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Hirotsu T.,Kumamoto University | Higashi T.,Kumamoto University | Hashim I.I.A.,Kumamoto University | Hashim I.I.A.,Mansoura University | And 4 more authors.
Molecular Pharmaceutics | Year: 2017

Polyethylene glycol (PEG) modification (PEGylation) is one of the best approaches to improve the stabilities and blood half-lives of protein drugs; however, PEGylation dramatically reduces the bioactivities of protein drugs. Here, we present "self-assembly PEGylation retaining activity" (SPRA) technology via a host-guest interaction between PEGylated β-cyclodextrin (PEG-β-CyD) and adamantane-appended (Ad) proteins. PEG-β-CyD formed stable complexes with Ad-insulin and Ad-lysozyme to yield SPRAinsulin and SPRA-lysozyme, respectively. Both SPRA-proteins showed high stability against heat and trypsin digest, comparable with that of covalently PEGylated protein equivalents. Importantly, the SPRA-lysozyme possessed ca. 100% lytic activity, whereas the activity of the covalently PEGylated lysozyme was ca. 23%. Additionally, SPRA-insulin provided a prolonged and peakless blood glucose profile when compared with insulin glargine. It also showed no loss of activity. In contrast, the covalently PEGylated insulin showed a negligible hypoglycemic effect. These findings indicate that SPRA technology has potential as a generic method, surpassing conventional PEGylation methods for proteins. © 2017 American Chemical Society.


Sato H.,Hokkaido University | Saburi W.,Hokkaido University | Ojima T.,Nihon Shokuhin Kako Co. | Taguchi H.,Hokkaido University | And 2 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2012

Cellobiose 2-epimerase (CE) efficiently forms epilactose which has several beneficial biological functions. A thermostable CE from Rhodothermus marinus was immobilized on Duolite A568 and packed into a column. Lactose (100 g/L) was supplied to the reactor, kept at 50 °C at a space velocity of 8 h -1. The epilactose concentration of the resulting eluate was 30 g/L, and this was maintained for 13 d.


Ojima T.,Nihon Shokuhin Kako Co. | Saburi W.,Nihon Shokuhin Kako Co. | Saburi W.,Hokkaido University | Yamamoto T.,Nihon Shokuhin Kako Co. | Kudo T.,Nagasaki University
Applied and Environmental Microbiology | Year: 2012

An α-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl α-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30°C and pH 6.5. Monovalent cations such as K +, Rb +, Cs +, and NH 4 + increased the enzymatic activity to 2- to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40°C in the presence of 10 mM NH 4 +, although temperature stability was not affected. The apparent K m and k cat values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k cat/K m for pNPG increased 372- to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of α-D-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger α-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 α-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 α-glucosidase, respectively. © 2012, American Society for Microbiology.


Ojima T.,Nihon Shokuhin Kako Co. | Aizawa K.,Nihon Shokuhin Kako Co. | Saburi W.,Hokkaido University | Yamamoto T.,Nihon Shokuhin Kako Co.
Carbohydrate Research | Year: 2012

6-Gingerol [(S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one] is a biologically active compound and is abundant in the rhizomes of ginger (Zingiber officinale). It has some beneficial functions in healthcare, but its use is limited because of its insolubility in water and its heat-instability. To improve these physical properties, the glucosylation of 6-gingerol was investigated using α-glucosidases (EC. 3.2.1.20) from Aspergillus niger, Aspergillus nidulans ABPU1, Acremonium strictum, Halomonas sp. H11, and Saccharomyces cerevisiae, and cyclodextrin glucanotransferases (CGTase, EC. 2.4.1.19) from Bacillus coagulans, Bacillus sp. No. 38-2, Bacillus clarkii 7364, and Geobacillus stearothermophilus. Among these, only α-glucosidase from Halomonas sp. H11 (HaG) transferred a glucosyl moiety to 6-gingerol, and produced glucosylated compounds. The chemical structure of the reaction product, determined by nuclear magnetic resonance spectroscopy and mass spectrometry, was (S)-5-(O-α-d-glucopyranosyl)-1-(4-hydroxy-3-methoxyphenyl)decan-3-one (5-α-Glc-gingerol). Notably, the regioisomer formed by glucosylation of the phenolic OH was not observed at all, indicating that HaG specifically transferred the glucose moiety to the 5-OH of the β-hydroxy keto group in 6-gingerol. Almost 60% of the original 6-gingerol was converted into 5-α-Glc-gingerol by the reaction. In contrast to 6-gingerol, 5-α-Glc-gingerol, in the form of an orange powder prepared by freeze-drying, was water-soluble and stable at room temperature. It was also more stable than 6-gingerol under acidic conditions and to heat. © 2012 Elsevier Ltd. All rights reserved.


Patent
Kumamoto University and Nihon Shokuhin Kako Co. | Date: 2016-10-12

Provided is a pharmaceutical composition for the treatment of disorders such as Niemann-Pick disease and GM1 gangliosidosis which are caused by the storage of cholesterol, such as lysosomal storage disease. Also provided is a method for screening for said pharmaceutical compositions that uses iPS cell strains that phenocopy phentotypes of these disorders. Provided is a pharmaceutical composition for the treatment and/or prevention of lysosomal storage disease, characterized by containing hydroxypropyl--cyclodextrin as an active ingredient. Also provided are an iPS cell strain derived from patients suffering from intractable disorders and prepared using a new temperature-sensitive Sendai virus vector, and a screening method for pharmaceuticals using said iPS cell strain.


Patent
Nihon Shokuhin Kako Co. | Date: 2015-12-02

The present invention addresses the problem of developing a fat-processed starch which is excellent in terms of all of three properties, i.e., workability during the preparation of a coating material for fries, function required of coating materials for fries (adhesion to ingredients) and moderate slurry viscosity. The fat-processed starch of the present invention is produced by a process comprising the steps: adding a fat with which an emulsifying agent has been mixed to a starch having a water content adjusted to 25 to 45 mass%, in an amount of 0.02 to 0.4 mass% in terms of the weight of the fat excluding the emulsifying agent relative to the weight of dry matter of the starch, and mixing the fat and the starch; adjusting the water content of the mixture is 0.2 to 0.5 time that of the starch before the addition of the fat; and aging the starch having the adjusted water content.


Ojima T.,Nihon Shokuhin Kako Co. | Saburi W.,Hokkaido University | Yamamoto T.,Nihon Shokuhin Kako Co. | Mori H.,Hokkaido University | Matsui H.,Hokkaido University
Bioscience, Biotechnology and Biochemistry | Year: 2013

Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of β-1,4-linked oligosaccharides to Dmannose residues. In this study, we characterized CElike proteins from various aerobes (Flavobacterium johnsoniae NBRC 14942, Pedobacter heparinus NBRC 12017, Dyadobacter fermentans ATCC 700827, Herpetosiphon aurantiacus ATCC 23779, Saccharophagus degradans ATCC 43961, Spirosoma linguale ATCC 33905, and Teredinibacter turnerae ATCC 39867), because aerobes, more easily cultured on a large scale than anaerobes, are applicable in industrial processes. The recombinant CE-like proteins produced in Escherichia coli catalyzed epimerization at the C2 position of cellobiose, lactose, epilactose, and β-1,4-mannobiose, whereas N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, D-glucose, and D-mannose were inert as substrates. All the CEs, except for P. heparinus CE, the optimum pH of which was 6.3, showed highest activity at weakly alkaline pH. CEs from D. fermentans, H. aurantiacus, and S. linguale showed higher optimum temperatures and thermostability than the other enzymes analyzed. The enzymes from D. fermentans, S. linguale, and T. turnerae showed significantly high kcat and Km values towards cellobiose and lactose. Especially, T. turnerae CE showed a very high kcat value towards lactose, an attractive property for the industrial production of epilactose, which is carried out at high substrate concentrations.


Patent
Nihon Shokuhin Kako Co. | Date: 2013-04-23

The present invention addresses the problem of developing a fat-processed starch which is excellent in terms of all of three properties, i.e., workability during the preparation of a coating material for fries, function required of coating materials for fries (adhesion to ingredients) and moderate slurry viscosity. The fat-processed starch of the present invention is produced by a process comprising the steps: adding a fat with which an emulsifying agent has been mixed to a starch having a water content adjusted to 25 to 45 mass %, in an amount of 0.02 to 0.4 mass % in terms of the weight of the fat excluding the emulsifying agent relative to the weight of dry matter of the starch, and mixing the fat and the starch; adjusting the water content of the mixture is 0.2 to 0.5 time that of the starch before the addition of the fat; and aging the starch having the adjusted water content.


Patent
Nihon Shokuhin Kako Co. | Date: 2013-11-13

An object of the present invention is to provide a method for producing a saccharide polycondensate which is inexpensive and is applicable to a food or beverage product. Disclosed is a method for producing a saccharide polycondensate, which comprises carrying out a saccharide polycondensation reaction in the presence of activated carbon.


Ojima T.,Nihon Shokuhin Kako Co. | Saburi W.,Nihon Shokuhin Kako Co. | Saburi W.,Hokkaido University | Sato H.,Hokkaido University | And 3 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2011

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80°C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80°C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.

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