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D-Glucose HK Assay Kit

Product code: K-GLUHK-220A



220 assays (manual) / 2200 assays (microplate) / 2000 assays (auto-analyser)

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Content: (K-GLUHK-110A)
110 assays (manual) / 1100 assays (microplate) / 1000 assays (auto-analyser)
220 assays (manual) / 2200 assays (microplate) / 2000 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: D-Glucose
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 4 to 80 µg of D-glucose per assay
Limit of Detection: 0.66 mg/L
Reaction Time (min): ~ 5 min
Application examples: Wine, beer, fruit juices, soft drinks, milk, jam, dietetic foods, bakery products, candies, fruit and vegetables, tobacco, cosmetics, pharmaceuticals (e.g. infusions), feed, paper (and cardboard) and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by AOAC, EN, NEN, NF, DIN, GOST, OIV, IFU, AIJN and MEBAK

The D-Glucose HK (Regular) test kit is a high purity reagent for the measurement and analysis of D-glucose in plant and food products. Can be used in combination with other Megazyme's products that require glucose determination.

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 more of our monosaccharide and oligosaccharide assay kits.

Scheme-K-GLUHK-220A GLUHK Megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • 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
Megazyme publication

Measurement of Starch: Critical evaluation of current methodology.

McCleary, B. V., Charmier, L. M. J. & McKie, V. A. (2018). Starch‐Stärke, 71(1-2), 1800146.

Most commonly used methods for the measurement of starch in food, feeds and ingredients employ the combined action of α‐amylase and amyloglucosidase to hydrolyse the starch to glucose, followed by glucose determination with a glucose oxidase/peroxidase reagent. Recently, a number of questions have been raised concerning possible complications in starch analytical methods. In this paper, each of these concerns, including starch hydrolysis, isomerisation of maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase, enzyme purity and hydrolysis of sucrose and β‐glucans have been studied in detailed. Results obtained for a range of starch containing samples using AOAC Methods 996.11 and 2014 .10 are compared and a new simpler format for starch measurement is introduced. With this method that employs a thermostable α-amylase (as distinct from a heat stable α-amylase) which is both stable and active at 100°C and pH 5.0, 10 samples can be analysed within 2 h, as compared to the 6 h required with AOAC Method 2014.10.

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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
Measurement of carbohydrates in grain, feed and food.

McCleary, B. V., Charnock, S. J., Rossiter, P. C., O’Shea, M. F., Power, A. M. & Lloyd, R. M. (2006). Journal of the Science of Food and Agriculture, 86(11), 1648-1661.

Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successfully subjected to interlaboratory evaluation. All methods are based on the use of enzymes either purified by conventional chromatography or produced using molecular biology techniques. Such methods allow specific, accurate and reliable quantification of a particular component. Problems in calculating the actual weight of galactosyl-sucrose oligosaccharides in test samples are discussed in detail.

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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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Megazyme publication
Measurement of total starch in cereal products by amyloglucosidase-alpha-amylase method: collaborative study.

McCleary, B. V., Gibson, T. S. & Mugford, D. C. (1997). Journal of AOAC International, 80, 571-579.

An American Association of Cereal Chemists/AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measurement of total starch in a range of cereal grains and products. The flour sample is incubated at 95 degrees C with thermostable alpha-amylase to catalyze the hydrolysis of starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze the dextrins to glucose. Glucose is measured with glucose oxidase-peroxidase reagent. Thirty-two collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included chicken feed pellets, white bread, green peas, high-amylose maize starch, white wheat flour, wheat starch, oat bran, and spaghetti. All samples were analyzed by the standard procedure as detailed above; 4 samples (high-amylose maize starch and wheat starch) were also analyzed by a method that requires the samples to be cooked first in dimethyl sulfoxide (DMSO). Relative standard deviations for repeatability (RSD(r)) ranged from 2.1 to 3.9%, and relative standard deviations for reproducibility (RSD(R)) ranged from 2.9 to 5.7%. The RSD(R) value for high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%; the value was reduced to 2.9% when the DMSO procedure was used, and the determined starch values increased from 86.9 to 97.2%.

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Preliminary evaluation of fenugreek (Trigonella foenum-graecum) seed gum as a potential prebiotic for growing rabbits in Tunisia: effects on in vivo faecal digestibility and in vitro fermentation.

Zemzmi, J., Ródenas, L., Blas, E., Abdouli, H., Najar, T. & Pascual, J. J. (2020). World Rabbit Science, 28(3), 113-122.

This study aims to determine the effect of dietary inclusion of fenugreek seed gum (FSG), rich in galactomannans, on nutrient apparent digestibility and caecal environment, as well as on in vitro caecal fermentation of Tunisian growing rabbits. Three experimental diets were formulated, including 0, 0.25 and 0.5% of FSG (FSG0, FSG0.25 and FSG0.5, respectively) for the in vivo trial and 0, 0.125, 0.25, 0.5 and 100% of FSG (FSG0, FSG0125, FSG0.25, FSG0.5 and FSG100, respectively) for the in vitro trial. In the in vivo trial, 45 weaned rabbits 31 d old (15 per treatment) were housed in individual cages until 94 d of age. Apparent digestibility coefficients were determined at two ages, from 38 to 41 and from 56 to 59 d old, and caecal traits were recorded after slaughtering. In the in vitro trial, the five experimental diets were incubated with a rabbit caecal inoculum. Gas production was measured and modelled until 72 h and the fermentation traits were measured. Apparent faecal digestibility coefficients of main nutrients and main caecal environment traits were not significantly affected by the dietary inclusion of FSG (P>0.05). However, animals fed with FSG showed lower caecal pH (-0.15; P<0.05) values. Regarding the in vitro fermentation, FSG100 increased asymptotic gas production (+11.25, P<0.001), sharpness of the switching characteristic of the profile (+1.98, P<0.001) and the maximum substrate degradation rate (RM) (+0.188, P<0.001), but decreasing the time after incubation at which half of the asymptotic amount of gas has been formed (-5.86, P<0.001) and at which RM occurs (-4.53, P<0.01). Likewise, FSG100 significantly decreased caecal pH (-1.035, P<0.001), lactic acid (-9.51, P<0.069) and N-NH3 concentrations (-12.81, P<0.001). Meanwhile, it increased the total volatile fatty acids (VFA) production (+43.15, P<0.001). Gradual dietary inclusion of FSG from 0 to 0.5% only significantly increased total VFA production in the caecum (+100 mmol/L per percentage point of FSG inclusion; P<0.05). In conclusion, FSG is highly and rapidly in vitro fermented by rabbit caecal bacteria. However, dietary inclusion of FSG up to 0.5%, might be insufficient to affect the apparent digestibility and fermentation profile of growing rabbits to a great extent.

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The Warburg Effect in Yeast: Repression of Mitochondrial Metabolism Is Not a Prerequisite to Promote Cell Proliferation.

Bouchez, C. L., Hammad, N., Cuvellier, S., Ransac, S., Rigoulet, M. & Devin, A. (2020). Frontiers in Oncology, 10, 1333.

O. Warburg conducted one of the first studies on tumor energy metabolism. His early discoveries pointed out that cancer cells display a decreased respiration and an increased glycolysis proportional to the increase in their growth rate, suggesting that they mainly depend on fermentative metabolism for ATP generation. Warburg's results and hypothesis generated controversies that are persistent to this day. It is thus of great importance to understand the mechanisms by which cancer cells can reversibly regulate the two pathways of their energy metabolism as well as the functioning of this metabolism in cell proliferation. Here, we made use of yeast as a model to study the Warburg effect and its eventual function in allowing an increased ATP synthesis to support cell proliferation. The role of oxidative phosphorylation repression in this effect was investigated. We show that yeast is a good model to study the Warburg effect, where all parameters and their modulation in the presence of glucose can be reconstituted. Moreover, we show that in this model, mitochondria are not dysfunctional, but that there are fewer mitochondria respiratory chain units per cell. Identification of the molecular mechanisms involved in this process allowed us to dissociate the parameters involved in the Warburg effect and show that oxidative phosphorylation repression is not mandatory to promote cell growth. Last but not least, we were able to show that neither cellular ATP synthesis flux nor glucose consumption flux controls cellular growth rate.

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High yield production of levan-type fructans by Gluconobacter japonicus LMG 1417.

Hövels, M., Kosciow, K., Kniewel, J., Jakob, F. & Deppenmeier, U. (2020). International Journal of Biological Macromolecules, 164, 295-303.

Levan, a β-2,6-glycosidic linked fructan, is a promising alternative for the inulin dominated fructan market. Although levan is already used in some cosmetic products, the commercial availability of the fructan is still limited. Here we show that Gluconobacter japonicus LMG 1417 is a potent levan-forming organism and a promising platform for the industrial production of levan. The levansucrase LevS1417, which is produced by G. japonicus LMG 1417 and secreted by a signal-peptide-independent pathway, exhibited extraordinary high activity (4726 ± 821 U mg−1 at 50 °C). A cell-free levan production based on the supernatant of the investigated strain led to a final levan yield of 157.9 ± 7.6 g L−1. The amount of secreted levansucrase was more than doubled by plasmid-mediated homologous overproduction of LevS1417 in G. japonicus LMG 1417. Accordingly, the space-time yield of cell-free levan production was doubled using the plasmid-bearing mutant.

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Characterisation and In Vitro Evaluation of Fenugreek (Trigonella foenum-graecum) Seed Gum as a Potential Prebiotic in Growing Rabbit Nutrition.

Zemzmi, J., Ródenas, L., Blas, E., Najar, T. & Pascual, J. J. (2020). Animals, 10(6), 1041.

A fenugreek seed gum, extracted from Trigonella foenum-graecum seeds and rich in galactomannan, was chemically and physically characterised and its prebiotic potential for young rabbits was evaluated in vitro, both as pure fenugreek seed gum and when included up to 20 g/kg in rabbit diets rich in soluble and insoluble fibre. Fenugreek seed gum was resistant to pepsin and pancreatin digestion but was totally fermented by rabbit caecal bacteria. Fenugreek seed gum linear inclusion up to 20 g/kg in diets rich in soluble fibre has led to a reduction in the solubility of some nutrients during in vitro enzymatic phase and an increase in the fermented fraction. Fenugreek seed gum satisfies two essential conditions of a prebiotic: resistance to enzymatic digestion and being totally fermented by caecal bacteria.

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Structural comparison of different galacto-oligosaccharide mixtures formed by β-galactosidases from lactic acid bacteria and bifidobacteria.

Kittibunchakul, S., van Leeuwen, S. S., Dijkhuizen, L., Haltrich, D. & Nguyen, T. H. (2020). Journal of Agricultural and Food Chemistry, 68(15), 4437-4446.

The LacLM-type β-galactosidase from Lactobacillus helveticus DSM 20075 expressed in both Escherichia coli (EcoliBL21Lhβ-gal) and Lactobacillus plantarum (Lp609Lhβ-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by β-galactosidase from L. helveticus, together with three GOS mixtures produced using β-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, L. reuteri (Lr-GOS), L. bulgaricus (Lb-GOS), and Streptococcus thermophilus (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using β-galactosidases (β-gal I and β-gal II) from Bifidobacterium breve, was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional 1H NMR spectroscopy. β-Galactosidases from lactic acid bacteria and B. breve displayed a preference to form β-(1→6)- and β-(1→3)-linked GOS. The GOS mixtures produced by these enzymes consisted of mainly DP2 and DP3 oligosaccharides, accounting for ~90% of all GOS components. GOS mixtures obtained with β-galactosidases from lactic acid bacteria and B. breve were quite similar to the commercial GOS III mixture in terms of product spectrum and showed a broader product spectrum than the commercial GOS V mixture. These GOS mixtures also contained a number of GOS components that were absent in the commercial Vivinal GOS (V-GOS).

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The loosening effect of tea polyphenol on the structure of octenyl succinic anhydride modified waxy maize starch.

Wang, X., Leng, X. & Zhang, G. (2020). Food Hydrocolloids, 99, 105367.

Octenyl succinic anhydride modified waxy maize starch after heat-moisture treatment (HT-OSAS) is a heat-stable slowly digestible starch (SDS), but the mechanism is poorly understood. Tea polyphenols (TP), as a probe with hydrophobic moiety, were employed to study the structural basis for its slow digestion. The content of RS (8.91%) in the HT-OSAS was significantly increased by TP in a dose-dependent manner (19.06% at TP of 15%) while there is a slight decrease of SDS content (34.85-30.63%). However, after retrogradation for one week, TP significantly reduced the SDS content (29.79%-15.67%). Additionally, TP increased the hydrodynamic radius of HT-OSAS (60-102 nm), but reduced its emulsifying activity (-35%), gelatinization enthalpy (-45%), and viscosity (-66%), demonstrating an inhibitory effect of TP on the association of HT-OSAS molecules, and a compact physical structure of HT-OSAS formed through OSA-mediated hydrophobic interaction is likely the basis to its slow digestion property. Novel strategies to densify the physical structure of starch could improve the nutritional quality of starch with a slow digestion property.

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Novel denitrifying bacteria Pseudomonas stutzeri strain D1-from isolation to the biomass production.

Vidaković, A., Šovljanski, O., Vučurović, D., Racić, G., Đilas, M., Ćurčić, N. & Markov, S. (2019). Chemical Industry and Chemical Engineering Quarterly, (00), 18-18.

An aerobic denitrifier was newly isolated and identified by VITEK® 2 Compact System and MALDI-TOF MS as P. stutzeri strain D1. Sequence amplification indicates that the denitrification genes napA, nirS, norB and nosZ are present in a novel strain D1, as well as in reference strain ATCC 17588. Strain D1 had capability to fully remove 3 g/L of nitrate (as KNO3) in 48 h, while the reference strain completed this task in 60 h. Single factor experiments indicate that the optimal conditions for biomass production were: temperature of 30°C, pH value of 7 and inoculum volume of 5 vol.%. Scaling up of biomass production of both denitrifiers was successfully performed in 3 and 7 L laboratory bioreactors by reaching 9 log CFU/mL of the viable cells. The results demonstrate the feasibility of using investigated P. stutzeri strains in denitrification processes and the simplicity of the up-scaling of biomass production for the treatment of large areas contaminated with nitrate.

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A simulated gastrointestinal tract study of texturized rice grains: Impact of texturization on starch digestibility.

Ye, J., Liu, C., Luo, S., Wu, J., Hu, X. & McClements, D. J. (2019). Journal of Cereal Science, 89, 102800.

The digestibility of starch in cooked ordinary rice grains and cooked texturized rice grains was compared using an in vitro gastrointestinal tract (GIT), which involved sequentially exposing samples to simulated oral, gastric, and small intestinal regions. The total extent of starch hydrolysis (%) of texturized rice was slightly lower than that of ordinary rice by the end of the GIT, probably because of its higher resistant starch content. Furthermore, the rate of starch hydrolysis in the small intestine was much slower for texturized rice than ordinary rice. Gel electrophoresis and microstructure analysis suggested that the proteins in the texturized rice formed a cross-linked network around the starch molecules. This network may have retarded the ability of digestive enzymes to access the starch, thereby reducing the hydrolysis rate. These results have important implications for the design of starch-based foods with a lower glycemic index, and therefore improved nutritional profiles.

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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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β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical characterization and recombinant expression for applications in dairy industry.

Kittibunchakul, S., Pham, M. L., Tran, A. M. & Nguyen, T. H. (2019). International Journal of Molecular Sciences, 20(4), 947.

β-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ~2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant β-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The Km and vmax values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol O-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of ONPG with Ki,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and ONPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55°C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of β-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.

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Influence of nano-fibrillated cellulose (NFC) on starch digestion and glucose absorption.

Liu, L., Kerr, W. L., Kong, F., Dee, D. R. & Lin, M. (2018). Carbohydrate Polymers, 196, 146-153.

Nano-fibrillated cellulose (NFC) is of interest in several fields due to its unique physical properties derived from its nanoscale dimensions. NFC has potential use in food systems as a dietary fiber that increases viscosity and may interact with and limit diffusion of glucose. This study focused on the effects of added NFC on solution viscosity, starch digestion and glucose absorption. NFC did not affect α-amylase and α-glucosidase activity, but significantly retarded glucose diffusion, delayed amylolysis and reduced the amount of glucose released during in vitro digestion of starch. Specifically, 1% NFC retarded ∼26.6% of glucose released during the amylolysis process. The greatly increased viscosity of NFC at concentrations >0.5% was thought to be the main mechanism for its potential hypoglycemic effects. NFC suspensions also had higher glucose adsorption capacity than those containing cellulose. In addition, NFC bound 35.6% of the initial glucose level (5–200 mM). These results suggest that NFC may be useful for building viscosity in food products and serve to inhibit glucose absorption in vivo in starch-containing products.

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Type and capacity of glucose transport influences succinate yield in two-stage cultivations.

Kyselova, L., Kreitmayer, D., Kremling, A. & Bettenbrock, K. (2018). Microbial cell factories,17(1), 1-15.

Background: Glucose is the main carbon source of E. coli and a typical substrate in production processes. The main glucose uptake system is the glucose specific phosphotransferase system (Glc-PTS). The PTS couples glucose uptake with its phosphorylation. This is achieved by the concomitant conversion of phosphoenolpyruvate (PEP) to pyruvate. The Glc-PTS is hence unfavorable for the production of succinate as this product is derived from PEP. Results: We studied, in a systematic manner, the effect of knocking out the Glc-PTS and of replacing it with the glucose facilitator (Glf) of Zymomonas mobilis on succinate yield and productivity. For this study a set of strains derived from MG1655, carrying deletions of ackA-pta, adhE and ldh  that prevent the synthesis of competing fermentation products, were constructed and tested in two-stage cultivations. The data show that inactivation of the Glc-PTS achieved a considerable increase in succinate yield and productivity. On the other hand, aerobic growth of this strain on glucose was strongly decreased. Expression of the alternative glucose transporter, Glf, in this strain enhanced aerobic growth but productivity and yield under anaerobic conditions were slightly decreased. This decrease in succinate yield was accompanied by pyruvate production. Yield could be increased in both Glc-PTS mutants by overexpressing phosphoenolpyruvate carboxykinase (Pck). Productivity on the other hand, was decreased in the strain without alternative glucose transporter but strongly increased in the strain expressing Glf. The experiments were complemented by flux balance analysis in order to check the observed yields against the maximal theoretical yields. Furthermore, the phosphorylation state of EIIAGlc was determined. The data indicate that the ratio of PEP to pyruvate is correlating with pyruvate excretion. This ratio is affected by the PTS reaction as well as by further reactions at the PEP/pyruvate node. Conclusions: The results show that for optimization of succinate yield and productivity it is not sufficient to knock out or introduce single reactions. Rather, balancing of the fluxes of central metabolism most important at the PEP/pyruvate node is important.

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Glucose biosensor based on disposable electrochemical paper-based transducers fully fabricated by screen-printing.

Lamas-Ardisana, P. J., Martínez-Paredes, G., Añorga, L. & Grande, H. J. (2018). Biosensors and Bioelectronics, 109, 8-12.

This paper describes a new approach for the massive production of electrochemical paper-based analytical devices (ePADs). These devices are fully fabricated by screen-printing technology and consist of a lineal microfluidic channel delimited by hydrophobic walls (patterned with diluted ultraviolet screen-printing ink in chromatographic paper grade 4) and a three-electrode system (printed with carbon and/or Ag/AgCl conductive inks). The printing process was characterised and optimized for pattern each layer with only one squeeze sweep. These ePADs were used as transducers to develop a glucose biosensor. Ionic strength/pH buffering salts, electrochemical mediator (ferricyanide) and enzyme (glucose dehydrogenase FAD-dependent) were separately stored along the microfluidic channel in order to be successively dissolved and mixed after the sample dropping at the entrance. The analyses required only 10 µl and the biosensors showed good reproducibility (RSD = 6.2%, n = 10) and sensitivity (0.426 C/M cm2), wide linear range (0.5-50 mM; r2 = 0.999) and low limit of detection (0.33 mM). Furthermore, the new biosensor was applied for glucose determination in five commercial soft-drinks without any sample treatment before the analysis. These samples were also analysed with a commercial enzymatic-kit assay. The results indicated that both methods provide accurate results.

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A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity.

Stressler, T., Reichenberger, K., Glück, C., Leptihn, S., Pfannstiel, J., Swietalski, P., Kuhm, A., Seitl, I. & Fischer, L. (2018). Applied microbiology and Biotechnology, 102(6), 2709-2721.

Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as β-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of β-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC Due to the action of arylsulfatase, an unpleasant “cowshed-like” off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.

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Competitive interactions moderate the effects of elevated temperature and atmospheric CO2 on the health and functioning of oysters.

Green, D. S., Christie, H., Pratt, N., Boots, B., Godbold, J. A., Solan, M. & Hauton, C. (2017). Marine Ecology Progress Series, 582, 93-103.

Global increases in sea temperatures and atmospheric concentrations of CO2 may affect the health of calcifying shellfish. Little is known, however, about how competitive interactions within and between species may influence how species respond to multiple stressors. We experimentally assessed separate and combined effects of temperature (12 or 16°C) and atmospheric CO2 concentrations (400 and 1000 ppm) on the health and biological functioning of native (Ostrea edulis) and invasive (Crassostrea gigas) oysters held alone and in intraspecific or interspecific mixtures. We found evidence of reduced phagocytosis under elevated CO2 and, when combined with increased temperature, a reduction in the number of circulating haemocytes. Generally, C. gigas showed lower respiration rates relative to O. edulis when the species were in intraspecific or interspecific mixtures. In contrast, O. edulis showed a higher respiration rate relative to C. gigas when held in an interspecific mixture and exhibited lower clearance rates when held in intraspecific or interspecific mixtures. Overall, clearance rates of C. gigas were consistently greater than those of O. edulis. Collectively, our findings indicate that a species’ ability to adapt metabolic processes to environmental conditions can be modified by biotic context and may make some species (here, C. gigas) competitively superior and less vulnerable to future climatic scenarios at local scales. If these conclusions are generic, the relative role of species interactions, and other biotic parameters, in altering the outcomes of climate change will require much greater research emphasis.

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