Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues

We replaced the single N‐terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non‐canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non‐canonical amino acid incorporation protocol involves a two‐step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of ～12% could be elevated to 40% by increasing the non‐canonical amino acid concentration in the medium by 10‐fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture. Copyright © 2008 John Wiley & Sons, Ltd.     

Effect of potassium,magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensisL)

Free amino acids in tea leaves are important chemical constituents that considerably influence the quality of tea, especially that of green tea. The present study was conducted to investigate the effects of the nutrients potassium, magnesium and sulphur on biomass production and amino acid accumulation in tea leaves in pot and field experiments. The results show that biomass production and the free amino acid contents in tea leaves increased considerably following the application of potassium and magnesium. Combined application of both nutrients produced the highest increment in free amino acids. The increased free amino acid content in tea leaves was accompanied by enhanced nitrate reductase activity. This indicates that potassium and magnesium application improved the nitrogen metabolism, leading to an increased synthesis of amino acids. A comparison between K₂CO₃ vs K₂SO₄ and MgO vs MgSO₄ revealed significantly higher amino acid contents in tea leaves when both potassium and magnesium were applied in the sulphate form, indicating that not only potassium and magnesium but also sulphur plays an important role in the accumulation of amino acids in tea leaves. A pot experiment using KCl and K₂SO₄ was carried out in order to compare the two commercially available potassium fertiliser forms in their effect on biomass production and the content of free amino acids in tea leaves. The results showed no difference on biomass production between the two fertiliser forms. However, whereas KCl tentatively depressed nitrate reductase activity and hence the accumulation of free amino acids, K₂SO₄ significantly increased the contents of these quality promoting constituents of tea leaves. The role of fertiliser composition, detrimental effects of accompanying anions, eg Cl⁻ and differences in nutrient availability (eg oxide≪sulphate form) and possible impact on soil chemistry as main reasons for the observations, being crucial aspects with regard to fertilisation of tea for good quality are discussed. © 1998 SCI.     

Glucosinolate content and amino acid composition of rapeseed (Brassica napus) meal as affected by sulphur and nitrogen nutrition

Rapeseed plants have been grown in soil-free culture with varying amounts of nitrogen fertiliser applied as nitrate, and with 50% of the nitrate-N exchanged for ammonium. The experiment was performed at two levels of sulphate fertiliser. Yield, protein content, glucosinolate content and amino acid composition were studied. The glucosinolate content was lower and the protein content was higher at a high level of nitrogen fertiliser. Exchange of 50% of the nitrate-N for ammonium caused no significant change in glucosinolate or protein content. The amount of glucosinolate was higher at the high sulphate level. There were no sulphur-nitrogen interactions. Content of total aspartic acid increased with nitrogen fertilisation, while total content of other amino acids did not change significantly with fertilisation. The effect of a wide variety of applications of sulphate fertiliser on glucosinolate and total amino acid content have been studied in a separate experiment. Although both protein content and methionine content were reduced at a low sulphate level, glucosinolate content was reduced considerably more. Experiments in the field revealed that although a reduction in glucosinolate content of rapeseed may be obtained from using fertilisers low in sulphur on sandy soils this does not seem possible on heavy soils.     

Oxidation of methionine. Effects of hydrogen peroxide alone and in combination with iodide and selenite

The oxidation of methionine by hydrogen peroxide, and the influence of iodide, pH, amino acids and selenite were studied with free methionine and with casein and fish fillet protein. The concentration levels tested ranged from 0·05 mm to 3·0 mm. Hydrogen peroxide oxidation was not influenced by pH in the range 5·0 to 8·0; at pH 8·5 the rate of oxidation was increased. When iodide was added in amounts equivalent to or less than the amounts of H₂O₂, the reaction was accelerated with free but not protein-bound methionine. At higher levels iodide inhibited the oxidation. An amino acid mixture and proteins inhibited the effect of iodide; this effect seemed to be due to tryptophan. Selenite also accelerated the effect of H₂O₂, both with free and with protein-bound methionine. Cu⁺⁺ catalysed the oxidation by H₂O₂ at low reactant concentration but not at the higher levels. The reaction between methionine and H₂O₂ seemed to be of first order with respect to both reactants.     

Ultradian metabolic oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide, a population synchronizer, is produced by sulphite reductase

We have reported that the consecutive cyclic production of H(2)S resulted in population synchrony of ultradian metabolic oscillation (Sohn et al., 2000). In order to understand the origin of H(2)S and its nature of periodic production, changes of sulphur compounds concentration and responsible enzymes were investigated. The concentrations of extracellular sulphate, intracellular glutathione and cysteine oscillated during metabolic oscillation but only the oscillation of sulphate concentration was out of phase with H(2)S production. The sulphate concentration in culture directly affected the amplitude and the period of metabolic oscillation: (a) the period of metabolic oscillation shortened from 50 min to 30 min when sulphate concentration in the medium was reduced from 46 mM to 2.5 mM; (b) the metabolic oscillation disappeared under sulphate-depletion conditions and arose again by the addition of sulphate. Pulse injection of sulphite (10 microM) perturbed metabolic oscillation with a burst production of H(2)S, while thiosulphate (up to 500 microM) was without apparent effect. Furthermore, addition of S-adenosyl methionine (100 microM) or azoxybacilin (3 mg/kg) decreased H(2)S production with perturbation of metabolic oscillation. The results presented here suggest that H(2)S, a population synchronizer, is produced by sulphite reductase in the sulphate assimilation pathway, and dynamic regulation of sulphate uptake plays an important role in ultradian metabolic oscillation.     

Inhibition of L-alanine uptake into bovine jejunal brush border membrane vesicles by L-amino acids

Jejunal epithelial cells from slaughtered Holstein cows were fractionated to obtain purified brush border membranes from which membrane vesicles were prepared for use in amino acid uptake studies. Uptake of alanine was determined by incubation of vesicles with a solution containing radiolabelled alanine, isolation of vesicles and accumulated alanine by filtration, and detection of accumulated alanine by liquid scintillation counting. Uptake studies were conducted under conditions shown to provide linear rates of accumulation. Sodium-dependent active transport was determined as the difference between uptake measured in the presence and absence of sodium in the extravesicular buffer. Inhibition of alanine uptake increased with increasing extra-vesicular inhibitor concentration until a plateau value was reached. Inhibition of sodium-dependent alanine uptake by 100 mM glycine was 72%; 25 mM isoleucine, valine, or methionine completely inhibited initial alanine uptake. These results indicate the existence of at least two sodium-dependent transport systems, one capable and one incapable of accepting glycine for transport. At concentrations designed to represent expected concentrations of free amino acids in intestinal digesta, several equimolar mixtures (.2 to 5 mM) of 20 amino acids inhibited alanine uptake, suggesting that significant interaction among amino acids for uptake may be occurring under in vivo conditions.     

Effect of insulin in conjunction with glucose, amino acids and potassium on net metabolism of glucose and amino acids in the goat mammary gland.

The hyperinsulinaemic euglycaemic insulin clamp technique was used to study the effect of insulin on the arterio-venous concentration differences of glucose and amino acids across the mammary gland in dairy goats. Insulin was given in conjunction with K to prevent insulin hypokalaemia. Appropriate amino acid infusion was used to blunt insulin-induced hypoaminoacidaemia or to create hyperaminoacidaemia and maintain this state under insulin treatment. Hyperaminoacidaemia alone only stimulated mammary leucine uptake but did not significantly modify the net metabolism of other amino acids and glucose. Insulin infusion at physiological level in conjunction with glucose, KCl-NaCl and amino acids failed to alter mammary uptake of glucose and essential amino acids; occasional increase in arginine extraction and decrease in tyrosine extraction were exceptions. Thus these new experimental conditions did not reveal any galactopoietic effect of insulin.     

Milk production,nutrient utilization,and endocrine responses to increased postruminal lysine and methionine supply in dairy cows

The effect of increased postruminal supply of lysine and methionine was investigated in a production trial involving 64 dairy cows in early lactation. Within each of two basal rations, based on either corn silage or grass silage, rations were either naturally deficient in lysine or fortified with 24 g of lysine in a rumen-protected form and naturally deficient in methionine or fortified with 12 g of methionine in a rumen-protected form. The data were analyzed separately for the four lysine and the four methionine treatment groups. Milk production, body weight gain, and plasma concentrations of insulin-like growth factor-I, bovine somatotropin, insulin, glucose, nonesterified fatty acids, and urea were monitored over a 12-wk period. Supplementation with protected methionine led to increases in milk fat and protein contents of 2.4 and 1.8 g/kg of milk, respectively. Supplementation with protected lysine or methionine numerically increased protein yield comparable to values reported in the literature, but the treatment effects were not statistically significant. Efficiency of utilization of absorbed amino acids for milk protein synthesis and efficiency of utilization of metabolizable energy for milk production were not significantly altered in response to increased postruminal lysine and methionine flow, but a numerically increased efficiency of utilization of total amino acids was observed. No significant effect of lysine or methionine supplementation was observed on endocrine parameters nor on plasma metabolite concentrations. However, across treatment groups, high milk yield was correlated with low plasma insulin-like growth factor-I concentrations (r = -0.44) and partially with low plasma nonesterified fatty acids concentration and insulin levels (r = -0.26), while body weight gain was negatively correlated (r = -0.33) with elevated plasma bovine somatotropin concentrations.     

Nutritive value of potato crude protein as influenced by manuring and amino acid composition.

Potatoes were grown in pots and fertilised with varying amounts of nitrogen (as nitrate or ammonium sulphate or urea), phosphorus, potassium and cow manure. The largest yield, 451 g DM per pot, was obtained with nitrogen in the form of urea. N-applications and P- and K-deficiency in the soil increased the total-N content, which varied from 1.14–3.07%. Concentrations of nitrate-N (0.001–0.016%) were negligible. Increasing N-concentrations were associated with decreases in the crude protein of most amino acids including lysine (6.26–4.17%), threonine (4.08–2.90%), methionine (1.98–1.46%) and tryptophan (1.74–0.86%). The concentrations of aspartic and glutamic acids (probably mainly present as their amides) increased. Differences in amino acid composition between boiled and unboiled potatoes were negligible. From 70 to 90% of the decrease in concentration of most essential amino acids could be accounted for by changes in N-content of dry matter. Phosphorus and potassium affected the amino acid composition indirectly through their effects on N-content. In rat feeding experiments increasing N-concentrations in potatoes increased the true digestibility of the crude protein from 79 to 91 but decreased the biological value from 82 to 59. The net protein utilisation did also decrease (from 67 to 54). These relationships were linear below 2.2% N in dry matter. Changes were mainly dependent on variations in N-content. Decreases in essential amino acid indices or chemical scores were closely reflected in the results of the feeding experiments. The true amino acid digestibilities were different for individual amino acids and increased with increasing N-concentration in dry matter.     

Digestive utilisation of protein and amino acids from raw and heated lentils by growing rats

The effect of autoclave treatment on the digestive utilisation of protein and amino acids from lentils was studied in growing rats. Twenty 3‐week‐old Wistar rats (mean live weight 59 ± 4.8 g) were fed two experimental diets (n = 10 rats per diet) consisting of raw lentils (Lens culinaris M, var vulgaris, cv magda‐20) (diet RL) or lentils autoclaved at 120 °C and 1 atm for 30 min (diet AL). An additional group of 10 animals was fed a low‐protein (4%) diet and used to estimate metabolic nitrogen and amino acid excretion. Autoclaving caused a 76% reduction in the levels of trypsin inhibitor activity, but failed to improve the faecal digestive utilisation of protein or total amino acids. Lowest essential amino acid true digestibility in diets RL and AL was found for cysteine (60.2 vs 60.1%) and methionine (68.9 vs 66.6%). The protein digestibility‐corrected amino acid score was 71.2 and 66.4% and the availability of sulphur amino acid‐corrected amino acid score was 57.1 and 52.1% for diets RL and AL respectively. Sulphur amino acids were the first limiting ones. Overall, autoclave treatment did not improve protein or total amino acid digestibility from lentils, but caused a significant improvement in leucine and lysine digestibility and a significant decrease in the digestive utilisation of tyrosine and methionine. © 2002 Society of Chemical Industry     

In situ activity of chymotrypsin in sugar‐salted herring during cold storage

In situ activity of intestinal chymotrypsin in sugar‐salted whole herring during cold storage was evaluated by analysing changes in the low‐molecular‐weight nitrogen fraction when a specific inhibitor was added. Addition of chymostatin (0.01 mM ) to sugar‐salted herring gave 100% inhibition of chymotrypsin activity compared to sugar‐salted herring without chymostatin. Inhibition of chymotrypsin did not affect the profile of low‐molecular‐weight peptides analysed by capillary zone electrophoresis, but resulted in 22% lower content after 26 weeks of storage. Inhibition of chymotrypsin resulted in 17% lower content of free amino acids. The relative amount of single free amino acids was unaffected by the presence of inhibitor, except for valine, leucine, isoleucine, methionine, phenylalanine and tyrosine, which were formed in lower relative amount. Removal of intestines (gutted herring) and thereby all intestinal proteolytic activity did not change the free amino acid profile, except for the concentration of histidine which decreased more when intestines were present. © 2000 Society of Chemical Industry     

QUANTITATIVE GAS CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS IN BEER AND WORT

Amino acids present in beer and wort samples were isolated using Dowex 50W-X8 ion exchange resin. These acids were converted to N-trifluoroacetyl methyl esters and were separated on a 10 ft. x 1/4 in. copper column packed with 3/4% Carbowax 1540 and 1/4% NPGS on Chromosorb W. Complete separation and quantitative estimation was obtained for eleven amino acids: valine, alanine, isoleucine, leucine, glycine, proline, aspartic acid, threonine, methionine, glutamic acid, and phenylalanine. Standards of the acids showed a relative standard deviation of 4% and less for nine acids while threonine and methionine gave deviations of 9% and 15% respectively. The other naturally-occurring amino acids did not interfere with the separation of the above eleven. Amino acid assimilation data is presented along with other beer and wort analyses.     

Whole blood and plasma amino acid uptakes by lactating bovine mammary glands

The objective of the experiment was to determine the contribution of red blood cells to transport of individual amino acids to lactating bovine mammary glands. Blood samples were collected from coccygeal and subcutaneous abdominal veins of 21 lactating Holstein cows on d 35, 70, 105, and 126 of lactation. Samples were collected every 20 min for 12 h. Subsamples of whole blood and plasma were pooled by hour and day. Hourly plasma samples and daily whole blood and plasma samples were analyzed for amino acid concentration. Plasma glutamate concentration was stable throughout the 12-h collection period, indicating that sample collection did not perturb amino acid homeostasis. Therefore, data from pooled daily samples were used for subsequent comparisons. Whole blood arteriovenous differences of phosphoserine, aspartate, glutamate, hydroxyproline, phosphoethanolamine, serine, asparagine, glycine, glutamine, taurine, histidine, citrulline, threonine, alanine, beta-aminoisobutyrate, carnosine, arginine, proline, alpha-aminobutyrate, tyrosine, valine, methionine, cystine, isoleucine, leucine, phenylalanine, tryptophan, ornithine, and lysine differed significantly from plasma arteriovenous differences. Uptakes of individual amino acids from plasma were poorly correlated with uptake from whole blood. These data clearly indicate that uptake data derived from plasma do not adequately represent whole blood amino acid uptake.     

Effects of nitrogen,phosphorus and potassium on amino acid composition and on relationships between nitrogen and amino acids in wheat and oat grain

During two seasons, pot experiments in which increasing applications of nitrogen, phosphorus and potassium were given to wheat and oats produced grain with widely varying N contents. The total amino acid composition of the grain was mainly dependent on N content of grain. Phosphorus and potassium affected the amino acid composition of wheat and oat grain only indirectly through their effects on nitrogen concentration. In wheat increasing grain N% was accompanied by a decrease in the amount (as g per 16 g N) of lysine, threonine, methionine, cystine and an increase in glutamic acid, proline, phenylalanine, and serine contents. Lysine, methionine, and cystine in oats decreased with increasing grain N content, but arginine, aspartic acid, glutamic acid and phenylalanine increased. Changes were less pronounced with oats than with wheat, and concentrations of most essential amino acids were higher in oats. When expressed as % dry matter all amino acids increased with increasing N concentration. Linear regression equations were calculated and significant correlations were found in both crops between concentrations of most amino acids and N content in grain. Some correlations between amino acids in the grain are also given. Correlations between amino acids and nitrogen within a variety were similar despite widely differing yields.     

PHENOLIC ACIDS IN BEERS AND WORTS

The total contents of phenolic acids measured by high-performance liquid-chromatography were 5–8 mg/litre in beers brewed in Ireland whereas 16–40 mg/litre were present in four other beers. In all beers the predominant phenolic acids were vanillic, p-coumaric and ferulic acids. Free phenolic acids were extracted from Emma barley grains and malt in very small amounts (15–28 mg/kg) but larger quantities (191 mg/kg) were released on mashing the malt. Little change occurred in the contents of phenolic acids on processing a lager wort through to the finished beer. Treatment with excess Polyclar AT removed astringent flavour and phenolic acids from an experimental ale but this flavour loss could not be accounted for by the adsorption of phenolic acids. The flavour threshold for a nine-component phenolic acid mixture in lager was between 50 mg/litre and 100 mg/litre.     

Effect of glutamine supplementation on splanchnic metabolism in lactating dairy cows

The suggestion that glutamine (Gln) might become conditionally essential postpartum in dairy cows has been examined through increased postruminal supply of Gln. Net nutrient flux through the splanchnic tissues and mammary gland was measured in 7 multiparous Holstein cows receiving abomasal infusions of water or 300 g/d of Gln for 21 d in a crossover design. Milk yield increased significantly (by 3%) in response to Gln supplementation, but the 2.4% increase in milk protein yield was not statistically significant. Glutamine treatment had no effect on portal or hepatic venous blood flows. Net portal appearance of Gln and Glu was increased by Gln supplementation, accounting for 83% of the infused dose with, therefore, only limited amounts available to provide additional energy to fuel metabolism of the portal-drained viscera. The extra net portal appearance of Gln was offset, however, by a corresponding increase in hepatic removal such that net Gln splanchnic release was not different between treatments. Nonetheless, the Gln treatment resulted in a 43% increase in plasma Gln concentration. Infusions of Gln did not affect splanchnic flux of other nonessential amino acids or of essential amino acids. Glutamine supplementation increased plasma urea-N concentration and tended to increase net hepatic urea flux, with a numerical increase in liver hepatic O₂ consumption. There were no effects on glucose in terms of plasma concentration, net portal appearance, net liver release, or postliver supply, suggesting that Gln supplementation had no sparing effect on glucose metabolism. Furthermore, mammary uptake of glucose and amino acids, including Gln, was not affected by Gln supplementation. In conclusion, this study did not support the hypothesis that supplemental Gln would reduce glucose utilization across the gut or increase liver gluconeogenesis or mammary glutamine uptake to increase milk protein synthesis.     

NUTRITIVE AND ORGANOLEPTIC CHARACTERISTICS OF NONDAIRY IMITATION MILKS

SUMMARY –13 nondairy imitation milk powders, concentrates and bottled beverages were analyzed for general composition, nutrients and organoleptic properties. These products were obtained from 11 domestic sources, which decreased to eight by the end of the study. Nondairy imitation milks were characterized by wide fluctuations in riboflavin (1.9–418.0 μ per 100 ml) and thiamine (0.2–54.7 μ per 100 ml), protein (0.76–3.67%) and amino acids like lysine (4.7–13.6 μmole per ml). Calcium and magnesium in all imitation milks were well below levels of the same elements in cow's milk reconstituted from whole milk powder, whereas sodium generally was much higher, almost threefold in several instances. The appearance of most nondairy imitation milks closely resembled fresh, pasteurized cow's milk, but the flavor, ascertained by three milk judges, was not similar nor as acceptable to them. Several imitation milks scored in flavor as well as or slightly better than cow's milk reconstituted from whole milk powder, but in all these instances, including the cow's milk, the flavor quality was only fair. Most imitation milks contained less protein than reconstituted cow's milk, but two contained concentrations of 3.26 and 3.67%, comparable to cow's milk. A number of nondairy imitation milks showed very low levels of calcium, riboflavin, thiamine and essential amino acids, lysine and methionine, in relation to cow's milk.     

Study of the White Film Developed on the Cut Surface of Vacuum-packed Dry-cured Ham Slices

Scanning electron microscopy of the white film developed on the cut surface of some vacuum-packed dry-cured ham slices showed a fibrous structure composed of protein materials. Gradient SDS-PAGE revealed the main components in the film were proteins with molecular weight in the range 26000–87000, but especially 37000, 41000, 56000, and 65000. Free amino acids were also in the film in low proportions (250 μg total free amino acids per mg of protein). The main amino acids were proline methionine, isoleucine, leucine, phenylalanine, histidine and tyrosine. 11.7% of the total free amino acids was tyrosine, thus indicating its minor role in formation of white film.     

Effect of sulphur deficiency on the growth and metabolism of sugar beet (Beta vulgaris cv Druid)

Sugar beet plants were grown in variations of a nutrient culture system to induce sulphur deficiency. The effect of sulphur deprivation on the growth and metabolism was investigated by measuring leaf area, chlorophyll content, fresh and dry weights, sulphur, nitrogen, sulphate and nitrate concentrations, glutathione and free amino acid concentrations. Both total sulphur and sulphate concentrations were markedly reduced in response to sulphur deficiency, while significant increases in arginine concentration in shoot tissue were observed. Increases were also observed in shoot nitrogen and nitrate concentrations and both shoot and root N/S ratios. These results demonstrate that total sulphur, sulphate S and sulphate as a percentage of total S are suitable indicators of sulphur deficiency in sugar beet. Arginine responds to sulphur deficiency, but its use as an indicator needs validation under field conditions. © 2000 Society of Chemical Industry     

Hydrogen sulphide production during cider fermentation is moderated by pre‐fermentation methionine addition

Yeast assimilable nitrogen (YAN) concentration and composition impact hydrogen sulphide (H₂S) production and fermentation kinetics during wine fermentation, but this phenomenon has not been extensively studied in cider fermentation. Our hypothesis was that H₂S production during cider fermentation could be decreased through pre‐fermentation modification of concentrations of individual amino acids. Apple juice (53 mg L⁻¹ YAN) was supplemented with asparagine, arginine, methionine or ammonium and fermented with EC1118 and UCD522 yeast strains. No difference in H₂S production among fermentations was observed with addition of asparagine, arginine or ammonium. Methionine addition of 5 mg L⁻¹ decreased H₂S production by yeast strain EC1118 at 53 mg L⁻¹ YAN. With 153 mg L⁻¹ initial YAN, only methionine addition of 50 mg L⁻¹ decreased H₂S production, and no tested methionine rates decreased H₂S production with 253 mg L⁻¹ initial YAN. Supplementation to 153 mg L⁻¹ YAN resulted in increased H₂S production at all methionine concentrations tested. Sensory differences in aroma were detected in samples supplemented with ammonium and methionine, and these differences were correlated with observed differences in H₂S production. Our results indicate that supplementing cider fermentations with methionine leads to lower H₂S formation, especially in apple juice containing low YAN. © 2017 The Authors Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling