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276 results on '"Ishibashi, Matsujiro"'

22. Catalytic mechanism and evolutional characteristics of thioredoxin from Halobacterium salinarum NRC-1

Author

Arai, Shigeki, Shibazaki, Chie, Shimizu, Rumi, Adachi, Motoyasu, Ishibashi, Matsujiro, Tokunaga, Hiroko, Tokunaga, Masao, Shigeki, Arai, Chie, Shibazaki, Rumi, Shimizu, and Motoyasu, Adachi

Abstract

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content ([Asp + Glu] / [Arg + Lys + His] = 9.0) among known TRXs, was chosen to elucidate a catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main chain structure of HsTRX-A is similar to those of homologous TRXs; e.g., root-mean-square deviations for Cα atoms were

Published
2020

23. Expression, Folding, and Activation of Halophilic Alkaline Phosphatase in Non-Halophilic Brevibacillus choshinensis

Author

Amreta Laksmi, Fina, Imamura, Hikari, Tsurumaru, Hirohito, Nakamura, Yoshitaka, Hanagata, Hiroshi, Arai, Shigeki, Tokunaga, Masao, Ishibashi, Matsujiro, and Shigeki, Arai

Abstract

Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments. We aimed to clarify the stabilization mechanism of halophilic enzymes. We previously attempted to express halophilic alkaline phosphatase from Halomonas (HaALP) in non-halophilic E. coli. However, the expressed HaALP showed little activity. Therefore, we overexpressed HaALP in Gram-positive non-halophilic Brevibacillus choshinensis in this study, which was successfully expressed and purified in its active form. HaALP was denatured in 6 M urea, refolded using various salts and the non-ionic osmolyte trimethylamine N-oxide (TMAO), and assessed by native polyacrylamide gel electrophoresis. HaALP refolded in 3M NaCl or 3 M TMAO containing Na+ ions. Hydrophobic interactions due to a high salt concentration or TMAO enhanced the formation of the folding intermediate (the monomer precursor), and only Na+ ions activated the dimer form. This insight into the stabilization mechanism of HaALP may lead to the development of industrial applications of halophilic enzymes under low-salt conditions.

Published
2019

30. Improved substrate specificity for D-galactose of L-arabinose isomerase for industrial application

Author

Amreta, Laksmi Fina, Tsurumaru, Hirohito, Nakamura, Yoshitaka, Saksono, Budi, Tokunaga, Masao, and Ishibashi, Matsujiro

Abstract

L-Arabinose isomerase isolated from Geobacillus stearothermophilus (GSAI) was modified to improve its substrate specificity for D-galactose for the production of D-tagatose, a potential reduced-energy sweetener. Among the selected residues, mutation at residue 18 produced a mutant strain, H18T, which exhibited increased activity for D-galactose compared with the wild-type (WT) enzyme. Analysis of the substrate specificity of H18T showed a 45.4% improvement for D-galactose. Replacing histidine with threonine at residue 18 resulted in approximately 2.7-fold and 1.8-fold higher substrate binding and catalytic efficiency, respectively, for D-galactose. Further enhancement of the specific activity and catalytic efficiency of H18T for D-galactose by up to 2.7-fold and 4.3- fold, respectively, was achieved by adding borate during L-arabinose isomerase catalysis. Moreover, H18T showed thermostability and no destabilization was detected, which is promising for the industrial production of D-tagatose

Published
2018

42. Expression and characterization of l-arabinose isomerase from Geobacillus stearothermophilus for improved activity under acidic condition

Author

Amreta Laksmi, Fina, Arai, Shigeki, Arakawa, Tsutomu, Tsurumaru, Hirohito, Nakamura, Yoshitaka, Sakson, Budi, Tokunaga, Masao, Ishibashi, Matsujiro, and Shigeki, Arai

Abstract

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.

Published
2020

48. Thermodynamic analysis of the activation mechanism of the GCSF reception induced by ligand binding

Author

Mine, Shouhei, Koshiba, Takumi, Honjo, Eijiro, Okamoto, Tomoyuki, Tamada, Taro, Yoshitake; Matsukura, Maeda, Yasuko, Horie, Akane, Ishibashi, Matsujiro, Sato, Miharu, Azuma, Mizue, Tokunaga, Masao, Nitta, Katsutoshi, and Kuroki, Ryota

Subjects
Binding proteins -- Analysis, Chemical structure -- Analysis, Granulocyte colony-stimulating factor -- Analysis, Biological sciences, Chemistry
Abstract

The quantitative and structural properties of the interaction of Granulocyte colony-stimulating factor (GCSF) and its receptor were investigated. The thermodyanmic data suggested that the activated structure of the Granulocyte colony-stimulating factor receptor (GCSFR) induced by GCSF binding to Ig-CRH results in the formation of a 2:2 complex structure and the Ig domain would bring each cytokine receptor homologous region (CRH) domain closer and facilitate the interaction of their intracellur domains.

Published
2004

49. Structure of a highly acidic β-lactamase from the moderate halophileChromohalobactersp. 560 and the discovery of a Cs+-selective binding site

Author

Arai, Shigeki, primary, Yonezawa, Yasushi, additional, Okazaki, Nobuo, additional, Matsumoto, Fumiko, additional, Shibazaki, Chie, additional, Shimizu, Rumi, additional, Yamada, Mitsugu, additional, Adachi, Motoyasu, additional, Tamada, Taro, additional, Kawamoto, Masahide, additional, Tokunaga, Hiroko, additional, Ishibashi, Matsujiro, additional, Blaber, Michael, additional, Tokunaga, Masao, additional, and Kuroki, Ryota, additional

Published
2015
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