ABSTRACT
Pyruvate carboxylase (Pyc) catalyzes formation of oxaloacetic acid from pyruvic acid by fixing one mole of CO2. Manyevidences have confirmed that biosynthesis of some different kinds of organic acids and intracellular and extracellular lipidsis driven by Pyc and over-expression of the PYC gene in the industrial microorganisms can promote production of thedifferent kinds of organic acids and intracellular and extracellular lipids. Therefore, the Pyc from different sources isregarded as a key enzyme in microbial biotechnology and is an important target for metabolic engineering of the industrialmicrobial strains. However, very little is known about the native Pycs and their functions and regulation in the industrialmicroorganisms
ABSTRACT
ALP2 gene encoding alkaline protease cloned from Aureobasidium pullulans HN2-3 was ligated into the surface display plasmid and expressed in the cells of the yeast Yarrowia lipolytica. The expressed alkaline protease was immobilized on the yeast cells. The activity of the immobilized enzyme with 6 His tag was found to be significantly higher than that of without 6 His tag. The immobilized enzyme showed lower optimal temperature and a lower affinity for azocasein than the free enzyme purified from A. pullulans HN2-3. The thermal stability of the immobilized enzyme enhanced and the pH stability decreased, compared to that of the free enzyme.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Caseins/chemistry , Cations , Cell Membrane/metabolism , Cloning, Molecular , Endopeptidases/chemistry , Endopeptidases/genetics , Enzymes, Immobilized/chemistry , Fungi/enzymology , Gene Expression Regulation, Fungal , Genetic Engineering/methods , Hydrogen-Ion Concentration , Ions , Kinetics , Models, Biological , Temperature , Yarrowia/enzymology , Yarrowia/geneticsABSTRACT
The effect of inositol supplementation on glucose derepression, invertase secretion and SUC2 gene expression in Saccharomyces sp. W4 was studied. Invertase secretion was repressed, when the yeast cells, grown the synthetic medium without inositol (I(-) medium) contained more than 0.2% (w/v) initial concentration of glucose. However, in the same medium plus inositol (I(+) medium, inositol conc. 100 microg/100 ml), invertase secretion was repressed only at glucose concentrations higher than 2.0% (w/v). Results showed that secreted invertase activity increased only in the I+ medium, whereas intracellular invertase activity remained constant in both media during the cell, growth. The mRNA encoding secreted invertase was higher in the glucose-derepressed cells grown in the I(+) medium than in the glucose-repressed cells grown in the I(-) medium. Similarly, phosphatidylinositol (PI) content was significantly higher in the cells grown in the I(+) medium than in the I(-) medium. These results indicated that PI might be involved in the glucose derepression, invertase secretion and SUC2 gene expression at the transcriptional level in the yeast.
Subject(s)
Cell Culture Techniques , Culture Media , Dose-Response Relationship, Drug , Gene Expression Regulation, Fungal , Glucose/metabolism , Inositol/metabolism , Phospholipids/metabolism , RNA/metabolism , RNA, Fungal/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Time Factors , beta-Fructofuranosidase/biosynthesisABSTRACT
Mutant A11, a mutant of Saccharomycopsis fibuligera Sdu with low acid and neutral trehalase was found to accumulate over 18% (w/w) trehalose from starch in its cells. In this study, trehalose-6-phosphate synthase (Tps1) was purified to homogeneity from this mutant, with a 30-fold increase in the specific enzyme activity, as compared to the concentrated cell-free extract, from initial cells. The molecular mass of the purified enzyme as determined by SDS-PAGE was 66 kDa. The optimum pH and temperature of the purified enzyme were 6.6 and 37 degrees C, respectively. The enzyme was activated by Ca2+, K+ and Mg2+, with K+ showing the highest activation at 35 mM. On the other hand, Mn2+, Cu2+, Fe3+, Hg2+ and Co2+ inhibited the enzyme. The enzyme was also strongly inhibited by protease inhibitors such as iodoacetic acid, EDTA and PMSF.
Subject(s)
Cell-Free System , Chromatography, Gel , Chromatography, Ion Exchange , Edetic Acid/chemistry , Glucosyltransferases/chemistry , Hydrogen-Ion Concentration , Iodoacetic Acid/chemistry , Ions , Mutation , Protease Inhibitors/pharmacology , Saccharomycopsis/enzymology , Temperature , Trehalose/chemistryABSTRACT
In this study, the effects of inositol addition on maltase activity and expression of MAL1+ gene encoding maltase in Schizosaccharomyces pombe were investigated. The maximum specific maltase activity was observed, when the concentration of inositol reached 6.0 microg/ml in the synthetic medium containing 2.0% glucose. At 1.0 microg/ml inositol concentration, the maltase activity continuously decreased, as initial glucose concentration was higher than 0.1%. mRNA encoding maltase and phosphatidylinositol (PI) content were higher in the cells grown in the synthetic medium with 6.0 microg/ml of inositol and 2.0% glucose than those with 1.0 microg/ml of inositol. These results demonstrated that higher inositol concentration in the synthetic medium could derepress MAL1+ gene expression in S. pombe and PI might be involved in derepression of MAL1+ gene expression in S. pombe probably by PI-type signalling pathway.