Pheromone responsiveness is regulated by components of the Gpr1p‐mediated glucose sensing pathway in Saccharomyces cerevisiae

Many fungi have evolved mechanisms to assess environmental nutrient availability prior to the energy‐intensive process of mating. In this study, we examined one such system in Saccharomyces cerevisiae, involving a glucose‐sensing pathway mediated by Gpr1p and the pheromone‐induced mating pathway. Initially we observed that the mating pathway in MATa cells is sensitive to environmental glucose depletion. This phenomenon can be partially reversed with a high glucose spike, but not with the addition of low levels of glucose. Deletion of the low‐affinity glucose receptor, Gpr1p, eliminated this glucose‐induced recovery of pheromone responsiveness. We then determined the impact of GPR1 deletion on the mating pathway and observed that, in all end points studied, the mating pathway response to pheromone is reduced in the absence of Gpr1p. Similarly, elimination of the Gα for Gpr1p, Gpa2p, resulted in reduction in pheromone sensitivity in all assays studied. The negative effect of removing Gpr1p on mating pathway activation could be recovered by overexpressing the mating receptor, Ste2p. Furthermore, Ste2p levels are reduced in the absence of glucose and GPR1. These data suggest that activity of the GPCR‐mediated mating pathway in S. cerevisiae is modulated by extracellular glucose concentrations through the only other GPCR in MATa cells, Gpr1p. Copyright © 2014 John Wiley & Sons, Ltd.     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

Flavan‐3‐ol C‐glycosides – Preparation and model experiments mimicking their human intestinal transit

In order to study the human intestinal transit of flavan‐3‐ol C‐glycosides, several C‐glycosyl derivatives were prepared by non‐enzymatic reaction of (+)‐catechin with α‐D ‐glucose, α‐D ‐galactose and α‐D ‐rhamnose, respectively. In contrast to literature data, we propose that the reaction mechanism proceeds in analogy to the rearrangement of flavan‐3‐ols during epimerization under alkaline conditions. Four of the 12 synthesized flavan‐3‐ol C‐glycosides were incubated under aerobic conditions at 37°C using saliva (2 min) and simulated gastric juice (3 h). To simulate human intestine, the C‐glycosides were also incubated under anaerobic conditions at 37°C both in human ileostomy fluid (10 h) and colostomy fluid (24 h), respectively. The flavan‐3‐ol C‐glycosides under study, i.e. (+)‐epicatechin 8‐C‐β‐D ‐glucopyranoside (1a), (+)‐epicatechin 6‐C‐β‐D ‐glucopyranoside (1d), (+)‐catechin 6‐C‐β‐D ‐galactopyranoside (2b), (+)‐catechin 6‐C‐β‐D ‐rhamnopyranoside (3b) were analyzed in the incubation samples by HPLC‐DAD and HPLC‐DAD‐MS/MS. They were found to be stable in the course of incubation in saliva, simulated gastric juice and ileostomy fluid and underwent degradation in colostomy fluid. While the 6‐C‐β‐D ‐glucopyranoside 1d was completely metabolized between 2 and 4 h, decomposition of the 6‐C‐β‐D ‐galactopyranoside 2b reached only 16±2% within 4 h of incubation. Linear degradation rates of 1d and 2b in colostomy fluid differed significantly. As microbial metabolism of flavan‐3‐ols is known not to be influenced by the stereochemistry of the aglycon, varying degradation rates are ascribed to the effect of the sugar moiety. Based on these results we assume that flavan‐3‐ol C‐glycosides pass through the upper gastrointestinal tract (oral cavity, stomach and small intestine) unmodified and are then metabolized by the colonic microflora.     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Purification and structural elucidation of a novel fucoglucan from the fruiting bodies of Phellinus baumii Pilát

BACKGROUND: Recently, much attention has focused on the biological properties of fungal polysaccharides. Polysaccharides extracted from Phellinus baumii Pilát are reported to have antitumor and immunostimulatory properties, as well as anti‐mutagenicity activity, hypoglycemic and free radical scavenging properties. In this paper, we report the chemical and structural characterization of a novel neutral polysaccharide isolated from the fruiting bodies of P. baumii Pilát. RESULTS: PBF4, a purified polysaccharide, was isolated from the fruiting bodies of P. baumii Pilát, and was shown to be composed of L ‐fucose and D ‐glucose in a ratio of 1:4. It was found to be a fucoglucan consisting of a α‐(1 → 4)‐D ‐glucopyranose backbone with some insertions of α‐(1 → 2)‐L ‐Fucp residues. It also contained a minor β‐(1 → 4,6)‐linked D ‐glucopyranosyl and β‐glycosidically linked nonreducing‐end D ‐glucopyranosyl residues. CONCLUSION: Our results add a new polysaccharide to those already described for different fungal groups. This kind of polysaccharide is useful for further study of the structure–function relationships and mechanism responsible for its biological activities. Copyright © 2008 Society of Chemical Industry     

Structural studies of an acidic polysaccharide of Mesona blumes gum

Mesona blumes gum can be isolated into a neutral polysaccharide (NMBG) and an acidic polysaccharide (AMBG). The yield of AMBG can be up to 90% (w/w). AMBG with a molecular weight of 6566 Da is composed of galactose, glucose, mannose, xylose, arabinose, rhamnose and galacturonic acid in the molar ratio 2.66:1:0.37:2.29:12.5:5.99:23.5. Structural features of the purified AMBG were investigated by a combination of chemical and instrumental analysis, such as periodate oxidation, Smith degradation, methylation, and ¹³C and ¹H NMR. It was found that AMBG possessed a (1 → 4)‐α‐galacturonan backbone with some insertions of (1 → 2)‐α‐L ‐Rhap residues. The branches of arabinogalactan, arabinan, galactan and xylan were all attached to the backbone via O‐4 of α‐L ‐Rhap residues. In addition, some α‐L ‐Rhap residues on the backbone terminated with α‐L ‐Araf and some O‐6 in galacturonic acid residues were acetylated or methyl esterified. The molecular structure of AMBG at different concentrations was observed by atomic force microscopy (AFM). It was found that AMBG showed spherical lumps at 1 µg mL^?1 but an irregular worm‐like shape at 10 µg mL^?1, which indicated that the viscosity of AMBG might be caused by its notable molecular aggregation. Copyright © 2007 Society of Chemical Industry     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

Changes in volatile compound composition of Antrodia camphorata during solid state fermentation

BACKGROUND: Although the volatiles present in mushrooms and fungi have been investigated by many researchers, including Antrodia camphorata in submerged fermentation, there are few data available regarding changes in volatile compounds during fermentation. Our research has revealed that solid state fermentation of A. camphorata is highly odiferous compared with submerged cultures and the odor changed with increasing culture time. Therefore the aim of this study was to investigate the changes in volatile compound composition of A. camphorata during solid state fermentation. RESULTS: Altogether, 124 major volatile compounds were identified. The volatile compounds produced by A. camphorata during growth in solid state fermentation were quite different. Oct‐1‐en‐3‐ol, octan‐3‐one and methyl 2‐phenylacetate were predominant in exponential growth phase production, while the dominant volatiles produced in stationary phase were octan‐3‐one and methyl 2‐phenylacetate. In stationary phase, lactone compounds in A. camphorata, such as 5‐butyloxolan‐2‐one, 5‐heptyloxolan‐2‐one, 6‐heptyloxan‐2‐one, contributed greatly to peach and fruit‐like flavor. Terpene and terpene alcohol compounds, such as 1‐terpineol, L ‐linalool, T‐cadinol, (E, E)‐farnesol, β‐elemene, cis‐α‐bisabolene and α‐muurolene, made different contributions to herbal fresh aroma in A. camphorata. Nineteen volatile sesquiterpenes were detected from solid state fermentation of A. camphorata. The compounds 5‐n‐butyl‐5H‐furan‐2‐one, β‐ionone, (?)‐caryophyllene oxide, aromadendrene oxide, diepi‐α‐cedrene epoxide, β‐elemene, α‐selinene, α‐muurolene, azulene, germacrene D, γ‐cadinene and 2‐methylpyrazine have not hitherto been reported in A. camphorata. CONCLUSION: The preliminary results suggest that the aroma‐active compounds produced by A camphorata in solid state fermentation might serve as an important source of natural aroma compounds for the food and cosmetic industries or antibiotic activity compounds. The sesquiterpenes could be identified as possible taxonomic markers for A. camphorata. Copyright © 2011 Society of Chemical Industry     

Beneficial effect of the unsaponifiable matter from rice bran on oxidative stress in vitro compared with α‐tocopherol

Unsaponifiable matter (UM) was prepared from rice bran using n‐hexane extraction followed by removal of its fatty acid methyl ester with supercritical CO₂ under heat‐stable conditions. The UM was made up of 1% of vitamin E isomers, 28% of γ‐oryzanol and 71% of uncharacterized compounds. The aim of this study was to determine the antioxidant activities of the UM, using α‐tocopherol (α‐T) as a positive control, by measuring the Fe³⁺‐reducing antioxidant power (FRAP), 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) free‐radical‐scavenging property and lipid peroxidation in rat liver microsomes. In addition, the effects of the UM on the tert‐butyl hydroperoxide (t‐BOOH)‐induced cytotoxicity in cultured rat hepatocytes were also investigated. In FRAP assay and DPPH^? free‐radical‐scavenging assay, the results were expressed g^?1α‐T or g^?1 UM. The amount of UM used in lipid peroxidation assay and cytotoxicity assay was the amount required to have equal amounts of total vitamin E isomers in the sample and the control α‐T. The UM, as well as α‐T, exhibited significant antioxidant activities in FRAP, radical‐scavenging and membrane‐lipid oxidation. The FRAP value for total vitamin E isomers in the UM (TVEIUM) was 9.1 times higher than that for α‐T. In terms of their capacities to perform radical‐scavenging and lipid peroxidation, both TVEIUM and α‐T showed similar antioxidant activities. In experiments using cultured rat hepatocytes, the t‐BOOH‐induced lactate dehydrogenase release was significantly inhibited by the addition of 63.5 and 160 µg ml^?1 of TVEIUM treatments (84 and 89%, respectively), and that of 63.5 and 160 µg ml^?1 of α‐T treatment (88 and 93%, respectively). The antioxidant function against oxidative stress of the UM prepared from rice bran may extend its use to being a potential dietary supplement. Copyright © 2004 Society of Chemical Industry     

Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation

We describe a rapid, sensitive and semi-quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non-switching, and are mutated in one of the early subfunctions (either mat1-Mc or mat1-Pc), so that meiosis is only induced after exposure to exogenous pheromone (M-factor or P-factor, respectively). Pheromone activity is assessed as an iodine-positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M-factor produced by an M mam1 strain, and its sensitivity towards P-factor is greatly increased by using a hyper-sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P-factor is very much stimulated by exposure of P cells to M-factor.     

α‐l‐Rhamnosidase from Aspergillus flavus MTCC‐9606 isolated from lemon fruit peel

An α‐l ‐rhamnosidase producing fungal strain has been isolated from decaying lemon fruit. The fungal strain has been identified as Aspergillus flavus. The α‐l ‐rhamnosidase has been purified from the culture filtrate of the fungal strain using ultra filtration and cation exchange chromatography on carboxy methyl (CM) cellulose. The molecular mass of the purified enzyme determined by SDS–PAGE analysis was 41 kDa. The K_m values of the enzyme using p‐nitrophenyl‐α‐l ‐rhamnopyranoside and naringin as the substrates were 1.89 and 1.6 mm respectively. The pH and temperature optima of the enzyme were 11.0 and 50 °C respectively. The effects of various chemical species present in grape fruit juice and wine on the activity of the enzyme have been determined.     

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria

GABA transaminase (GABA‐T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA‐T from Saccharomyces cerevisiae (ScGABA‐TKG) is an α‐ketoglutarate‐dependent enzyme encoded by the UGA1 gene, while higher plant GABA‐T is a pyruvate/glyoxylate‐dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA‐T). The GABA‐T from A. thaliana is localized in mitochondria and mediated by an 18‐amino acid N‐terminal mitochondrial targeting peptide predicated by both web‐based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA‐TKG and AtGABA‐T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA‐TKG and AtGABA‐T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA‐TKG deletion mutant Δuga1 with AtGABA‐T or ScGABA‐TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat‐induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA‐T is functionally interchangeable with ScGABA‐TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA‐T is about half as efficient in doing so as ScGABA‐TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate‐limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.     

XV. Yeast sequencing reports. Isolation and DNA sequence of the STE13 gene encoding dipeptidyl aminopeptidase

We have isolated a mutant which exhibits partial constitutivity for a -specific gene expression in α cells. The wild-type gene was cloned and demonstrated to be allelic to the STE13 gene, which encodes the dipeptidyl aminopeptidase involved in processing of the α-factor prepropheromone. Thus, the mating defect of the ste13 mutations in α cells may result both from the production of incompletely processed α-factor and from the increased expression of a -specific genes. The STE13 open reading frame of 931 amino acids contains a putative membrane-spanning segment near its amino terminus and is 31% identical to a second yeast dipeptidyl aminopeptidase (DAP2). A null mutant of STE13 has been constructed: it is viable and sporulation-proficient. The sequence has been deposited in the GenBank data library under Accession Number L21944.     

Hydrolytic enzyme activities as indicators of fungal spoilage in bakery products

Cake analogues were prepared at different a_w (0.80, 0.85 and 0.90) and pH (6 and 7.5) levels and inoculated with a range of seven representative moulds of spoilage mycoflora of bakery products. The responses recorded were colony radii after 1, 2, 3 and 4 weeks as well as microbial counts and total and specific activities of six hydrolytic enzymes after 4 weeks. Multivariate analysis was applied in order to determine correlations among the responses and thus assess the suitability of using enzyme activities as estimators for fungal growth. The results showed that enzyme activities correlated with growth parameters for Aspergillus and Penicillium isolates, although the useful enzymes were not the same for the three isolates tested. The correlation was poor, however, for Eurotium isolates. In general, specific activities of some enzymes showed better correlation with growth parameters, with α‐D ‐galactosidase, β‐D ‐galactosidase, β‐D ‐glucosidase and N‐acetyl‐β‐D ‐glucosaminidase being the more useful enzymes among those assayed. © 2003 Society of Chemical Industry