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.
The metabolic potential of Escherichia coli BL21 in defined and rich medium.
Li, Z., Nimtz, M. & Rinas, U. (2014). Microbial Cell Factories, 13(1), 45.
Background: The proteome reflects the available cellular machinery to deal with nutrients and environmental challenges. The most common E. coli strain BL21 growing in different, commonly employed media was evaluated using a detailed quantitative proteome analysis. Results: The presence of preformed biomass precursor molecules in rich media such as Luria Bertani supported rapid growth concomitant to acetate formation and apparently unbalanced abundances of central metabolic pathway enzymes, e.g. high levels of lower glycolytic pathway enzymes as well as pyruvate dehydrogenase, and low levels of TCA cycle and high levels of the acetate forming enzymes Pta and AckA. The proteome of cells growing exponentially in glucose-supplemented mineral salt medium was dominated by enzymes of amino acid synthesis pathways, contained more balanced abundances of central metabolic pathway enzymes, and a lower portion of ribosomal and other translational proteins. Entry into stationary phase led to a reconstruction of the bacterial proteome by increasing e.g. the portion of proteins required for scavenging rare nutrients and general cell protection. This proteomic reconstruction during entry into stationary phase was more noticeable in cells growing in rich medium as they have a greater reservoir of recyclable proteins from the translational machinery. Conclusions: The proteomic comparison of cells growing exponentially in different media reflected the antagonistic and competitive regulation of central metabolic pathways through the global transcriptional regulators Cra, Crp, and ArcA. For example, the proteome of cells growing exponentially in rich medium was consistent with a dominating role of phosphorylated ArcA most likely a result from limitations in reoxidizing reduced quinones in the respiratory chain under these growth conditions. The proteomic alterations of exponentially growing cells into stationary phase cells were consistent with stringent-like and stationary phase responses and a dominating control through DksA-ppGpp and RpoS.
Comparative transcriptomic profile analysis of fed-batch cultures expressing different recombinant proteins in Escherichia coli.
Sharma, A. K., Mahalik, S., Ghosh, C., Singh, A. B. & Mukherjee, K. J. (2011). AMB Express, 1(1), 33.
There is a need to elucidate the product specific features of the metabolic stress response of the host cell to the induction of recombinant protein synthesis. For this, the method of choice is transcriptomic profiling which provides a better insight into the changes taking place in complex global metabolic networks. The transcriptomic profiles of three fed-batch cultures expressing different proteins viz. recombinant human interferon-beta (rhIFN-β), Xylanase and Green Fluorescence Protein (GFP) were compared post induction. We observed a depression in the nutrient uptake and utilization pathways, which was common for all the three expressed proteins. Thus glycerol transporters and genes involved in ATP synthesis as well as aerobic respiration were severely down-regulated. On the other hand the amino acid uptake and biosynthesis genes were significantly repressed only when soluble proteins were expressed under different promoters, but not when the product was expressed as an inclusion body (IB). High level expression under the T7 promoter (rhIFN-β and xylanase) triggered the cellular degradation machinery like the osmoprotectants, proteases and mRNA degradation genes which were highly up-regulated, while this trend was not true with GFP expression under the comparatively weaker ara promoter. The design of a better host platform for recombinant protein production thus needs to take into account the specific nature of the cellular response to protein expression.
Organic carbon supplementation of sterilized municipal wastewater is essential for heterotrophic growth and removing ammonium by the microalga Chlorella Vulgaris.
Perez‐Garcia, O., Bashan, Y. & Esther Puente, M. (2011). Journal of Phycology, 47(1), 190-199.
Heterotrophic growth of the microalga Chlorella vulgaris Beij. in synthetic as well as sterilized municipal wastewater of a nonindustrialized city was measured. The city wastewater contained high levels of ammonium and nitrate, medium levels of phosphate, and low levels of nitrite and organic molecules and could not support heterotrophic growth of C. vulgaris. Evaluation of 11 known carbon sources for this microalga that were added to standard synthetic wastewater containing the same levels of nitrogen and phosphorus as the municipal wastewater revealed that the best carbon sources for heterotrophic growth were Na-acetate and D-glucose. These provided the highest growth rates and the largest removal of ammonium. Growth increased with concentration of the supplement to an optimum at 0.12 M Na-acetate. This carbon source was consumed completely within 10 d of incubation. Higher concentrations inhibited the growth of C. vulgaris. The microalgal populations under heterotrophic growth conditions were one level of magnitude higher than that under autotrophic growth conditions that served as a comparison. No growth occurred in the dark in the absence of a carbon source. Na-acetate was superior to D-glucose. In municipal wastewater, when Na-acetate or D-glucose was added, C. vulgaris significantly enhanced ammonium removal under heterotrophic conditions, and its capacity was equal to ammonium removal under autotrophic growth conditions. This study showed that sterilized wastewater can be treated by C. vulgaris under heterotrophic conditions if supplemented with the appropriate organic carbon source for the microalgae.
Large-scale metabolome analysis and quantitative integration with genomics and proteomics data in Mycoplasma pneumonia.
Maier, T., Marcos, J., Wodke, J. A. H., Paetzold, B., Liebeke, M., Gutiérrez-Gallego, R. & Serrano, L. (2013). Molecular BioSystems, 9(7), 1743-1755.
Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.
Decline in mammary translational capacity during intravenous glucose infusion into lactating dairy cows.
Curtis, R. V., Kim, J. J. M., Bajramaj, D. L., Doelman, J., Osborne, V. R. & Cant, J. P. (2014). Journal of Dairy Science, 97(1), 430-438.
The objective of this study was to determine effects of glucose on milk protein yield and mammary mammalian target of rapamycin (mTOR) activity in dairy cattle in early lactation. Eight multiparous cows at 73 ± 8 d in milk were randomly assigned to 2 treatments in a crossover design for two 6-d periods. Treatments were jugular infusion of either saline (Sal) or 896 g/d glucose (Glc). All cows were fed a total mixed ration with 42% neutral detergent fiber, had free access to water, and were milked twice a day. Within each period, blood samples were taken (d 5) and mammary tissue was collected by biopsy (d 6) from each hindquarter for Western blot analysis. In addition to Sal and Glc treatments, on d 6, rapamycin dissolved in 50% dimethyl sulfoxide was administered via the teat canals into the left quarters, with a control solution administered into the right quarters. Rapamycin had no effect on milk protein yields or phosphorylation state of mTOR signaling proteins. Infusions of Glc significantly increased milk yield but only tended to increase milk protein yields. Milk fat tended to be decreased in cows infused with Glc, whereas lactose yields were significantly increased. Glucose infusion did not increase plasma glucose levels, but insulin and nonessential AA concentrations increased by 21 and 16%, respectively, branched-chain AA concentrations decreased 24%, and essential AA concentrations tended to decrease by 14%. Infusion of Glc significantly decreased abundances of both phosphorylated and total ribosomal S6 kinase 1 (S6K1) in mammary tissue by 27 and 11%, respectively. Abundance of phosphorylated eukaryotic initiation factor 4E-binding protein 1 (4EBP1) decreased significantly by 25%, whereas total 4EBP1 exhibited a tendency to decrease by 16%. We conclude that the mTOR signaling pathway is not the only regulator of milk protein synthesis. Decreases in essential AA concentrations in plasma suggest that protein synthesis was stimulated in nonmammary tissues of the body, presumably skeletal muscle.
A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus.
Alalouf, O., Balazs, Y., Volkinshtein, M., Grimpel, Y., Shoham, G. & Shoham, Y. (2011). Journal of Biological Chemistry, 286(49), 41993-42001.
Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for Kcat and Kcat/Km suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-β-D-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family.
Microbial carbohydrate esterases deacetylating plant polysaccharides.
Biely, P. (2012). Biotechnology Advances, 30(6), 1575-1588.
Several plant polysaccharides are partially esterified with acetic acid. One of the roles of this modification is protection of plant cell walls against invading microorganisms. Acetylation of glycosyl residues of polysaccharides prevents hydrolysis of their glycosidic linkages by the corresponding glycoside hydrolases. In this way the acetylation also represents an obstacle of enzymatic saccharification of plant hemicelluloses to fermentable sugars which appears to be a hot topic of current research. We can eliminate this obstacle by alkaline extraction or pretreatment leading to saponification of ester linkages. However, this task has been accomplished in a different way in the nature. The acetyl groups became targets of microbial carbohydrate esterases that evolved to overcome the complexity of the plant cell walls and that cooperate with glycoside hydrolases in plant polysaccharide degradation. This article concentrates on enzymes deacetylating plant hemicelluloses excluding pectin. They are currently grouped in at least 8 families, specifically in CE families 1–7 and 16, originally assigned as acetylxylan esterases, the enzymes acting on hardwood acetyl glucuronoxylan and its fragments generated by endo-β-1,4-xylanases. There are esterases deacetylating softwood galactoglucomannan, but they have not been classified yet. The enzymes present in CE families 1–7 differ in structure and substrate and positional specificity. There are families behaving as endo-type and exo-type deacetylates, i.e. esterases deacetylating internal sugar residues of partially acetylated polysaccharides and also esterases deacetylating non-reducing end sugar residues in oligosaccharides. With one exception, the enzymes of all mentioned CE families belong to serine type esterases. CE family 4 harbors enzymes that are metal-dependent aspartic esterases. Three-dimensional structures have been solved for members of the first seven CE families, however, there is still insufficient knowledge about their substrate specificity and real physiological role. Current knowledge on catalytic properties of the selected families of CEs is summarized in this review. Some of the families are emerging also as new biocatalysts for regioselective acylation and deacylation of carbohydrates.
Assay for peptidoglycan O-acetyltransferase: A potential new antibacterial target.
Moynihan, P. J. & Clarke, A. J. (2013). Analytical Biochemistry, 439(2), 73-79.
The O-acetylation of peptidoglycan occurs at the C-6 hydroxyl group of muramoyl residues in many human pathogens, both gram positive and gram negative, such as Staphylococcus aureus and species of Campylobacter, Helicobacter, Neisseria, and Bacillus, including Bacillus anthracis. The process is a maturation event being catalyzed either by integral membrane O-acetylpeptidoglycan transferase (Oat) of gram-positive bacteria or by a two-component peptidoglycan O-acetyltransferase system (PatA/PatB) in gram-negative cells. Here, we describe the development of the first in vitro assay for any peptidoglycan O-acetyltransferase using PatB from Neisseria gonorrhoeae as the model enzyme. This assay is based on the use of chromogenic p-nitrophenyl acetate as the donor substrate and chitooligosaccharides as model acceptor substrates in place of peptidoglycan. The identity of the O-acetylated chitooligosaccharides was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rates of transacetylations were determined spectrophotometrically by monitoring p-nitrophenol release after accounting for both spontaneous and enzyme-catalyzed hydrolysis of the acetate donor. Conditions were established for use of the assay in microtiter plate format, and its applicability was demonstrated by determining the first Michaelis–Menten kinetic parameters for PatB. The assay is readily amenable for application in the high-throughput screening for potential inhibitors of peptidoglycan O-acetyltransferases that may prove to be leads for novel classes of antibiotics.
Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a heteroxylan containing mucilaginous tissue.
Jensen, J. K., Johnson, N. & Wilkerson, C. G. (2013). Frontiers in Plant Science, 4(183).
The exact biochemical steps of xylan backbone synthesis remain elusive. In Arabidopsis, three non-redundant genes from two glycosyltransferase (GT) families, IRX9 and IRX14 from GT43 and IRX10 from GT47, are candidates for forming the xylan backbone. In other plants, evidence exists that different tissues express these three genes at widely different levels, which suggests that diversity in the makeup of the xylan synthase complex exists. Recently we have profiled the transcripts present in the developing mucilaginous tissue of psyllium (Plantago ovata Forsk). This tissue was found to have high expression levels of an IRX10 homolog, but very low levels of the two GT43 family members. This contrasts with recent wheat endosperm tissue profiling that found a relatively high abundance of the GT43 family members. We have performed an in-depth analysis of all GTs genes expressed in four developmental stages of the psyllium mucilagenous layer and in a single stage of the psyllium stem using RNA-Seq. This analysis revealed several IRX10 homologs, an expansion in GT61 (homologs of At3g18170/At3g18180), and several GTs from other GT families that are highly abundant and specifically expressed in the mucilaginous tissue. Our current hypothesis is that the four IRX10 genes present in the mucilagenous tissues have evolved to function without the GT43 genes. These four genes represent some of the most divergent IRX10 genes identified to date. Conversely, those present in the psyllium stem are very similar to those in other eudicots. This suggests these genes are under selective pressure, likely due to the synthesis of the various xylan structures present in mucilage that has a different biochemical role than that present in secondary walls. The numerous GT61 family members also show a wide sequence diversity and may be responsible for the larger number of side chain structures present in the psyllium mucilage.
Structural changes and cellular localization of resuscitation-promoting factor in environmental isolates of Micrococcus luteus.
Koltunov, V., Greenblatt, C. L., Goncharenko, A. V., Demina, G. R., Klein, B. Y., Young, M. & Kaprelyants, A. S. (2010). Microbial Ecology, 59(2), 296-310.
Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in “spent culture medium.” Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.