Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.     

Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.     

Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.Glutamine is one of the most important compounds in nitrogen metabolism; it is not only a constituent of proteins but is also a donor of the amino (amido) moiety in the biosynthesis of other amino acids, purines, pyrimidines, pyridine coenzymes, and complex carbohydrates. Glutamine is also used in the treatment of gastric ulcers and has been produced commercially by direct fermentation with certain bacteria (6–10).In recent years, enzymatic synthesis has come to rival direct fermentation as a means of producing amino acids. In the case of glutamine, however, the need for a stoichiometric supply of ATP for the endoergonic reaction of glutamine synthetase (GS) precludes the development of an economically valuable method, unless ATP can be regenerated and recycled.Processes for the production of various substances using dried yeast cells as an enzyme source were established by Tochikura and colleagues (2, 4, 16, 18–20). The processes are driven by the chemical energy of ATP released by the alcoholic fermentation by the yeast, which has been wasted in alcoholic brewing (17). Tochikura and colleagues also designed a process in which the yeast fermentation of sugar is combined with an endoergonic reaction catalyzed by an enzyme from a different microorganism (3). The results suggest that the process offers the possibility of producing many compounds at a high yield by using various biosynthetic reactions and high concentrations of substrates. Tochikura et al. introduced the general idea of coupled fermentation with energy transfer for the process; its principle is indicated in Fig. ​Fig.1,1, with glutamine production as an example. Open in a separate windowFIG. 1Scheme of glutamine production by the coupled fermentation with energy transfer method. ∗1, glycolytic pathway is abridged. ∗2, inorganic phosphate (P_i) is recycled.In the process of coupled fermentation with energy transfer, a catalytic amount of ATP is regenerated with the energy of sugar fermented by yeast, in the form of baker’s yeast (4, 16, 18, 19, 23). The energy-utilizing system for the synthesis can involve the enzyme(s) of yeast itself or those of other organisms. It should be noted that, from another point of view, the use of the energy-utilizing system results in ADP regeneration to complete the fermentation of glucose, and that, if there is no ADP regeneration, the yeast fermentation of sugar can proceed only as follows, in the presence of inorganic phosphate (the Harden-Young effect of inorganic phosphate [1]), 2 · glucose + 2 · inorganic phosphate → fructose 1,6-bisphosphate (FBP) + 2 · C₂H₅OH + 2 · CO₂ (Harden-Young equation), where ADP regeneration for the fermentation of 1 mol of glucose is carried out by the phosphorylation of another mole of glucose to FBP.We previously reported glutamine production, obtained by employing a combination of baker’s yeast cells and GS from Gluconobacter suboxydans, as the first application of the coupled fermentation with energy transfer method for the production of a nonphosphorylated compound (12, 13). In addition, we achieved high-yield glutamine production by using the Corynebacterium glutamicum (Micrococcus glutamicus) enzyme and larger amounts of the substrates (15). The maximum amounts of glutamine formed (23 to 25 g/liter) and the yield based on glutamate (50 to 100%) were to some extent satisfactory, but the yield based on the energy source (glucose) for ATP regeneration was not satisfactory (about 40% of the theoretical value; 2 mol of glutamine can be formed when 1 mol of glucose is consumed).In the present study, we examined the characteristics of glutamine production regarding product yield based on the energy source for ATP regeneration and regarding the reactivity of GS during glutamine production, which is closely related to the product yield. The results of preliminary attempts to improve glutamine production are also described. In these experiments, a yeast mutant which has a low assimilating ability for glycerol and/or ethanol was used.     

Gene transfection of multicellular spheroid of hepatocytes on an artificial substrate

The handling of hepatocytes, a major cell population in the liver, is an important technique in both liver tissue engineering and hepatology. However, these cells are so fragile that it has been impossible to harvest hepatocytes with high viability from tissue culture dishes after a period of culture in vitro. In this study, we employed an artificial substrate for transfection of multilayer hepatocytes and harvested these cells with high viability after transfection. Hepatocytes cultured on an amphiphilic artificial substrate form multilayer aggregates (spheroids) in the presence of growth factors during gene transfection with cation liposomes. Compared to cells cultured on a collagen-coated plate, these spheroids are easily harvested with high viability by pipetting in EDTA solution. In addition, these spheroids rapidly spread on collagen after transfer from the artificial substrate, demonstrating that hepatocytes in the center of the spheroids were viable. Epidermal growth factor (EGF) increased the transfection efficiency into hepatocytes while hepatocyte growth factor (HGF) alone did not increase the efficiency. However, HGF synergestically increased the effect of EGF on transfection. Interestingly, this transfection required the process of spheroid formation because the gene was not transfected once the spheroid formation completed or under conditions where hepatocytes did not form spheroids. This method using spheroidal hepatocytes for in vitro transfection is promising for the development of ex vivo gene therapy.     

Distribution of the Argentine ant, Linepithema humile, along the Seto Inland Sea, western Japan: Result of surveys in 2003–2005

The distribution of the Argentine ant, Linepithema humile, was investigated in 65 cities or towns along the Seto Inland Sea, western Japan in 2003–2005. Our results include all available information of their distribution in Japan until 2005. Argentine ants have invaded Aichi Prefecture (Tahara‐shi), Hyogo Prefecture (Kobe‐shi), Hiroshima Prefecture (Hiroshima‐shi, Fuchu‐cho, Hatsukaichi‐shi, Ono‐cho and Otake‐shi), and Yamaguchi Prefecture (Iwakuni‐shi and Yanai‐shi). The most widespread distribution was found around Hatsukaichi‐shi including the westernmost part of Hiroshima‐shi and the easternmost of Ono‐cho.     

Efficacy and Safety of Sitagliptin Added to Insulin in Japanese Patients with Type 2 Diabetes: The EDIT Randomized Trial

Aims

To clarify the efficacy and safety of adding sitagliptin to insulin therapy in Japanese patients with suboptimally controlled type 2 diabetes (T2DM).

Study Design and Methods

This was a 24-week, prospective, randomized, open-labeled, controlled trial. Patients with T2DM who were suboptimally controlled despite receiving at least twice daily injection of insulin were enrolled in the study. The patients were randomized to continuation of insulin treatment (Insulin group) or addition of sitagliptin 50 to 100 mg daily to insulin treatment (Ins+Sita group). The primary outcome was change in HbA1c at week 24.

Results

Adding sitagliptin to insulin significantly reduced HbA1c from 7.9 ± 1.0% at baseline to 7.0 ± 0.8% at week 24 (P <0.0001), while there was no significant change in HbA1c in the Insulin group (7.8 ± 0.7% vs. 7.8 ± 1.1%, P = 0.32). The difference in HbA1c reduction between the groups was 0.9% (95% confidence interval, 0.4 to 1.5, P = 0.01). There was no significant weight gain in either group. Incidence of hypoglycemia was significantly reduced in the Ins+Sita group compared with the Insulin group. Treatment satisfaction was improved in the Ins+Sita group. Baseline HbA1c level and beta cell function were associated with the magnitude of reduction in HbA1c in the Ins+Sita group.

Conclusion

Adding sitagliptin to insulin reduced HbA1c without weight gain or increase in hypoglycemia, and improved treatment satisfaction in Japanese patients with T2DM who were suboptimally controlled despite at least twice daily injection of insulin.

Trial Registration

The University Hospital Medical Information Network (UMIN) Clinical Trials Registry UMIN000004678     

Characterization of Endoplasmic Reticulum-Localized UDP-d-Galactose: Hydroxyproline O-Galactosyltransferase Using Synthetic Peptide Substrates in Arabidopsis

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses. HGT required a divalent cation of manganese for maximal activity and consumed UDP-d-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35°C. The favorable substrates for the activity seemed to be peptides containing two alternating imino acid residues including at least one acceptor Hyp residue, although a peptide with single Hyp residue without any other imino acids also functioned as a substrate. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec61 and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum. To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway.O-glycosylation is the addition of a sugar to hydroxy amino acids such as Thr, Ser, Hyp, Hyl, or Tyr (Lehle et al., 2006). This type of protein modification occurs in many organisms to modify a large variety of proteins. Several types of sugars can be linked to proteins via O-glycosylation, including Man, N-acetylgalactosamine, Glc, Xyl, N-acetylglucosamine, Fuc, Gal, and arabinofuranose (Araf). In addition, elongation of the added sugar residues yields a large variety of oligo- and polysaccharide extensions on the substrate proteins. These modifications are known to play important roles in various phenomena, including pathways required to maintain biological systems and basic cellular functions.Structural analysis of oligo- and polysaccharides in plant cell walls has revealed the presence of three types of O-linked structures, Gal-O-Hyp, Araf-O-Hyp, and Gal-O-Ser (Kieliszewski and Shpak, 2001; Seifert and Roberts, 2007). A part of these three structures has been found on proteins in the super family that includes arabinogalactan protein (AGP) and extensin, which are localized to the cell surface. AGPs contain O-linked arabinogalactan oligo- or polysaccharides attached to Hyp residues (Gal-O-Hyp). It is known that arabinogalactan polysaccharides mainly consist of β-1,3 linkages of Gal polymers (Seifert and Roberts, 2007). Extensin contains short arabino-oligosaccharide chains attached to Hyp residues (Araf-O-Hyp) and single Gal residues linked to Ser residues (Gal-O-Ser). It has been suggested that these O-linked structures play an important role in many stages of growth and development in plants, including signaling, embryogenesis, and programmed cell death (Knox, 2006; Seifert and Roberts, 2007). However, our understanding of the biosynthesis of these O-linked structures is limited at present.Shpak et al. described a novel strategy to elucidate O-glycosylation of AGPs via introduction of synthetic genes encoding a protein substrate of glycosyltransferases into plant cells (Shpak et al., 1999; Estevez et al., 2006). This strategy provided good evidence for the substrate specificities of Hyp O-galactosyltransferase (HGT). Hyp galactosylation occurs on clustered noncontiguous Hyp residues such as Xaa-Hyp-Xaa-Hyp repeats of AGPs (where Xaa is any amino acid except Hyp; Tan et al., 2003). However, the arabinogalactosylation site is not limited to clustered noncontiguous Hyp residues, as isolated Hyp residues with appropriate surrounding sequences can be modified with arabinogalactan (Matsuoka et al., 1995; Shimizu et al., 2005). Therefore, the mechanism of glycosylation to Hyp residues seems complex in plants, while we have little information about the glycosyltransferase(s) involved in arabinogalactan biosynthesis. To examine the enzymatic properties and to identify genes involved in arabinogalactan biosynthesis, we first attempted to establish an in vitro assay for HGT activity, which catalyzes the initial step in arabinogalactan biosynthesis in plants.Here, we report a novel assay for HGT activity based on the use of endoplasmic reticulum (ER)-enriched cell lysates extracted from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of the enzyme and chemically synthesized fluorescent peptides as enzyme substrates. The method enabled us to characterize the enzymatic properties of HGT and to determine the localization of HGT in Arabidopsis cells. Properties of the enzyme and the usefulness of our assay for various studies are discussed.     

Efficient extraction of thioreodoxin from Saccharomyces cerevisiae by ethanol

Thioredoxin, an antioxidant protein, is a promising molecule for development of functional foods because it protects the gastric mucosa and reduces the allergenicity of allergens. To establish a method for obtaining an ample amount of yeast thioredoxin, we found here that thioredoxin is released from Saccharomyces cerevisiae by treatment with 20% ethanol. We also found that Japanese sake contains a considerable amount of thioredoxin.