A Model Mechanism for the Enzymatic Synthesis of Lupin Alkaloids

1979 ◽  
Vol 34 (9-10) ◽  
pp. 704-708 ◽  
Author(s):  
Michael Wink ◽  
Thomas Hartmann ◽  
Hans-M artin Schiebel
Keyword(s):  
Enzyme System ◽  
Cell Suspension Cultures ◽  
Gas Liquid Chromatography ◽  
Alkaloid Biosynthesis ◽  
Tracer Studies ◽  
Lupinus Polyphyllus ◽  
Crude Enzyme Preparation ◽  
Enzymatic Model ◽  
Hypothetical Mechanism

Abstract A crude enzyme preparation obtained from cell suspension cultures of Lupinus polyphyllus catalyzes the pyruvate dependent conversion of cadaverine into the tetracyclic lupin alkaloids. As the first reaction product 17-oxosparteine could be identified by gas-liquid chromatography and mass spectroscopy. In some experiments sparteine was found additionally. A participation of di­amine oxidase could be ruled out. The cadaverine-pyruvate transaminating enzyme system (17-oxosparteine synthase) catalyzes the formation of 17-oxosparteine from three cadaverine units without releasing free intermediates. These results are inconsistent with the hypothetical mechanism thus far formulated for the lupin alkaloid biosynthesis. A new enzymatic model mechanism is proposed regarding both the results of the enzymatic experiments and those of the in vivo tracer studies.

Diurnal Fluctuation of Quinolizidine Alkaloid Accumulation in Legume Plants and Photomixotrophic Cell Suspension Cultures

1982 ◽  
Vol 37 (5-6) ◽  
pp. 369-375 ◽  
Author(s):  
Michael Wink ◽  
Thomas Hartmann
Keyword(s):  
Cell Suspension ◽  
Cell Suspension Cultures ◽  
Early Morning ◽  
Suspension Cultures ◽  
Diurnal Fluctuation ◽  
Alkaloid Biosynthesis ◽  
Lupinus Polyphyllus ◽  
Quinolizidine Alkaloid ◽  
Early Afternoon ◽  
Alkaloid Contents

Abstract Fluctuations of quinolizidine alkaloid content in leaflets of Lupinus polyphyllus, L. hartwegii, Baptisia australis, and Sarothamnus scoparius were studied over a 36 h period. The alkaloid contents reached their maximum at noon or early afternoon, and their minimum during the night. The amplitudes of diurnal variations in alkaloid levels lay within a range of 60 to 470% of the early morning contents of alkaloids (= 100%) in leaves. In photomixotrophic cell suspension cultures of L. polyphyllus and S. scoparius cultured under day-night regime, a similar increase of the alkaloid levels was observed within 5 to 8 h after onset of illumination. A subsequent excretion of the alkaloids produced, which also followed a diurnal rhythm, was found. S. scoparius cells excreted lupanine, L. polyphyllus cells 13-cinnamoyloxylupanine. From the cell culture and plant experiments it can be assumed that the alkaloids which are formed during illumination are translocated to some degree afterwards. In cell suspension cultures the alkaloids are subjected to rapid and rhythmic turnover. Exogenous alkaloids, fed to the cultures, are taken up by the cells and disappear usually within the first 72 h. A possible mechanism for a light-mediated regulation of quinolizidine alkaloid biosynthesis which was found to be localized in leaf chloroplast, is discussed.

Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2257-2266 ◽  
Author(s):  
Helmuth Adelsberger ◽  
Christian Hertel ◽  
Erich Glawischnig ◽  
Vladimir V. Zverlov ◽  
Wolfgang H. Schwarz
Keyword(s):  
Extracellular Enzymes ◽  
Enzyme System ◽  
Thermophilic Bacterium ◽  
Recombinant Enzymes ◽  
Type Mode ◽  
Complete Set ◽  
First Time ◽  
Hydrolysis Of

Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), β-xyloside (bxlA, bxlB, bglZ) and α-arabinofuranoside (arfA, arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo. The active enzyme system was reconstituted in vitro from recombinant enzymes.

scholarly journals Mining the Microbiota of the Neonatal Gastrointestinal Tract for Conjugated Linoleic Acid-Producing Bifidobacteria

2004 ◽  
Vol 70 (8) ◽  
pp. 4635-4641 ◽  
Author(s):  
E. Rosberg-Cody ◽  
R. P. Ross ◽  
S. Hussey ◽  
C. A. Ryan ◽  
B. P. Murphy ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Bifidobacterium Bifidum ◽  
Genomic Diversity ◽  
Gas Liquid Chromatography ◽  
Fecal Material ◽  
Single Strain ◽  
Different Strains

ABSTRACT This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml−1 by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/μg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.

scholarly journals Paenibacillus curdlanolyticus Strain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides

2006 ◽  
Vol 72 (4) ◽  
pp. 2483-2490 ◽  
Author(s):  
Patthra Pason ◽  
Khin Lay Kyu ◽  
Khanok Ratanakhanokchai
Keyword(s):  
Growth Phase ◽  
Enzyme Preparation ◽  
Enzyme System ◽  
Crude Enzyme ◽  
Cellulolytic Enzyme ◽  
Crude Enzyme Preparation ◽  
Multienzyme Complexes ◽  
Sds Page ◽  
Paenibacillus Curdlanolyticus ◽  
Cellulose Binding

ABSTRACT A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, β-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, β-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.

Biosynthesis of Nitrogenous Phospholipids in Spinach Leaves

1974 ◽  
Vol 52 (6) ◽  
pp. 469-482 ◽  
Author(s):  
M. O. Marshall ◽  
M. Kates
Keyword(s):  
Microsomal Fraction ◽  
Enzyme System ◽  
Supernatant Fraction ◽  
Exchange Reactions ◽  
Methylation Pathway ◽  
Ethanolamine Kinase ◽  
Spinach Leaves

Pathways for biosynthesis of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), in spinach leaves have been studied both in vivo (whole leaves and leaf slices) and in vitro (cell-free leaf fractions). Biosynthesis of PS was shown to occur by the action of a particle-bound CDP-diglyceride: serine phosphatidyltransferase, and PE by the action of a PS-decarboxylase localized in the 100 000 × g supernatant fraction. PE was also formed by the operation of the CDP-ethanolamine:diglyceride phosphorylethanolamine transferase, localized in the microsomal fraction. The presence of ethanolamine kinase required for formation of phosphorylethanolamine was demonstrated in vitro, but not the presence of CTP:phosphorylethanolamine cytidyltransferase; however, the latter is presumed present on the basis of in vivo results. Operation of the methylation pathway for biosynthesis of PC was established in vivo, and direct methylation of phosphatidyl-N-methylethanolamine to phosphatidyl-N,N-dimethylethanolamine (PE-diMe) and of PE-diME to PC by S-adenosylmethionine was demonstrated with a particulate enzyme system localized in the microsomal fraction; direct methylation of PE itself could not be shown in this system. PC was also synthesized by the CDP-choline:diglyceride phosphorylcholine transferase system localized in the microsomal fraction. Synthesis of PE and PC by Ca2+-stimulated exchange reactions with ethanolamine and choline, respectively, could be demonstrated, but at low rates. However, no synthesis of PS by exchange reactions with serine could be detected.

scholarly journals In vitro Development of Cauliflower Synthetic Seeds and Development of Plantlets In vivo

2014 ◽  
Vol 24 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Zahida Qamar ◽  
Md. Belal Hossain ◽  
Idrees A. Nasir ◽  
Bushra Tabassum ◽  
Tayyab Husnain
Keyword(s):  
Cell Suspension ◽  
Somatic Embryos ◽  
Cell Suspension Cultures ◽  
Synthetic Seeds ◽  
Embryogenic Calli ◽  
Globular Cells ◽  
Vitro Development ◽  
Direct Use

Synthetic seeds of cauliflower cv. Chillout were developed by encapsulating mature somatic embryos in neutral gel media. Somatic embryos were obtained by optimizing callus and cell suspension cultures of cauliflower. Friable, yellowish embryogenic calli were obtained on MS supplemented with 2 mg/l  2,4-D and 0.5 mg/l BAP using hypocotyl as explants, while calli were regenerated in media consisting of 5 mg/l BAP, 2 mg/l Kn and 6 mg/l GA3. Somatic embryo-genesis was induced in cell suspension culture where auxins were removed in successive steps triggering  conversion of globular cells into the heart, torpedo stage (71%) and finally into cotyledonary/somatic embryos (28%). The mature somatic embryos were encapsulated by mixing mature cell suspension with sodium alginate and calcium chloride mixture (1 : 4). Developed synthetic seeds germinated into complete plantlets when placed in neutral gel media.  Germination efficiency of synthetic seeds decreased to about 50 per cent after 12 weeks of storage at 4ºC followed by a rapid decrease to zero per cent after 16 weeks. It was also observed that cauliflower plantlets from synthetic seeds survived successfully when transferred to soil demonstrating  that cauliflower synthetic seeds is a promising step towards their  in vivo direct use. Plant Tissue Cult. & Biotech. 24(1): 27-36, 2014 (June) D. O. I. http://dx.doi.org/10.3329/ptcb.v24i1.19193

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