scholarly journals Functional Expression and Characterization of Trichome-Specific (-)-Limonene Synthase and (+)-α-Pinene Synthase from Cannabis sativa

2007 ◽  
Vol 2 (3) ◽  
pp. 1934578X0700200 ◽  
Author(s):  
Nils Günnewich ◽  
Jonathan E. Page ◽  
Tobias G. Köllner ◽  
Jörg Degenhardt ◽  
Toni M. Kutchan
Keyword(s):  
Phylogenetic Analysis ◽  
Natural Products ◽  
Cannabis Sativa ◽  
Functional Expression ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Limonene Synthase ◽  
Monoterpene Synthases

Two recombinant, stereospecific monoterpene synthases, a (-)-limonene synthase (CsTPS1) and a (+)-α-pinene synthase (CsTPS2), encoded by Cannabis sativa L. cv. ‘Skunk’ trichome mRNA, have been isolated and characterized. Recombinant CsTPS1 showed a Km value of 6.8 μM, a Vmax of 1.1 × 10−4 μmol/min and Vmax/Km of 0.016; the pH optimum was determined at pH 6.5, and a temperature optimum at 40°C. Recombinant CsTPS2 showed a Km value of 10.5 μM, a Vmax of 2.2 × 10−4 μmol/min and Vmax/Km of 0.021; the pH optimum was determined at pH 7.0, and a temperature optimum at 30°C. Phylogenetic analysis showed that both CsTPSs group within the angiosperms and belong to the Tpsb subgroup of monoterpene synthases. The enzymatic products (-)-limonene and (+)-α-pinene were detected as natural products in C. sativa trichomes.

Gene cloning, functional expression and characterization of a novel GH46 chitosanase from Streptomyces avermitilis (SaCsn46A)

2021 ◽  
Author(s):  
Jing Guo ◽  
Yi Wang ◽  
Wenjun Gao ◽  
Xinrou Wang ◽  
Xin Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Functional Expression ◽  
Streptomyces Avermitilis ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Glycosidic Bonds ◽  
Sds Page ◽  
Mature Enzyme ◽  
Layer Chromatography

Abstract A novel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acids signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 oC and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 oC. The Km and Vmax values of this enzyme were 1.32 mg∙mL− 1, 526.32 µM∙mg− 1∙min− 1, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3 mM concentration. The enzyme can hydrolyze a variety of polysaccharides which linked by β-1,4-glycosidic bonds such as chitin, xylan and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

Partial Purification and Characterization of S-Adenosyl-ʟ-Methionine: Norreticuline N-Methyltransferases from Berberis Cell Suspension Cultures

1986 ◽  
Vol 41 (1-2) ◽  
pp. 126-134 ◽  
Author(s):  
Chi-Kit Wat ◽  
Paul Steffens ◽  
Meinhart H. Zenk
Keyword(s):  
Cell Suspension ◽  
Cell Suspension Cultures ◽  
Apparent Molecular Weight ◽  
Suspension Cultures ◽  
Partial Purification ◽  
Enzymatic System ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Ph Optimum

Abstract Two new N-methyltransferases (NMT-I and NMT-II) were found to occur in Berberis vulgaris cell suspension cultures. One of these enzymes (NMT-I) was partially purified (100-fold) and characterized. This enzyme is specific for tetrahydrobenzylisoquinoline alkaloids and S-adenosyl-ʟ-methionine serves as the methyl donor. The apparent molecular weight of the enzyme is 68,000. The pH optimum of the enzyme is 7.6, the temperature optimum 35 °C. Apparent KM values for (R)-tetrahydropapaverin as substrate were 0.2 mᴍ and for SAM 0.04 mᴍ. The preparation of the same type of enzyme from B. wilsoniae var. subcaulialata was utilized as an efficient enzymatic system for the synthesis of stereochemically pure (R)-as well as (S)-reticuline labelled with tritium or 14C at the N-CH3 group. Enzymes catalyzing this type of reactions are named S-adenosyl-ʟ-methionine: norreticuline N-methyltransferases.

scholarly journals Characterization and Phylogenetic Analysis of a Novel GH43 β-Xylosidase From Neocallimastix californiae

2021 ◽  
Vol 2 ◽  
Author(s):  
Marcus Stabel ◽  
Julia Hagemeister ◽  
Zacharias Heck ◽  
Habibu Aliyu ◽  
Katrin Ochsenreither
Keyword(s):  
Phylogenetic Analysis ◽  
Hydrolytic Enzymes ◽  
Lignocellulosic Materials ◽  
Anaerobic Fungi ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Highly Active ◽  
Beechwood Xylan ◽  
Hemicellulose Degradation ◽  
Huge Variety

Degradation of lignocellulosic materials to release fermentable mono- and disaccharides is a decisive step toward a sustainable bio-based economy, thereby increasing the demand of robust and highly active lignocellulolytic enzymes. Anaerobic fungi of the phylum Neocallimastigomycota are potent biomass degraders harboring a huge variety of such enzymes. Compared to cellulose, hemicellulose degradation has received much less attention; therefore, the focus of this study has been the enzymatic xylan degradation of anaerobic fungi as these organisms produce some of the most effective known hydrolytic enzymes. We report the heterologous expression of a GH43 xylosidase, Xyl43Nc, and a GH11 endoxylanase, X11Nc, from the anaerobic fungus Neocallimastix californiae in Escherichia coli. The enzymes were identified by screening of the putative proteome. Xyl43Nc was highly active against 4-Nitrophenol-xylopyranosides with a Km of 0.72 mM, a kcat of 29.28 s−1, a temperature optimum of 32°C and a pH optimum of 6. When combined, Xyl43Nc and X11Nc released xylose from beechwood xylan and arabinoxylan from wheat. Phylogenetic analysis revealed that Xyl43Nc shares common ancestry with enzymes from Spirochaetes and groups separately from Ascomycete sequences in our phylogeny, highlighting the importance of horizontal gene transfer in the evolution of the anaerobic fungi.

scholarly journals Preparation and Characterization of 5′-Phosphodiesterase from Barley Malt Rootlets

2010 ◽  
Vol 5 (2) ◽  
pp. 1934578X1000500
Author(s):  
Jie Hua ◽  
Ke-long Huang
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Barley Malt

Two 5′- phosphodiesterases (5′-PDE-a and 5′-PDE-b) were isolated from barley malt rootlets, and further purified by gel filtration on Sephadex G-25 and Sephadex G-75. 5′-PDE-a had a pH optimum of 5.0, temperature optimum of 70oC, and specific activity of 0.0143 mM ·mg−1-min−1. 5′-PDE –b had a pH optimum of 6.0, temperature optimum of 65°C and specific activity of 0.0125 mM ·mg−1·min−1. Both enzymes can be used to hydrolyze RNA to form 5′-nucleotides. The enzymes were quite stable at 70oC for 420 minutes. The Km was 0.24 mM for 5′-PDE-a and 0.16 mM for 5′-PDE-b with t-RNA (yeast) as substrate.

scholarly journals Purification, Properties, and Characterization of Recombinant Streptomyces sp. Strain C5 DoxA, a Cytochrome P-450 Catalyzing Multiple Steps in Doxorubicin Biosynthesis

1999 ◽  
Vol 181 (1) ◽  
pp. 298-304 ◽  
Author(s):  
Robbie J. Walczak ◽  
Michael L. Dickens ◽  
Nigel D. Priestley ◽  
William R. Strohl
Keyword(s):  
Substrate Specificity ◽  
Soluble Protein ◽  
Biosynthetic Pathway ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Streptomyces Sp ◽  
Properties And Characterization ◽  
Cytochrome P 450 ◽  
Late Stages

ABSTRACT DoxA is a cytochrome P-450 monooxygenase involved in the late stages of daunorubicin and doxorubicin biosynthesis that has a broad substrate specificity for anthracycline glycone substrates. Recombinant DoxA was purified to homogeneity from Streptomyces lividanstransformed with a plasmid containing the Streptomyces sp. strain C5 doxA gene under the control of the strong SnpR-activated snpA promoter. The purified enzyme was a monomeric, soluble protein with an apparent M rof 47,000. Purified DoxA catalyzed the 13-hydroxylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin and 13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The pH optimum for heme activation was pH 7.5, and the temperature optimum was 30°C. The k cat/Km values for the oxidation of anthracycline substrates by purified DoxA, incubated with appropriate electron-donating components, were as follows: for 13-deoxydaunorubicin, 22,000 M−1 · s−1; for 13-dihydrodaunorubicin, 14,000 M−1 · s−1; for 13-dihydrocarminomycin, 280 M−1 · s−1; and for daunorubicin, 130 M−1 · s−1. Our results indicate that the conversion of daunorubicin to doxorubicin by this enzyme is not a favored reaction and that the main anthracycline flux through the late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed through the 4-O-methyl series of anthracyclines.

Characterization of Glutamine Synthetase of Roots, Etiolated Cotyledons and Green Leaves from Sinapis alba (L.)

1986 ◽  
Vol 41 (7-8) ◽  
pp. 712-716 ◽  
Author(s):  
Remigius Manderscheid ◽  
Aloysius Wild
Keyword(s):  
Glutamine Synthetase ◽  
Crude Extract ◽  
Substrate Binding ◽  
Sinapis Alba ◽  
Kinetic Properties ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Green Leaves ◽  
Sinapis Alba L

Abstract Glutamine synthetase of roots, etiolated cotyledons and green leaves from mustard plants cannot all clearly be separated by DEAE-Sephacel chromatography. However, the enzyme of the roots, etiolated cotyledons and green leaves, respectively, differed in the kinetic properties deter­mined in the crude extract. The root enzyme showed a pH-optimum of about 6.9, a Km value of 3 mᴍ for glutamate and a temperature optimum at 48 °C. Glutamine synthetase of etiolated cotyledons possessed a Km for glutamate of 6 or 12 mᴍ, depending on the dithioerythritol con­centration in the homogenisation buffer and a temperature optimum at 46 °C. The enzyme of green leaves was characterized by a temperature optimum at 40 °C, a pH-optimum at about 7.4 and a low glutamate affinity with positive cooperative substrate binding. Based on isolation of chloroplasts and identification of glutamine synthetase the enzyme of green leaves seems to be the chloroplastic form. This enzyme was purified by DEAE-Sephacel, hydroxylapatite and Sephacryl S-300 chromatography. Affinity for glutamate and MgSO4 of the purified enzyme differed from that found in the crude extract. The function of the different isoenzymes is discussed.

scholarly journals Isolation and partial characterization of protease from Pseudomonas aeruginosa ATCC 27853

2010 ◽  
Vol 75 (8) ◽  
pp. 1041-1052 ◽  
Author(s):  
Lidija Izrael-Zivkovic ◽  
Gordana Gojgic-Cvijovic ◽  
Ivanka Karadzic
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Zinc Ion ◽  
Metal Chelators ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Sds Page ◽  
Potential Use

Enzymatic characteristics of a protease from medically important, referent strain of Pseudomonas aeruginosa ATCC 27853 were determined. According to SDS PAGE and gel filtration it was estimated that molecular mass of the purified enzyme was about 15 kDa. Other enzymatic properties were found to be: pH optimum 7.1, pH stability between pH 6.5 and pH 10; temperature optimum around 60?C while the enzyme was stable at 60?C for 30 min. The inhibition of the enzyme was observed with the metal chelators such as EDTA and 1,10- phenanthroline, suggesting that the protease is a metalloenzyme. Further more it was determined that enzyme contains one mole of zinc ion per mole of enzyme. The protease is stable in the presence of different organic solvents, which enable potential use for synthesis of peptides.

scholarly journals The Plasma Membrane H+-ATPase from the Biotrophic Rust Fungus Uromyces fabae: Molecular Characterization of the Gene (PMA1) and Functional Expression of the Enzyme in Yeast

1998 ◽  
Vol 11 (6) ◽  
pp. 458-465 ◽  
Author(s):  
Christine Struck ◽  
Claudia Siebels ◽  
Oliver Rommel ◽  
Marcus Wernitz ◽  
Matthias Hahn
Keyword(s):  
Plasma Membrane ◽  
Rust Fungus ◽  
Single Gene ◽  
Functional Expression ◽  
Wild Type ◽  
Ph Optimum ◽  
Uromyces Fabae ◽  
Mutant Derivative ◽  
Regulatory Properties

To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H+-ATPase from Uromyces fabae was isolated. Uf-PMA1 exists probably as a single gene. However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae. Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed in haustoria and rust-infected leaves. This is in contrast to elevated enzyme activity in haustoria compared to germinated spores (C. Struck, M. Hahn, and K. Mendgen. Fungal Genet. Biol. 20:30–35, 1996). Unexpectedly, the PMA1-encoded rust protein is more similar to H+-ATPases from plants (55% identity) than from ascomycetous fungi (36% identity). When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae, both the wild-type enzyme and a mutant derivative (Δ76) deleted for the 76 C-terminal amino acids were able to support growth of a yeast strain lacking its own H+-ATPases. Compared to the wild-type, the Δ76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity, and a more alkaline pH optimum. These results indicate that the C-terminal region of the rust enzyme exhibits auto-regulatory properties.

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