D-Gluconic Acid/D-Glucono-δ-lactone Assay Kit

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.

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.

The presence of sodium gluconate in Cheddar cheese, which can be added as a processing aid during manufacture, has been shown as a contributing factor to gas production and gas defects in aged cheese. A survey of gluconate content in retail Cheddar cheese was performed. Fifty-three retail packs of Cheddar cheese were purchased from stores in Utah as blocks, shreds, sliced cheeses, or individual-sized cuts and sticks. This included mild, medium, and sharp cheeses. Analysis of gluconate content was performed using an enzyme assay. Levels of gluconate ≥0.1% were found in 7 samples with the highest level being 0.45% (wt/wt).

Miscanthus sinensis Andersson naturally grows at mining sites and accumulates heavy metals, suggesting heavy metal tolerance. This study aimed to elucidate the heavy metal tolerance mechanisms of M. sinensis, which naturally grew at a mine site, considering root endophytes. Elemental analysis by inductively coupled plasma-optical emission spectroscopy showed that M. sinensis highly accumulated zinc and lead in adventitious roots and dead root bark, exhibiting tolerance via these elemental removals in dead root bark. Phenolic analysis by high-performance liquid chromatography revealed chlorogenic acid production in adventitious roots. Phialocephala fortinii, which showed the highest appearance, and Talaromyces verruculosus, which produced gluconic acid as a zinc detoxicant, were isolated from adventitious roots of M. sinensis. In inoculation tests, P. fortinii and T. verruculosus significantly enhanced seedling growth by increasing potassium uptake and possibly indole-3-acetic acid production. Our results provide fundamental knowledge for using M. sinensis and root endophytes for revegetation at mining sites.

2-hydroxy acids are organic carboxylic acids ubiquitous in the living world and are important building blocks in organic synthesis. Recently, the lactate racemase (LarA) superfamily, a diverse superfamily of 2-hydroxy acid racemases and epimerases using the nickel-pincer nucleotide (NPN) cofactor, has been uncovered. In this study, we performed a taxonomic analysis of the LarA superfamily, showing the distribution of lactate racemase homologs (LarAHs) sequences across the three domains of life. Subsequently, we overexpressed and purified nine LarAHs and investigated their biochemical properties and substrate specificities. We show that LarAHs from the lactate racemases group are more promiscuous than previously thought, with some members showing high specificity towards glycerate or 2-hydroxybutyrate. In other phylogenetic groups, we identified a new malate racemase and 2-hydroxyglutarate racemase, as well as a new 2-gluconate epimerase from an eukaryotic organism. We show that some LarAHs are able to isomerize up to 16 different substrates, mostly 2-hydroxy acids with hydrophobic side chains, thereby identifying 14 novel 2-hydroxy acid racemization and epimerization reactions catalyzed by LarAHs. These include the racemization of glycerate, 2-hydroxybutyrate, 2,4-dihydroxybutyrate, 2-hydroxyvalerate, 2-hydroxycaproate, 2,3-dihydroxyisovalérate, 2-hydroxy-3,3-dimethylbutyrate, 3-(4-hydroxyphenyl)lactate, 2-hydroxy-4-phenylbutyrate, and 2-hydroxy-4-oxo-4-phenylbutyrate. Additionally, we observed the C2-epimerization of all 2,3-dihydroxybutyrate stereoisomers (4-deoxy-DL-threonate and 4-deoxy-DL-erythronate) and the C2-epimerization of D-arabinarate epimers. Finally, through comparative analysis of Alphafold structural predictions, we identified key residues likely involved in substrate specificity and predicted the function of half of the LarAHs from the LarA superfamily. In conclusion, this study widely expands the scope of substrates isomerized by NPN-dependent enzymes.

Bacterial Cellulose (BC) is a biopolymer with numerous applications. The growth of BC-producing bacteria, Komagataeibacter intermedius, could be stimulated by Dekkera bruxellensis, however, the effect on BC yield needs further investigation. This study investigates BC production and biochemical changes in the K. intermedius-D. bruxellensis co-culture system. D. bruxellensis was introduced at various concentrations (10³ and 10⁶ CFU/mL) and inoculation times (days 0 and 3). BC yield was ~24% lower when D. bruxellensis was added at 10³ CFU/mL compared to K. intermedius alone (0.63 ± 0.11 g/L). The lowest BC yield was observed when 10³ CFU/mL yeast was added on day 0, which could be compromised by higher gluconic acid production (10.08 g/L). In contrast, BC yields increased by ~88% when 10⁶ CFU/mL D. bruxellensis was added, regardless of inoculation time. High BC yield might correlate with faster sugar consumption or increased ethanol production when 10⁶ CFU/mL D. bruxellensis was added on day 0. These results suggest that cell concentration and inoculation time have crucial impacts on species interactions in the co-culture system and product yield.

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099^T, Kozakia baliensis DSM 14400^T and Gluconobacter cerinus DSM 9533^T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099^T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400^T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of A. carbonarius to produce and accumulate OTA in grapes.

Isomerization reactions are fundamental in biology. Lactate racemase, which isomerizes L- and D-lactate, is composed of the LarA protein and a nickel-containing cofactor, the nickel-pincer nucleotide (NPN). In this study, we show that LarA is part of a superfamily containing many different enzymes. We overexpressed and purified 13 lactate racemase homologs, incorporated the NPN cofactor, and assayed the isomerization of different substrates guided by gene context analysis. We discovered two malate racemases, one phenyllactate racemase, one α-hydroxyglutarate racemase, two D-gluconate 2-epimerases, and one short-chain aliphatic α-hydroxyacid racemase among the tested enzymes. We solved the structure of a malate racemase apoprotein and used it, along with the previously described structures of lactate racemase holoprotein and D-gluconate epimerase apoprotein, to identify key residues involved in substrate binding. This study demonstrates that the NPN cofactor is used by a diverse superfamily of α-hydroxyacid racemases and epimerases, widely expanding the scope of NPN-dependent enzymes.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. Aspergillus carbonarius is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in A. carbonarius pathogenicity using designed pacC gene knockout mutant. ΔAcpacC mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The ΔAcpacC mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of AcpacC esulted in a complete inhibition of OTA production at pH 7.0. Additionally, ΔAcpacC mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of pacC gene into ΔAcpacC mutant restored the WT phenotype. Our results demonstrate important roles of PacC of A. carbonarius in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Objective: The development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs). Results: In combination with a β-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1-oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation. Conclusions: We conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

Aim: A study was carried out in 2013 and 2014 to determine the key environmental and viticultural variables affecting the concentration of rotundone, the black pepper aroma compound, in Vitis vinifera L. cv. Duras red wines at 10 different vineyard blocks. Methods and Results: For each block, data for fruit quality attributes, as well as climatic and agronomical variables, were collected. Rotundone was quantified in wines prepared by microvinification techniques (in a 1-L Erlenmeyer flask). Rotundone concentration varied across blocks from 63 ng/L to 239 ng/L in 2013 and from 25 ng/L to 115 ng/L in 2014. Three separate partial least squares regression models were constructed to predict rotundone concentration in wines in 2013, in 2014, and in both vintages. Gluconic acid, a secondary metabolite of Botrytis cinerea, had a substantial contribution to the 2013 and multivintage models, with a negative regression coefficient with rotundone concentration. Other predictors were associated with abiotic factors such as cumulative rainfall, thermal index, hours of sunshine and mean daily irradiation. Conclusions: Our results indicate that mesoscale climatic variables are the key factors determining rotundone concentration, and also suggest that Botrytis cinerea may be involved in rotundone degradation. Significance and impact of the study: Our findings may assist grape growers producing Duras red wines to select specific vineyard blocks with the aim of producing wines with a desired rotundone concentration. They also open up new fields of investigation into mechanisms involved in possible rotundone degradation by Botrytis cinerea.

Andean tropical glaciers are disappearing rapidly and, consequently, the microbes immured in these frozen environments will be lost forever. Some of these microbes are thought to be potentially useful to develop biotechnological products or processes. Among these microbes, plant-growth promoting (PGP) bacteria have been proposed as valuable tools to develop cold-active biofertilizers and/or biopesticides. A few years ago, we hypothesized that bacteria immured within glacial ice could be effective in promoting plant growth and/or in protecting plants from pathogen infection, at low temperatures. In this study, we aimed at testing some of these traits, with a suitable plant model (Triticum aestivum). In the present study, from a collection of bacteria isolated from Venezuelan tropical glaciers, we selected four Pseudomonas isolates and tested their PGP effects at low temperatures, both in vitro and on wheat plantlets. The isolates grew well over a wide range of low temperatures and were thus classified as eurypsychrophilic. They also displayed well-known PGP traits: solubilization of inorganic phosphates, production of phytohormones and antagonism against a phytopathogenic oomycete (Pythium ultimum). Inoculation of T. aestivum seeds with some of these Pseudomonas spp. isolates promoted a significant elongation of their roots and shoots. This was also the case when wheat plantlets were grown in sterile sand or soil, at 15°C. Inoculation of wheat seeds also protected plantlets against the damage caused by P. ultimum. Together, our results suggest that some of these Pseudomonas spp. isolates could act as cold-active biofertilizers and/or biocontrol agents.

Under peculiar climatic conditions, the beneficial form of the necrotrophic fungus Botrytis cinerea can develop on grape berries as a latent infection, known as noble rot, which induces positive biochemical and metabolic changes in the berries, including an increase in the sugar content and the production of aromatic compounds that improve wine quality. The infected berries undergo rapid withering, which is required to produce famous sweet white wines such as Sauternes and Tokaj. To gain insight into the molecular interactions between grapevine berries (Vitis vinifera) and B. cinerea during the establishment of noble rot, we prepared a large-scale transcriptomics dataset representing noble rot development and carried out a comparative meta-analysis with gray mold infection and natural post-harvest withering. In particular, we artificially induced berry botrytization of two grape varieties (Garganega and Möller-Thurgau) and sampled them at different stages of noble rot for comparative whole-transcriptome analysis, highlighting important common transcriptional reprogramming in both varieties reflecting an accelerated withering process. Simultaneously, we analyzed the modulation of B. cinerea genes and compared the expression profile during noble rot development with the previously reported gray mold infection profile, revealing the onset of an infection process by the fungus in its beneficial form associated with reduced virulence. This, together with the restrained plant defense response observed in botrytized berries, may favour the development of noble rot instead of gray mold. Finally, the comprehensive meta-analysis of gene expression during noble rot infection, gray mold and post-harvest withering led to the identification of key genes specifically modulated during noble rot infection.

Ethanol exerts a strong positive effect on the cellulose yields from the widely exploited microbial producers of the Komagataeibacter genus. Ethanol is postulated to provide an alternative energy source, enabling effective use of glucose for cellulose biosynthesis rather than for energy acquisition. In this paper, we investigate the effect of ethanol supplementation on the global gene expression profile of Komagataeibacter xylinus E25 using RNA sequencing technology (RNA-seq). We demonstrate that when ethanol is present in the culture medium, glucose metabolism is directed towards cellulose production due to the induction of genes related to UDP-glucose formation and the repression of genes involved in glycolysis and acetan biosynthesis. Transcriptional changes in the pathways of cellulose biosynthesis and c-di-GMP metabolism are also described. The transcript level profiles suggest that Schramm-Hestrin medium supplemented with ethanol promotes bacterial growth by inducing protein biosynthesis and iron uptake. We observed downregulation of genes encoding transposases of the IS110 family which may provide one line of evidence explaining the positive effect of ethanol supplementation on the genotypic stability of K. xylinus E25. The results of this study increase knowledge and understanding of the regulatory effects imposed by ethanol on cellulose biosynthesis, providing new opportunities for directed strain improvement, scaled-up bionanocellulose production, and wider industrial exploitation of the Komagataeibacter species as bacterial cellulose producers.