Purification and characterization of thermostableD-hydantoinase from thermophilicbacillus stearothermophilus SD-1

A thermostable D-hydantoinase of thermophilicBacillus stearothermophilus SD-1 was purified to homogeneity using an immuno-affinity chromatography. The affinity chromatography that employed polyclonal antibody immobilized on Sepharose 4B was simple to operate and gave a purification yield of 60% of enzyme activity. Molecular mass of the enzyme was determined to be about 133.9 kDa by gel filtration chromatography and the molecular mass of the subunit was 54 kDa on SDS-PAGE. Mass spectrometric analyses were also performed for the determination of the molecular mass of the native enzyme and its subunit. The apparent molecular masses were 51.1 and 102.1 kDa for the subunit and native enzyme, respectively. Based on the molecular masses determined by these two methods, it is suggested that the D-hydantoinase exists as a dimeric conformation in the cell. Isoelectric pH of the enzyme was observed to be 4.47. It was found that the enzyme requires one manganese ion per molecule of enzyme for the activity. The optimal pH and temperature for the catalytic activity were about 8.0 and 65‡C., respectively. The half-life of the enzyme was estimated to be 30 min at 80‡C., confirming that the enzyme purified is one of the most thermostable D-hydantoinase reported so far. Kinetic constants of the enzyme for different substrates were also determined.     

Purification and characterization of thermostable d-hydantoinase from Bacillus thermocatenulatus GH-2

A thermostable D-hydantoinase was isolated from thermophilic Bacillus thermocatenulatus GH-2 and purified to homogeneity by using immunoaffinity chromatography. The molecular mass of the enzyme was determined to be about 230 kDa, and a value of 56 kDa was obtained as a molecular mass of the subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that oligomeric structure of the enzyme is tetrameric. Isoelectric pH of the enzyme was found to be approx 4.3. The enzyme required Mn2+ for the activity and exhibited its highest activity with phenylhydantoin as a substrate. The optimal pH and temperature for catalytic activity were about 7.5 and 65 degrees C, respectively. The half-life of the enzyme was estimated to be about 45 min at 80 degrees C.     

Fast high-performance liquid chromatographic purification of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

A procedure was established for the rapid isolation of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (PEPCK) from an overproducing strain. Overexpression was achieved by the transformation of yeast cells with the multicopy plasmid YEp352 harbouring the PEPCK structural gene. The enzyme was purified to homogeneity using first anion-exchange chromatography on Q-Sepharose followed by hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration on Sephacryl S200. The purified phosphoenolpyruvate carboxykinase was further characterized with respect to the molecular mass, displaying an apparent molecular mass corresponding to a tetrameric form.     

Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources

The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane. A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described. This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique. Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T. aquaticus. The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.     

Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA

A solvent tolerant Pseudomonas aeruginosa PseA strain was isolated from soil. It secreted a novel alkaline protease, which was stable and active in the presence of range of organic solvents, thus potentially useful for catalysis in non-aqueous media. The protease was purified 11.6-fold with 60% recovery by combination of ion exchange and hydrophobic interaction chromatography using Q-Sepharose and Phenyl Sepharose 6 Fast Flow matrix, respectively. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 35,000 Da. The enzyme was stable in the pH range of 6.0-9.0, the optimum being 8.0. The Km and Vmax towards caseinolytic activity were found to be 2.7 mg/ml and 3 micromol/min, respectively. The protease was most active at 60 degrees C and characterized as a metalloprotease because of its sensitivity to EDTA and 1,10-phenanthroline. It was tested positive for elastase activity towards elastin-orcein, thus appears to be an elastase, which is known as pseudolysin in other strains of P. aeruginosa. The protease withstands range of detergents, surfactants and solvents. It is stable and active in all the solvents having log P above 3.2, at least up to 72 h. These two properties make it an ideal choice for applications in detergent formulations and enzymatic peptide synthesis.     

Hydroxyapatite-based immobilized metal affinity adsorbents for protein purification

The employment of metal ion-charged hydroxyapatite for the one-step purification of poly(His)-tagged recombinant proteins was investigated. Fe(III) showed the highest selectivity toward the poly(His)-tagged D-hydantoinase and the best operation stability. The optimal selectivity was observed in 20 mM pH 8.0 buffer containing 150 mM NaCl and 50 mM NaF. The adsorbed poly(His)-tagged enzyme could be quantitatively recovered from hydroxyapatite with 150 mM pH 8.0 phosphate buffer. The capacity of Fe(III)-loaded hydroxyapatite for poly(His)-tagged D-hydantoinase was 4.9 mg/g hydroxyapatite, comparable to commercial agarose-based Ni-NTA adsorbents. Under optimal conditions, a D-hydantoinase preparation with a purity above 95% from crude cellular lysate could be obtained with the one-step purification process employing Fe(III)-loaded hydroxyapatite. The application of Fe(III)-loaded hydroxyapatite for the purification of poly(His)-tagged N-acetyl-D-glucosamine 2-epimerase under denaturing conditions was also demonstrated. These results demonstrate that hydroxyapatite is a promising adsorbent for immobilized metal affinity chromatography.     

2,3-吡啶二甲酰亚胺及其工程菌转化产物的高效液相色谱检测

建立了利用含D-海因酶的基因工程菌转化吡啶二甲酰亚胺（PDI）生成3-氨基甲酰基-α-吡啶甲酸（α-3CP）的高效液相色谱（HPLC）检测方法。将工程菌pET3a-hyd/BL21（DE3）诱导表达后收集菌体,以PDI为底物,37℃摇床反应30 min后,以13000 r/min离心,取上清液进行HPLC检测。色谱条件：Hypersil^TM GOLD C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为H₂O-乙腈（体积比为90：10,含0.1%（体积分数）三氟乙酸）;检测波长为254 nm。当底物PDI达到饱和浓度时,测得工程菌pET3a-hyd/BL21（DE3）的比活力为0.61 U/（mL·10OD_{600 nm}）。该研究为今后利用生物法制备复杂半酰胺有机物提供了坚实的理论基础。     

限进介质烷基-二醇基硅胶(ADS)的制备

 烷基 二醇基硅胶 (ADS)系限进介质的一种 ,可用于含生物大分子的复杂生物样品的直接进样与分析。一种新的、非常经济的方法可用于烷基 二醇基硅胶的制备。首先 ,将γ 环氧丙基氧丙基三甲氧基硅烷基键合至微孔硅胶上 (粒度 5 μm ,孔径 6nm)以制备环氧基硅胶 ,再令环氧基硅胶与硬脂酸在有机溶液中进行反应以制备酯型十八烷基反相填料。将制得的反相填料填充至色谱柱中 ,并令含有酯酶的溶液通过色谱柱。通过酶解作用可将硅胶表面的酯基除去 ,而硅胶的内孔表面仍保持疏水特性不变 ,这是由于硅胶上的小孔对酶分子具有体积排除作用。     

Isolation and characterization of ACTX-6: a cytotoxic L-amino acid oxidase from Agkistrodon acutus snake venom

The characterization of an L-amino acid oxidase purified from Agkistrodon acutus snake venom was investigated. An L-amino acid oxidase (LAAO) was purified from A. acutus snake venom through DEAE Sepharose F.F. and Source 30 S chromatography. The molecular mass of this enzyme was determined by SDS-PAGE, size exclusion chromatography, and mass spectrometry. Substrate specificity, cytotoxicity, antitumor activity in vivo, and apoptosis-inducing activity were assayed. The LAAO purified from A. acutus snake venom was designated as ACTX-6. It is a covalently bound homodimer and its molecular mass is about 96 kDa. This enzyme preferred to oxidize hydrophobic L-amino acids; the best substrates were L-Met, L-Leu, L-Trp, and L-Phe. ACTX-6 demonstrated cytotoxicity in vitro and could inhibit tumor growth in vivo. Flow cytometry analysis showed that it could markedly increase accumulation of sub-G1 phase, which suggested that this enzyme could induce apoptosis. ACTX-6 could effectively inhibit tumor growth and it is a potential substance to develop into an antitumor drug.     

Screening,isolation and selection of a potent lipase producing microorganism and its use in the kinetic resolution of drug intermediates

Lipases are ubiquitous enzymes that belong to family of serine hydolases with a wide variety of industrial applications. This study reports isolation, screening and identification of enantioselctive lipase producing microorganism for kinetic resolution of racemic alcohols. For this, we collected soil samples from different oil rich environments and we performed primary screening that was by carried out by using MSM-tributryin clear zone assay. The selected samples from first screen were subjected to secondary screening to distinguish lipase producing strains from esterase producing strains using p-nitrophenyl palmitate lipase assay. In tertiary screening, 16 lipase producing strains that were identified in secondary screening were employed for resolution of 5 different (RS)-alcohols. Out of all 16 lipase producing strains, only one strain selectively converted 3 racemic alcohols. Based on morphological, biochemical and physiological characteristics, and 16S rRNA gene sequencing, the strain was identified as Pseudomonas beteli. The strain was found to be S-selective and there been no reports on use of Pseudomonas beteli lipase for kinetic resolution of alcohols. The lipase activity was further increased by media optimization and by improving growth conditions, and production of lipase in shake flask study as well as in laboratory scale fermenter. The optimum time for enzyme production by Pseudomonas beteli was 96 ​h whereas cell mass growth was highest at 72 ​h. Optimum temperature and pH were 30 ​°C and 6, respectively. Beef extract (5 ​g/L), peptone (5 ​g/L), sodium chloride (5 ​g/L), yeast extract (1 ​g/L) and glucose (5 ​g/L) were found as optimum nutrition sources for the cell mass growth and lipase production by Pseudomonas sp. Overall, 3.4 times higher enzyme activity and 2.75 times higher cell mass growth were achieved in bioreactor in comparison to the shake flask study. Lipase having high titer was employed successfully for the kinetic resolution of several drug intermediates.     

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.     

Evaluating the ability of polyhydroxyalkanoate synthase mutants to produce P(3HB-co-3HA) from soybean oil

Polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp 61-3 (PhaC1(Ps)) is able to synthesize P(3HB-co-3HA), consisting of a 3HB unit and medium-chain-length 3HA units of 6-12 carbon atoms. Expression vectors encoding 76 PhaC1(Ps) mutants with an amino acid replacement at position 130, 325, 477 or 481 were individually introduced into Ralstonia eutropha. The mutant enzyme genes were evaluated in terms of their abilities to synthesize P(3HB-co-3HA) using soybean oil as a carbon source. 20 mutants showed significantly high accumulation levels of PHA exceeding 30 wt.-% and as high as 57 wt.-%. It was found that hydrophobic amino acids at the positions are more likely to enhance accumulation of PHA in R. eutropha.     

Antibiofilm Activity of α-Amylase from Bacillus subtilis S8-18 Against Biofilm Forming Human Bacterial Pathogens

The extracellular α-amylase enzyme from Bacillus subtilis S8-18 of marine origin was proved as an antibiofilm agent against methicillin-resistant Staphylococcus aureus (MRSA), a clinical strain isolated from pharyngitis patient, Vibrio cholerae also a clinical isolate from cholera patient and Pseudomonas aeruginosa ATCC10145. The spectrophotometric and microscopic investigations revealed the potential of α-amylase to inhibit biofilm formation in these pathogens. At its BIC level, the crude enzyme caused 51.81–73.07% of biofilm inhibition. Beyond the inhibition, the enzyme was also effective in degradation of preformed mature biofilm by disrupting the exopolysaccharide (EPS), an essential component in biofilm architecture. Furthermore, the enzyme purified to its homogeneity by chromatographic techniques was also effective in biofilm inhibition (43.83–61.68%) as well as in degradation of EPS. A commercial α-amylase enzyme from B. subtilis was also used for comparative purpose. Besides, the effect of various enzymes and temperature on the antibiofilm activity of amylase enzymes was also investigated. This study, for the first time, proved that α-amylase enzyme alone can be used to inhibit/disrupt the biofilms of V. cholerae and MRSA strains and beholds much promise in clinical applications.     

Analysis of bacterial lipodepsipeptides by matrix-assisted laser desorption/ionisation time-of-flight and high-performance liquid chromatography with electrospray mass spectrometry

Strains of certain plant pathogenic bacteria, in particular several pathovars of Pseudomonas syringae, are known to produce cyclic lipodepsipeptides (LDPs) endowed with peculiar structural features and noticeable biological activities. In this study, a mass spectrometry procedure is proposed for screening LDP-producing bacterial strains and for identifying and assessing individual LDPs. After matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) screening of thirteen P. syringae strains for LDP production, the extracts from culture filtrates of eight positive strains were subjected to electrospray mass spectrometry for the identification of LDPs. Five strains were found to produce two forms of syringomycins (SR-E and SR-G) and two forms of syringopeptin 25 (SP25A and SP25B); two strains produced SR-E, SR-G and a new form of SP22; one strain produced syringotoxin (ST) and syringostatin A (SS-A) in addition to SP25A and SP25B. The yield in culture of two major LPDs: SR-G (3.2-13.8 mg x L(-1)) and SP25A (41.6-231.5 mg x L(-1)) was assessed by and high-performance liquid chromatography with electrospray mass spectrometry (HPLC/ESI-MS) in both scan and single ion monitoring (SIM) modes. Results of this investigation showed that the mass spectrometry protocol developed here is a precise and reliable method for screening bacterial strains for LDP production and for assessing the amount of each metabolite under various culture conditions. This could be of practical value in view of potential applications, e.g. biocontrol of post-harvest fungal diseases.     

Identification,Cloning and Biochemical Characterization of Pseudomonas putida A (ATCC 12633) Monooxygenase Enzyme necessary for the Metabolism of Tetradecyltrimethylammonium Bromide

This study presents the first report of the purification and characterization of a monooxygenase enzyme from Pseudomonas putida A (ATCC 12633) that is responsible for the oxidation of physiologically relevant quaternary ammonium compounds, the tetradecyltrimethylammonium bromide. The degradation of tetradecyltrimethylammonium bromide by P. putida A (ATCC 12633) is initiated by N-dealkylation and catalysed by tetradecyltrimethylammonium monooxygenase (TTABMO), resulting in the formation of tetradecylalkanal and trimethylamine. Based on sequence analysis, the gene for TTABMO (ttbmo) corresponded to an ORF named PP2033 in the genome of P. putida KT2440. Mutation in ttabmo blocked the utilization of tetradecyltrimethylammonium bromide by Pseudomonas putida A (ATCC 12633) as carbon and nitrogen sources. The enzyme can be highly overexpressed in P. putida Δttabmo-T7 in active form and purified as a hexahistidine fusion protein. Like the native enzyme, the his-TTABMO was found to be a monomer with molecular mass of 40 kDa, the isoelectric point 7.3, that catalyses the breakdown of tetradecyltrimethylammonium bromide and utilized NADPH and FAD as cofactor. The biochemical properties and the analysis of the respective protein sequence revealed that TTABMO represents a typical flavoprotein monooxygenase, which is member of a flavoprotein family that is distinct from Baeyer–Villiger monooxygenases.     

Formation and purification of nitrile hydratase fromCorynebacterium pseudodiphteriticum ZBB-41

A strain of nitrile hydratase-forming microorganism,Corynebacterium pseudodiphteriticum ZBB-41, was isolated from soil and the conditions for the enzyme formation have been studied. Addition of ferric or ferrous ions and the use of methacrylamide as an inducer greatly enhanced enzyme formation. When the strain was cultivated at 27°C for 64 h in an optimum medium, the specific activity of the enzyme was 83U/mg of dry cell weight. The enzyme was purified from the crude extract of methacrylamide induced cells in 6 steps with 9.5-fold purification and 8.8% recovery. The enzyme has a molecular mass of 80,000 and consists of two subunits with molecular mass of 28,000 and 25,000 respectively. The isoelectric point was 4.2. The Km value to acrylonitrile was 0.21 M. The enzyme was strongly inhibited by Hg²⁺, Cu²⁺, Ag⁺, iodoacetate, and phenyl mercuric acetate and can be protected by 2-mercaptoethanol. The enzyme was noncompetitively inhibited by sodium cyanide, the Ki value was 0.13 mM.     

The size of adenylate cyclase and guanylate cyclase from the rat renal medulla.

The size distribution of adenylate cyclase from the rat renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant form in Triton X-100 are s20,w, 5.9S; Strokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are: s20w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value for V for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrane components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrane. Similar studies have been performed on the soluble guanylate cyclase of the rat renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20w, 6.3S; Stokes radius, 54 A, V, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; Axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases it activity two- to fourfold and changes the physical properties to: s20,w, 5.5S; Stokes radius, 62 A; V, 0.74 ml/g; mass, 148,000 daltons, f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregate with s20,w, 10S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.     

Influence of encapsulated enzyme on the surface properties of freeze-dried liposomes in trehalose

The interfacial properties of liposomes in which a soluble protease from Mucor meihie was encapsulated and subjected to freeze-drying procedure in the presence of trehalose, have been studied. Enzyme encapsulation in liposomes subjected to freeze-drying produces changes in the membrane interfacial properties as a consequence of penetration of some of the protein hydrophobic portion into the lipid bilayer. This membrane perturbation can be explained when considering that the presence of trehalose during the freeze-drying process brings about membrane hydrophobic defects where the protein can be accommodated without inducing any additional disorder. These interfacial changes in the membrane were detected by a decreased partition of Merocyanine 540, loss in the lytic effect of lysoderivatives and an increase in ANS binding sites in comparison to freeze-dried liposomes in the absence of protease. This information can be useful to modify the enzyme release of liposomes in a controlled way as a function of the interacting molecules present in the outside environment.     

Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.     

Magnetic octadecyl‐based matrix solid‐phase dispersion coupled with gas chromatography with mass spectrometry in a proof‐of‐concept determination of multi‐class pesticide residues in carrots

A novel procedure is put forward based on the combination of the well‐established matrix solid‐phase dispersion and the magnetic and sorption properties of magnetic octadecyl in the presence of n‐octanol and was employed in a proof‐of‐concept sample preparation and determination of several classes of pesticide residues in carrots. The procedure does not require the transfer of blend to cartridge and subsequent packing, nor any co‐sorbent for extract clean up. The hydrophobic magnetic nanoparticles utilized as a sorbent, can be retrieved by n‐octanol under the application of a magnetic field due to hydrophobic interactions. Elution of pesticide residues is then carried out with an organic solvent. A total of 26 pesticides were included in this procedure and the target compounds were analyzed using gas chromatography with mass spectrometry in the selective ion monitoring mode. The average extraction recoveries obtained from carrot samples fortified at three different concentrations (20, 50, and 500 μg/kg) were 77–107%. The estimated limits of quantitation for most target analytes were in the low μg/kg level. The study demonstrates that the proposed extraction procedure is simple and effective, avoiding a clean‐up step for the sample preparation of vegetable.