scholarly journals Expression and characterization of recombinant trehalose synthase in Bacillus subtilis

2020 ◽  
Vol 42 (4) ◽  
Author(s):  
Nguyen Ngoc Trieu ◽  
Nguyen Thi Quynh ◽  
Nguyen Duc Hoang ◽  
Nguyen Manh Dat ◽  
Tran Duc Long ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Bacillus Subtilis ◽  
Specific Activity ◽  
Optimal Conditions ◽  
Trehalose Synthase ◽  
Optimal Activity ◽  
His Tag ◽  
C Terminus

Trehalose synthase (TreS, EC 2.4.1.245) is a potential catalyst for synthesis of trehalose, an important natural disaccharide. In this study, the treS gene of Pseudomonas putida (VTCC 12263) was cloned into pHT01 plasmid at BamHI-XbaI position, expressed in Bacillus subtilis (B. subtilis) 1012, and characterized. The recombinant TreS had molecular weight of 68 kDa when fused with 8xHis tag at the C-terminus. catalyzed conversion of maltose to trehalose in optimal conditions had specific activity of 1.664 U/g. Expression of TreS was highest when B. subtilis 1012 harboring pHT01-treS was cultured in TB medium at 30 oC, induced with 1.0 mM IPTG when OD600 reached 0.8 and harvested after 10 hours of induction. The recombinant TreS purified by Ni-sepharose chromatography had specific activity of 41.700 U/g and formed a single band on Western blot with monoclonal antibody against His-tag. The recombinant TreS had optimal activity at 37 oC in 100 mM pH 7.4 PBS and 300 mM maltose. It was inhibited by NaCl, KCl and MgCl2 (retaining 45% or 75% specific activity in buffer containing 5 mM KCl or 5 mM MgCl2, respectively) and stimulated by imidazol (with specific activity increasing by 30–200%).

scholarly journals Immunohistochemical and immunochemical characterization of a new endothelial cell-specific antigen.

1986 ◽  
Vol 34 (2) ◽  
pp. 209-214 ◽  
Author(s):  
J U Alles ◽  
K Bosslet
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Specific Antigen ◽  
Similar Molecular Weight ◽  
Immunochemical Characterization ◽  
Sds Page

A new monoclonal antibody (moab BW 200) of IgG3 kappa-isotype was generated which recognizes an epitope located on an antigen molecule restricted to human neoplastic and non-neoplastic endothelial cells. The molecular weight of the antigen was determined using immunoprecipitation analysis followed by SDS-PAGE. Despite its similar molecular weight to FVIII-RAG, the antigen detected by moab BW 200 was shown to be different from FVIII-RAG.

scholarly journals Production optimization using Plackett-Burman and Box-Behnken designs with partial characterization of amylase from marine actinomycetes

2021 ◽  
Author(s):  
Sarah I Bukhari ◽  
Mohamed H Al-Agamy ◽  
Mahmoud S Kelany ◽  
Mohammad R Al Hazani ◽  
Moaz M Hamed
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Fermentation Medium ◽  
Culture Conditions ◽  
Production Optimization ◽  
Activity Measurement

Abstract Amylase is an industrial enzyme that is used in the food and biofuel industries. We screened four actinomycetes strains for amylase biosynthesis. The Streptomyces rochei strain had a larger hydrolytic zone (24 mm) on starch agar plates, than the other isolates. Plackett-Burman’s experimental design was implemented to optimize the conditions for amylase production by the selected strains. Growth under optimized culture conditions led to 1.7, 9.8, 7.7, and 3.12 -fold increases for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specific activity measurement in comparison with growth under primary conditions. When applying the Box-Behnken design on S. rochei using the most significant parameters starch, K2HPO4, pH, and temperature, there was a 12.22-fold increase in the specific activity measurement: 7.37 U/mg. The optimal fermentation medium formula was kept at 30.6°C for seven days. The amylase from S. rochei was partially purified, and its molecular weight was determined using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was found to be 45, 43, and 53 kDa. Amylase was particularly active at pH 6 and 65°C. The purified enzyme was most active at 65°C and a pH of 6, thermal stability of 70°C for 40 min and salt concentration of 1 M with a Km and Vmax of 6.58 mg/ml and 21.93 mg/ml/min, respectively. The amylase improved by adding Cu + 2, Zn + 2, and Fe + 2 (152.21%, 207.24%, and 111.89%). Increased production of amylase enzyme by Streptomyces rochei KR108310 attracts the production of industrially significant products.

scholarly journals Characterization of Inhibin from Culture and Non Culture of Granulose Cells for Monoclonal Antibody of Inhibin Production

2010 ◽  
Vol 4 (1) ◽  
Author(s):  
Amiruddin Amiruddin ◽  
Tongku Nizwan Siregar ◽  
Amalia Sutriana ◽  
Dwinna Aliza ◽  
T. Armansyah
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Western Blot ◽  
Total Amino Acid ◽  
Multiple Ovulation ◽  
Sds Page ◽  
Isoelectric Points ◽  
Granulose Cells

This study has long-term objectives to obtain immunogenic prototype that can be used to induce multiple ovulation in goats. Working steps of this study were begun with the collection of ovarium from goats, collection of granulose cells, culture of granulose and characterization of molecular weight and isoelectric point (pI) of inhibin protein of granulose cells obtained from culture and non-culture of granulose cells, and followed by preparation of monoclonal antibody toward inhibin. The results showed that inhibin isolated either from culture or non-culture of granulose cells produced a 32 kDa band. Molecular weight of inhibin was measured by Western Blot. The 32 kDa band of SDS PAGE product appeared on Western Blot result was inhibin molecules produced by granulose cells collected fom culture and non-culture of granulose cells that can be identified by Mab-inhibin. Product of IEF gel electrophoresis suggested that inhibin molecule collected from culture of granulose cells has no charge at isoelectric points ranging from 5-6, depends on its total amino acid composition.

Purification and characterization of catalase-1 from Bacillus subtilis

1987 ◽  
Vol 65 (11) ◽  
pp. 939-947 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala
Keyword(s):  
Hydrogen Peroxide ◽  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Catalase Activity ◽  
Purification And Characterization ◽  
Ph Range ◽  
Sulfhydryl Compounds ◽  
Native Molecular Weight

The catalase activity produced in vegetative Bacillus subtilis, catalase-1, has been purified to homogeneity. The apparent native molecular weight was determined to be 395 000. Only one subunit type with a molecular weight of 65 000 was present, suggesting a hexamer structure for the enzyme. In other respects, catalase-1 was a typical catalase. Protoheme IX was identified as the heme component on the basis of the spectra of the enzyme and of the isolated hemochromogen. The ratio of protoheme/subunit was 1. The enzyme remained active over a broad pH range of 5–11 and was only slowly inactivated at 65 °C. It was inhibited by cyanide, azide, and various sulfhydryl compounds. The apparent Km for hydrogen peroxide was 40.1 mM. The amino acid composition was typical of other catalases in having relatively low amounts of tryptophan and cysteine.

Production and characterization of a murine monoclonal antibody against human thromboxane synthetase

1987 ◽  
Author(s):  
P Wernet ◽  
M Haurand ◽  
W Nüsing ◽  
E M Schneider ◽  
K Jaschonek ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Specific Activity ◽  
Serum Antibody ◽  
Leukemic Cells ◽  
Lung Fibroblasts ◽  
Tissue Blood Flow ◽  
Specific Staining ◽  
Thromboxane Synthetase ◽  
Serum Antibody Titer

Eicosanoids appear to have an important role in the actual momentary regulation of tissue blood flow. The function of constricting blood vessels by affecting the vascular tone has been assigned to thromboxane. Thromboxane synthetase, the enzyme responsible for the conversion of Prostaglandin-H2 into thromboxane A2, has been shown to be present in platelets, lung fibroblasts and the brain. Recently, thromboxane synthetase has been totally purified. The enzyme isolated from platelets appears to have a molecular weight of 58,800 Dalton and to belong to the group of cytochrome P450 proteins. In order to make a monoclonal antibody against thromboxane synthetase, BALB/c mice were injected four times i.m. with 10, 5, 5 and 4 μg of the platelet purified enzyme in complete Freund's adjuvant. The serum antibody titer against thromboxane synthetase in an ELISA was higher than 1:1000 after the second boost. One mouse received a fifth i.v. injection of 10 μg of the purified enzyme. One monoclonal antibody of the several hundreds of hybridomas screened in an ELISA revealed specific activity against thromboxane synthetase with a titer of 1:512 present in the culture supernatant. After recloning this reagent, called T0300, was used for the preparation of an immunoaffinity column, where it also reacted specifically. In immunoprecipitation experiments T0300 was able to precipitate a 58,000 D molecule. Also the biological activity of thromboxane synthetase could be blocked by monoclonal antibody T0300. In addition this reagent was employed in indirect immunofluorescence on leukemic cells employing a FACS IV cytofluorometer. Here specific staining of two megacaryocytic blast cell populations could be demonstrated. Thus T0300 appears to be a monoclonal antibody against human thromboxane synthetase.

scholarly journals Preliminary Characterization of a Monoclonal Antibody (AS-2) against Cell Cycle Related Proteins

1998 ◽  
Vol 17 (2) ◽  
pp. 93-101
Author(s):  
Stefano Nigro ◽  
Anna Rapallo ◽  
Angela Di Vinci ◽  
Elio Geido ◽  
Roberto Orecchia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Dna Content ◽  
Staining Pattern ◽  
Isolated Nuclei ◽  
Erythroleukemia Cell ◽  
Related Proteins

A monoclonal antibody (AS-2) raised by using isolated nuclei from a human erythroleukemia cell line as immunogen is described.AS-2 was of IgM type and recognized proteins present in both isolated cytoplasms and nuclei. The molecular weight of the AS-2 recognized proteins in the cytoplasm was 200 kDa and 70 and 60 kDa in the nucleus. The relative amount of these proteins were measured simultaneously with DNA content by flow cytometry. We found the highest protein content (or stainability) for both cells and nuclei in late-G1, S and G2, at approximately the same level, and the lowest content in M and early-G1. Sorting based on DNA content and AS-2 associated fluorescence helped identifying the staining pattern of cells and nuclei. Interphase isolated nuclei and cell cytoplasms were characterized by interdispersed staining over the entire surfaces while mitoses showed two dots only. The present preliminary data indicate that the proteins recognized by the AS-2 monoclonal are cell cycle related and suggest that in mitoses they are associated with the centrosomes.

Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5

Biologia ◽  
2011 ◽  
Vol 66 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Farrukh Jamil ◽  
Naeem Rashid ◽  
Qurra-tul-Ann Gardner ◽  
Muhammad Akhtar
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Specific Activity ◽  
Subtilis Strain ◽  
Amino Acid Residues ◽  
Expression And Purification ◽  
Gene Encoding ◽  
Glycine Oxidase ◽  
Bacillus Subtilis Strain R5

AbstractThe gene encoding the glycine oxidase from Bacillus subtilis strain R5 (goxR) was cloned and expressed in Escherichia coli. The gene consisted of 1,110 nucleotides that encoded a protein (GoxR) of 369 amino acid residues with a molecular mass of 40,761 Da. The GoxR exhibited 98.6% identity with glycine oxidase from B. subtilis strain 168. Gene expression and purification of the recombinant GoxR were performed. The recombinant GoxR existed in a homotetramer form. The recombinant protein effectively catalyzed the oxidation of glycine and d-alanine. The specific activity of the purified recombinant GoxR was 0.96 U/mg when glycine was used as a substrate and 1.0 U/mg when d-alanine was substrate. The enzyme displayed its highest activity at pH 8.0 and at a temperature of 50°C. The activation energy of the reaction catalyzed by the enzyme was calculated to be 26 kJ/mol. The enzyme activity was significantly inhibited in the presence of organic solvents. No enhancement of enzyme activity was observed in the presence of metal cations. The experimental results presented in this study demonstrate that the enzyme was a bonafide glycine oxidase.

Characterization of SPβ: a temperate bacteriophage from Bacillus subtilis 168M

1977 ◽  
Vol 23 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Fred D. Warner ◽  
Gary A. Kitos ◽  
Martin P. Romano ◽  
H. Ernest Hemphill
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Mitomycin C ◽  
Temperate Bacteriophage ◽  
Low Levels

Bacillus subtilis 168M is lysogenic for a temperate bacteriophage called SPβ. The virus head is 76 m wide by 82 m long and the tail measures 12 by 358 m. The DNA molecular weight is 62 million. SPβ is spontaneously released at low levels in cultures of B. subtilis 168M, and can be induced at higher levels by treatment with mitomycin C or N-methyl-N′-nitro-N-nitroso-guanidine.

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