3-Hydroxybenzoate:Coenzyme A Ligase from Cell Cultures of Centaurium erythraea: Isolation and Characterization

2000 ◽  
Vol 381 (2) ◽  
pp. 155-160 ◽  
Author(s):  
Wagner Barillas ◽  
Ludger Beerhues
Keyword(s):  
Medicinal Plant ◽  
Cell Cultures ◽  
Hydroxybenzoic Acid ◽  
Isolation And Characterization ◽  
Apparent Homogeneity ◽  
Coa Ligase ◽  
Molecular Masses ◽  
The Difference ◽  
Temperature Optima ◽  
Centaurium Erythraea

Abstract In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme A ligase (3HBL) supplies the starter substrate for the formation of an intermediate benzophenone. 3HBL from cell cultures of the medicinal plant Centaurium erythraea was purified to apparent homogeneity using a sevenstepprocedure. The enzyme was an AMPforming CoA ligase with a K = 14.7 for 3-hydroxybenzoic acid, 8.5 for coenzyme A and 229 for ATP. The pH and temperature optima were 7.5 and 35C, respectively. In SDSPAGE, two polypeptides of M 41500 and 40500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their Ntermini were blocked. The difference in their apparent molecular masses could not be attributed to glycosylation. 3HBL had a native M of approx. 50000 and is thus active as a monomer.

A truncated β-xylosidase from the anaerobic fungus Neocallimastix patriciarum 27

1994 ◽  
Vol 40 (6) ◽  
pp. 484-490 ◽  
Author(s):  
Hong Zhu ◽  
K.-J. Cheng ◽  
Cecil W. Forsberg
Keyword(s):  
Ion Exchange ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Barley Straw ◽  
Anaerobic Fungus ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Neocallimastix Patriciarum ◽  
Molecular Masses ◽  
Temperature Optima

Two extracellular β-xylosidases, xylosidase I and II, were isolated from the ruminal fungus Neocallimastix patriciarum 27 after growth in a barley straw medium. Xylosidase I was purified 88-fold to apparent homogeneity by ion-exchange, affinity, and gel filtration chromatography. The purified xylosidase I had an isoelectric point (pI) of 4.7 and was a monomelic protein with a molecular mass of 39.5 kDa as estimated by both SDS-PAGE and gel filtration. Xylosidase II was partially purified to approximately 95% purity. Xylosidase II had the same pI (4.7) as xylosidase I, and appeared to be a dimeric enzyme composed of two polypeptides with molecular masses of 85 and 45 kDa, respectively, on SDS-PAGE. Peptide mapping of the three proteins suggested that xylosidase I was a truncated product originating from xylosidase II. Xylosidases I and II had similar pH optima of 6.0, but different temperature optima of 50 and 40 °C, respectively. The Km and Vmax for xylosidase I were 0.59 mM of p-nitrophenyl-β-D-xylopyranoside and 38.04 U∙mg protein−1, respectively, and those for xylosidase II were 0.13 mM and 8.9 U∙mg protein−1, respectively. Both enzymes hydrolysed pNPX and xylobiose with the production of xylose, but only xylosidase I exhibited activity toward p-nitrophenyl-α-L-arabinofuranoside.Key words: xylosidase, Neocallimastix, patriciarum, glycosidase.

Isolation and HPLC Determination of the Chemical Components of Gentianella acuta (Michx.) Hulten

2018 ◽  
Vol 15 (1) ◽  
pp. 21-33
Author(s):  
Ying Wei ◽  
Yongqiao Liu ◽  
Yifan Hele ◽  
Weiwei Sun ◽  
Yang Wang ◽  
...  
Keyword(s):  
Medicinal Plant ◽  
High Speed ◽  
Chemical Constituents ◽  
Folk Medicine ◽  
Gradient Elution ◽  
Chemical Components ◽  
Isolation And Characterization ◽  
Major Chemical ◽  
Main Components ◽  
First Time

Background: Gentianella acuta (Michx.) Hulten is an important type of medicinal plant found in several Chinese provinces. It has been widely used in folk medicine to treat various illnesses. However, there is not enough detailed information about the chemical constituents of this plant or methods for their content determination. Objective: The focus of this work is the isolation and characterization of the major chemical constituents of Gentianella acuta, and developing an analytical method for their determination. Methods: The components of Gentianella acuta were isolated using (1) ethanol extraction and adsorption on macroporous resin. (2) and ethyl acetate extraction and high speed countercurrent chromatography. A HPLC-DAD method was developed using a C18 column and water-acetonitrile as the mobile phase. Based on compound polarities, both isocratic and gradient elution methods were developed. Results: A total of 29 compounds were isolated from this plant, of which 17 compounds were isolated from this genus for the first time. The main components in this plant were found to be xanthones. The HPLC-DAD method was developed and validated for their determination, and found to show good sensitivity and reliability. Conclusion: The results of this work add to the limited body of work available on this important medicinal plant. The findings will be useful for further investigation and development of Gentianella acuta for its valuable medicinal properties.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Cinnamate‐CoA ligase is involved in biosynthesis of benzoate‐derived biphenyl phytoalexin in Malus  ×  domestica ‘Golden Delicious’ cell cultures

2019 ◽  
Vol 100 (6) ◽  
pp. 1176-1192 ◽  
Author(s):  
Deepa Teotia ◽  
Mariam Gaid ◽  
Shashank S. Saini ◽  
Aparna Verma ◽  
Ragothaman M. Yennamalli ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Malus Domestica ◽  
Coa Ligase ◽  
Golden Delicious

Biotechnological Production of Natural Calorie Free Steviol Glycosides in Stevia rebaudiana: An Update on Current Scenario

2020 ◽  
Vol 8 (2) ◽  
pp. 70-84
Author(s):  
Abeer Kazmi ◽  
Mubarak Ali Khan ◽  
Sher Mohammad ◽  
Amir Ali ◽  
Huma Ali
Keyword(s):  
Medicinal Plant ◽  
Cell Cultures ◽  
Metabolic Pathways ◽  
Plant Material ◽  
Research Work ◽  
Stevia Rebaudiana ◽  
Steviol Glycosides ◽  
Market Demand ◽  
Biotechnological Production ◽  
Current Scenario

Stevia rebaudiana is a vital medicinal plant of the genus Stevia and family Asteraceae. It is commonly used as a natural sweetener plant and its products are 300 times sweeter than the commonly used sugarcane. The sweetening potential is due to the presence of calorie-free steviol glycosides (SGs). The plant species has been extensively profiled to identify steviol glycosides (SGs) with intensity sweetening properties. However, the limited production of plant material is not fulfilling the higher market demand worldwide. Researchers are working worldwide to enhance the production of important SGs through the intervention of different biotechnological approaches in S. rebaudiana. In this review, the research work conducted in the last twenty years, on the different aspects of biotechnology to enhance the production of SGs has been precisely reviewed. Biotechnological methods such as micropropagation, callus and cell cultures, elicitation and the metabolomics and transcriptomic elucidation of the biosynthetic metabolic pathways for the production of steviol glycosides have been concisely reviewed and discussed.

scholarly journals Isolation and characterization of a novel haemoprotein b-559 from Bengal gram (Cicer arietinum)

1987 ◽  
Vol 243 (3) ◽  
pp. 723-728 ◽  
Author(s):  
C S Ramadoss ◽  
B C Shenoy ◽  
A Borthakur
Keyword(s):  
Gel Filtration ◽  
Molecular Size ◽  
Soret Band ◽  
Beta Band ◽  
Photosynthetic Membrane ◽  
Isolation And Characterization ◽  
Bengal Gram ◽  
Apparent Homogeneity ◽  
Reduced State

A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.

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