ABSTRACT
AIMS: Artificial genes, which encode 48 or 64 repeats of a tripeptide, glutamyl-tryptophanyl-lysine have been cloned to the yeast expression vector pAM82 containing the PHO5 promoter and expressed in Saccharomyces cerevisiae AH22. METHODS AND RESULTS: When the yeast cells harbouring recombinant plasmids pALTG6-2 and pALTG4-4 were derepressed in Burkholder minimal medium (Toh-e, A., Ueda, Y., Kakimoto, S.I. and Oshima, Y. (1973) Journal of Bacteriology113, 727-738) containing low phosphate (0.03 g l-1 KH2PO4 and 1.5 g l-1 KCl), the expression was the highest after 24 h induction and the artificial polypeptides were synthesized to about 10% (pALTG6-2) and 14% (pALTG4-4) of the total cell protein. CONCLUSIONS: The artificial polypeptides produced in yeast were made to react with the rabbit antiserum against the polypeptide purified from Escherichia coli and found only in the pellet fraction of cell lysates, indicating the formation of inclusion body. Artificial polypeptide consisting of Glu-Trp-Lys may be useful as partial supplement in food and feeds. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of single cell enriched with homopolymers of an essential amino acid in yeast might be an important tool of supplementing cereal diets and feed grain rations and could be used as means for improvement of the amino acid profile of single cell protein and production of pharmaceutical peptides.
Subject(s)
Amino Acids, Essential/genetics , Genes, Synthetic , Oligopeptides/genetics , Saccharomyces cerevisiae/genetics , Amino Acids, Essential/chemistry , Cloning, Molecular , Culture Media , Electrophoresis, Polyacrylamide Gel , Glutamic Acid/genetics , Lysine/genetics , Models, Genetic , Oligopeptides/biosynthesis , Oligopeptides/chemistry , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Repetitive Sequences, Amino Acid , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/ultrastructure , Tryptophan/geneticsABSTRACT
The angiotensin-converting enzyme (ACE) inhibitory effect was tested in the culture broth from submerged mycelial cultures of 20 basidiomycetes. The ACE inhibitory effect of culture broth from Flammulina velutipes strain 414 was the highest (52.8%), followed by Lentinus edodes strains 2 (44.4%) and 16 (41.3%). Nutritional requirements for the production of ACE inhibitory substance from F. velutipes were studied. Sucrose, ammonium acetate, and glutamic acid were chosen for the maximum production of ACE inhibitory substance. The optimal medium composition was (g/l): sucrose 20, ammonium acetate 5, glutamic acid 2, KH(2)PO(4) 3, MgSO(4).7H(2)O 0.8, and yeast extract 0.5. Under optimal culture conditions, the ACE inhibitory effect was more than 80%.
Subject(s)
Agaricales/metabolism , Angiotensin-Converting Enzyme Inhibitors/metabolism , Agaricales/growth & development , Culture Media , Hydrogen-Ion Concentration , TemperatureABSTRACT
A water-soluble crude polysaccharide fraction (BR-1) prepared from the root of Bupleurum falcatum L. (Japanese name = Saiko) prevented HCl/ethanol induced ulcerogenesis in mice significantly. BR-1 was fractionated into four polysaccharide fractions (BR-2, BR-3, BR-4, and BR-5) by the addition of cetyltrimethylammonium bromide, and the strongly acidic polysaccharide fraction BR-2 showed the most potent inhibition of gastric lesion formation. When BR-2 was further fractionated by anion-exchange chromatography, the most potent anti-ulcer activity was observed in the pectin-like polysaccharide, bupleuran 2IIc. Bupleuran 2IIc was homogeneous as determined by electrophoresis and gel filtration. Bupleuran 2IIc was composed mainly of galacturonic acid with small proportions of arabinose, rhamnose, and galactose, and its average relative molecular mass was estimated to be 63,000 d. BR-2 lost most of its activity after treatment with periodate or digestion with endo-polygalacturonase indicating that the polygalacturonan region and/or the molecular mass may contribute to activity.
Subject(s)
Anti-Ulcer Agents/pharmacology , Drugs, Chinese Herbal/pharmacology , Plant Extracts/pharmacology , Polysaccharides/pharmacology , Animals , Anti-Ulcer Agents/isolation & purification , Bupleurum , Drugs, Chinese Herbal/chemistry , Male , Mice , Mice, Inbred ICR , Plant Extracts/chemistry , Polysaccharides/isolation & purificationABSTRACT
An anti-complementary pectic polysaccharide (BR-2-IIb), isolated from the roots of Bupleurum falcatum L., has an average molecular weight of 36,000 (gel filtration), and was subjected to methylation analysis before and after carboxyl-reduction, digestion with endo-polygalacturonase, base-catalysed beta-elimination, and partial acid hydrolysis. BR-2-IIb consisted mainly of galacturonic acid, arabinose, rhamnose, and galactose in the molar ratios 13.0:2.1:1.4:1.0 and contained a large enzyme-sensitive polygalacturonan region. The enzyme-resistant region (PG-1) was rich in neutral sugars and contained a backbone of 4-linked GalA and 2-linked Rha to which a highly branched arabinogalactan was attached to position 4 of some 2-linked Rha units. Partial acid hydrolysis of BR-2-IIb gave Ara-(1----3)-Ara, Ara-(1----4)-Arap, Ara-(1----5)-Araf, Ara-(1----6)-Gal, Gal-(1----4)-Gal, GalA-(1----2)-Rha, GalA-(1----4)-Rha, GalA----Rha----Rha, Gal----Rha----Rha, and GlA-(1----6)-Gal in addition to (1----4)linked oligogalacturonides. The anticomplementary activity of BR-2-IIb was enhanced by de-esterification, but carboxyl-reduction decreased the activity.