The heterogeneity of brewer's yeast old yellow enzyme

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.     

Preparation and evaluation of an enzyme which degrades yeast cell walls

Summary A method for the production and preparation of an enzyme which degrades yeast cell walls from a species of aRhizoctonia (tentatively identified asR. solani) was established on a commercial scale. The production of crude enzyme powder, having a lytic activity of 100 units/mg, in batches of 80 kg is feasible.The enzyme preparation was evaluated for industrial use. When yeast cells were treated with this enzyme, the digestibility of feed yeast was improved 1.4–2 fold in vitro; the efficiency of a mechanical disintegrator in extracting cellular substances was increased 35–50%; the release of soluble glucans having widely varying degrees of polymerization was induced; the extraction of cellular protein by alkali was facilitated 2–3 fold; an 80% release of cell-bound invertase was induced and 2–3 times more yeast extract could be prepared.Studies on Fungal Enzymes Active in Hydrolysing Yeast Cell Wall (VII)     

Isolation of the inorganic pyrophosphatase from brewer's yeast and studies on the localization of this enzyme in brewer's and baker's yeast.

• 1.1. Inorganic pyrophosphatase is mainly located in the hyaloplasm for both brewer's and baker's yeast.
• 2.2. Extensive purification of brewer's yeast pyrophosphatase results in the separation of three active forms of the enzyme.
• 3.3. The isoelectric point of all the three enzymes is close to 5.0.
• 4.4. The molecular weight of the most active pyrophosphatase is about 60,000.

     

Accumulation of toxic metal ions on cell walls ofDatura innoxia suspension cell cultures

Summary Rapidly dividing cell suspension cultures derived fromDatura innoxia (Mill.) selectively remove certain toxic metal ions from nutrient and waste solutions. Many ions, including necessary micronutrients, bind tightly to different components of the primary cell wall. Cell viability is not required for metal chelation to the extracellular matrix, and biopolymers purified from these cultures can be used to selectively remove metal ions from waste streams. Binding of normally toxic metals to the primary cell wall significantly reduces their toxicity. Chemical and metal luminescence methods that generate information about metal binding and cell-wall components responsible for this are presented. The feasibility of using plant cells and their components for bioremediation is discussed. Presented in the Session-in-Depth Bioremediation through Biotechnological Means at the Congress on Cell and Tissue Culture, San Diego, CA, June 5–9, 1993.     

Adsorption of metal ions on yeast cells at varied cell concentrations

Adsorption isotherms for adsorption of metal ions on yeast cellswere determined at varied cell concentrations. The mass of adsorbedmetal per unit weight of the cells at an equilibrium metal concentrationwas largely dependent on the cell concentration. An experimentalequation was presented, relating the included parameters. (Received August 19, 1975; )     

Enzymatic lysis of yeast cell walls

Lysis of yeast cell walls using zymolase and lysozyme was studied. During coupled zymolase–lysozyme treatment, nearly three times more reducing sugars were released from the yeast cells compared to controls. Enzyme treatment followed by extraction at pH 9 resulted in a yield of more than 80% of the total nitrogen of the yeast cell. Protein degradation occurred during enzyme treatment. The precipitation of proteins was significantly increased by succinylation after enzyme treatment. This also reduced the nucleic acid content of the yeast proteins to less than 2% and enhanced the extractability of nitrogenous material.     

Mechanical properties of Bacillus subtilis cell walls: effects of ions and lysozyme.

Bacterial threads of Bacillus subtilis have been immersed in, and redrawn from, water of various pH values, in solutions of (NH4)2SO4 and NaCl of various concentrations, and in lysozyme solutions. The changes in the tensile strength, elastic modulus, and other mechanical properties of the bacterial cell wall due to these treatments were obtained. The data show that change in pH has little effect but that as the salt concentration is increased, the cell walls become more ductile. A high salt concentration (1 M NaCl) can reduce the modulus by a factor of 26 to 13.5 MPa at 81% relative humidity and the strength by a factor of only 2.5. Despite attacking the septal-wall region of the cellular filaments, lysozyme has no effect on the mechanical properties. There is no significant change in the stress relaxation behavior due to any of the treatments. The dependence of mechanical properties on the salt concentration is discussed in terms of the polyelectrolyte nature of cell walls. The evidence presented in this and the accompanying paper (J. J. Thwaites and U.C. Surana, J. Bacteriol., 173:197-203, 1991) supports the idea that the peptidoglycan in bacterial cell wall is an entanglement network with a large degree of molecular flexibility, with some order but no regular structure.     

Preparation of zymosan from yeast cell walls

Cell walls of the yeastSaccharomyces cerevisiae after disintegration and protoplasm removal by centrifugation and repeated washing were suspended in 0.5m Na₂HPO₄, pH 7.8–8.0, as a 5% or 10% suspension, depending on the mode of heating. The suspension was boiled for 3h, purified by repeated washing with water and ethanol and dried. The yield was approximately 1.8% of the starting amount of pressed commercial baker’s yeast.     

Enzynatic method of disrupting yeast cell walls]

The influence of the preparation of mucolytic enzymes obtained from the culture liquid filtrate of Actinomyces griseinus on the yeast Candida guilliermondii was investigated. Lipids, hydrocarbons and proteins were extracted and free amino acids were separated from the biomass made of disrupted yeast cells. The extract contained 73% of cytoplasmic substances whereas normal yeast (enzymically untreated) contain on the average 13.7%. Lipids and hydrocarbons were measured.     

Effects of brewer's yeast cell wall on constipation and defecation in experimentally constipated rats

Brewer's yeast cell wall (BYC) was tested on constipated male Sprague-Dawley rats that had been induced by loperamide (2 mg/kg of body weight). The preventive effect of BYC on constipation was examined and compared with that of a non-fiber diet (NF) as the control. The dose-response of BYC and the effect on defecation by constipated experimental rats were also compared with the characteristics of cellulose diet (CE) group which served as a control. Defecation was observed to be greater by the rats fed with BYC than by those fed with NF or CE. The fecal water content and level of volatile fatty acids (VFA) in the cecal contents were likewise higher in the rats fed with BYC. These results indicate that the administration of BYC was effective for improving defecation and other parameters related to defecation. These favorable effects of BYC supplemented to the diet are attributed to the fermentation ability, water holding capacity and swelling force in the large intestine.     

Insulin activity: stimulatory effects of cinnamon and brewer's yeast as influenced by albumin.

Cinnamon and Brewer's yeast extracts have been shown to potentiate the action of insulin in isolated adipocytes. In this study, isolated rat epididymal adipocytes were used to evaluate the influence of bovine serum albumin on insulin activity as affected by cinnamon and Brewer's yeast extracts. Albumin at 0.01-0.1% decreased the insulin stimulatory effects of cinnamon from 11.8- to 5.3-fold and 2% albumin decreased this effect to near control levels. Conversely, the insulin-enhancing properties of Brewer's yeast remained low in the presence of less than 0.25% albumin but subsequently increased 2.8-, 4.8- and 5.6-fold in the presence of 0.25, 0.50 and 1.0% albumin, respectively. In the absence of added insulin, increased activity of the insulin-stimulated utilization of glucose by both extracts was observed but only Brewer's yeast extract displayed additive effects when tested at higher insulin levels. Due to the inhibitory and enhancing effects of albumin on the insulin activity of cinnamon and Brewer's yeast, respectively, it is suggested that the effects of albumin be assessed when evaluating the insulin-enhancing effects of other substances using isolated adipocytes.     

Construction and analysis of recombinant glucanolytic brewer's yeast strains

Summary Four different types of endo--1,4-glucanase active bottom-fermenting brewer's yeast strains were constructed using recombinant DNA technology. To study the effects of different promoters, copy numbers and integration sites, the egl1 gene of the filamentous fungus Trichoderma reesei was inserted between the promoter and terminator regions of either the PGK1 or ADH1 gene of yeast. The egl1 gene was transferred to the industrial brewer's yeast on a multicopy plasmid or alternatively integrated into the LEU2, PGK1 or ADH1 locus of the yeast. Integration into the PGK1 or ADH1 locus did not affect the brewing properties of the yeast or the quality of the finished beer. Integration into the LEU2 locus, however, decreased the metabolic activity of yeast and prolonged fermentation was needed. In pilot brewing conditions the PGK1 promoter was stronger than that of ADH1. Even a single copy of the egl1 gene in the PGK1 integrant strains gave rise to sufficient enzyme activity for the hydrolysis even of unusually high total amounts of -glucans in worts. Offprint requests to: M.-L. Suihko     

Composition of the cell walls of several yeast species

Cell walls, representing 26%–32% of the cell dry weight, were prepared from several strains of the yeasts Kloeckera apiculata, Debaryomyces hansenii, Zygosaccharomyces bailii,Kluyveromyces marxianus and Saccharomyces cerevisiae. Extraction of the walls with potassium hydroxide at 4 °C, followed by saturation of the alkali-soluble extract with ammonium sulphate gave fractions of mannoprotein, alkali-soluble glucan and alkali-insoluble glucan. Chitin was associated with the alkali-insoluble glucan. The proportions of the different fractions within the walls varied with the species and strain. Mannoprotein comprised between 25% and 34% of the walls, the content of alkali-insoluble glucan ranged from 15% to 48%, and the content of alkali-soluble glucan ranged from 10% to 48%. There was significant variation in the physical appearance of the alkali-soluble glucans and the relative viscosity of suspensions of these glucans. The yeasts could represent novel sources of polysaccharides with industrial and medical applications. Received: 30 December 1997 / Received revision: 24 March 1998 / Accepted: 27 March 1998