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Ethanol Assay Kit

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Ethanol Assay Kit K-ETOH Scheme
Product code: K-ETOH

60 assays (manual) / 600 assays (microplate) / 600 assays (auto-analyser)

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Content: 60 assays (manual) / 600 assays (microplate) / 600 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: Ethanol
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.25 to 12 µg of ethanol per assay
Limit of Detection: 0.093 mg/L
Reaction Time (min): ~ 5 min
Application examples: Wine, beer, cider, alcoholic fruit juices, spirits, liqueurs, low-alcoholic / non-alcoholic beverages, pickles, fruit and fruit juice, chocolate products, vinegar, jam, bread and bakery products, honey, soy sauce, dairy products, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by AOAC (AOAC Method 2019.08, First Action), IFU, EBC, MEBAK and ASBC

The Ethanol test kit is a simple, reliable and accurate method for the measurement and analysis of ethanol in beverages and foodstuffs.

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).

View our full range of alcohol assay kits.

Scheme-K-ETOH ETOH megayzme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Simple format – aldehyde dehydrogenase supplied as stable suspension 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation 
  • Rapid reaction 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats
Validation of Methods
Megazyme publication

Determination of ethanol concentration in Kombucha beverages: Single-laboratory validation of an enzymatic method, First Action Method 2019.08.

Ivory, R., Delaney, E., Mangan & McCleary, B. V. (2020). Journal of AOAC International, qsaa122.

The Ethanol Assay Kit is an enzymatic test kit developed by Megazyme for the determination of ethanol in a variety of samples. The kit has been validated in a single laboratory for use with Kombucha fermented drinks, fruit juices and low-alcohol beer samples. The commercially available Ethanol Assay Kit (Megazyme catalogue no. K-ETOH) contains all components required for the analysis. Quantification is based on the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase and further oxidation of acetaldehyde by acetaldehyde dehydrogenase with conversion of NAD+ to NADH. The single laboratory validation (SLV) outlined in this document was performed on a sample set of eight different commercial Kombucha products purchased in Ireland, a set of five Cerilliant aqueous ethanol solutions, two BCR low-alcohol beer reference materials, two alcohol-free beer samples and two fruit juice samples against SMPR 2016.001 (1). Parameters examined during the validation included Working range, Selectivity, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness (bias), Precision (reproducibility and repeatability), Robustness and Stability.

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Megazyme publication

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.

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Megazyme publication

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.

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Rapid colorimetric detection of genome evolution in SCRaMbLEd synthetic Saccharomyces cerevisiae strains. 

Wightman, E. L., Kroukamp, H., Pretorius, I. S., Paulsen, I. T. & Nevalainen, H. K. (2020).  Microorganisms, 8(12), 1914.

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

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Dendropanax morbifera Leaf Extracts Improved Alcohol Liver Injury in Association with Changes in the Gut Microbiota of Rats.

Eom, T., Ko, G., Kim, K. C., Kim, J. S. & Unno, T. (2020). Antioxidants, 9(10), 911.

This study evaluated the protective effects of Dendropanax morbifera leaf (DML) extracts in the liver due to excessive ethanol consumption. Our results showed that the ethanol extract had better antioxidant activity than the water extract, likely due to the higher levels of total flavonoid and phenolic compounds in the former. We found that the main phenolic acid was chlorogenic acid and the major flavonoid was rutin. Results from the animal model experiment showed concentration-dependent liver protection with the distilled water extract showing better liver protection than the ethanol extract. Gut microbiota dysbiosis induced by alcohol consumption was significantly shifted by DML extracts through increasing mainly Bacteroides and Allobaculum. Moreover, predicted metabolic activities of biosynthesis of beneficial monounsaturated fatty acids such as oleate and palmitoleate were enhanced. Our results suggest that these hepatoprotective effects are likely due to the increased activities of antioxidant enzymes and partially promoted by intestinal microbiota shifts.

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Factors influencing the production of the antioxidant hydroxytyrosol during alcoholic fermentation: Yeast strain, initial tyrosine concentration and initial must.

Rebollo-Romero, I., Fernández-Cruz, E., Carrasco-Galán, F., Valero, E., Cantos-Villar, E., Cerezo, A. B., Troncosso, A. M. & Garcia-Parrilla, M. C. (2020). LWT, 130, 109631.

Hydroxytyrosol is well known for its potent antioxidant activity and anticarcinogenic, antimicrobial, cardioprotective and neuroprotective properties. Main food sources are olive oil (formed from the hydrolysis of oleuropein) and wine. One possible explanation to its origin in wines is the synthesis from tyrosol, which in turn is produced from the Ehrlich pathway by yeasts. This work aims to explore the factors that could increase the content as the strain of yeast, the initial tyrosine concentrations as precursor and the effect of synthetic and sterilized natural grape musts. Alcoholic fermentations in synthetic must showed that hydroxytyrosol is produced by all the yeast strains under study. Commercial Saccharomyces cerevisiae yeasts were those which produced higher concentrations, being the Red Fruit strain the biggest producer (6.12 ng/mL). Once the strain was selected, alcoholic fermentations were performed in synthetic must, with different tyrosine concentrations. The amount of hydroxytyrosol did not increase in a proportional way as tyrosine does. On the other hand, higher concentrations of hydroxytyrosol were obtained in natural grape musts (10.46 ng/mL) than in synthetic must (4.03 ng/mL). This work confirms the capacity of winemaking yeasts to produce the bioactive hydroxytyrosol.

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Activated sludge denitrification in marine recirculating aquaculture system effluent using external and internal carbon sources.

Letelier-Gordo, C. O., Huang, X., Aalto, S. L. & Pedersen, P. B. (2020). Aquacultural Engineering, 90, 102096.

Stringent environmental legislation in Europe, especially in the Baltic Sea area, limits the discharge of nutrients to natural water bodies, limiting the aquaculture production in the region. Therefore, cost-efficient end-of-pipe treatment technologies to reduce nitrogen (N) discharge are required for the sustainable growth of marine land-based RAS. The following study examined the potential of fed batch reactors (FBR) in treating saline RAS effluents, aiming to define optimal operational conditions and evaluate the activated sludge denitrification capacity using external (acetate, propionate and ethanol) and internal carbon sources (RAS fish organic waste (FOW) and RAS fermented fish organic waste (FFOW)). The results show that between the evaluated operation cycle times (2, 4, and 6 h), the highest nitrate/nitrite removal rate was achieved at an operation cycle time of 2 h (corresponding to a hydraulic retention time of 2.5 h) when acetate was used as a carbon source. The specific denitrification rates were 98.7 ± 3.4 mg NO3-N/(h g biomass) and 93.2 ± 13.6 mg NOx-N/(h g biomass), with a resulting volumetric denitrification capacity of 1.20 kg NO3-N/(m3 reactor d). The usage of external and internal carbon sources at an operation cycle time of 4 h demonstrated that acetate had the highest nitrate removal rate (57.6 ± 6.6 mg N/(h g biomass)), followed by propionate (37.5 ± 6.3 mg NO3-N/(h g biomass)), ethanol (25.5 ± 6.0 mg NO3-N/(h g biomass)) and internal carbon sources (7.7 ± 1.6-14.1 ± 2.2 mg NO3-N/(h g biomass)). No TAN (Total Ammonia Nitrogen) or PO43- accumulation was observed in the effluent when using the external carbon sources, while 0.9 ± 0.5 mg TAN/L and 3.9 ± 1.5 mg PO43--P/L was found in the effluent when using the FOW, and 8.1±0.7 mg TAN/L and 7.3 ± 0.9 mg PO43--P/L when using FFOW. Average sulfide concentrations varied between 0.002 and 0.008 mg S2-/L when using the acetate, propionate and FOW, while using ethanol resulted in the accumulation of sulfide (0.26 ± 0.17 mg S2-/L). Altogether, it was demonstrated that FBR has a great potential for end-of-pipe denitrification in marine land-based RAS, with a reliable operation and a reduced reactor volume as compared to the other available technologies. Using acetate, the required reactor volume is less than half of what is needed for other evaluated carbon sources, due to the higher denitrification rate achieved. Additionally, combined use of both internal and external carbon sources would further reduce the operational carbon cost.

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Fermentative Microbes of Khadi, a Traditional Alcoholic Beverage of Botswana.

Motlhanka, K., Lebani, K., Boekhout, T. & Zhou, N. (2020). Fermentation, 6(2), 51.

Khadi is a popular traditional alcoholic beverage in rural households in Botswana. The product is produced by fermentation of ripened sun-dried Grewia flava (Malvaceae) fruits supplemented with brown table sugar. Despite its popularity, its growing consumer acceptance, its potential nutritional value, and its contribution to the socio-economic lifestyle of Botswana, the production process remains non-standardized. Non-standardized production processes lead to discrepancies in product quality and safety as well as varying shelf life. Identification of unknown fermentative microorganisms of khadi is an important step towards standardization of its brewing process for entrance into commercial markets. The aim of this study was to isolate and identify bacteria and yeasts responsible for fermentation of khadi. Yeasts and bacteria harbored in 18 khadi samples from 18 brewers in central and northern Botswana were investigated using classic culture-dependent techniques and DNA sequencing methods. Additionally, we used the same techniques to investigate the presence of bacteria and yeasts on six batches of ripened-dried G. flava fruits used for production of the sampled brews. Our results revealed that Saccharomyces cerevisiae closely related to a commercial baker’s yeast strain sold locally was the most predominant yeast species in khadi suggesting a possible non-spontaneous brewing process. However, we also detected diverse non-Saccharomyces yeasts, which are not available commercially in retail shops in Botswana. This suggests that spontaneous fermentation is partially responsible for fermentation of khadi. This study, presenting the first microbiological characterization of a prominent traditional alcoholic beverage in Botswana, is vital for development of starter cultures for the production of a consistent product towards the commercialization of khadi.

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A microbubble-sparged yeast propagation–fermentation process for bioethanol production.

Raghavendran, V., Webb, J. P., Cartron, M. L., Springthorpe, V., Larson, T. R., Hines, M., Mohammed, H., ZimmermaN, W. B., K Poole, R., GreeN, J. & Green, J. (2020). Biotechnology for Biofuels, 13, 1-16.

Background: Industrial biotechnology will play an increasing role in creating a more sustainable global economy. For conventional aerobic bioprocesses supplying O2 can account for 15% of total production costs. Microbubbles (MBs) are micron-sized bubbles that are widely used in industry and medical imaging. Using a fluidic oscillator to generate energy-efficient MBs has the potential to decrease the costs associated with aeration. However, little is understood about the effect of MBs on microbial physiology. To address this gap, a laboratory-scale MB-based Saccharomyces cerevisiae Ethanol Red propagation-fermentation bioethanol process was developed and analysed. Results: Aeration with MBs increased O2 transfer to the propagation cultures. Titres and yields of bioethanol in subsequent anaerobic fermentations were comparable for MB-propagated and conventional, regular bubble (RB)-propagated yeast. However, transcript profiling showed significant changes in gene expression in the MB-propagated yeast compared to those propagated using RB. These changes included up-regulation of genes required for ergosterol biosynthesis. Ergosterol contributes to ethanol tolerance, and so the performance of MB-propagated yeast in fed-batch fermentations sparged with 1% O2 as either RBs or MBs were tested. The MB-sparged yeast retained higher levels of ergosteryl esters during the fermentation phase, but this did not result in enhanced viability or ethanol production compared to ungassed or RB-sparged fermentations. Conclusions: The performance of yeast propagated using energy-efficient MB technology in bioethanol fermentations is comparable to that of those propagated conventionally. This should underpin the future development of MB-based commercial yeast propagation.

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Inferring active metabolic pathways from proteomics and essentiality data.

Montero-Blay, A., Piñero-Lambea, C., Miravet-Verde, S., Lluch-Senar, M.  & Serrano, L. (2020). Cell Reports, 31(9), 107722.

Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways.

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Assessing population diversity of Brettanomyces yeast species and identification of strains for brewing applications.

Colomer, M. S., Chailyan, A., Fennessy, R. T., Olsson, K. F., Johnsen, L., Solodovnikova, N. & Forster, J. (2020). Frontiers in Microbiology, 11, 637.

Brettanomyces yeasts have gained popularity in many sectors of the biotechnological industry, specifically in the field of beer production, but also in wine and ethanol production. Their unique properties enable Brettanomyces to outcompete conventional brewer’s yeast in industrially relevant traits such as production of ethanol and pleasant flavors. Recent advances in next-generation sequencing (NGS) and high-throughput screening techniques have facilitated large population studies allowing the selection of appropriate yeast strains with improved traits. In order to get a better understanding of Brettanomyces species and its potential for beer production, we sequenced the whole genome of 84 strains, which we make available to the scientific community and carried out several in vitro assays for brewing-relevant properties. The collection includes isolates from different substrates and geographical origin. Additionally, we have included two of the oldest Carlsberg Research Laboratory isolates. In this study, we reveal the phylogenetic pattern of Brettanomyces species by comparing the predicted proteomes of each strain. Furthermore, we show that the Brettanomyces collection is well described using similarity in genomic organization, and that there is a direct correlation between genomic background and phenotypic characteristics. Particularly, genomic patterns affecting flavor production, maltose assimilation, beta-glucosidase activity, and phenolic off-flavor (POF) production are reported. This knowledge yields new insights into Brettanomyces population survival strategies, artificial selection pressure, and loss of carbon assimilation traits. On a species-specific level, we have identified for the first time a POF negative Brettanomyces anomalus strain, without the main spoilage character of Brettanomyces species. This strain (CRL-90) has lost DaPAD1, making it incapable of converting ferulic acid to 4-ethylguaiacol (4-EG) and 4-ethylphenol (4-EP). This loss of function makes CRL-90 a good candidate for the production of characteristic Brettanomyces flavors in beverages, without the contaminant increase in POF. Overall, this study displays the potential of exploring Brettanomyces yeast species biodiversity to find strains with relevant properties applicable to the brewing industry.

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Dihydromyricetin protects the liver via changes in lipid metabolism and enhanced ethanol metabolism.

Silva, J., Yu, X., Moradian, R., Folk, C., Spatz, M. H., Kim, P., Bhatti, A. A., Davies, D. L. & Liang, J. (2020). Alcoholism: Clinical and Experimental Research, 44(5), 1046-1060.

Background: Excess alcohol (ethanol, EtOH) consumption is a significant cause of chronic liver disease, accounting for nearly half of the cirrhosis‐associated deaths in the United States. EtOH‐induced liver toxicity is linked to EtOH metabolism and its associated increase in proinflammatory cytokines, oxidative stress, and the subsequent activation of Kupffer cells. Dihydromyricetin (DHM), a bioflavonoid isolated from Hovenia dulcis, can reduce EtOH intoxication and potentially protect against chemical‐induced liver injuries. But there remains a paucity of information regarding the effects of DHM on EtOH metabolism and liver protection. As such, the current study tests the hypothesis that DHM supplementation enhances EtOH metabolism and reduces EtOH‐mediated lipid dysregulation, thus promoting hepatocellular health. Methods: The hepatoprotective effect of DHM (5 and 10 mg/kg; intraperitoneal injection) was evaluated using male C57BL/6J mice and a forced drinking ad libitum EtOH feeding model and HepG2/VL‐17A hepatoblastoma cell models. EtOH‐mediated lipid accumulation and DHM effects against lipid deposits were determined via H&E stains, triglyceride measurements, and intracellular lipid dyes. Protein expression of phosphorylated/total proteins and serum and hepatic cytokines was determined via Western blot and protein array. Total NAD+/NADH Assay of liver homogenates was used to detect NAD + levels. Results: DHM reduced liver steatosis, liver triglycerides, and liver injury markers in mice chronically fed EtOH. DHM treatment resulted in increased activation of AMPK and downstream targets, carnitine palmitoyltransferase (CPT)‐1a, and acetyl CoA carboxylase (ACC)‐1. DHM induced expression of EtOH‐metabolizing enzymes and reduced EtOH and acetaldehyde concentrations, effects that may be partly explained by changes in NAD+. Furthermore, DHM reduced the expression of proinflammatory cytokines and chemokines in sera and cell models. Conclusion: In total, these findings support the utility of DHM as a dietary supplement to reduce EtOH‐induced liver injury via changes in lipid metabolism, enhancement of EtOH metabolism, and suppressing inflammation responses to promote liver health.

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Tandem integration of aerobic fungal cellulase production, lignocellulose substrate saccharification and anaerobic ethanol fermentation by a modified gas lift bioreactor.

Xue, D., Yao, D., Sukumaran, R. K., You, X., Wei, Z. & Gong, C. (2020). Bioresource Technology, 302, 122902.

Cellulase production, lignocellulose saccharification and bioethanol fermentation were integrated to efficiently produce bioethanol. A modified gas lift bioreactor was developed for bioethanol production by the integrated process. Cellulase production was achieved using Aspergillus niger mycelia immobilized within the reactor in wire meshes, and Saccharomyces cerevisiae cells were immobilized in resin beads. During four repeated batches fermentation, cellulase activities were more than 6.28 U/mL and bioethanol production was over 45.9 g/L for 48 h. The factual bioethanol conversion efficiency was 86.8%. By the modification of the modified gas lift bioreactor, immobilization of Aspergillus niger mycelia and Saccharomyces cerevisiae cells, aerobic cellulase production, substrate saccharification and anaerobic bioethanol fermentation were successfully integrated in tandem. The integrated processes is of great significance in bioethanol production.

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Clostridium cellulovorans metabolism of cellulose as studied by comparative proteomic approach.

Usai, G., Cirrincione, S., Re, A., Manfredi, M., Pagnani, A., Pessione, E. & Mazzoli, R. (2020). Journal of Proteomics, 216, 103667.

Clostridium cellulovorans is among the most promising candidates for consolidated bioprocessing (CBP) of cellulosic biomass to liquid biofuels (ethanol, butanol). C. cellulovorans metabolizes all the main plant polysaccharides and mainly produces butyrate. Since most butyrate and butanol biosynthetic reactions from acetyl-CoA are common, introduction of single heterologous alcohol/aldehyde dehydrogenase can divert the branching-point intermediate (butyryl-CoA) towards butanol production in this strain. However, engineering C. cellulovorans metabolic pathways towards industrial utilization requires better understanding of its metabolism. The present study aimed at improving comprehension of cellulose metabolism in C. cellulovorans by comparing growth kinetics, substrate consumption/product accumulation and whole-cell soluble proteome (data available via ProteomeXchange, identifier PXD015487) with those of the same strain grown on a soluble carbohydrate, glucose, as the main carbon source. Growth substrate-dependent modulations of the central metabolism were detected, including regulation of several glycolytic enzymes, fermentation pathways (e.g. hydrogenase, pyruvate formate lyase, phosphate transacetylase) and nitrogen assimilation (e.g. glutamate dehydrogenase). Overexpression of hydrogenase and increased ethanol production by glucose-grown bacteria suggest a more reduced redox state. Higher energy expenditure seems to occur in cellulose-grown C. cellulovorans (likely related to overexpression and secretion of (hemi-)cellulases), which induces up-regulation of ATP synthetic pathways, e.g. acetate production and ATP synthase.

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Heterologous secretory expression of β-glucosidase from Thermoascus aurantiacus in industrial Saccharomyces cerevisiae strains.

Smekenov, I., Bakhtambayeva, M., Bissenbayev, K., Saparbayev, M., Taipakova, S. & Bissenbaev, A. K. (2020). Brazilian Journal of Microbiology, 51(1), 107-123.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer β-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a β-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.

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Deletion of Voltage-Dependent Anion Channel 1 knocks mitochondria down triggering metabolic rewiring in yeast.

Magri, A., Di Rosa, M. C., Orlandi, I., Guarino, F., Reina, S., Guarnaccia, M., Morello, G., Spampinato, A., Cavallaro, S., Messina, A., Vai, M. & De Pinto, V. (2020). Cellular and Molecular Life Sciences, 77(16), 3195-3213.

The Voltage-Dependent Anion-selective Channel (VDAC) is the pore-forming protein of mitochondrial outer membrane, allowing metabolites and ions exchanges. In Saccharomyces cerevisiae, inactivation of POR1, encoding VDAC1, produces defective growth in the presence of non-fermentable carbon source. Here, we characterized the whole-genome expression pattern of a VDAC1-null strain (Δpor1) by microarray analysis, discovering that the expression of mitochondrial genes was completely abolished, as consequence of the dramatic reduction of mtDNA. To overcome organelle dysfunction, Δpor1 cells do not activate the rescue signaling retrograde response, as ρ0 cells, and rather carry out complete metabolic rewiring. The TCA cycle works in a “branched” fashion, shunting intermediates towards mitochondrial pyruvate generation via malic enzyme, and the glycolysis-derived pyruvate is pushed towards cytosolic utilization by PDH bypass rather than the canonical mitochondrial uptake. Overall, Δpor1 cells enhance phospholipid biosynthesis, accumulate lipid droplets, increase vacuoles and cell size, overproduce and excrete inositol. Such unexpected re-arrangement of whole metabolism suggests a regulatory role of VDAC1 in cell bioenergetics.

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Hangover relieving and antioxidant effects of Gynostemma pentaphyllum (Thunb.) Makino and/or Hovenia dulcis Thunb. extracts.

Park, E. M. & Kim, M. Y. (2019). Journal of Applied Pharmaceutical Science, 9(10), 116-119.

The present study was an attempt to study alcohol metabolizing and antioxidant properties of Gynostemma pentaphyllum (Thunb.) Makino distillate (GPD), and combination effects with Hovenia dulcis Thunb. extract (HDE) on these activities. The alcohol-metabolizing activity of GPD with/without HDE was evaluated by assessing alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities. To define the effect of GPD with/without HDE on alcohol metabolism, antioxidant activities and total phenolic content of GPD with/without HD extract were evaluated using 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, ferrous chelating assays and the Folin-Ciocalteu method. Cytotoxicity against human normal liver CHANG cells was also evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. GPD treatment alone or in combination with HDE significantly increased ADH and ALDH activities; combined treatment was most effective. The contents of total phenolic and flavonoid were greater in combination than the level found in GPD alone. GPD revealed synergistic antioxidant effect when combined with HDE. GPD and/or HDE had no antiproliferative activity against the normal liver cell line. These results suggest that GPD-HDE combination is the potential natural resource for the management of ethanol-induced liver toxicity.

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Acetate metabolism and the inhibition of bacterial growth by acetate.

Pinhal, S., Ropers, D., Geiselmann, J. & de Jong, H. (2019). Journal of Bacteriology, 201(13).

During aerobic growth on glucose, Escherichia coli excretes acetate, a mechanism called “overflow metabolism.” At high concentrations, the secreted acetate inhibits growth. Several mechanisms have been proposed for explaining this phenomenon, but a thorough analysis is hampered by the diversity of experimental conditions and strains used in these studies. Here, we describe the construction of a set of isogenic strains that remove different parts of the metabolic network involved in acetate metabolism. Analysis of these strains reveals that (i) high concentrations of acetate in the medium inhibit growth without significantly perturbing central metabolism; (ii) growth inhibition persists even when acetate assimilation is completely blocked; and (iii) regulatory interactions mediated by acetyl-phosphate play a small but significant role in growth inhibition by acetate. The major contribution to growth inhibition by acetate may originate in systemic effects like the uncoupling effect of organic acids or the perturbation of the anion composition of the cell, as previously proposed. Our data suggest, however, that under the conditions considered here, the uncoupling effect plays only a limited role.

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Assessment of β-glucans, phenols, flavor and volatile profiles of hulless barley wine originating from highland areas of China.

Zhang, K., Yang, J., Qiao, Z., Cao, X., Luo, Q., Zhao, J., Wang, F. & Zhang, W. (2019). Food Chemistry, 293, 32-40.

Low alcohol hulless barley wine (HW) is a popular beverage among the highland areas in China. It is known to have several health benefits due to the presence of β-glucan and antioxidant compounds. Therefore, the total β-glucan content, total phenols and flavonoids of HW samples from the highland areas of Sichuan province and Tibet were determined in this study. The results indicated that HW is abundant in both β-glucan (54-76 mg/L) and phenolic compounds (131-178 mg/L). Moreover, this study also investigated the flavor and aroma characteristics of HW samples. A total of forty six volatile aroma substances were identified by GC-MS. The HWs could be classified into three distinct groups in terms of the region of origin according to the results of PCA based on the GC-MS data. These findings provide a useful foundation for further study of the health benefits and the flavor characteristics of HW in highland areas.

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An engineered GH1 β-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

Santos, C. A., Morais, M. A., Terrett, O. M., Lyczakowski, J. J., Zanphorlin, L. M., Ferreira-Filho, J. A., Tonoli, C. C. C., Murakami, M. T., Dupree, P. & Souza, A. P. (2019). Scientific Reports, 9(1), 1-10.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

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Catalytic decomposition of the oleaginous yeast Cutaneotrichosporon oleaginosus and subsequent biocatalytic conversion of liberated free fatty acids.

Braun, M. K., Lorenzen, J., Masri, M., Liu, Y., Baráth, E., Brück, T. & Lercher, J. A. (2019). ACS Sustainable Chemistry & Engineering, 7(7), 6531-6540.

A single step catalytic cell wall lysis and triglyceride hydrolysis combined with the enzymatic conversion of lipids using the oleaginous yeast Cutaneotrichosporon oleaginosus (ATCC 20509) as a model is described. Catalytic decomposition of yeast cells resulted in hydrolysis of about a third of cellular polysaccharides and all triglycerides. Enzymatic processing of the lipid fraction with an oleate hydratase from Stenotrophomonas maltophilia led to conversion of oleic acid to 10-hydroxystearic acid (10-HSA) (50%) without additional purification. Cell wall polysaccharides were depolymerized by in situ formed amino acids from cell protein fragments. The activity of the in situ generated, free amino acids was higher compared to that of additionally added acids. Studies with the cellobiose and β-(1→3)-glucan indicated that glutamic and aspartic acids, which are the dominant amino acids in yeast cells, are surprisingly more effective in hydrolysis in aqueous phase than sulfuric acid. This points to a concerted mechanism of glycosidic ether bond cleavage catalyzed by amino acids rather than to a pathway catalyzed by hydronium ions. The overall yield of the presented downstream process at 453 K resulted in the release of 80% of total lipids.

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Signal Word : Danger
Hazard Statements : H302, H319, H334, H412
Precautionary Statements : P261, P264, P270, P273, P280, P284, P301+P312, P304+P340, P305+P351+P338, P330, P337+P313, P342+P311, P501
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