Yoshihiko Maekawa

Selective enzymatic preparation of vinyl sugar esters using DMSO as a denaturing co-solvent

The protease-catalyzed transesterifications between hexoses and divinyladipate were examined. In dimethylformamide hexoses such as d-glucose, d-mannose, d-galactose and α-methyl d-galactoside were esterified with divinyladipate by alkaline protease from Streptomyces sp. to give corresponding 6-O-vinyl adipoyl sugars. When the denaturing cosolvent, DMSO, was added to the solvent, galactose was selectively esterified at only the C-2 position.

Production and characterization of active soluble human β1,4-galactosyltransferase in Escherichia coli as a useful catalyst in synthesis of the Gal β1→4 GlcNAc linkage

An active and soluble human beta1,4-galactosyltransferase (beta-GT) was produced in Escherichia coli using a maltose-binding protein fusion system. The purified recombinant beta-GT has a K(m) value of 0.035 mM for UDP-galactose and a V(max) of 643 x 10(3) nmol/mg/h. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-linked oligosaccharides. The properties of the purified enzyme were identical to those of bovine milk beta-GT.

Regio-selective preparation of vinyl adenosine ester catalyzed by alkaline protease

The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3′-position of hydroxyl group of ribofuranose in adenosine to give 3′-O-vinyladipoyl adenosine without other products.

The relaxation of specificity of BanI restriction endonuclease from Bacillus aneurinolyticus IAM 1077

Abstract The restriction endonuclease Ban I from Bacillus aneurinolyticus IAM 1077, which recognizes 5′-GGPyPuCC-3′ and cleaves between G and G within this sequence, has decreased substrate specificity at high nuclease concentrations. The relaxation of its specificity was enhanced during modified reactions: digestion of pBR322 DNA or lambda DNA in the presence of high glycerol and dimethyl-sulfoxide (DMSO) produced additional fragments in addition to the inherent fragments. Therefore, it is required to check the reaction conditions carefully for generation of inherent fragments.

CLONING AND EXPRESSION OF THE BAMHI RESTRICTION-MODIFICATION SYSTEM IN BACILLUS SUBTILIS

Abstract The genes of the GGATCC-specific Bam HI restriction-modification system of Bacillus amyloliquefaciens H have been cloned and expressed in Bacillus subtilis MT-2. B. subtilis MT-2 carrying the recombinant plasmid (pBamHIRM22) produced about 10-fold more Bam HI restriction endonuclease and Bam HI methylase than B. amyloliquefaciens H did. B. subtilis MT-2 (pBamHIRM22) restricted unmodified phage. Restriction and modification genes were stably maintained in B. subtilis MT-2 on one plasmid and the produced Bam HI endonuclease remained stable even in the stationary phase of the culture. Bam HI endonuclease from B. subtilis MT-2 (pBamHIRM22) had the same molecular weight and N-terminal amino acid sequence as that from B. amyloliquefaciens H.

Cloning and expression in Escherichia coli of the BanI and BanIII restriction-modification systems from Bacillus aneurinolyticus

Abstract The genes coding for the GGPyPuCC-specific ( Ban I) and ATCGAT-specific ( Ban III) restriction-modification systems of Bacillus aneurinolyticus IAM1077 were cloned and expressed in Escherichia coli using pBR322 as a vector. The plasmids carrying the Ban I and Ban III restriction-modification genes were designated pBanIRM8 and pBanIIIRM12, respectively. The restriction maps of these recombinant plasmids were constructed. These two plasmids were stably maintained in E. coli HB101. However, when E. coli JM109 was used as a host, pBanIIIRM12 was efficiently propagated but pBanIRM8 was not. The HB101 cells carrying only the restriction gene of Ban III were viable, but the Ban I restriction gene carrier could not form colonies on agar plates. The growth of bacteriophage λ was strongly restricted only in the F. coli HB101 cells harboring pBanIRM8. These facts indicate that the Ban I restriction enzyme is expressed and functions more efficiently than Ban I modification enzyme in E. coli .

Chemical and Enzymatic Synthesis of Polymer Containing Reducing Sugar Branches.

バチルス属由来のプロテアーゼを用いて, ジメチルホルムアミド (DMF) 中においてグルコースとアジピン酸ジビニルの間でエステル交換反応を行い, 重合性糖エステル, 6-ο-ビニルアジポイル-D-グルコースを合成した. ひき続きアゾ系の重合開始剤を用いてDMFあるいは水中で, 重合性糖エステルのラジカル重合を行いポリ (6-ο-ビニルアジポイル-D-グルコース) を得た. この還元糖の分岐を有するポリマーはニトロブルーテトラゾリウム法においてはモノマーよりも高い還元力を示したが, シトクロームc法においては変化はなく, 測定方法の違いにより異なった還元力を示した.