Preparing D-glucose with comparatively high yield and purity from many kinds of lignocelluloses through a basically unified method is an important technology because almost all of the yeasts show limitative use of carbon sources in ethanol fermentation. Although cellulase is of great interest in the production of D-glucose, the enzyme degradation demands a pre-treatment for loosening the hard structure of lignocelluloses. A typical acid hydrolysis of lignocelluloses digesting directly to monosaccharides is also used; however, the hydrolysate contains not only a mixture of pentose and hexose but also some chemicals inhibiting the growth of yeast. Therefore, representative samples of hardwood (Japanese oak), softwood (Japanese cedar) and annual plants (sugar cane bagasse) were examined in order to look for a convenient method of collecting oligosaccharides from these biomasses employing the concept that high-molecular-weight molecules with hard structure are gradually loosened and then degraded into low-molecular-weight molecules with strong alkaline or strong acid under mild conditions. Potassium hydroxide was suitable for the annual plant but insufficient for wood in the segregation of hemicellulose and cellulose. Concentrations of sulfuric acid for extracting selectively the majority of hemicelluloses from annual plants and wood were 42.5 and 44% (v/v), respectively. Lignocellulose (100 mg) was treated with the concentrated sulfuric acid (10 mL) at 20°C for 5 h under 120 rpm shaking condition. A treatment with 55% (v/v) sulfuric acid of the residual material under the same conditions was appropriate for preparation of oligosaccharides composed chiefly of D-glucose.
Cellulosic oligosaccharides (720-920 mg) were prepared by a two-step extraction using 43.5% sulfuric acid and then 55% sulfuric acid from lignocelluloses (2 g each) of Japanese oak and eucalyptus as hardwood, Japanese cedar and cypress as softwood, and sugar cane bagasse and rice straw as annual plant residues. After the complete saccharification of the oligosaccharide and treatment with the appropriate amount of activated carbon, D-glucose with a comparatively high purity was prepared. As the D-glucose was in a strong sulfuric acid solution, the sugar solution was applied on a column packed with an ion exchanging resin (Diaion DSR01) in order to remove almost all of the acid. The fractions eluting the sugar were collected and concentrated with a rotary evaporator. Although the concentrated sugar solution showed an acidity of pH 2-3, acid tolerant Issatchenkia orientalis MF 121 could ferment ethanol successfully in YPD medium using the sugar as the sole source of carbon.
1,4-β-Endoglucanase was purified from the hepatopancreas of the marine mollusc, Patinopecten yessoensis, by cellulose column chromatography. The enzyme (EG66) showed the Mr of 66K on SDS-PAGE, and the hydrolytic activity was maximal at pH 6.0. EG66 readily hydrolyzed glucomannan and β-1,3-1,4-glucan as well as CMC and hydroxyethyl cellulose. On the other hand, crystalline cellulose, xylan, xyloglucan, laminarin, β-1,3-1,6-glucan, galactomannan, alginate, methyl cellulose and hydroxypropylmethyl cellulose were not hydrolyzed at all. N-Terminal amino acid sequence of EG66, which was determined as GATNVQIT NEWPGGFQGTF, had a motif common to the other molluscan endoglucanases.
Award Address: The Award of Merit of the 2008's JSAG
The structure-function relationship of the polysaccharides from the view point of the rheological characteristics in aqueous solutions has been insighted into when I was listening to the first movement of the piano sonata No. 14, Op. 27-2, entitled Moon-light which was composed by Ludwig van Beethoven on July in 1973. At that time, I was a doctoral student at the Department of Food Science and Technology, Kyushu University. After getting job at the University of the Ryukyus in 1982, it has been investigated. Gelation mechanism for κ-carrageenan, ι-carrageenan, agarose (agar), gellan gum, alginic acid, curdlan and amylose has been analyzed, respectively, at the molecular level where sulfate oxygen, ring oxygen, hemiacetal oxygen, cations, hydroxyl groups oriented at axial, and methyl groups take part in the intra- and intermolecular associations with ionic bonding, electrostatic forces of attraction, hydrogen bonding and van der Waals forces of attraction. Co-gelation mechanism of xanthan with galactomannan (locust bean gum, tara bean gum, guar gum and Leucaena galactomannan), and with konjac glucomannan, where trisaccharide side-chains of the former molecules contribute with hydrogen bonding has also been analyzed, respectively. The structure-function relationship for non-gelling welan, rhamsan, S-657, S-88, S-198, shizophyllan and amylopectins (rice, potato and wheat), the structure of which is similar to that of gelling gellan, curdlan and amylose has been analyzed. The gellan family of the polysaccharides, as metioned above, have given a good opportunity to investigate the molecular origin for their rheological characteristics. Recently, we have analyzed a gelatinization and retrogradation mechanisms of rice, potato and wheat starches. From the results and discussions, the principles of the polysaccharide gels have been established. Though many investigations concerning the gelling properties of the polysaccharides have been done to discuss structure-function relationship, no one has established gelation mechanisms at the molecular level except the author. There is reasonable consistency in our investigations. Thus, rheological analysis is one of the most significant methods to realize the structure-function relationships of polysaccharides in aqueous solutions. We have isolated a novel fucoidan, which substituted an acetyl group, from commercially cultured Cladosiphon okamuranus in Okinawa, Japan and has been covered by a patent. The fucoidan has an excellent flow characteristics and immunological activity. The fucoidan has now been used in food and cosmetic industries, production of which increases year by year.
Award Address: Distinguished Young Scientist Award of the 2008's JSAG
This paper describes the study on the structure and function of enzymes hydrolyzing a polysaccharide, pullulan and the related enzymes. The paper is composed of the following three major sections. (1) A thermophilic actinomycete, Thermoactinomyces vulgaris R-47, produces two pullulan hydrolyzing-enzymes, TVA I and TVA II. TVA II forms a dimer, and this is the first observation of a dimeric enzyme in the α-amylase family. In contrast, TVA I functions as a monomer, and domain N of TVA I is identified as a novel carbohydrate-binding module, CBM34. (2) The crystal structure and function of the isopullulanase were determined. The isopullulanase forms a unique β-helix fold, which is not found in other pullulan-hydrolyzing enzymes. (3) A glucoamylase gene was found downstream of the TVA II gene, and the primary structure of this glucoamylase resembled that of glucodextranase. The glucoamylase and related enzymes were further studied, and the crystal structures and functions of Arthrobacter globiformis I42 glucodextranase (GDase) and Escherichia coli glucosidase, YgjK, have been determined. Although GDase and YgjK scarcely hydrolyzed starch and their substrate specificities were completely different from that of glucoamylase, these enzymes have a common (α/α)6-barrel domain like glucoamylase.
Functional and processing properties of novel wheat flours, such as polished, waxy and high-amylose wheat flours were determined. Polished flours increased the amounts of minerals and anti-oxidant activity, and the amounts of dietary fibers, phytic acids, ferulic acids and pentosans were 2, 3, 10 and 2 times more, and large amounts of damaged starches and higher maltose values were obtained, as compared with the common flour of CW. The distributions of proteins in polished flours were different depending on the portions (fractions) of wheat grains, and proteins of the innermost fraction showed low-allergic reactions. Baking method including long fermentation significantly improved the baking properties of polished flours, and especially sourdough method increased the free amino acids, reducing sugars and organic acids during fermentation. The CO2 gas in polished-flour-sourdoughs distinctly increased, and their pH, total titratable acidity levels and buffering capacity were significantly better than the CW-sourdough. As a result, the growth of lactic acid bacteria and yeasts in polished-flour-sourdoughs were accelerated during fermentation, and its substituted breads with middle or innermost fractions improved baking qualities having favorable volatile compounds, such as iso-butanol and β-phenyl ethyl alcohol, more than CW-sourdough bread. In contrast, waxy wheat flours improved the staleness of breadcrumbs during storage and its freshness after reheating, while high-amylose wheat flours increased amounts of resistant starch in breadcrumbs during storage. Moreover, waxy wheat flours increased viscous and sticky textures of boiled-noodles, and high-amylose wheat flours suppressed the damage of noodles during boiling with the similar qualities to the durum pasta. Consequently, these novel wheat flours would be sufficient foodstuffs with excellently nutritious, functional and processing properties to give additional values of new taste or functionality to their final products.
Award Address: Research and Development Award of the 2008's JSAG
Phosphate ester groups are known to link to some glucosyl residues in starch molecule. We focused on the utilization of esterified phosphoryl group in potato starch components. In starch processing, the actions of the amylolytic enzymes were hindered by the phosphoryl groups linked to the glucosyl residue, and phosphoryl oligosaccharides (POs) were obtained as indigestible components by the enzymes. The POs were composed of two fractions, PO-1 and PO-2. Fraction PO-1 was the main fraction, and it was composed of maltotriose, maltotetraose and maltopentaose to which one phosphoryl group was attached. Fraction PO-2 was predominantly composed of maltopentaose and maltohexaose to which at least two phosphoryl groups were attached. The POs had the ability to form a soluble complex with calcium and had inhibitory effect on the formation of a calcium-phosphate precipitate. Based on the function of the POs, described above, we applied the POs of calcium (POs-Ca®) as a food ingredient. The POs-Ca was an advantageous food ingredient as a soluble calcium source. Relating to prevent of dental caries, POs can not be fermented by cariogenic microorganisms, mutans streptococci, and reduces the plaque pH fall in vitro. Moreover, POs-Ca was effectively enhanced remineralization of enamel lesion. The enhanced remineralization in the POs-Ca group was considered to be caused by increased soluble Ca contents in saliva. The chewing gum containing POs-Ca can produce the effective environment in saliva. That is, it can be said that POs-Ca is an ingredient which cannot be easily influenced of flavor and coloring matter, is the substance which performs re-calcification. The present results suggested that daily chewing of a sugar-free gum containing POs-Ca could be one effective approach to stimulate remineralization of enamel and thereby to prevent dental caries.