Akira Ohtakara

Hypocholesterolemic action of chitosans with different viscosity in rats.

The relationship between hypocholesterolemic efficacy and average molecular weight of chitosan was studied in rats fed a cholesterol-enriched (0.5%) diet. Several chitosan preparations with a comparable degree of deacetylation but differing widely in average molecular weight, as demonstrated by viscosity, almost completely prevented the rise of serum cholesterol at the 5% dietary level. At the 2% level, chitosans with viscosities at both extremes exerted a comparable cholesterol-lowering action. The glucosamine oligomer composed mainly of three to five aminosugar residues was not effective. The results indicate that the hypocholesterolemic action of chitosans is independent of their molecular weight within the tested viscosity range.

Immobilization of thermostable α-galactosidase from Pycnoporus cinnabarinus on chitosan beads and its application to the hydrolysis of raffinose in beet sugar molasses

Abstract α-Galactosidase (EC 3.2.1.22) from Pycnoporus cinnabarinus was immobilized on chitosan beads, BCW 1000, and crosslinked chitosan beads, BCW 3000 and 3500, of three different sizes, which were untreated or previously treated with glutaraldehyde. The activity yields of the immobilized enzymes were between 25 to 45%, except for glutaraldehyde-untreated B BCW 1000. Leakage of the enzyme with increasing ionic strength was observed in glutaraldehyde-untreated BCW 1000 and 3000. The α-galactosidases immobilized on glutaraldehyde-treated BCW 3000 and 3500 were active at pH 3–6 and at 70–80°C, and stable between pH 3 and 9, and below 70°C. The immobilized α-galactosidase was continuously used for 30 days to hydrolyze raffinose in beet sugar molases.

Chitosanase from Streptomyces griseus

Publisher Summary This chapter describes the assay method and procedure for the purification of chitosanase from Streptomyces griseus. The assay is based on the estimation of amino sugars produced in the hydrolysis of glycol chitosan, a water-soluble derivative of chitosan, by the method of Rondle and Morgan, s using glucosamine as a reference compound. The purified chitosanases are classified into two groups: the enzymes hydrolyzing only chitosan and the enzymes hydrolyzing both chitosan and carboxymethylcellulose. A strain of Streptomyces griseus has produced chitosanase in culture broth with chitosan as the single carbon and nitrogen source. The purified enzyme is able to hydrolyze chitosan and carboxymethylcellulose, and produces glucosamine oligomers in the hydrolysis of chitosan.

Structure-activity relationships in the induction of single-strand breakage in plasmid pBR322 DNA by amino sugars and derivatives

Structure-activity relationships in the induction of strand breakage in plasmid pBR322 DNA by amino sugars and their derivatives were investigated using agarose gel electrophoresis. The coexistence of a potential free aldehyde group at the C-1 position and a free amino group at the C-2 position in the molecules was indispensable for the display of DNA strand-breaking activity in both mono- and oligo-aminosaccharides. The activity was increased by the introduction of an acidic group, especially a phosphate group, at the C-6 position. The activity was also increased by the addition of Cu2+. The order of activity of the amino monosaccharides tested was D-isoglucosamine > D-mannosamine > D-galactosamine > D-glucosamine, and it is suggested that this order is correlated with the portion of acyclic (aldehydo) form in the solution of each sugar. The possible chemical basis for DNA strand breakage by amino sugars is discussed.

Viscosimetric assay for chitinase

Publisher Summary This chapter describes the procedures for the viscosimetric assay of chitinase activity in which water-soluble derivatives of chitin are used as substrates. The viscosimetric assay for chitinase is a more sensitive and effective procedure to detect a flight activity. However, this assay procedure is somewhat troublesome and too time consuming to determine the chitinase activity of numerous samples. The viscosity of a glycol chitin solution varies depending on the degree of polymerization. Some differences have been observed among the values of chitinase activity determined by procedure using different batches of substrates. Therefore, the viscosimetric assay of chitinase activity should be carried out using the same batch of glycol chitin in the same experiment. This assay has been applied to the determination of chitinase activity in the purification of chitinases from Aspergillus niger and Vibrio sp., and those from goat serum and bovine serum.

PURIFICATION AND CHARACTERIZATION OF CHITOSANASE FROM STREPTOMYCES GRISEUS

Chitosanase from Streptomyces griseus was purified from the culture filtrate by precipitation with ammonium sulfate, column chromatography on CM-Sephadex C-25 and gel filtration on Sephadex G-75. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of chitosanase was estimated to be about 35,000 by sodium dodecyl sulfate gel electrophoresis and the isoelectric point was approximately at pH 9.7. The enzyme hydrolyzed carboxymethyl cellulose as well as colloidal chitosan, soluble chitosan, glycol chitosan and carboxymethyl chitosan. The optimum pH of the enzyme was about at 8.0 for glycol chitosan and about at 6.5 for carboxymethyl cellulose. Chitosanase was stable over a range of pH 6.0 to 8.0 at 37°C for 3 hours and below 40°C or less after heating for 15 minutes at pH 7.0. The enzyme activities were strongly inhibited by metal ions such as Ag+ and Hg 2+ , and p-chloromercuribenzoate. Chitosanase of Streptomyces griseus differed from that of Streptomyces sp. No.6 in its activity against carboxymethyl cellulose.

Purification and Enzymatic Properties of α-Galactosidase from Pycnoporus cinnabarinus

α-Galactosidase (EC 3.2.1.22) from Pycnoporus cinnabarinus was purified by precipitation with ammonium sulfate, column chromatographies on DEAE-Sephadex A-50, Sephadex G-100, CM-Sephadex C-50, and SP-Sephadex C-50, and isoelectric focusing. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis, and the molecular weight was estimated to be about 210,000 by gel filtration on Sephacryl S-200 and about 52,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme exhibited the optimum pH at 5.0 and was stable between pH 3 and 9. The optimum temperature of the enzyme was 75°C. The enzyme was thermostable at pH 5.0 and completely lost its activity after heating at 90°C or at pH 3.5. The Michaelis constants were 0.31 mM for p -nitrophenyl α-D-galactoside, 0.80 mM for melibiose, 2.16mM for raffinose, and 1.15 mM for stachyose. The α-galactosidase activity was strongly inhibited by Ag+, Hg++, p -chloromercuribenzoate, galactose, and melibiose. The enzyme also seemed to catalyse a glycos...

Analysis of chitooligosaccharides and reduced chitooligosaccharides by high-performance liquid chromatography

Publisher Summary This chapter describes a rapid and sensitive procedure for simultaneous determination of chitooligosaccharides and reduced chitooligosaccharides by HPLC and its application for studies on the mode of action of chitinase and β-N-acetylhexosaminidase. Chitooligosaccharides and reduced chitooligosaccharides can be hydrolyzed with chitinase and β-N-acetylhexosaminidase from Pycnoporus cinnabarinus . These oligosaccharides are rapidly separated by reversed-phase HPLC on amine-modified silica columns. A technique for separating chitooligosaccharides using HPLC has been recently applied to investigate the lysozyme-catalyzed hydrolysis and transglycosylation, and the action pattern of endo- and exochitinase from Munduca sexta. The chapter shows the products produced in the hydrolysis of reduced (GIcNAc) 5 with β-N-acetylhexosaminidase. This result indicates that the β-N-acetylhexosaminidase is an exo-type enzyme acting on reduced (GlcNAc) 5 at the nonreducing end to release N-acetylglucosamine.