Purification and Characterization of β-Glucosidase from a Newly Isolated Strain Tolypocladium cylindrosporum Syzx4

A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer(ITS) rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27...     

Isolation and Characterization of Galloylglucoses Effective against Multidrug-Resistant Strains of Escherichia coli and Klebsiella pneumoniae

The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid–liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-β-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-β-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-β-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-β-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2–256 µg/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria.     

Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

To analyze whether expressed amyloid precursor protein(APP) existed in hydrophilic(cytoplasmid) or hy-drophobic(lipid bilayer) environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coli. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hy-drophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni2. agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coil cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.     

Characterization of a Novel Mesophilic Bacterial Amylase Secreted by ZW2531-1, a Strain Newly Isolated from Soil

A novel mesophilic bacterial amylase, named oligosaccharide-producing multifunctional amylase(OPMA), was discovered and characterized. OPMA is an extracellular enzyme secreted by ZW2531-1, a strain newly isolated from Chinese soil. It could be purified to homogeneity from the culture supernatant of ZW2531-1 by 30%―60% saturated ammonium sulfate precipitation, followed by twice Sephadex gel filtration chromatography. OPMA is a 66 kDa protein based on SDS-PAGE and has an isoelectric point(pI) at pH=5.3 by Iso...     

Purification, Molecular Cloning, and Biochemical Characterization of Subtilisin JB1 from a Newly Isolated Bacillus subtilis JB1

An extracellular gelatinolytic enzyme obtained from the newly isolated Bacillus subtilis JB1, a thermophilic microorganism relevant to the aerobic biodegradation process of fish-meal production, was purified via ammonium sulfate precipitation, Sephadex G-200 Gel filtration chromatography, and one-dimensional gel electrophoresis separation and subsequently identified via peptide mass fingerprinting and chemically assisted fragmentation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The subtilisin JB1 gene was sequenced and its recombinant protein prosubtilisin JB1 was expressed in Escherichia coli, and the purified prosubtilisin JB1 (62 kDa) protein was digested with gelatin, bovine serum albumin, azocasein, fibrinogen, and the fluorogenic peptide substrate Ala-Ala-Phe-7-amido-4-methylcoumarin hydrochloride, whereas the serine protease inhibitors phenylmethylsulfonyl fluoride and chymostatin completely inhibited its enzyme activity at an optimal pH of 7.5. Thus, our results show that subtilisin JB1 may serve as a potential source material for use in industrial applications of proteolytic enzymes and microorganisms for fishery waste degradation and fish by-product processing.     

Purification and Biochemical Characterization of a Novel Alkaline (Phospho)lipase from a Newly Isolated Fusarium solani Strain

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH₂-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca²⁺ and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5–10 and at temperatures below 45 °C.     

Production and Characterization of a Novel Thermostable Extracellular Agarase from Pseudoalteromonas hodoensis Newly Isolated from the West Sea of South Korea

A Gram-negative, aerobic, motile, rod-shaped, agarolytic bacterium, designated as H7, was isolated from a coastal seawater sample. This strain grows at pH 6.0–8.0, temperature of 15–40 °C, and at an NaCl concentration of 1–7 % (w/v). Ubiquinone-8 was the predominant respiratory quinone, and the DNA G+C content was 45.82 mol%. Analysis of the 16S rRNA sequence suggests that strain H7 belongs to the genus Pseudoalteromonas. DNA-DNA hybridization analysis showed DNA relatedness of as low as 55.42 and 40.27 % with its nearest phylogenetic neighbors Pseudoalteromonas atlantica IAM12927^T and Pseudoalteromonas espejiana NCIMB2127^T, respectively, which led us to name H7 Pseudoalteromonas hodoensis sp. nov. The type strain is H7^T (=DSM25967^T = KCTC23887^T). An agarase (AgaA7) was purified to homogeneity from the cell-free culture broth of H7 through many steps of chromatography. Purified AgaA7 had an apparent molecular weight of 35 kDa, with a distinct NH₂-terminal sequence of Ala-Asp-Ala-Thr-X-Pro (X, any amino acid) from the reported proteins, implying that it is a novel enzyme. The optimum pH and temperature for agarase activity were 7.0 and 45 °C, respectively. Thin-layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that AgaA7 is both an exo- and endo-type β-agarase that degrades agarose into neoagarotetraose, neoagarohexaose, and neoagarooctaose (minor).     

Expression and Purification of Bioactive High-Purity Recombinant Mouse SPP1 in Escherichia coli

Secreted phosphoprotein 1 (SPP1) is a phosphorylated acidic glycoprotein. It is broadly expressed in a variety of tissues, and it is involved in a number of physiological and pathological events, including cancer metastasis, tissues remodeling, pro-inflammation regulation, and cell survival. SPP1 has shown its function of protecting tissues and organs against injury and wound, giving itself potentials to become a therapy target or giving its antibodies of other counter-acting reagents potentials to become drug candidates. Non-tagged (native) recombinant SPP1 would be valuable in therapeutic and pharmaceutical researches. In our study, mouse Spp1 DNA fragment without signal peptide was built in pET28a(+) vector and transformed into Escherichia coli BL21 (DE3). The recombinant mouse SPP1 (rmSPP1) was then expressed in bacteria upon induction by isopropyl β-d-thiogalactopyranoside (IPTG). The abundance of rmSPP1 was increased using isoelectric precipitation and ammonium sulfate fractionation methods, and anion and cation exchange chromatography was employed to further purify rmSPP1. Finally, we got rmSPP1 product with 12.8 % productivity, 97 % purity, satisfactory bioactivity, and low endotoxin content.     

Expression of Recombinant Human Epidermal Growth Factor in Escherichia coli and Characterization of its Biological Activity

Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.     

Purification and Characterization of Alkaline Pectin Lyase from a Newly Isolated Bacillus clausii and Its Application in Elicitation of Plant Disease Resistance

Alkaline pectin lyase (PNL) shows potential as a biological control agent against several plant diseases. We isolated and characterized a new Bacillus clausii strain that can produce 4,180?U/g of PNL using sugar beet pulp as a carbon source and inducer. The PNL was purified to apparent homogeneity using ultrafiltration, ammonium sulfate fractionation, DEAE Sepharose Fast Flow, and Sephadex G-75 gel filtration. The purified PNL was found to be a monomeric protein with a molecular weight of 35?kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It demonstrated optimal activity with K _m of 0.87?mg/ml at pH?10.0 and 60?°C. The enzyme is stable in the pH range of 8.0?C10.0 and temperature ??40?°C. Ca²⁺ was found to stimulate the enzymatic activity of the PNL by up to 410?%. Mass spectrometric results gave 38?% match coverage with pectate lyase from B. clausii KSM-K16 (gi|56961845). The PNL was found to elicit disease resistance in cucumber seedlings, suggesting that it may have applications in biocontrol and sustainable agriculture.     

Synthesis and Expression in Escherichia coli of a Human Neutrophil Activating Protein-1/Interleukin-8 Gene

The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was sho     

Biosynthesis of Zinc Oxide Nanoparticles Using Leaf Extract of Passiflora subpeltata: Characterization and Antibacterial Activity Against Escherichia coli Isolated from Poultry Faeces

The current study was undertaken to investigate the antibacterial (against molecular characterized E. coli isolated from poultry faeces) potential of biosynthesized zinc oxide nanoparticles (ZnO-NPs) from Passiflora subpeltata Ortega aqueous leaf extract. The biosynthesized nanoparticles were subjected to physico-chemical characterization to study shape, size and purity by UV–Vis spectroscopy, X-Ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS) and Transmission Electron Microscopy (TEM). The molecular identification of isolated E. coli from faeces samples was carried out by using 16–23s rRNA primers. The results of the physico-chemical characterization revealed that the biosynthesized nanoparticles were of 93.7% purity with an average size between 45 and 50 nm. The ZnO-NPs offered significant inhibition against the isolated Gram-negative E. coli with MIC at 62.5 µg mL^?1 concentration. The antibacterial potential of ZnO NPs against E. coli has also been investigated by the cell viability test, and further the effects of ZnO NPs on bacterial morphological structures was analysed by SEM and TEM.

     

Production and Characteristics of a Bioflocculant by Klebsiella pneumoniae YZ-6 Isolated from Human Saliva

The production and characterization of a bioflocculant, MBF-6, by Klebsiella pneumoniae was investigated. Optimum culture conditions for bioflocculant production were an initial medium pH of 7, an incubation temperature of 30 °C, and an inoculum size of 1 % (v/v) of cell density 1.0?×?10⁸ cfu/mL. The carbon, nitrogen, and cation sources for optimum bioflocculant production were glucose, peptone, and ZnCl₂. The bioflocculant mainly consisted of protein (3.4 %) and sugar (95.1 %). Fourier transform infrared (FTIR) spectrum revealed the presence of carboxyl and hydroxyl groups while the thermogravimetric analysis (TGA) showed a degradation temperature (T _d) of 81.4 °C. MBF-6 had a good flocculating rate in kaolin suspension without cation addition and was stable over a wide range of pH and temperature. Investigation on the flocculation efficacy of the characterized MBF-6 for wastewater treatment of dairy, woolen, brewery, and sugar industries suggested it to be effective.     

Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production,Characterization, and Thermal Stability

A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g^?1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g^?1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K _M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.     

人野生型MBL基因在大肠杆菌中的表达

为获得人MBL蛋白，并对其功能进行初步研究，用DNA重组法构建了组氨到标签融合原核表达质粒pET28(b)-MBL。将重组质粒转入大肠杆菌BL21（DE3），经IPTG在37℃条件下诱导培养，利用SDS－PAGE，Westem-blot检测目的蛋白的表达，用IMAC金属螯合层析柱对其进行纯化。成功地表达了重组MBL蛋白，纯化的MBL浓度约为844μg/mL，为制备MBL的基因工程抗体奠定了基础。     

信号肽对OPMA在枯草杆菌中的分泌表达及其催化活性的影响

将编码枯草杆菌BS168的amyE信号肽(S)的基因与编码多功能淀粉酶OPMA-N和OPMA-G的基因重组, 分别构建了分泌表达多功能淀粉酶OPMA-N和OPMA-G的重组载体pMA5-OPMA-SN和pMA5-OPMA-SG, 并分别在枯草杆菌BS168中实现了OPMA-SN和OPMA-SG的有效分泌表达.SDS-PAGE分析表明, 该信号肽的引入提高了异源蛋白的分泌量, OPMA-SN和OPMA-SG的分泌水平(53%和67%)比OPMA-N和OPMA-G(45%和58%)明显提高, 但此信号肽在分泌表达OPMA-N或OPMA-G后不能被有效切除.活力分析表明, OPMA-SN(5.17 U/mg)和OPMA-N(4.57 U/mg)的活力水平与分泌水平一致, 但OPMA-SG(4.50 U/mg)和OPMA-G(4.65 U/mg)的活力水平却与分泌水平呈反相关性.通过分析OPMA-SN和OPMA-SG分子的空间构象发现, 信号肽的存在不影响OPMA-N的活性部位构象, 但影响OPMA-G的活性部位构象, 从而导致OPOMA-G的催化活性下降.     

An Extremely Alkaline Novel Xylanase from a Newly Isolated Streptomyces Strain Cultivated in Corncob Medium

Streptomyces sp. CS802, recently isolated from Korean soil, produced xylanase in corncob medium. An extracellular xylanase (Xyn802) was purified by a single-step gel filtration and biochemical properties were studied. It showed high activity in extremely alkaline condition with optimum pH at 12.0 and exhibited stability between pH?7.5 and 13.0. It produced xylobiose and xylotriose as the major products from xylan, suggesting its endoxylanase nature. N-terminal amino acid sequences of Xyn802 were ADRNANRD which are significantly different from the reported xylanase. The activity was enhanced by various detergents and a reducing agent and stable in various organic solvents. Xyn802 produced by utilizing corncob, an agro-waste material, might be a novel xylanase based on its peculiar biochemical characteristics, and it can be a suitable candidate for the production of xylooligosaccharides including other useful products.