Glucose(xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues

Streptomyces sp. CH7 was found to efficiently produce glucose(xylose) isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose) isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its Km values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its Vmax values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85ºC and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60ºC after 30 min. These findings indicate that glucose(xylose) isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

Simultaneous saccharification and fermentation process of different cellulosic substrates using a recombinant Saccharomyces cerevisiae harbouring the beta-glucosidase gene

In Brazil, the production of ethanol from sugarcane produces large amounts of lignocellulosic residues (bagasse and straw), which have been driving research and development for the production of second generation ethanol. In the present work, a recombinant Saccharomyces cerevisiae strain expressing the beta-glucosidase gene from Humicola grisea was used for ethanol production from three different cellulosic sources by simultaneous saccharification and fermentation. Initially, a enzymatic pre-hydrolysis step was done with a solid:liquid ratio of 1:4, and an enzymatic load of 25 filter paper activity (FPU).g-1 of cellulosic substrate. Using sugarcane bagasse pretreated cellulignin, crystalline cellulose and carboxymethyl cellulose, 51.7 g L-1, 41.7 g L-1 and 13.8 g L-1 of ethanol was obtained, respectively, at the end of 55 hrs of fermentation. The highest ethanol productivity (0.94 g L-1 hrs-1) was achieved using sugarcane bagasse pretreated cellulignin. The use of a recombinant S. cerevisiae led to extremely low glucose concentrations when compared to other works reported in literature.

The Role of Tyrosine 207 in the Reaction Catalyzed by Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

The functional signifcance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fuorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in Km CO2, a 32fold decrease in Vmax, and a 6-fold decrease in Km for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO2 and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.

Estudo e caracterização do processo de glutatiolação e desglutatiolação da unidade 20S do proteassomo da levedura Saccharomyces cerevisiae: Implicações na regulação do metabolismo redox intracelular e na geração de peptídeos / Study and characterization of the S-glutathiolation and deglutathiolation of the 20S proteasome core from the yeast Saccharomyces cerevisiae: Implications on the intracellular redox metabolism and peptide generation

O proteassomo é o componente do sistema Ubiquitina-Proteassomo (UPS), responsável pela degradação de proteínas intracelulares marcadas com cauda de ubiquitina. No entanto, a unidade catalítica do proteassomo (20SPT), destituída de unidades regulatórias, é capaz de degradar proteínas de maneira ubiquitina-independente. Diversas modificações pós-traducionais já foram descritas para o 20SPT, incluindo a S-glutatiolação. De acordo com Demasi e col., (2003) o 20SPT da levedura Saccharomyces cerevisiae possui a atividade tipo-quimiotripsina modulada por glutationa e o mecanismo de glutatiolação implica na formação do intermediário ácido sulfênico. No presente trabalho, identificamos por espectrometria de massas (MS/MS) um total de sete resíduos diferentes de cisteína glutatiolados no 20SPT, sendo seis in vitro por incubação com GSH e três in vivo, extraído de células crescidas até atingir fase estacionária tardia em meio rico. Analisando a estrutura 3D do 20SPT, observou-se que os resíduos de cisteína glutatiolados não estão localizados na entrada da câmara catalítica nem próximos aos sítios-ativos, indicando um mecanismo alostérico da modulação da atividade proteassomal...

The proteasome is the protease of the Ubiquitin-Proteasome System (UPS) responsible for the breakdown of intracellular ubiquitin-tagged proteins. However, the catalytic particle of the proteasome (20SPT) is capable of hydrolyzing some substrates in an ubiquitin-independent fashion. The S-glutathiolation of the 20SPT was described among several post-translational modifications and according to Demasi et. al. (2003), the chymotrypsin-like activity of proteasome from yeast Saccharomyces cerevisiae is regulated by glutathione. The mechanism of S-glutathiolation is dependent on the formation of the sulfenic acid intermediate in the cisteine residues of the 20SPT. In this present work, we identified in vitro and in vivo, a total of seven different S-glutathiolated proteasomal cysteine residues by mass spectrometry studies (MS/MS) and, by analyzing the 3D structure of the 20SPT, the modified cysteine residues are not located either on the entrance of the catalytic core or near to the active sites, indicating an allosteric mechanism of proteasomal modulation. During protein degradation, the natively S-glutathiolated 20SPT produces different patterns of peptide products when compared to the DTT-reduced particle through distinct site-specific cleavage of the protein substrates, as herein demonstrated by HPLC and MS/MS analyses...

Boty-II, a novel LTR retrotransposon in Botrytis cinerea B05.10 revealed by genomic sequence

Botrytis cinerea is a necrotrophic pathogen causing pre- and post-harvest diseases in at least 235 plant species. It manifests extraordinary genotype and phenotype variation. One of the causes of this variation is transposable elements. Two transposable elements have been discovered in this fungus, the retrotransposon (Boty), and the transposon (Flipper). In this work, two complete (Boty-II-76 and Boty-II-103) and two partial (Boty-II-95 and Boty-II-141) long terminal repeat (LTR) retrotransposons were identified by an in silico genomic sequence analysis. Boty-II-76 and Boty-II-103 contain 6439 bp nucleotides with a pair of LTRs at both ends, and an internal deduced pol gene encoding a polyprotein with reverse transcriptase and DDE integrase domains. They are flanked by 5 bp direct repeats (ACCAT, CTTTC). In Boty-II-141, two LTRs at both ends, and a partial internal pol gene encoding a protein with a DDE integrase domain were identified. In Boty-II-95, a right LTR and a partial internal pol gene encoding a protein with no conserved domains were identified. Boty-II uses a self-priming mechanism to initiate synthesis of reverse transcripts. The sequence of the presumed primer binding site for first-strand reverse transcription is 5’-TTGTACCAT-3’. The polypurine-rich sequence for plus-strand DNA synthesis is 5’-GCCTTGAGCGGGGGGTAC-3’. Fourteen Boty-II LTRs that contain 125-158 bp nucleotides and share 69.1 ~ 100 percent identities with the short inverted terminal repeats of 5 bp (TGTCA…TGACA) were discovered. Analysis of structural features and phylogeny revealed that Boty-II is a novel LTR retrotransposon. It could potentially be used as a novel molecular marker for the investigation of genetic variation in B. cinerea.

Microbial succession in a fermenting of wild forest noni (Morinda coreia Ham) fruit plus molasses and its role in producing a liquid fertilizer

The numbers of lactic acid bacteria (LAB) and yeasts that were present during a wild forest noni (Morinda coreia Ham) fermentation, the changes in its physico-chemical properties and levels of plant nutrients were investigated. LAB increased rapidly during the first 7 days and were the dominant population until after day 21 when the LAB were declining and the yeasts began to dominate. Identification of the LAB and yeasts to species level showed that the dominant LAB throughout was Lactobacillus plantarum while Lactobacillus pentosus was found but only at day 21. Saccharomyces cerevisiae was the most dominant species of yeast throughout but was slowly replaced by Pichia membranifaciens and then Pichia anomala. Rhodotolura mucilaginosa, an aerobic yeast, was only detected at the beginning of the fermentation process. It is suggested that the Pichia spp. were responsible for consuming lactic acid. After 56 days, the values of pH, acetic acid, ethanol and electrical conductivity in the fermented product were 3.66, 3.34 g L-1, 16.98 g L-1 and 14.47 mS cm-1, respectively. Increased amounts of plant nutrients were present at day 56 mostly derived from the degradation of plant material. At day 56 the amounts were as follows (in mg L-1): N 633, P 1210, K 4356, Ca 693, Mg 536, Mn 7, B 51, Zn 169, and total carbon/total nitrogen ratio (C/N ratio) 18. Based on the seed germination index (GI) of cherry tomato (Lycopersicon esculentum Mill), the extract diluted 256-fold gave the best GI of 157 percent.

Cloning and heterologous expression of Cryptococcus neoformans CnSRB1 cDNA in Saccharomyces cerevisiae.

In this study, we report the results of cloning, sequencing and functional analysis by complementation test of the putative Cryptococcus neoformans homolog CnSRB1. The nucleotide sequence revealed 63% identity, and the deduced amino acid sequence showed 66 and 64% identity to its respective homolog of Saccharomyces cerevisiae and Candida albicans, respectively. Functional complementation test indicated that the putative CnSRB1 gene could compensate the defect caused by a mutation in ScSRB1 in the S. cerevisiae srb1 mutant. Taken together, these results suggest that the putative CnSrblp is a functional homolog of ScSrb1p.

Identificación preliminar in vitro de propiedades probióticas en cepas de S. cerevisiae / In vitro preliminary identification of probiotic properties of S. cerevisiae strains

Objetivo. Evaluar preliminarmente in vitro algunas propiedades probióticas de dos cepas nativas de S.cerevisiae. Materiales y métodos. Las cepas fueron utilizadas en ensayos de tolerancia a sales biliares, pH, temperatura, adherencia a Salmonella spp., E.coli y Shigella spp., y antagonismo. Se realizó un diseño factorial 33 x 3, con tres niveles de cada factor (cepa, pH y concentración inicial de sustrato) por triplicado, para establecer las condiciones de cultivo de cada cepa. Como control se empleó una cepa comercial (B). La cepa seleccionada se empleó para la producción en biorreactor de 2L; la biomasa fue sometida a secado por temperatura; al producto resultante se le determinó concentración de N2 y la viabilidad celular. Resultados. La cepa A (obtenida de caña de azúcar), toleró pH 3 ± 0.2, 0.3% (p/v) de sales biliares y 42oC. El ANOVA del diseño factorial reportó diferencias significativas entre los 27 ensayos (p≤0.05), el análisis de superficies reportó que la interacción entre los factores cepa y Sustrato (S0) son significativos, sugiriendo para la optimización la cepa A y concentraciones crecientes de S0. Los resultados se reprodujeron en biorreactor con mx 0.31h-1, td 2.18h y Y(x/s) 0.126g/g; la biomasa seca obtenida fue viable y reportó entre 6.3 y 6.9% N2/g. Conclusiones. Se identificaron levaduras nativas con propiedades probióticas como tolerancia a pH, sales biliares, temperatura y adherencia a Salmonella spp., E.coli y Shigella spp.

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55ºC, and, in the substratum absence, the thermostability was for 1h at 50ºC. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65ºC was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme's preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action.

A glucoamilase é amplamente utilizada na indústria de alimentos no processamento do amido para a produção de xarope com alto teor de glicose e também muito empregada nos processos de fermentação para produção de cerveja e etanol. Neste trabalho a glucoamilase de Aspergillus awamori expressa em Saccharomyces cerevisiae produzida sob fermentação líquida foi avaliada quanto à produtividade em diferentes amidos e caracterizada físico-quimicamente. A enzima apresentou alta atividade específica de 13,8 U/mg proteína e de 2,9 U/mg biomassa ao final de 48 h de fermentação em meio contendo amido solúvel. A glucoamilase apresentou temperatura ótima de atividade a 55ºC, e temperatura de desnaturação térmica na ausência de substrato por 1h a 50ºC. O pH ótimo de atividade foi na faixa de 3,5 - 4,0 e a estabilidade ao pH entre os valores 5,0 e 7,0. A meia vida a 65ºC foi 30,2 min., e a energia de desnaturação foi de 234.3 KJ mol-1. A hidrólise em diferentes substratos mostrou a preferência da enzima pelos substratos com maior grau de polimerização, sendo o amido de milho gelatinizado o substrato preferencial à ação enzimática.

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.

DNA repair by polymerase delta in Saccharomyces cerevisiae is not controlled by the proliferating cell nuclear antigen-like Rad17/Mec3/Ddc1complex

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186 percent, L-leucine _ 155 percent and L-phenylalanine - 168 percent) and with 1 mM levamisole (122 percent; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20 percent, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166 percent) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90 percent, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

Formal TCA cycle description based on elementary actions.

Many databases propose their own structure and format to provide data describing biological processes. This heterogeneity contributes to the difficulty of large systematic and automatic functional comparisons. To overcome these problems, we have used the BioPsi formal description scheme which allows multi-level representations of biological process information. Applied to the description of the tricarboxylic acid cycle (TCA), we show that BioPsi allows the formal integration of functional information existing in current databases and make them available for further automated analysis. In addition such a formal TCA cycle process description leads to a more accurate biological process annotation which takes in account the biological context. This enables us to perform an automated comparison of the TCA cycles for seven different species based on processes rather than protein sequences. From current databases, BioPsi is able to unravel information that are already known by the biologists but are not available for automated analysis tools and simulation software, because of the lack of formal process descriptions. This use of the BioPsi description scheme to describe the TCA cycle was a key step of the MitoScop project that aims to describe and simulate mitochondrial metabolism in silico.

Produção de glicose-6-fosfato desidrogenase de Saccharomyces cerevisiae geneticamente modificada através de processo descontínuo alimentado / The glucose-6-phosphate dehydrogenase production in fed-batch culture of recombinant Saccharomyces cerevisiae

Este trabalho tem como finalidade estudar e estabelecer alguns parâmetros no processo fermentativo descontínuo alimentado de uma levedura recombinante Saccharomyces cerevisiae W303-181, visando aumentar a obtenção da enzima glicose-6-fosfato desidrogenase (G6PDH). Foram feitas a padronização e otimização do preparo de inóculo de S. cerevisiae recombinante. Foram três as condições estudadas. Reduziu-se o tempo de preparo do inóculo de 114 h, da primeira condição, para e 64 e 48 h para a segunda e terceira condições, respectivamente. Essas duas últimas condições mostraram-se adequados para dar continuidade com os processos fermentativos. Foi feito um estudo de otimização da concentração de micronutrientes (adenina, histidina, triptofano e uracila) no meio de cultivo usando a metodologia de superfície de resposta. Concluiu-se que, ao empregar o meio de cultivo cujas concentrações dos micronutrientes tenham sido otimizadas, a atividade específica de G6PDH atingiu 7927 U/L, 3,2 vezes maior que para o meio controle. Estudou-se a influência da constante de tempo (K), na síntese de G6PDH em processo descontínuo alimentado com vazão de alimentação exponencialmente crescente e decrescente, utilizando meio de cultivo otimizado e não otimizado. Os valores estudados de K para vazão de alimentação exponencialmente crescente foram 0,2, 0,3, 0,5, 0,7 e 0,8 'h POT.MENOS'1' e, para a decrescente, foram 0,2, 0,5 e 0,8 'h POT.MENOS'1'. Dentre os cultivos com vazão exponencialmente crescente e com meio de cultivo não otimizado, encontrou-se para K 'IGUAL'0,2 'h POT.MENOS'1' uma atividade enzimática (558 U/L) 4,1 vezes maior que para a levedura selvagem. Dentre os cultivos nas vazões exponencialmente crescente e decrescente, encontrou-se para a vazão crescente e K'IGUAL'0,2 'h POT.MENOS'1' os maiores níveis de produção de G6PDH (847 U/L)...

Aspectos toxicológicos do bissulfito e outros agentes tóxicos sobre a síntese de glicerol-3-fosfato desidrogenase citoplasmática de levedura de panificação / Toxicological effects of bisulfite and other toxic agents on the cytoplasmic glycerol-3-phosphate dehydrogenase of baker's yeast

A síntese de glicerol-3-fosfato desidrogenase intracelular (EC 1.1.1.8) foi investigada a partir de fermento de panificação em cultivos submersos, contendo glicose ou glicerol como únicas fontes de carbono. Agentes inibitórios da via glicolítica, do ciclo de Krebs e da Cadeia Respiratória inibiram a síntese da enzima quando adicionados em baixas concentrações até 7,5 x 10-4 mol/L. A repressão exercida pela glicose sobre a síntese da glicerol-3-fosfato desidrogenase em meio YP glicose foi reduzida, com a adição de produtos de fermentação e de bissulfito de sódio. Observou-se aumentos de 22-110% na síntese da enzima. Entretanto, em meio YP glicerol, a adição de bissulfito de sódio 0,06 % (p/v) reduziu a síntese da enzima em 29%, enquanto, o acetaldeído 0,012 % (v/v) estimulou a síntese de glicerol-3-fosfato desidrogenase em 12%...

Conservation of glycolytic oscillations in Saccharomyces cerevisiae and human pancreatic b-cells: a study of metabolic robustness

The present study compares two computer models of the first part of glucose catabolism in different organisms in search of evolutionarily conserved characteristics of the glycolysis cycle and proposes the main parameters that define the stable steady-state or oscillatory behavior of the glycolytic system. It is suggested that in both human pancreatic b-cells and Saccharomyces cerevisiae there are oscillations that, despite differences in wave form and period of oscillation, share the same robustness strategy: the oscillation is not controlled by only one but by at least two parameters that will have more or less control over the pathway flux depending on the initial state of the system as well as on extra-cellular conditions. This observation leads to two important interpretations: the first is that in both S. cerevisiae and human b-cells, despite differences in enzyme kinetics and mechanism of feedback control, evolution seems to have kept an oscillatory behavior coupled to the glucose concentration outside the cytoplasm, and the second is that the development of drugs to regulate metabolic dysfunctions in more complex systems may require further study, not only determining which enzyme is controlling the flux of the system but also under which conditions and how its control is maintained by the enzyme or transferred to other enzymes in the pathway as the drug starts acting.

Regulation of trehalose metabolism by Adox and AdoMet in Saccharomyces cerevisiae.

Effect of a potent methylation inhibitor oxidized adenosine (Adox), and a universal methyl group donor S-adenosyl-L-methionine (AdoMet) on trehalose metabolism was studied in two haploids of S. cerevisiae of mating types MATalpha, met3 (6460 -8D) and MATa, leu2, ura3, his4 (8534 -10A). Trehalose level decreased in presence of Adox in both strains. Both neutral trehalase (NT) and trehalose-6-phosphate (tre-6-p) synthase activities increased in presence of Adox in -8D strain. Decrease in trehalose level in -8D thus could not be explained in the light of increased tre-6-p synthase activity; however, it could be correlated with increased NT activity. In strain -10A, NT activity was reduced in presence of Adox while tre-6-p synthase activity increased. Enzyme activity profiles in -10A thus do not explain the reduced trehalose level on Adox treatment. Effect of AdoMet was not very prominent in either strain, though in -8D a small increase in trehalose level was seen on treatment. Intracellular AdoMet level of untreated cells of -10A was seen to be almost six times higher than that of -8D. Further, AdoMet treatment caused increase in its level compared to untreated cells, suggesting AdoMet uptake. No effect of either compound was seen on acid trehalase (AT) activity in any strain. The results suggest that there was a possible effect of demethylation on trehalose metabolism (particularly in the synthetic direction) in both strains, though effect of methylation was not very prominent, the reason for which is not very clear.

Effect of sodium on Saccharomyces cerevisiae invertase activity.

Effect of Na+ ions on yeast invertase activity has been studied as a function of pH. At acidic pH, Na+ (5-100 mM) had no effect but invertase activity was inhibited (38-44%) by Na+ ions (100 mM) with an increase in pH (6.8 and 8.0). Kinetic analysis revealed that invertase inhibition by Na+ ions was non-competitive and reversible in nature. Value of K(m) remained unaltered (33.3 mM) in presence of Na+ (20-100 mM) while Vmax decreased by 21-44% under these conditions. Value of Ki was of the order of 85 mM. Mechanism of observed inhibition of invertase activity as a consequence of Na+ ions interactions at the active site of the enzyme has been described.

Harmaline interactions with yeast invertase.

The effect of harmaline, a plant alkaloid has been studied on yeast invertase activity in the absence and presence of 50mM Na+ as a function of pH. Harmaline (1-3 mM) inhibited the invertase activity at pH 5.2, 6.8 and 8 both in the absence (44-92%) and (22-85%) of Na+ ions. Kinetic analysis revealed that harmaline is a non-competitive inhibitor of invertase, at pH 5.2 and 6.8 but at pH 8, it produced a mixed type of inhibition, Km increased by 450% and 175% and Vmax decreased by 82% and 63% in the absence and presence of 50mM Na ions respectively. The observed inhibition of invertase by harmaline was reversible in nature. These findings suggest that the presence of Na+ site is not a prerequisite for the inhibition of enzyme by harmaline.