Isolation and Characterization of Wild-Type Lipoxygenase LOXPsa1 from Pleurotus sapidus

2014 ◽  
Vol 69 (3-4) ◽  
pp. 149-154 ◽  
Author(s):  
Ina Plagemann ◽  
Ulrich Krings ◽  
Ralf G. Berger
Keyword(s):  
De Novo ◽  
Specific Activity ◽  
Maximal Activity ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Peptide Mass ◽  
Purification Scheme ◽  
Pleurotus Sapidus ◽  
Nano Liquid Chromatography

The lipoxygenase LOXPsa1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS=MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOXPsa1 were identified by de novo sequencing, and the postulated molecular mass of 72:5 kDa was confirmed. With linoleic acid as the substrate, the LOXPsa1 showed a specific activity of 113 U mg-1 and maximal activity at pH 7.0 and 30 °C, respectively.

scholarly journals Characterization of a Mannose-6-Phosphate Isomerase fromThermus thermophilusand Increased l-Ribose Production by Its R142N Mutant

2010 ◽  
Vol 77 (3) ◽  
pp. 762-767 ◽  
Author(s):  
Soo-Jin Yeom ◽  
Eun-Sun Seo ◽  
Bi-Na Kim ◽  
Yeong-Su Kim ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Wild Type ◽  
Active Site Residues ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Mannose 6 Phosphate

ABSTRACTAn uncharacterized gene fromThermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed inEscherichia coli. The maximal activity of the recombinant enzyme forl-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion ofl-ribulose tol-ribose, a potential starting material for manyl-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase inl-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. Thekcat/Kmof the R142N mutant was 3.8-fold higher than that ofGeobacillus thermodenitrificansmannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reportedkcat/Km. The R142N mutant enzyme produced 213 g/literl-ribose from 300 g/literl-ribulose for 2 h, with a volumetric productivity of 107 g liter−1h−1, which was 1.5-fold higher than that of the wild-type enzyme.

scholarly journals Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Saleem Farooq ◽  
Ruqeya Nazir ◽  
Shabir Ahmad Ganai ◽  
Bashir Ahmad Ganai
Keyword(s):  
Specific Activity ◽  
Temperature Range ◽  
Psychrotrophic Bacteria ◽  
Isolation And Characterization ◽  
Cold Active ◽  
Active Protease ◽  
Quantitative Screening ◽  
First Time ◽  
Low K

AbstractAs an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4–30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5–35 °C and pH 6.0–12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low Km value of 1.0 mg/ml and Vmax 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

scholarly journals Isolation and characterization of the SPT2 gene, a negative regulator of Ty-controlled yeast gene expression.

1985 ◽  
Vol 5 (7) ◽  
pp. 1543-1553 ◽  
Author(s):  
G S Roeder ◽  
C Beard ◽  
M Smith ◽  
S Keranen
Keyword(s):  
Gene Product ◽  
Regulatory Region ◽  
Negative Regulator ◽  
Transcriptional Level ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Insertion Mutations ◽  
His4 Gene

The his4-917 mutation of Saccharomyces cerevisiae results from the insertion of the Ty element Ty917 into the regulatory region of the HIS4 gene and renders the cell His-. The hist4-912 delta mutant, which carries a solo delta in the 5'-noncoding region of HIS4, is His+ at 37 degrees C but His- at 23 degrees C. Both these mutations interfere with HIS4 expression at the transcriptional level. The His- phenotype of both insertion mutations is suppressed by mutations at the SPT2 locus. The product of the wild-type SPT2 gene apparently represses HIS4 transcription in these mutant strains; this repression is relieved when the SPT2 gene is destroyed by mutation. The repression of transcription by SPT2 presumably results from an interaction between the SPT2+ gene product and Ty or delta sequences. In this paper, we report the cloning and DNA sequence analysis of the wild-type SPT2 gene and show that the gene is capable of encoding a protein of 333 amino acids in length. In addition, we show that a dominant mutation of the SPT2 gene results from the generation of an ochre codon which is presumed to lead to a shortened SPT2 gene product.

Isolation and characterization of Schizosaccharomyces pombe mutants with defective NAD-dependent malic enzyme

1986 ◽  
Vol 32 (6) ◽  
pp. 481-486 ◽  
Author(s):  
C. Osothsilp ◽  
R. E. Subden
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Malic Enzyme ◽  
Pool Analysis ◽  
Starch Gel Electrophoresis ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Starch Gel ◽  
Colony Color ◽  
Color Indicator

To obtain NAD-dependent malic enzyme mutants of Schizosaccharomyces pombe, a colony color indicator screening system was developed. Mutants defective for malic acid utilization (mau mutants) are yellow, while wild-type colonies are blue on the defined bromcresol green based indicator medium. NAD-dependent malic enzyme mutants were distinguished from other mau mutants by subsequent, starch gel electrophoresis, spectrophotometry, complementation tests, and intermediate pool analysis with cell-free extracts.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

scholarly journals ISOLATION AND CHARACTERIZATION OF CARBENDAZIM DEGRADING Trichoderma harzianum RIFAI MUTANTS

2010 ◽  
Vol 20 ◽  
Author(s):  
ITAMAR SOARES DE MELO ◽  
CÉLIA MARIA MAGANHOTTO DE S. SILVA ◽  
JANE L. FAULL
Keyword(s):  
Trichoderma Harzianum ◽  
Uv Light ◽  
Light Exposure ◽  
Wild Type ◽  
Pesticide Degradation ◽  
Isolation And Characterization ◽  
Degradation Ability

Mutants of Trichoderma harzianum Rifai, obtained after ultraviolet (UV) light exposure, showed high resistant to the fungicide benomyl. A mutant (2B6) was capable of degrading carbendazim, other fungicide of the benzimidazole fungicide. This mutant degraded 41.5% of the molecule within five days. This and others mutants (2B1 and 2B2) presented variation in size and frequency of uni-nucleated and/or bi-nucleated spores compared to the wild type. Four primers generated RAPDs patterns that allowed the mutant to be differentiated from the wild-type. It is concluded that using UV mutagenization, it is feasible to obtain strains of T. harzianum with improved pesticide degradation ability.

Sign in / Sign up

Export Citation Format

Share Document