Optimization of accessible catalase activity in polyacrylamide gel-immobilized Saccharomyces cerevisiae

The permeabilization of Saccharomyces cerevisiae (baker's yeast), either before or after immobilization in polyacrylamide gel (PAG), has been examined as a means to increase the catalase activity of PAG-immobilized yeast cells. Prior permeabilization of the cells resulted in large losses of catalase activity during immobilization, but permeabilization after immobilization produced increases in the catalase activity of yeast/PAG particles. A dependence of the accessible catalase activity on the concentration of polyacrylamide in permeabilized yeast/PAG particles, and on the method of permeabilization of the immobilized cells, was observed. Optimal levels of stable catalase activity (1000-2000 IU/g PAG particles; ca. 5%-10% of total available activity) were obtained by immobilizing yeast cells (0.5 g wet cells/mL gel) in 10% (w/v) PAG, followed by permeabilization of the entrapped cells with either cetyltrimethylammonium bromide, Triton X-100 and one freeze-thaw, or five freeze-thaw cycles. (c) 1992 John Wiley & Sons, Inc.     

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae

The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead.     

Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production

For development of theanine production from glutamic acid and ethylamine by coupling yeast sugar fermentation as an ATP-regenerating system, several strains were selected from among about 200 methylamine- and/or methanol-assimilating bacteria depending on the theanine-forming activity of their permeated cells. The amount of theanine formed by the cells of the selected strains was much larger than that by the cells of Escherichia coli AD494 (DE3) expressing Pseudomonas taetrolens Y-30 glutamine synthetase (GS), which has been found to be a usable enzyme for theanine production. A GS-like enzyme responsible for the theanine-forming reaction was obtained from an obligate methylotroph isolate, Methylovorus mays No. 9. The enzyme was induced by methylamine in the culture medium. A molecular mass of 410-470 kDa was obtained by gel filtration of the enzyme, and 51 kDa by SDS-PAGE analysis. The enzyme showed high activity toward methylamine rather than ammonia, which indicates that it is similar to known gamma-glutamylmethylamide synthetase. The isolated enzyme also had high reactivity to ethylamine in a neutral pH range, and formed theanine from glutamic acid and ethylamine in a reaction mixture containing a yeast sugar fermentation system for ATP-regeneration.     

Cell wall glycoproteins of Candida albicans as released by different methods

Different methods of extraction frequently used in other studies were used to release glycoproteins from both intact cells and isolated cell walls of yeast and hyphal forms of Candida albicans. Extracts were obtained from whole cells by treatment (i) with 2-mercaptoethanol (beta ME) at pH 8.6 and 37 C degrees and (ii) with zymolyase after treatment with beta ME. Extracts were obtained from isolated and washed cell walls (i) by boiling with beta ME and sodium dodecyl sulphate (SDS), (ii) by boiling with SDS and (iii) by treatment with zymolyase after SDS. The extracts were separated by SDS-polyacrylamide gel electrophoresis and analysed by Western blotting with four reagents. Analysis with concanavalin A (ConA) revealed different glycoprotein populations depending on the treatment. Three possible germ-tube-specific constituents were observed; and 80 kDa component released by beta ME from both intact cells and cell walls, and 47 kDa and 43 kDa moieties released by zymolyase only from intact cells. MAb 4C12, specific for the protein portion of a large germ tube constituent, recognized polydisperse material which just entered the gel in beta ME extracts and in the region extending up from 200 kDa to near the top of the gel in zymolyase extracts. MAb 24.17, specific for a carbohydrate determinant of yeast phase cells, reacted with disperse material in the region from the top of the gel to one-third to two-thirds the distance to the 220 kDa mass marker. Antiserum specific for the serotype A determinant of mannan reacted with large disperse component(s) migrating in the region from the top of the gel to about two-thirds the distance to the 220 kDa mass marker and with a 180 kDa component. The components recognized by MAb 4C12, but not those recognised by MAb 24.17 and serotype A antiserum, were effected by treatment with endo-beta-N-acetylglucosamidase H. The various analyses revealed that the method of extraction affected the composition and size of the constituents recognized by the reagents.     

Fragmentation radio-induite de l'ADN évaluée par immunomarquage anti poly(ADP-ribose) dans les CHO. Étalonnage par électrophorèse en champ pulsé

The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos) ylation to follow and estimate γ-ray-induced DNA fragmentation at the level of isolated cells after γ-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immunolabelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after γ-irradiation.     

α-galactosidase from the yeast Candida javanica

Summary α-Galactosidase (Ec 3.2.1.22) was obtained from the yeast C. javanica grown on a mineral culture medium supplemented with melibiose plus raffinose. The cell-bound α-galactosidase and the preparations from the culture filtrate and from the cells exhibited maximum activity at pH 4 and 70°C. After 15 min at 70°C, 70% of the α-galactosidase activity was recovered, and after 15 min at 80°C complete inactivation was observed. The Michaelis constant (Km) of α-galactosidase on pNPG and on melibiose was about 11 mM. Galactose as a reaction product had no effect on α-galactosidase activity of the preparation from the cells, whereas glucose (2 and 10 mM) increased the activity. On the basis of polyacrylamide gel electrophoresis, the preparation from the cell extract, obtained after ultrafiltration and ethanol precipitation, showed only an active band which stained with Coomassie-blue but not with Schiff-periodate. The α-galactosidase from C. javanica is α-galactosidase A, it lacks invertase and it reduces group B specificity of erythrocytes.     

Purification and properties of yeast ATP (CTP):tRNA nucleotidyltransferase from wild type and overproducing cells

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) has been purified from wild type cells of the yeast Saccharomyces cerevisiae, as well as from a strain that overproduces the activity. Purification from the wild type strain was accomplished with a multistep protocol including ammonium sulfate fractionation, anion exchange chromatography, gel filtration, and affinity chromatography. The purified enzyme is near homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 59,000 Da is smaller than reported previously. A similar molecular mass is obtained by gel filtration demonstrating that the enzyme is active as a monomer. The pH optimum for the enzyme is around 9.5. The apparent KM values for ATP and CTP were determined to be 5.6 x 10(-4) M and 1.8 x 10(-4) M, respectively. Purification of the enzyme from the overproducing cells was accomplished by a three step protocol with high yield. The nucleotidyltransferase activity from the overproducing cells had a KM for CTP indistinguishable from that of the wild type enzyme, and the mobility of the protein on sodium dodecyl sulfate gels was the same regardless of the source. Thus, the overproducing strain appears to be a good source for large amounts of yeast nucleotidyltransferase for further biochemical and structural studies.     

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.     

Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans

NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems. Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s. The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast. Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin. Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.     

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.     

Ethanol tolerance of immobilized brewers' yeast cells

A method based on the survival of yeast cells subjected to an ethanol or heat shock was utilized to compare the stress resistance of free and carrageenan-immobilized yeast cells. Results demonstrated a significant increase of yeast survival against ethanol for immobilized cells as compared to free cells, while no marked difference in heat resistance was observed. When entrapped cells were released by mechanical disruption of the gel beads and submitted to the same ethanol stress, they exhibited a lower survival rate than entrapped cells, but a similar or slightly higher survival rate than free cells. The incidence of ethanol- or heat-induced respiratory-deficient mutants of entrapped cells was equivalent to that of control or non-stressed cells (1.3 ± 0.5%) whereas ethanol- and heat-shocked free and released cells exhibited between 4.4% and 10.9% average incidence of respiration-deficient mutants. It was concluded that the carrageenan gel matrix provided a protection against ethanol, and that entrapped cells returned to normal physiological behaviour as soon as they were released. The cell growth rate was a significant factor in the resistance of yeast to high ethanol concentrations. The optimum conditions to obtain reliable and reproducible results involved the use of slow-growing cells after exhaustion of the sugar substrate.     

Influence of yeast quality on performance of gnotobiotically grown Artemia

Using axenically grown Artemia, a model system was developed to evaluate the effect of bacteria on the survival and development of this crustacean. Two strains of baker's yeast (Saccharomyces cerevisiae) were used in all experiments as feed for Artemia: a wild-type strain and its mnn9 mutant, defective in the synthesis of mannoproteins in the outer cell wall. The genetic background, yeast growth phase and growth medium appeared to be important parameters determining the quality of yeast cells as feed for Artemia. A strong positive correlation between Artemia performance and the yeast cell wall chitin and glucan content was obtained, while the mannoprotein content was negatively correlated. Mnn9 yeast cells grown till exponential phase in minimal medium proved to be excellent feed for Artemia, yielding an average 95% survival and 4-mm growth after 6 days at 28 °C, which is comparable to the best results obtained with algal feed. The standard growth test yields highly reproducible results and can become an excellent tool to study the mode of action of bacteria. Furthermore, yeast cell viability and the method used to kill/sterilize the cells are important parameters influencing nauplii performance.     

Application of the single cell gel electrophoresis on yeast cells.

In the present paper, we have applied the single cell gel electrophoresis (SCGE) assay on yeast cells treating Saccharomyces cerevisiae cells with hydrogen peroxide and methyl methanesulfonate (MMS), two DNA damaging agents. In order to overcome the problem with the yeast cell wall that prevented DNA to be extended by the electric field, we disintegrated the cell wall after embedding the cells in agarose. A characteristic picture of comets with residual nuclei and tails was observed and the length of the comet tails was dependent on the concentration of the damaging agents. Yeast cells developed comets at concentrations at least 10 times lower than the concentrations at which comets begin to appear in mammalian cells after treatment with the two genotoxic agents. The higher sensitivity of the yeast comet assay and the fact that S. cerevisiae is one of the most thoroughly studied and easy to work with eukaryotic model system suggest that the proposed method could be an useful tool for investigation of the DNA damaging activity of potential genotoxins.     

Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification

Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttraslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by the dprl mutation which has recently been shown to affect the processing of yeast RAS proteins. To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.     

Extractive Synthesis of Ethyl‐Oleate Using Alginate Gel Co‐Entrapped Yeast Cells and Lipase Enzyme

To synthesize ethyl‐oleate ester, a complex Ca‐alginate gel co‐entrapped system was prepared. The gel beads contained two kinds of biocatalysts (living yeast cells and a lipase enzyme) and various amounts of glucose (100–400 g/L). These alginate beads dispersed directly in pure oleic acid. To follow the bioconversion of the cell growth, the glucose uptake of yeast cells, the concentration of ethanol inside the gel beads and the ethyl‐oleate concentration in oleic acid phase was monitored. The glucose was quantitatively taken up by yeast cells during 24–72 h, depending on the concentration of glucose. After this 24–72‐hour period, the glucose uptake was stopped. In accordance with changes in glucose concentration, the concentration of ethanol and ethyl‐oleate increased rapidly during the first day of fermentation and thereafter slowed down. It is supposed that the inhibitory effect of produced ethanol would be resolved by co‐immobilization of lipase in the same gel particles. Using lipase, one is able to transform ethanol to ethyl‐oleate, which is soluble in oleic acid. According to the data obtained a minimum of 4 U/mL lipase is required to increase ethyl‐oleate production significantly. Summing up it can be concluded that by means of this system a maximum yield of ethanol and ethyl‐oleate was achieved when gel beads containing 100 g/L glucose and 4 U/mL lipase enzyme were used.     

Detection of NAD:arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. and Romig, W.R. (1979) J. Biol. Chem. 254, 5849-5854) was utilized to identify NAD:arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and 125I-labeled guanyltyramine as ADPribose acceptor, formation of ADPribosyl 125I-guanyltyramine was linear with time and enzyme concentration. The product was indistinguishable on both thin-layer and high-performance liquid chromatography from that formed by choleragen. Using 125I-guanyltyramine, ADPribosyltransferase activity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractionated, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase product by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free 125I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl 125I-guanyltyramine and other metabolites of 125I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl 125I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, 125I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Poly(ADP-ribosyl)ation of DNA polymerase beta in vitro

DNA polymerase beta purified from bovine thymus is markedly inhibited when incubated in a reconstituted poly(ADP-ribosyl)ating reaction system. Analyses of the reaction product synthesized in this system by SDS-polyacrylamide gel electrophoresis and subsequent fluorography of the gel indicated that ADP-ribose is covalently attached to DNA polymerase beta molecule (Mr = 44,000).     

Functional analysis of plasma prophenoloxidase system in the marine mussel Perna viridis

The role of prophenoloxidase (proPO) system in recognition and phagocytosis of yeast cells by hemocytes was examined in vitro using whole plasma and proPO system isolated from the plasma of the marine mussel, Perna viridis. The proPO was isolated from the plasma by ammonium sulphate precipitation and gel filtration. The final proPO preparation was homogeneous in native PAGE, and could be activated by trypsin, α-chymotrypsin and pronase-E. Laminarin (a polymer of β-1, 3-glucan) and lipopolysaccharides (LPS) from diverse bacterial species effectively activated the isolated proPO, demonstrating the ability of this proenzyme to interact directly with microbial surface components. The susceptibility of proPO activation to inhibition by serine protease inhibitors such as soybean trypsin inhibitor (STI) or p-nitrophenyl-p′-guanidinobenzoate (p-NPGB), indicates that the isolated fraction may contain an integral serine protease domain in an inactive state. The presence of laminarin- or LPS-activated whole plasma of P. viridis facilitated adherence of yeast cells to hemocyte surface as well as eventually stimulated phagocytic uptake of the target cells by hemocytes, and no such hemocytic response was recorded with STI controls. This and other results strongly suggest that the intermediary factors generated during activation of plasma proPO system by non-self molecules play a key role in recognition and opsono-phagocytosis by hemocytes. However, the proPO system isolated from P. viridis plasma, after activation with microbial surface components, failed to show an opsonic effect.     

限磷对产甘油假丝酵母甘油合成与胞内磷积累的影响

研究了磷酸盐限量对产甘油假丝酵母甘油合成与胞内磷积累的影响。结果表明, 当酵母细胞从适磷或富磷培养基转接入低磷培养基时, 发酵过程中胞内积累的磷逐渐减少; 而当菌体从低磷培养基转接入适磷或富磷培养基时, 发酵过程中胞内聚磷酸盐的积累量迅速增加。当细胞在第14小时和第38小时从适磷培养基转接入低磷培养基时甘油得率分别高达60.9%和61.4%, 而甘油产率则分别为2.03 g/(L·h)和2.23 g/(L·h)。这些现象说明限制发酵培养基中的磷浓度是产甘油假丝酵母高产甘油的必要条件, 并为其反复分批发酵法生产甘油提供了重要依据。