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.
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.
Aldose reductase is implicated in high glucose‐induced oxidative stress in mouse embryonic neural stem cells.
Fu, J., Tay, S. S. W., Ling, E. A. & Dheen, S. T. (2007). Journal of Neurochemistry, 103(4), 1654-1665.
Oxidative stress caused by hyperglycemia is one of the key factors responsible for maternal diabetes-induced congenital malformations, including neural tube defects in embryos. However, mechanisms by which maternal diabetes induces oxidative stress during neurulation are not clear. The present study was aimed to investigate whether high glucose induces oxidative stress in neural stem cells (NSCs), which compose the neural tube during development. We also investigated the mechanism by which high glucose disturbs the growth and survival of NSCs in vitro. NSCs were exposed to physiological D-glucose concentration (PG, 5 mmol/L), PG with L-glucose (25 mmol/L), or high D-glucose concentration (HG, 30 or 45 mmol/l). HG induced reactive oxygen species production and mRNA expression of aldose reductase (AR), which catalyzes the glucose reduction through polyol pathway, in NSCs. Expression of glucose transporter 1 (Glut1) mRNA and protein which regulates glucose uptake in NSCs was increased at early stage (24 h) and became down-regulated at late stage (72 h) of exposure to HG. Inhibition of AR by fidarestat, an AR inhibitor, decreased the oxidative stress, restored the cell viability and proliferation, and reduced apoptotic cell death in NSCs exposed to HG. Moreover, inhibition of AR attenuated the down-regulation of Glut1 expression in NSCs exposed to HG for 72 h. These results suggest that the activation of polyol pathway plays a role in the induction of oxidative stress which alters Glut1 expression and cell cycle in NSCs exposed to HG, thereby resulting in abnormal patterning of the neural tube in embryos of diabetic pregnancy.
Assessment of xylitol serum levels during the course of parenteral nutrition including xylitol in intensive care patients: A case control study.
Schneider, A. S., Schettler, A., Markowski, A., Luettig, B., Momma, M., Seipt, C., Hadem, J. & Wilhelmi, M. (2014). Clinical Nutrition, 33(3), 483-488.
Background & aims: Xylitol has been approved for parenteral nutrition and may be beneficial in catabolic situations. The aim was to establish an easy method to monitor xylitol serum levels in patients receiving xylitol and to determine whether xylitol is safe. Methods: A commercially available xylitol test was validated and used to measure serum levels in 55 patients admitted to our intensive care unit with an indication for parenteral nutrition with xylitol for at least 24 h. Controls consisted of the most recent 56 patients admitted to the intensive care unit who received parenteral nutrition without xylitol for at least 2 days. Xylitol serum levels were determined using the test. Adverse events, liver enzymes, lactate, bilirubin, γ-glutamyl transpeptidase, and insulin requirement were secondary endpoints. Results: Patients receiving xylitol received 32.6% less insulin than controls. The amount of energy they received was comparable (xylitol: 810.1; controls: 789.8 kcal). Mean liver enzymes and lactate levels were similar in both groups. Adverse events considered attributable to xylitol did not occur. Xylitol did not accumulate in patients' blood and returned to near baseline values one day after parenteral nutrition was stopped. Conclusions: Parenteral nutrition with xylitol appears to be safe for critical care patients. There were no signs of hepatoxicity.
The peach (Prunus persica [L.] Batsch) homeobox gene KNOPE3, which encodes a class 2 knotted-like transcription factor, is regulated during leaf development and triggered by sugars.
Testone, G., Condello, E., Verde, I., Caboni, E., Iannelli, M. A., Bruno, L., Mariotti, D., Bitonti, M. B. & Giannino, D. (2009). Molecular Genetics and Genomics, 282(1), 47-64.
Class 1 KNOTTED1-like transcription factors (KNOX) are known to regulate plant development, whereas information on class 2 KNOX has been limited. The peach KNOPE3 gene was cloned, belonged to a family of few class 2 members and was located at 66 cM in the Prunus spp. G1 linkage-group. The mRNA localization was diversified in leaf, stem, flower and drupe, but recurred in all organ sieves, suggesting a role in sap nutrient transport. During leaf development, the mRNA earliest localized to primordia sieves and subsequently to mesophyll cells of growing leaves. Consistently, its abundance augmented with leaf expansion. The transcription was monitored in leaves responding to darkening, supply and transport block of sugars. It peaked at 4 h after darkness and dropped under prolonged obscurity, showing a similar kinetic to that of sucrose content variation. Feeding leaflets via the transpiration stream caused KNOPE3 up-regulation at 3 h after fructose, glucose and sucrose absorption and at 12 h after sorbitol. In girdling experiments, leaf KNOPE3 was triggered from 6 h onwards along with sucrose and sorbitol raise. Both the phloem-associated expression and sugar-specific gene modulation suggest that KNOPE3 may play a role in sugar translocation during the development of agro-relevant organs such as drupe.
A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.
Wiebe, M. G., Nygård, Y., Oja, M., Andberg, M., Ruohonen, L., Koivula, A., Penttilä, M. & Toivari, M. (2015). Applied Microbiology and Biotechnology, 99(22), 9439-9447.
An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l-1 and 11.5 g D-xylitol l-1 from 26 g D-xylose l-1), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l-1). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.
Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains.
Verhoeven, M. D., de Valk, S. C., Daran, J. M. G., van Maris, A. J. & Pronk, J. T. (2018). FEMS Yeast Research, In Press.
D-Glucose, D-xylose and L-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three ‘specialist’ Saccharomyces cerevisiae strains. A D-glucose- and L-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented D-xylose in the presence of 20 g L-1 of D-glucose and L-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose-non-fermenting laboratory strain, rapidly and simultaneously converted D-glucose and L-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved ‘generalist’ strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1 D-glucose, 10 g L-1 D-xylose and 5 g L-1 L-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.