In this study, we developed a method to synthesize sophorose using three enzymes-sucrose phosphorylase from Leuconostoc mesenteroides, 1,2-β-oligoglucan phosphorylase from Enterococcus italicus, and exo β-1,2-glucooligosaccharide sophorohydrolase from Parabacteroides distasonis-in a one-pot reaction, employing inexpensive starting materials. After optimization, a reaction was carried out using 5 mM glucose, 250 mM sucrose, 10 mM inorganic phosphate, and enzyme concentrations of 5 µg/mL sucrose phosphorylase, 20 µg/mL 1,2-β-oligoglucan phosphorylase, and 50 µg/mL exo β-1,2-glucooligosaccharide sophorohydrolase at 30 °C for 48 h, yielding 108 mM sophorose. Following yeast treatment, sophorose was purified by size-exclusion chromatography with a final yield of 45 % based on the amount of sucrose used as the donor substrate.

The cold-shock expression system in Escherichia coli was developed on a manual induction approach using optical density at 600 nm (OD600) measurements and isopropyl β-D-1-thiogalactopyranoside (IPTG) addition. In this study, we show that cold-shock expression performs equally well using an autoinduction approach wherein OD600 measurements and IPTG addition may be eliminated. We further demonstrate that cold-shock expression with autoinduction can better facilitate high-throughput experiments.

The present work evaluates starch from 29 diverse pea accessions for structural (amylose content and amylopectin chain length distribution), granule size distribution, thermal, pasting, and retrogradation properties. Amylose content and the proportion of amylopectin chains with the degree of polymerization (DP) of 6–10, 11–20, and >20 vary in the range from 21.4% to 57.0%, 17.9% to 25.8%, 64.0% to 66.5%, and 9.4% to 16.7%, respectively. Starches with a higher proportion of small granules (<5 µm) have a greater proportion of amylopectin chains with DP > 10 and exhibit higher transition temperatures/enthalpy and vice versa. Retrogradation in starch pastes relates negatively to the proportion of amylopectin chains with DP < 10. Paste viscosities relate negatively with amylopectin chains with DP > 10, amylose content, and retrogradation while positively with transition temperatures/enthalpy and the potential of granules to swell.

YcjT is a kojibiose phosphorylase found in Escherichia coli. We found that sucrose was a good acceptor of YcjT in reverse phosphorolysis using β-d-glucose 1-phosphate as a donor. The product was identified as β-d-fructofuranosyl α-d-glucopyranosyl-(1→2)-α-d-glucopyranoside. This sugar was also synthesized from sucrose and maltose using YcjT and maltose phosphorylase and promoted the growth of the probiotic Bifidobacterium breve.

Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of β-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl β-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-β-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl β-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-β-d-glucopyranosyl β-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be β-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-β-d-glucopyranosyl β-d-glucuronide structure released from bacterial and plant acidic carbohydrates.

N -acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae , a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The K m values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.

Soluble sugars in feeds are important for ruminant production; however, performing numerous sugar analyses within a short period is a laborious task. Here, we developed a phenol-sulfuric acid (PSA) assay in a microplate format to quantify soluble sugars in ruminant feeds. This method is easy and quick and requires only a small quantity of harmful reagents. We found that assay measurements were not affected by the representative organic acids and sugar alcohol contained in feeds. The treatment of activated charcoal with ethanol extract prior to the PSA assay was effective in removing interfering compounds for a more accurate determination of soluble sugars in certain feeds. Furthermore, the inter-day and intra day repeatability of the present method was acceptable. Hence, we conclude that the method developed in this study is suitable for routine analysis of soluble sugars content in ruminant feeds.

The present study evaluated Amaranthus caudatus (AC) and A. hypochondriacus (AH) starches obtained as coproduct during protein extraction for composition, granule size, amylopectin fine structure, thermal, retrogradation, pasting and dynamic rheological-properties to elucidate structure-function relationships. The starches exhibited unimodal particle size distribution with mean granule size of 1.26–3.12 μm. AC starch with larger granules (mean granule size 3.12 μm) than AH starches (1.26–1.59 μm) gelatinized at lower temperatures (lower DSC transition and pasting temperatures), showed higher paste viscosities and produced more elastic gels (lower tan δ and higher Gʹ). Starch granule size related positively with the proportion of amylopectin chains with DP < 12, paste viscosities and dynamic rheological moduli while negatively with non-starch components, gel tan δ and the proportion of amylopectin chains with DP > 12. Starches with greater proportion of amylopectin chains with DP > 12 showed higher gelatinization temperatures, while shorter chains (DP < 12), lipids and proteins contributed to reduced retrogradation tendencies (lower percent retrogradation).

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg 2+ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.

Sago palm (Metroxylon sagu Rottb.) is distributed in Southeast Asia and Melanesia, and it produces comparatively high yield of starch (more than 200 kg per plant). This species can grow in wetland swamps where other food crops cannot grow economically and adapt to problem soils such as acid or saline with low input. The Society of Sago Palm Studies and the authors have published books entitled “The Sago Palm: The Food and Environmental Challenges of the 21st Century (Kyoto Univ. Press 2015)” and “Sago Palm: Multiple Contributions to Food Security and Sustainable Livelihoods (Springer 2018)”. This paper provides a brief review on (1) characteristics of seed germination and preparation of planting materials utilizing germinated seeds, (2) symbiosis of sago palm and microbes (nitrogen fixing bacteria or arbuscular mycorrhizal fungi), (3) creating new value from pith waste after conventional starch extraction (recovery of starch, ethanol production or preparation of biodegradable foam from the waste) and (4) utilization of leaf or bark from our former publications and the achievements published in our journal ‘SAGO PALM’ or the proceedings of the International Sago Symposium to discuss sustainable production and utilization of sago palm resource in Asia and Pacific.

Sago residue after starch extraction contains a considerable amount of starch. In this study, we aimed to develop a simple and effective method for producing bioethanol from sago residue. Starch in sago residue was efficiently liquefied with thermostable α-amylase at 90 ° C using a mass ratio of sago residue to α-amylase solution of 1:6. The liquefied solution (approximately 100 g/L sugar) was subjected to both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes to produce ethanol. In SHF, glucose prepared with α-amylase and amyloglucosidase was almost consumed by yeast after 36 h of fermentation at 37 ° C, and 34.2 g/L ethanol was produced with a yield of 66.0%. In SSF, 43.2 g/L ethanol was obtained with a yield of 86.4% after 72 h of saccharification and fermentation at 37 ° C using the liquefied solution with added amyloglucosidase and yeast. This has been the highest ethanol concentration ever reported in bioethanol production from sago residue.

Starches from 42 diverse Indian potato cultivars are evaluated for diversity in structural (amylose content and amylopectin chain length distribution), morphological (granules size distribution), thermal, and pasting properties. Amylose content varied between 6.5 and 32.2% while the proportion of short (DP 6–12), medium (DP 13–18), and long (DP 19–30) amylopectin chains varied in the range from 37.2 to 45.4%, 35.6 to 39.1%, and 17.8 to 24.5%, respectively. Starches with higher transition temperature showed lower enthalpy of gelatinization. The proportion of small granules (<10 μm) correlated negatively to short amylopectin chains (DP 6–12), peak viscosity, and breakdown viscosity. Transition and pasting temperature related negatively to the proportion of short and medium chains of amylopectin (DP 6–12 and 13–18, respectively), while positively to that of long chains (DP 19–30). Peak viscosity and breakdown viscosity has a negative relation while the final and setback viscosity have a positive relation with long amylopectin chains.

1,3-β-グルカンホスホリラーゼ(BGP)は1,3-β-グルコシド結合の加リン酸分解を可逆的に触媒する酵素である。BGPは40年以上前に発見された酵素であるが,研究報告がほとんどなく,詳細な性質が明らかにされてこなかった。本研究ではOchromonas danica由来BGP(OdBGP)の特性と一次構造を調べた。本酵素は三糖以上のラミナリオリゴ糖からラミナリンまで,幅広い分子量の糖質の加リン酸分解とその逆反応(合成反応)を触媒した。反応平衡は合成方向に傾いていることがわかった。α-グルコース1-リン酸とラミナリビオースを基質としてOdBGPの反応を行ったところ,直鎖1,3-β-グルカンが合成された。また,OdBGPとスクロースホスホリラーゼを併用した反応では,スクロースとグルコースを出発材料として1,3-β-グルカンをワンポット合成することができた。酵素合成グルカンを高眼圧(緑内障)モデルラットの硝子体に投与したところ,網膜神経節細胞の保護効果が認められた。OdBGPの一次構造中には既知の保存領域は認められなかったが,予測二次構造はGH 94ファミリーのホスホリラーゼと類似していた。

Structural, thermal, and rheological properties of starches from 13 Amaranthus hypochondriacus lines and 8 Amaranthus caudatus lines were evaluated and related using principal component analysis. Amylopectin chains with DP 6–12, DP 13–18, DP 19–24, and DP 25–30 ranged from 43.60 to 55.72%, 31.59 to 34.49%, 10.14 to 16.37%, and 2.56 to 5.53%, respectively, for A. hypochondriacus and 50.67 to 57.53%, 29.30 to 32.02%, 10.04 to 13.09%, and 2.75 to 4.2%, respectively for A. caudatus. A. hypochondriacus showed higher proportion of granules <1 µm (14.76–35.64%) than A. caudatus (0.16–6.92%). A. hypochondriacus and A. caudatus starches showed the granules of size 1–10 µm between 57.09 to 77.48% and 88.67 to 99.66%, respectively. The transition temperatures (To, Tp, and Tc) were negatively correlated with amylopectin short chains (DP 6–12). The pasting properties (PV, BV, FV, and SB) were positively correlated with DP 6–12 and negatively with DP 13–24. Starches with higher proportion of granules of 1–10 µm showed higher amount of DP 6–12, crystallinity, and paste viscosities. Starches with higher proportion of 11–30 µm had high DP 13–30 and gelatinization transition temperatures. The results reflected that the change in structure from liquid‐like to solid‐like was dependent upon chain length, starch with higher DP 6–10 showed higher Tcrossover (temperature at which G′ became higher than G″ during heating) and those with higher DP 13–24 showed lower Tcrossover.

We prepared biodegradable foam by pressing and baking a mixture containing native sago residue, cellulose, polyvinyl alcohol, magnesium stearate, and water. The foam was opaque and brown; it had a rough surface and a porous internal structure. The foam was denser than commercial polystyrene foam. However, the modulus of elasticity and modulus of rupture in flexure of the foam prepared from native sago residue were higher than those of polystyrene foam, indicating that the foam from sago residue was stiff, had high strength, and would be suitable for holding and protecting heavy materials. We also prepared another foam using acidtreated sago residue instead of native sago residue. The foam from the acid-treated residue was thinner and more porous than the foam from the native residue. A higher modulus of elasticity in flexure of the foam from the acid-treated residue was also observed. Thus, we found that partial acid hydrolysis of starch in sago residue modifies the structural and physico-mechanical properties of the resulting foam.

Ethanol fermentation from food wastes containing mainly starch without carrying out sterilization was investigated by using wild and tolerant yeast, Issatchenkia orientalis MF-121. The MF-121 strain is not a suitable choice for ethanol fermentation from lignocellulosic biomass because it is only capable of fermenting hexoses of glucose, mannose, and fructose to ethanol. Therefore, we first isolated acid- and salt-tolerant yeast that are capable of fermenting various monosaccharides to ethanol, and the isolated yeast that showed the ability to ferment ethanol from glucose, mannose, galactose, fructose, and xylose, was identified as Zygoascus hellenicus LK-5G on the basis of the 26S rRNA sequence analysis.

The neuroprotective effect of intravitreally injected beta-1,3-glucan was examined in a rat model of transient retinal ischemia. Retinal function and morphology were assessed in both control rats (Intact), and in rats administered beta-1,3-glucan after ischemic damage induced by elevation of intraocular pressure (IOP) to 130 mmHg for 45 min. beta-1,3-Glucan was intravitreally injected into male SD rats, with a final vitreal concentration of 5 mu g/eye. Retinal function was assessed by electroretinography, and retinal morphology was assessed by light microscopy and TUNEL assay (n = 3 for Intact, and n = 6 for beta-1,3-glucan and Vehicle). Morphometric analysis of retinal ganglion cells (RGCs) and the inner plexiform layer (IPL) showed decreased RGC number and thinner IPL in the eyes of Intact rats compared with those injected with beta-1,3-glucan. TUNEL assay results showed that the retinas of eyes injected with beta-1,3-glucan were similar to those of Intact eyes (the left eyes of the SD rats). However, the retinas of Vehicle eyes differed in RGC and IPL layer from beta-1,3-glucan injected eyes after transient retinal ischemia. Electroretinograms (ERGs) showed that the beta-1,3-glucan-administered rats had significantly more protective b-wave amplitudes compared with the Vehicle group. beta-1,3-Glucan exhibits a neuroprotective effect in a transient retinal ischemia model of retinal injury in rats.

1,3-β-D-Glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-β-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C–30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-β-glucans.

Native starches from twenty-six botanical sources were determined for their structural features and stability against freeze-thaw treatments. Starch gels (5%, w/w) were prepared and repeatedly freeze-thawed up to five cycles by storing at -18 degrees C for 21 h and then at 30 degrees C for 3 h. Water release (syneresis) from the thawed gel after the 1st, 3rd and 5th cycle was measured gravimetrically, and evaluated in relation to apparent amylose content (AAC) and distribution of amylopectin branch chains with degree of polymerization 6-12 (APC ratio). Syneresis was not observed for starch gels of cassava, normal and waxy japonica rice up to the 1st, 3rd and 5th cycle, respectively. On the other hand, syneresis rapidly occurred for starch gels of elephant yam, new cocoyam, potato, edible canna. and water yam. Optimal multiple linear regression models were generated to predict individual effect of AAC and APC ratio on syneresis of starch gels. The prediction models illustrated the positive unit-contribution of AAC and negative unit-contribution of APC ratio to syneresis (P<0.001). (C) 2011 Elsevier Ltd. All rights reserved.

The ability of 13 strains of multi-stress-tolerant Issatchenkia orientalis yeast to produce ethanol was examined under different stress conditions, including conditions of elevated H2SO4 and Na2SO4 concentrations and increased heat. The MF-121 strain produced a significant amount of ethanol after the incubation in acidic media containing high concentrations of salt, e.g., 50 g/l Na2SO4 at pH 2.0, or at high temperatures, e.g., 43°C, when compared with other strains.