the beta-lactamase from Ochrobactrum tritici that mediates the biodegradation of penicillin V was identified and characterized. When searching the proteins of Ochrobactrum tritici, the ß-lactamase (OtLac) was identified. OtLac consists of 347 amino acids, and predicted isoelectric point is 7.0. It is a class C ß-lactamase according to BLAST analysis. The coding gene of OtLac was amplified from the genomic DNA of Ochrobactrum tritici. The OtLac was overexpressed in E. coli BL21 (DE3) and purified with Ni2+ column affinity chromatography. The biodegradation ability of penicillin V by OtLac was identified in an in vitro study and analyzed by HPLC. The optimal temperature for OtLac is 32 â„ƒ and the optimal pH is 7.0. Steady-state kinetics showed that OtLac was highly active against penicillin V with a Km value of 17.86 µM and a kcat value of 25.28 s-1 respectively. CONCLUSIONS: OtLac demonstrated biodegradation activity towards penicillin V potassium, indicating that OtLac is expected to degrade penicillin V in the future.

The wheat growth-promoting traits of Ochrobactrum and Pantoea species, responsible for solubilization of different P sources, are ensured by genes encoding enzymes of multiple P-releasing pathways.

Production and release of organic acids and phosphatase enzymes by microbes are important for inorganic and organic phosphorus cycling in soil. The presence of microorganisms with corresponding traits in the plant rhizosphere lead to improved plant P uptake and ultimately growth promotion. We studied the potential of two rhizosphere-competent strains, Pantoea sp. MR1 and Ochrobactrum sp. SSR, for solubilization of different organic and inorganic P sources in vitro. In a pot experiment we further revealed the impact of the two strains on wheat seedling performance in soil amended with either phytate, rock phosphate or K2HPO4 as solely P source. To directly link P-solubilizing activity to the strain-specific genetic potential, we designed novel primers for glucose dehydrogenase (gcd), phosphatase (pho) and phytase (phy) genes, which are related to the organic and inorganic P solubilization potential. Quantitative tracing of these functional genes in the inoculated soils of the conducted pot experiment further allowed to compare strain abundances in the soil in dependency on the present P source. We observed strain- and P source-dependent patterns of the P solubilization in vitro as well as in the pot experiment, whereby P release, particularly from phytate, was linked to the strain abundance. We further revealed that the activity of microbial phosphatases is determined by the interplay between functional gene abundance, available soil P, and substrate availability. Moreover, positive impacts of microbial seed inoculation on wheat root architecture and aboveground growth parameters were observed. Our results suggest that screening for rhizosphere-competent strains with gcd, pho and phy genes may help to identify new microbial taxa that are able to solubilize and mineralize inorganic as well as organic bound P. Subsequently, the targeted use of corresponding strains may improve P availability in agricultural soils and consequently reduce fertilizer application.

Production and characterization of keratinase by Ochrobactrum intermedium for feather keratin utilization.

A newly isolated bacterium producing 55.5 U/mL keratinase on feather meal minimal medium was identified as Ochrobactrum intermedium. Optimization of process parameters by one-variable-at-a-time (OVAT) approach (substrate concentration 0.5% w/v, inoculum size 5% w/v, pH 7.0, 200 rpm for 96 h at 40 °C) resulted in 2.1-fold increase in keratinase secretion (117 U/mL). Keratinase was optimally active at pH 9.0 and 40 °C and was stable at pH 9.0 and 60 °C for 120 min. Calcium ions enhanced keratinase activity (158%) significantly, while it was strongly inhibited by both PMSF and EDTA, indicating it to be a metallo-serine protease. Keratinase degraded native chicken feathers efficiently resulting in 97.9% weight loss along with release of 745.5 µg/mL soluble proteins and 4196.69 µg/mL amino acids. Feather hydrolysate generated by NKIS 1 exhibited significant anti-oxidant and free-radical scavenging activity (90.46%). The present study revealed that O. intermedium NKIS 1 has potential applications in the biodegradation of chicken feathers and the value-addition of poultry waste.

Biochemical and structural insights into an Ochrobactrum sp. CSL1 ribose-5-phosphate isomerase A and its roles in isomerization of rare sugars.

Rare sugars have received increasing attention due to their important applications as sweeteners and building blocks. The substrate specificity and catalytic properties of ribose-5-phosphate isomerase A (RpiA) in isomerization of rare sugars have not been extensively explored. In this study, an RpiA from Ochrobactrum sp. CSL1 was cloned and expressed in Escherichia coli. The biochemical and reaction features were explored and its broad substrate specificity was identified. A higher reaction rate in isomerizing l-rhamnose to l-rhamnulose by OsRpiA, compared with OsRpiB found in the same strain indicated higher efficiency in preparing rare sugars, which was verified by kinetics study. The 2.8 Šresolution structure of OsRpiA was then solved and used in subsequent molecular dynamics experiments, providing a possible explanation for its distinct substrate specificity. The present study highlighted the unique role of microbial RpiA in preparing rare sugars, and its structural information provided a reliable reference for further reaction mechanism research and enzyme engineering work.

Purification, molecular characterization and metabolic mechanism of an aerobic tetrabromobisphenol A dehalogenase, a key enzyme of halorespiration in Ochrobactrum sp. T.

Due to the detoxification of tetrabromobisphenol A (TBBPA) varies from different bacterial strains and depends on their specific enzymatic machinery, it is necessary to understand them for potential in situ bioremediation application. The special ability of our previously isolated Ochrobactrum sp. T to simultaneously debrominate and aerobic mineralize TBBPA urgent us to continuously study its degradation molecular mechanism. Herein, the purification and characterization of the dehalogenase which can debrominate TBBPA was investigated based on its corresponding encoding gene tbbpaA. Results showed that an enzyme with molecular mass of 117 kDa, Km of 26.6 µM and Vmax of 0.133 µM min-1 mg-1 was purified and designated as bromophenol dehalogenase. It was the only detected dehalogenase which exhibited TBBPA degradation ability (78%). Moreover, its activity was significantly enhanced by adding NADPH or methyl viologen to the reaction solution. The high similarity of substrate spectrum between the dehalogenase from the recombinant strain and the wild strain further indicated that it was the main dehalogenase responsible for the debromination in wild strain. Based on three identified metabolites, a metabolic pathway of TBBPA by purified enzyme under oxic condition was proposed. This study provides an excellent dehalogenase candidate for mechanistic study of aerobic dehalogenation of brominated aromatic compound.

Expression of alcohol oxidase gene from Ochrobactrum sp. AIU 033 in recombinant Escherichia coli through the twin-arginine translocation pathway.

We cloned a set of genes encoding alcohol oxidase from Ochrobactrum sp. AIU 033 (OcAOD), which exhibits the appropriate substrate specificity for glyoxylic acid production from glycolic acid. The set of genes for OcAOD contained two open reading frames consisting of 555-bp (aodB) and 1572-bp (aodA) nucleotides, which encode the precursor for the ß-subunit and α-subunit of OcAOD, respectively. We expressed the cloned genes as an active product in Escherichia coli BL21(DE3). The recombinant OcAOD oxidized glycolic acid and primary alcohols with C2-C8 but not glyoxylic acid (as is the case for native OcAOD), whereas the Km and Vmax values for glycolic acid and the pH stability were higher than those of native OcAOD. A consensus sequence for the twin-arginine translocation (Tat) pathway was identified in the N-terminal region of the precursor for the ß-subunit, and the active form of OcAOD was localized in the periplasm of recombinant E. coli, which indicated that OcAOD would be transported from the cytoplasm to the periplasm by the hitchhiker mechanism through the Tat pathway. The OcAOD productivity of the recombinant E. coli was 24-fold higher than that of Ochrobactrum sp. AIU 033, and it was further enhanced by 1.2 times by the co-expression of additional tatABC from E. coli BL21(DE3). Our findings thus suggest a function of the ß-subunit of OcAOD in membrane translocation, and that the recombinant OcAOD has characteristics that are suitable for the enzymatic synthesis of glyoxylic acid as well as native OcAOD.

Characterization of an arylamidase from a newly isolated propanil-transforming strain of Ochrobactrum sp. PP-2.

Propanil, one of the most extensively used post-emergent contact herbicides, has also been reported to have adverse effect on environmental safety. A bacterial strain of Ochrobactrum sp. PP-2, which was capable of transforming propanil, was isolated from a propanil-contaminated soil collected from a chemical factory. An arylamidase gene mah responsible for transforming propanil to 3,4-dichloroaniline (3,4-DCA) was cloned from strain PP-2 by shotgun method and subsequently confirmed by function expression. The arylamidase Mah shares low amino acid sequence identity (27-50%) with other biochemically characterized amidases and shows less than 30% identities to other reported propanil hydrolytic enzymes. Mah was most active at pH 8 and 35 °C. Mah had a remarkable activity toward propanil (Km = 6.3 ±â€¯1.2 µM), showing the highest affinity efficiency for propanil as compared with other reported propanil hydrolytic enzymes. Our study also provides a new arylamidase for the hydrolysis of propanil.

Characterization of Ribose-5-Phosphate Isomerase B from Newly Isolated Strain Ochrobactrum sp. CSL1 Producing L-Rhamnulose from L-Rhamnose.

In this study, we attempted to find new and efficient microbial enzymes for producing rare sugars. A ribose-5-phosphate isomerase B (OsRpiB) was cloned, overexpressed, and preliminarily purified successfully from a newly screened Ochrobactrum sp. CSL1, which could catalyze the isomerization reaction of rare sugars. A study of its substrate specificity showed that the cloned isomerase (OsRpiB) could effectively catalyze the conversion of L-rhamnose to L-rhamnulose, which was unconventional for RpiB. The optimal reaction conditions (50°C, pH 8.0, and 1 mM Ca2+) were obtained to maximize the potential of OsRpiB in preparing L-rhamnulose. The catalytic properties of OsRpiB, including Km, kcat, and catalytic efficiency (kcat/Km), were determined as 43.47 mM, 129.4 sec-1, and 2.98 mM/sec. The highest conversion rate of L-rhamnose under the optimized conditions by OsRpiB could reach 26% after 4.5 h. To the best of our knowledge, this is the first successful attempt of the novel biotransformation of L-rhamnose to L-rhamnulose by OsRpiB biocatalysis.

Structural and functional characterisation of multi-copper oxidase CueO from lignin-degrading bacterium Ochrobactrum sp. reveal its activity towards lignin model compounds and lignosulfonate.

The identification of enzymes responsible for oxidation of lignin in lignin-degrading bacteria is of interest for biotechnological valorization of lignin to renewable chemical products. The genome sequences of two lignin-degrading bacteria, Ochrobactrum sp., and Paenibacillus sp., contain no B-type DyP peroxidases implicated in lignin degradation in other bacteria, but contain putative multicopper oxidase genes. Multi-copper oxidase CueO from Ochrobactrum sp. was expressed and reconstituted as a recombinant laccase-like enzyme, and kinetically characterized. Ochrobactrum CueO shows activity for oxidation of ß-aryl ether and biphenyl lignin dimer model compounds, generating oxidized dimeric products, and shows activity for oxidation of Ca-lignosulfonate, generating vanillic acid as a low molecular weight product. The crystal structure of Ochrobactrum CueO (OcCueO) has been determined at 1.1 Å resolution (PDB: 6EVG), showing a four-coordinate mononuclear type I copper center with ligands His495, His434 and Cys490 with Met500 as an axial ligand, similar to that of Escherichia coli CueO and bacterial azurin proteins, whereas fungal laccase enzymes contain a three-coordinate type I copper metal center. A trinuclear type 2/3 copper cluster was modeled into the active site, showing similar structure to E. coli CueO and fungal laccases, and three solvent channels leading to the active site. Site-directed mutagenesis was carried out on amino acid residues found in the solvent channels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223, and Asp462 for catalytic activity. The work identifies a new bacterial multicopper enzyme with activity for lignin oxidation, and implicates a role for bacterial laccase-like multicopper oxidases in some lignin-degrading bacteria. DATABASE: Structural data are available in the PDB under the accession number 6EVG.

Lipase and biosurfactant from Ochrobactrum intermedium strain MZV101 isolated by washing powder for detergent application.

BACKGROUND: Alkaline thermostable lipase and biosurfactant producing bacteria are very interested at detergent applications, not only because of their eco-friendly characterize, but alsoproduction lipase and biosurfactant by using cheap materials. Ochrobactrum intermedium strain MZV101 was isolated as washing powder resistant, alkaline thermostable lipase and biosurfactant producing bacterium in order to use at detergent applications. METHODS: O. intermedium strain MZV101 produces was lipase and biosurfactant in the same media with pH 10 and temperature of 60 °C. Washing test and some detergent compatibility character of lipase enzyme and biosurfactant were assayed. The antimicrobial activity evaluated against various bacteria and fungi. RESULTS: Lipase and biosurfactant produced by O. intermedium strain MZV101 exhibited high stability at pH 10-13 and temperature of 70-90 °C, biosurfactant exhibits good stability at pH 9-13 and thermostability in all range. Both lipase and biosurfactant were found to be stable in the presence of different metal ions, detergents and organic solvents. The lipase enzyme extracted using isopropanol with yield of 69.2% and biosurfactant with ethanol emulsification index value of 70.99% and yield of 9.32 (g/l). The single band protein after through from G-50 Sephadex column on SDS-PAGE was calculated to be 99.42 kDa. Biosurfactant O. intermedium strain MZV101 exhibited good antimicrobial activity against Gram-negative bacteria and against various bacterial pathogens. Based upon washing test biosurfactant and lipase O. intermedium strain MZV101considered being strong oil removal. CONCLUSION: The results of this study indicate that isolated lipase and biosurfactant with strong oil removal, antimicrobial activity and good stability could be useful for detergent applications.

Two superoxide dismutases from TnOtchr are involved in detoxification of reactive oxygen species induced by chromate.

BACKGROUND: Superoxide dismutases (SOD) have been reported as the most relevant bacterial enzymes involved in cells protection from reactive oxygen species (ROS). These toxic species are often the product of heavy metal stress. RESULTS: Two genes, chrC and chrF, from TnOtchr genetic determinant of strain Ochrobactrum tritici 5bvl1 were cloned in Escherichia coli in order to overexpress the respective proteins. Both proteins were purified and characterized as superoxide dismutases. ChrC was confirmed as being a Fe-SOD, and the enzymatic activity of the ChrF, not inhibited by hydrogen peroxide or potassium cyanide, suggested its inclusion in the Mn-SOD family. This identification was supported by chemical quantification of total metal content in purified enzyme. Both enzymes showed a maximum activity between pH 7.2-7.5. ChrF retained nearly full activity over a broader range of pH and was slightly more thermostable than ChrC. The genes encoding these enzymes in strain O. tritici 5bvl1 were inactivated, developing single and double mutants, to understand the contribution of these enzymes in detoxification mechanism of reactive oxygen species induced by chromate. During chromate stress, assays using fluorescent dyes indicated an increase of these toxic compounds in chrC, chrF and chrC/chrF mutant cells. CONCLUSIONS: In spite of the multiple genes coding for putative superoxide dismutase enzymes detected in the genome of O. tritici 5bvl1, the ChrC and ChrF might help the strain to decrease the levels of reactive oxygen species in cells.

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization.

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U mg(-1) protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4-80°C for 6 h. The enzymatic activity was not influenced by metal ions and chemical agents (K(+), Na(+), Zn(2+), Fe(3+), Mn(2+), Mg(2+), glucose, urea, lactate) commonly found in serum and urine, with Cu(2+) being the exception. The enzyme appears to be a metalloprotein stimulated by Ca(2+) and Fe(2+). Its Km and Kcat for oxalate were found to be 0.45 mM and 85 s(-1), respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50 mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.

Molybdenum-containing nicotine hydroxylase genes in a nicotine degradation pathway that is a variant of the pyridine and pyrrolidine pathways.

Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppAS and vppAL [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.

Improved biodegradation of textile dye effluent by coculture.

The present study demonstrates the de-colorization and degradation of textile effluent by coculture consisting of three bacterial species isolated from textile effluent contaminated environment with an aim to reduce the treatment time. The isolates were identified as Ochrobactrum sp., Pseudomonas aeruginosa and Providencia vermicola by 16S rRNA analysis. Their secondary structure was predicted and GC content of the sequence was found to be 54.39, 52.10, and 52.53%. The co-culture showed a prominent increase in the degradation activity due to the action of oxidoreductase enzymatic mechanism of laccase, NADH-DCIP reductase and azoreductase activity. The biodegradability index of 0.75 was achieved with 95% chemical oxygen demand (COD) reduction in 16 h and 78 and 85% reduction in total organic carbon (TOC) and total solids was observed. Bioaccumulation of metals was identified by X-ray diffraction (XRD) analysis. The effective decolorization was confirmed from the results of UV-vis spectroscopy, high performance liquid chromatography and Fourier transformed infrared spectrometer analyzes. The possible degradation pathway was obtained from the analysis of liquid chromatography-mass spectroscopy analysis and the metabolites such as 2-amino naphthalene and N-phenyl-1.3,5 triazine were observed. The toxic nature of the effluent was analyzed using phyto-toxicity, cell-death assay and geno-toxicity tests.

Partial purification and characterization of chromate reductase of a novel Ochrobactrum sp. strain Cr-B4.

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (Ea) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (Ed) was found to be 79.6 kJ/mol over a temperature range of 50-80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s(-1), and for deactivation of chromate reductase it was found to be 265.66 × 10(3) s(-1). The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN3, Pb(2+), Ni(2+), Zn(2+), and Cd(2+)) inhibited the enzyme activity, while metals like Cu(2+) and Fe(3+) significantly enhanced the reductase activity. The enzyme followed Michaelis-Menten kinetics with Km of 104.29 µM and a Vmax of 4.64 µM/min/mg.

Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative.

Biodegradation of pyrene and phenanthrene by bacterial consortium and evaluation of role of surfactant.

High molecular weight poly aromatic hydrocarbons (HMW PAHs) are well known for their hydrophobicity and they get strongly adsorbed onto the soil particles. Generally, surfactants facilitate the biodegradation of PAH by enhancing their solubility and desorption of hydrophobic compounds from soil particles. To investigate the role of synthetic surfactant in biodegradation of PAHs, two bacterial strains BP10 and P2 were incubated in soil spiked with pyrene and phenantherene (100 µg g-1of soil each) in isolation and in combination with/without Tween 80. After 14 days of incubation, pyrene and phenantherene were degraded by a combination of BP10 and P2 to the extent of 98% and 99%, respectively. Addition of tween 80 reduced the degradation of pyrene and phenantherene by 35 and 10%, respectively. Biosurfactant produced by selected strains i.e. BP10 and P2 could enhance desorption of pyrene (100 µg g-1of soil) by about 27% and 12%, respectively. However, desorption activity was relatively higher (32 and 29%, respectively) in case of phenanthrene (100 µg g-1of soil) from the spiked soil. Present study showed that in spite of additional chemical surfactant, bioaugmentation of highly petroleum hydrocarbon degrading bacterial combination was very effective in boosting the bioremediation of PAHs- contaminated sites.

Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequence.

Pichia pastoris is commonly used to express and secrete target proteins, although not all recombinant proteins can be successfully produced. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as a model to study the importance of the N-terminus of the protein for its secretion. While MPH can be efficiently expressed intracellularly in P. pastoris, it is not secreted into the extracellular environment. Three MPH mutants (N66-MPH, D10-MPH, and N9-MPH) were constructed through modification of its N-terminus, and the secretion of each by P. pastoris was improved when compared to wild-type MPH. The level of secreted D10-MPH was increased to 0.21 U/mL, while that of N9-MPH was enhanced to 0.16 U/mL. Although N66-MPH was not enzymatically active, it was secreted efficiently, and was identified by SDS-PAGE. These results demonstrate that the secretion of heterologous proteins in P. pastoris may be improved by modifying their N-terminal structures.

A novel phospholipase D constitutively secreted by Ochrobactrum sp. ASAG-PL1 capable of enzymatic synthesis of phosphatidylserine.

Phospholipase D (PLD) plays a crucial role in the enzyme-mediated preparation of phosphatidylserine (PS). A new PLD constitutively secreted by Ochrobactrum sp. ASAG-PL1 is now reported for the first time. After purification, the MW of the enzyme was ~37 kDa by SDS-PAGE. Its activity was highest at 40 °C and pH 7.0 and more than 90 % of the initial activity was maintained after 30 days at 4 °C and pH 7.0. This enzyme may then be useful as a potential biocatalyst for PS production.

High-resolution structures of AidH complexes provide insights into a novel catalytic mechanism for N-acyl homoserine lactonase.

Many pathogenic bacteria that infect humans, animals and plants rely on a quorum-sensing (QS) system to produce virulence factors. N-Acyl homoserine lactones (AHLs) are the best-characterized cell-cell communication signals in QS. The concentration of AHL plays a key role in regulating the virulence-gene expression and essential biological functions of pathogenic bacteria. N-Acyl homoserine lactonases (AHL-lactonases) have important functions in decreasing pathogenicity by degrading AHLs. Here, structures of the AHL-lactonase from Ochrobactrum sp. (AidH) in complex with N-hexanoyl homoserine lactone, N-hexanoyl homoserine and N-butanoyl homoserine are reported. The high-resolution structures together with biochemical analyses reveal convincing details of AHL degradation. No metal ion is bound in the active site, which is different from other AHL-lactonases, which have a dual Lewis acid catalysis mechanism. AidH contains a substrate-binding tunnel between the core domain and the cap domain. The conformation of the tunnel entrance varies with the AHL acyl-chain length, which contributes to the binding promiscuity of AHL molecules in the active site. It also supports the biochemical result that AidH is a broad catalytic spectrum AHL-lactonase. Taken together, the present results reveal the catalytic mechanism of the metal-independent AHL-lactonase, which is a typical acid-base covalent catalysis.