70 assays (manual) / 700 assays (microplate)
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|Content:||70 assays (manual) / 700 assays (microplate)|
Short term stability: 2-8oC,
Long term stability: See individual component labels
|Stability:||> 2 years under recommended storage conditions|
|Assay Format:||Spectrophotometer, Microplate|
|Linear Range:||0.8 to 35 µg of glycerol per assay|
|Limit of Detection:||0.34 mg/L|
|Reaction Time (min):||~ 5 min|
|Application examples:||Wine (and grape juice), beer, spirits, vinegar, marzipan, fruit juices, soft drinks, toothpaste, honey, tobacco, paper (and cardboard), cosmetics, pharmaceuticals, soap and other materials (e.g. biological cultures, samples, etc.).|
|Method recognition:||Methods based on this principle have been accepted by OIV and MEBAK|
The Glycerol test kit is a simple, reliable, rapid and accurate method for the measurement and analysis of Glycerol in beverages, foodstuffs and other materials.
Note for Content: The number of manual tests per kit can be doubled if all volumes are halved. This can be readily accommodated using the MegaQuantTM Wave Spectrophotometer (D-MQWAVE).
Browse our wide range of alcohol assay kit products.
- Novel format for increased stability
- Very competitive price (cost per test)
- All reagents stable for > 2 years as supplied
- Very rapid reaction
- Mega-Calc™ software tool is available from our website for hassle-free raw data processing
- Standard included
- Suitable for manual and microplate formats
Megazyme “advanced” wine test kits general characteristics and validation.
Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.
Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.Hide Abstract
Grape and wine analysis: Oenologists to exploit advanced test kits.
Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.
It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.Hide Abstract
Coordinated Transcriptional Control of Adipocyte Triglyceride Lipase (Atgl) by transcription factors Sp1 and PPARγ during Adipocyte Differentiation.
Roy, D., Farabaugh, K. T., Wu, J., Charrier, A., Smas, C., Hatzoglou, M., Thirumurgan, K. & Buchner, D. A. (2017). Journal of Biological Chemistry, jbc-M117.
The breakdown of stored fat deposits into its components is a highly regulated process that maintains plasma levels of free fatty acids to supply energy to cells. Insulin-mediated transcription of Atgl, the enzyme that mediates the rate-limiting step in lipolysis, is a key point of this regulation. In conditions such as obesity or insulin resistance, Atgl transcription is often misregulated, which can contribute to overall disease progression. The mechanisms by which Atgl is induced during adipogenesis are not fully understood. We utilized computational approaches to identify putative transcriptional regulatory elements in Atgl and then tested the effect of these elements and the transcription factors that bind to them in cultured pre- and mature adipocytes. Herein, we report that Atgl is downregulated by the basal transcription factor Sp1 in preadipocytes, and that the magnitude of downregulation dependents on interactions between Sp1 and PPARγ. In mature adipocytes, when PPARγ is abundant, PPARγ abrogated the transcriptional repression by Sp1 at the Atgl promoter and upregulated Atgl mRNA expression. Targeting the PPARγ-Sp1 interaction could be a potential therapeutic strategy to restore insulin sensitivity by modulating Atgl levels in adipocytes.Hide Abstract
Yang, F., Heit, C. & Inglis, D. L. (2017). Fermentation, 3(4), 61.
Acetic acid is undesired in Icewine. It is unclear whether its production by fermenting yeast is linked to the nicotinamide adenine dinucleotide (NAD+/NADH) system or the nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) system. To answer this question, the redox status of yeast cytosolic NAD(H) and NADP(H) were analyzed along with yeast metabolites to determine how redox status differs under Icewine versus table wine fermentation. Icewine juice and dilute Icewine juice were inoculated with commercial wine yeast Saccharomyces cerevisiae K1-V1116. Acetic acid was 14.3-fold higher in Icewine fermentation than the dilute juice condition. The ratio of NAD+ to total NAD(H) was 24-fold higher in cells in Icewine fermentation than the ratio from the dilute juice condition. Conversely, the ratio of NADP+ to total NADP(H) from the dilute fermentation was 2.9-fold higher than that in the Icewine condition. These results support the hypothesis that in Icewine, increased NAD+ triggered the catalysis of NAD+-dependent aldehyde dehydrogenase(s) (Aldp(s)), which led to the elevated level of acetic acid in Icewine, whereas, in the dilute condition, NADP+ triggered NADP+-dependent Aldp(s), resulting in a lower level of acetic acid. This work, for the first time, analyzed the yeast cytosolic redox status and its correlation to acetic acid production, providing a more comprehensive understanding of the mechanism of acetic acid production in Icewine.Hide Abstract
Miljić, U., Puškaš, V., Velićanski, A., Mašković, P., Cvetković, D. & Vujić, J. (2016). Journal of the Institute of Brewing, 122(2), 342-349.
A moderate intake of wine is associated with a positive impact on human health owing to the effects of important biologically active components present in the wine in large amounts. The aim of this study was to examine the chemical composition and to assess antimicrobial and cytotoxic activities of fruit wines produced from three plum varieties (Čačanska rana, Čačanska lepotica and Požegača) commonly grown in Serbia as an approach to assess the quality and acceptability of these wines as a functional food. Furthermore, the activity of a series of control samples was assessed in order to determine components from the wine that are responsible for its functional properties. The plum wines produced showed considerable antimicrobial activity against six bacterial and two yeast strains used in this study. In addition to antimicrobial activity, the plum wines showed a significant cytotoxic effect (IC50 < 50 µg Ml-1) on the growth of three tested cancer cell lines (Hep2c, RD and L2OB). Regarding the determined activities, Čačanska rana plum wine achieved the best results. The results indicated that the antimicrobial activity of the plum wines was, in large part, based on the effects of the total acids and the pH value, while the contribution of ethanol and the content of the phenolic compounds were not significant. Similar conclusions were drawn regarding the cytotoxic activity of this fruit wine. The results can be seen as a contribution to the global acceptance of fruit wines as a functional food, with the accent placed on moderate consumption. An important advantage of fruit wines (in particular plum wine), compared with traditional grape wine, is their lower alcohol content.Hide Abstract
Baur, C., Krewinkel, M., Kutzli, I., Kranz, B., von Neubeck, M., Huptas, C., Wenning, M., Scherer, S., Stoeckel, M., Hinrichs, J., Stressler, T. & Stressler, T. (2015). International Dairy Journal, 49, 46-55.
A secreted peptidase from Pseudomonas panacis was identified and purified. Genome sequencing of the producer strain allowed identification of the peptidase as AprA based on a comparison to peptide sequences of mass spectra obtained from the purified enzyme. The amino acid sequence of the 49.4 kDa peptidase was 98% similar to the metallopeptidase AprX from a Pseudomonas fluorescens strain. The peptidase showed maximum activity at pH 8 and 40°C and withstood general ultra-high temperature (UHT) processing (138°C for 18 s) in skim milk, with 88.0 ± 7.7% of the initial enzyme activity remaining after heating. The peptidase showed considerable enzyme activity under storage conditions of UHT milk. The potential for spoilage of milk might during storage was verified by adding very low enzyme activities to UHT-treated milk. The addition of 1 pkat mL-1 peptidase activity resulted in a destabilisation of the milk during four weeks storage.Hide Abstract
Cinquanta, L., Albanese, D., De Curtis, F., Malvano, F., Crescitelli, A. & Di Matteo, M. (2015). American Journal of Enology and Viticulture, ajev-2015.
Botrytis cinerea is responsible for the gray mold disease, which causes considerable economic losses for winemakers. Its evaluation in wine grapes is commonly performed through visual estimation, which was demonstrated to be prone to assessor bias. Rapid and simple enzymatic carbon screen printed amperometric biosensors were here used to evaluate gluconic acid and glycerol content on wine grapes at different B. cinerea infection degrees. The lower concentrations measurable by screen-printed amperometric biosensors were 3 mg/L for gluconic acid (corresponding to an infection degree lower than 1%) and 35 mg/L for glycerol; the response times with a flow rate of 0.5 mL/min were in a range of 0.5 to 2 min in the linear ranges. This study demonstrates the effectiveness of the biosensors for rapid analysis of gluconic acid and glycerol in grapes, confirming their high correlation with B. cinerea degree of infection (R2 = 0.98). Thus, the biosensor developed to measure gluconic acid in grapes (or must), was more precise, and gave a faster response than methods that currently exist allowing the percentage of infection of grape berries by B. cinerea to be evaluated.Hide Abstract
Bowen, A. J. & Reynolds, A. G. (2015). Food Research International, 76, 540-549.
Icewine is a sweet dessert wine made from pressing grapes naturally frozen on the vines. It is likely that freeze/thaw cycles endured by icewine grapes change their chemical and sensory profiles due to climatic events. Our objective was to determine the influence of harvest date on icewine must and wine basic chemical variables and aroma compounds. Riesling and Vidal icewines were made from grapes picked between December 2004 and February 2005; Harvest 1 (H1): 19 December; Harvest 2: 29 December; Harvest 3 (H3): 18 January; and Harvest 4 (H4): 11 February (Vidal only). Icewine musts differed in titratable acidity and pH (Vidal only). All basic wine chemical analytes differed across harvest dates. All aroma compounds differed in Vidal and Riesling wines. Highest concentrations for most aroma compounds were in the last harvest date; 16 of 24 for Vidal and 17 of 23 for Riesling. The latest harvest date had highest ethyl isobutyrate, ethyl 3-methylbutyrate, 1-hexanol, 1-octen-3-ol, 1-octanol, cis-rose oxide, nerol oxide, ethyl benzoate, ethyl phenylacetate, γ-nonalactone and β-damascenone. H1 had highest ethyl butyrate, ethyl hexanoate, linalool, 4-vinylguaiacol and ethyl octanoate. Based on odor activity values, the most odor-potent compounds were β-damascenone, cis-rose oxide, 1-octen-3-ol, ethyl octanoate, ethyl hexanoate, and 4-vinylguaiacol across harvest dates. PCA found most aroma compounds associated with the last harvest date, 4-vinylguaicol excepted, which was associated with H1. Harvest date was considered a discriminating dimension using canonical variant analysis for volatile compounds.Hide Abstract
Mai-Gisondi, G., Turunen, O., Pastinen, O., Pahimanolis, N. & Master, E. R. (2015). Enzyme and Microbial Technology, 79, 27-33.
The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE-CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE-CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.Hide Abstract
Bellon, J. R., Yang, F., Day, M. P., Inglis, D. L. & Chambers, P. J. (2015). Applied Microbiology and Biotechnology, 99(20), 8597-8609.
To remain competitive in increasingly overcrowded markets, yeast strain development programmes are crucial for fermentation-based food and beverage industries. In a winemaking context, there are many yeast phenotypes that stand to be improved. For example, winemakers endeavouring to produce sweet dessert wines wrestle with fermentation challenges particular to fermenting high-sugar juices, which can lead to elevated volatile acidity levels and extended fermentation times. In the current study, we used natural yeast breeding techniques to generate Saccharomyces spp. interspecific hybrids as a non-genetically modified (GM) strategy to introduce targeted improvements in important, wine-relevant traits. The hybrids were generated by mating a robust wine strain of Saccharomyces cerevisiae with a wine isolate of Saccharomyces bayanus, a species previously reported to produce wines with low concentrations of acetic acid. Two hybrids generated from the cross showed robust fermentation properties in high-sugar grape juice and produced botrytised Riesling wines with much lower concentrations of acetic acid relative to the industrial wine yeast parent. The hybrids also displayed suitability for icewine production when bench-marked against an industry standard icewine yeast, by delivering icewines with lower levels of acetic acid. Additionally, the hybrid yeast produced wines with novel aroma and flavour profiles and established that choice of yeast strain impacts on wine colour. These new hybrid yeasts display the desired targeted fermentation phenotypes from both parents, robust fermentation in high-sugar juice and the production of wines with low volatile acidity, thus establishing their suitability for wine styles that are traditionally troubled by excessive volatile acidity levels.Hide Abstract
Milczarek, R. R. & Liu, K. (2015). Food and Bioproducts Processing, 94, 208-217.
Condensed distillers solubles (CDS) is a viscous, syrupy co-product of ethanol production from corn or other starchy grains; CDS exhibits strong recalcitrance to drying due to its chemical composition, which includes a substantial amount of glycerol. The objectives of this study were to determine the drum drying performance of CDS and to compare it to that of a physically modified CDS (MCDS) having a reduced glycerol level. Material type (CDS vs. MCDS), dwell time, drum internal steam temperature, and gap width were evaluated for their effects on the final moisture content, water activity, and color of the dried product. While both CDS and MCDS could be dried to a range of endpoint moisture contents, dried CDS exhibited a narrow range of water activity compared to that of MCDS. Gap width was found to be the predominant factor affecting dried product color. This work demonstrates that drum drying can effectively reduce the moisture content of CDS, even though the water activity of the dried product cannot be reduced beyond ∼0.45. In contrast, MCDS can be readily drum-dried into a shelf-stable, flaked product with a pleasing appearance.Hide Abstract
Abad, S., Pérez, X., Planas, A. & Turon, X. (2014). Talanta, 121, 210-214.
Recently, the need for crude glycerol valorisation from the biodiesel industry has generated many studies for practical and economic applications. Amongst them, fermentations based on glycerol media for the production of high value metabolites are prominent applications. This has generated a need to develop analytical techniques which allow fast and simple glycerol monitoring during fermentation. The methodology should be fast and inexpensive to be adopted in research, as well as in industrial applications. In this study three different methods were analysed and compared: two common methodologies based on liquid chromatography and enzymatic kits, and the new method based on a DotBlot assay coupled with image analysis. The new methodology is faster and cheaper than the other conventional methods, with comparable performance. Good linearity, precision and accuracy were achieved in the lower range (10 or 15 g/L to depletion), the most common range of glycerol concentrations to monitor fermentations in terms of growth kinetics.Hide Abstract
Vlassis, T., Stamatelatou, K., Antonopoulou, G. & Lyberatos, G. (2013). Journal of Chemical Technology and Biotechnology, 88(11), 2000-2006.
BACKGROUND: Biodiesel is an alternative to fossil fuels and can be used directly in internal combustion engines when mixed with diesel. The economic feasibility of biodiesel production necessitates the valorisation of glycerol, which is produced in large quantities (equal to 10% of the biodiesel produced). Anaerobic digestion is applicable to a variety of organic residues yielding biogas rich in methane. In order to estimate the net potential of glycerol to yield methane, pure glycerol was selected to avoid any effect from the impurities in crude glycerol. RESULTS: The anaerobic digestion of pure glycerol was studied in two types of bioreactors: a continuous stirred tank reactor (CSTR) and a baffled reactor (periodic anaerobic baffled reactor, PABR). Both reactors were operated in mesophilic conditions (35°C) at various organic loading rates. The maximum glycerol loading achieved in a CSTR was 0.25 g COD L-1 d-1, yielding 0.074 ± 0.009 L CH4 L-1 d-1. On the other hand, PABR allowed glycerol degradation at a loading of 3 g COD L-1 d-1 yielding 0.993 ± 0.102 L CH4 L-1 d-1. CONCLUSION: PABR was proved to be more efficient since it was subjected to a 10-fold higher organic loading rate than CSTR. Moreover, its performance was much higher in terms of COD removal and methane productivity.Hide Abstract
Cutzu, R., Coi, A., Rosso, F., Bardi, L., Ciani, M., Budroni, M., Zara, G., Zara, S. & Mannazzu, I. (2013). World Journal of Microbiology and Biotechnology, 29(6), 1009-1017.
In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in β-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R2 = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l-1) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l-1) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.Hide Abstract
Chaudhary, N., Ngadi, M. O. & Simpson, B. (2012). Journal of Bioprocessing & Biotechniques.
Hydrogen and ethanol production from glucose, glycerol and crude glycerol fermentation using Escherichia coli was investigated. Crude glycerol used in this study contained 80% glycerol, 2.6% ash, 12.3% moisture, 1.7% free fatty acid, 3.4% MONG (matter organic non-glycerol), 2519 mg/kg sulphur and 9000 ppm sodium. The maximum yield of ethanol from crude glycerol of 0.36 g/g, corresponding to an ethanol concentration of 3.6 g/L was obtained at 10 g/L initial glycerol concentration, 5 g/L tryptone concentration and 100 rpm mixing speed. Comparable yields were obtained at the mixing speeds of 150 and 200 rpm. On comparison, this yield corresponded to 105% of the yield (0.34 g/g) obtained from pure glycerol at the same conditions and 85% of the maximum yield (0.42 g/g) of ethanol obtained from pure glycerol at 10 g/L initial glycerol concentration, 10 g/L tryptone concentration and 200 rpm mixing speed. Additionally, Escherichia coli growth for glycerol was characterized and compared to that for glucose with 10 % substrate concentration at 37°C and 200 rpm mixing speed. The net growth rate for glucose and glycerol were 0.43 and 0.26 h-1, respectively. The maximum dry weight attained for glucose and glycerol were 0.12 and 0.04 g/L, respectively.Hide Abstract
Puligundla, P., Poludasu, R. M., Rai, J. K. & Obulam, V. S. R. (2011). Annals of Microbiology, 61(4), 863-869.
The main objective of this study was to evaluate the effect of yeast immobilization on ethanolic fermentation of very high gravity (VHG) medium and to determine the concentrations of yeast storage carbohydrates like trehalose and glycogen during the process. Repeated batch ethanolic fermentation of VHG medium was carried out using Saccharomyces cerevisiae immobilized separately within Ca-alginate and κ-carrageenan polymers. Immobilization yields (Y1) were between 80 and 90% and ethanol yields (YP/S) were more than 0.41 with both carriers. An average fermentation efficiency of nearly 70% was observed in 48-h fermentation batches. Compared to free cells, a reduction of more than 50% in the accumulated trehalose, and a two-fold increase in intracellular glycogen levels were observed in immobilized yeast cells at 24 and 48 h of fermentation, respectively, with both carriers. The increased viability (up to four-fold higher) upon 18% ethanol treatment for 2 h, and the sustained viability over four successive batches of immobilized cells showed the protective nature of the polymer carriers. The chemical nature of the carriers was not found to have any adverse effect on ethanol yields. Application of immobilized yeast in porous matrices may serve as a feasible and better technique for ethanol production, at both pilot and industrial scale.Hide Abstract
Sharma, A. K., Mahalik, S., Ghosh, C., Singh, A. B. & Mukherjee, K. J. (2011). AMB Express, 1(1), 33.
There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.Hide Abstract
Chaudhary, N., Ngadi, M. O., Simpson, B. K. & Kassama, L. S. (2011). Advances in Chemical Engineering and Science, 1, 83-89.
Production of high value products from glycerol via anaerobic fermentation is of utmost importance for the biodiesel industry. The microorganism Escherichia coli (E. coli) K12 was used for fermentation of glycerol. The effects of glycerol concentration and headspace conditions on the cell growth, ethanol and hydrogen production were investigated. A full factorial experimental design with 3 replicates was conducted in order to test these factors. Under the three headspace conditions tested, the increase of glycerol concentration accelerated glycerol fermentation. The yields of hydrogen and ethanol were the lowest when glycerol concentration of 10 g/L was used. The maximum production of hydrogen was observed with an initial glycerol concentration of 25 g/L at a final concentration of hydrogen was 32.15 mmol/L. This study demonstrated that hydrogen production negatively affects cell growth. Maximum ethanol yield was obtained with a glycerol concentration of 10 g/L and was up to 0.40 g/g glycerol under membrane condition headspace. Statistical optimization showed that optimal conditions for hydrogen production are 20 g/L initial glycerol with initial sparging of the reactor headspace. The optimal conditions for ethanol production are 10 g/L initial glycerol with membrane.Hide Abstract
Pham, T. K. & Wright, P. C. (2008). Journal of Proteome Research, 7(11), 4766-4774.
Ethanol yield by Saccharomyces cerevisiae in very high glucose (VHG) media with an amino acid supplement was investigated. Amino acid supplementation led to positive cell responses, including reduced lag time and increased cell viability in VHG media. A quantitative shotgun proteomic analysis was used to understand how amino acid supplemented S. cerevisiae responds to high osmotic conditions. iTRAQ data revealed that most proteins involved in glycolysis and pentose phosphate pathways were up-regulated under high glucose shock. Reactivation of amino acid metabolism was also observed at the end of the lag phase. The relative abundance of most identified proteins, including aminoacyl-tRNA biosynthesis proteins, and heat-shock proteins, remained unchanged in the hours immediately following application of glucose shock. However, the expression of these proteins increased significantly at the end of the lag phase. Furthermore, the up-regulation of trehalose and glycogen biosynthesis proteins, first maintaining then latterly increasing glycolysis pathway activity was also observed. This was verified by enhanced ethanol yields at 10 and 12 h (0.43 and 0.45 g ethanol/g glucose) compared to 2 h (0.32 g ethanol/g glucose). These data combined with relevant metabolite measurements demonstrates that enhanced ethanol fermentation under VHG conditions can be achieved with the aid of amino acid supplementation.Hide Abstract
Pham, T. K., Chong, P. K., Gan, C. S. & Wright, P. C. (2006). Journal of Proteome Research, 5(12), 3411-3419.
Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQ-mediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering.Hide Abstract