We studied the structure of alginates extracted from five commercial Saccharina (Laminaria) species including three varieties (eight samples in all) harvested in Hokkaido, Japan. The algae used were Saccharina japonica, S. japonica var. diabolica, S. japonica var. ochotensis, S. japonica var. religiosa, S. longissima, S. coriacea, S. angustata, and S. sculpera (Kjellmaniella crassiforia). These alginates have molar fractions of mannuronic acid (FM) ranging from 0.68 to 0.76 and weight average molecular weights (Mws) ranging from 511,000 to 616,000. Alginate samples from both S. angustata (FM = 0.76) and S. longissima (FM = 0.68) showed intestinal immunological activity through Peyer’s patch cells of C3H/HeJ mice. Low molecular weight S. angustata alginate (FM = 0.75, Mw = 70,000) degraded using a wet pulverizing system showed higher activity than the native one.
Pedobacter heparinus heparin lyase II (PhHepII) is composed of N-terminal, central, and C-terminal domains. A long surface loop, designated loop-A, is in the N-terminal domain and is composed of amino acids 84-89. In this study, we deleted two, three, or four residues in loop-A to create Δ86-87, Δ85-87, and Δ84-87 PhHepII deletion mutants. We hypothesized that the deletions would increase PhHepII thermostability. After heating purified PhHepII enzymes at 45 °C for 5 min, 6.1 % of the enzyme activity remained in wild-type PhHepII, whereas 10.6 % of the enzyme activity remained in Δ86-87 PhHepII. The results indicated that the deletion caused a significant decrease in the activity, although Δ86-87 PhHepII is slightly more thermostable than wild-type PhHepII. In addtion, Δ85-87 and Δ84-87 PhHepII had weak or no enzyme activity, even when unheated. Circular dichroism spectra showed that Δ85-87 PhHepII was properly folded. These results suggest that the flexibility of loop-A is important for PhHepII enzyme activity.
Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated Verticillium dahliae strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that V. dahliae XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 184.108.40.206).
We examined a method to produce bread from crystalline rice flour without using thickening agents such as gluten, polysaccharide thickening, and amorphous rice flour. Rice grains were pulverized by a jet mill to produce flour. Samples of rice flours of various particle size distributions were prepared by using a size shifter. The degree of starch damage and the dynamic viscoelasticity of rice batter were measured in this work. We also baked bread of the flour of each size distribution to study processability for making bread. The batter made by the pulverized flour of rice particle size ranging from 75 to 106 μm had the highest expansion ratio and a good processability for baking breads compared to other particle size batters. The rice bread with high expansion ratio was produced by controlling particle size of crystalline rice flour without using thickening agents.
Sugarcane vinegar is produced in various countries of southern Asia. It is also a niche product of the Kagoshima and Okinawa Prefectures in Japan. Turbid compounds are sometimes found in sugarcane vinegar, thereby lowering the market value. In this study, the turbid compounds were precipitated with a 1:2 (v/v) volume of ethanol, and they were identified as α-1,6-glucan using enzymatic digestion tests and 13C nuclear magnetic resonance analysis. Moreover, Lactobacillus nagelii was isolated from sugarcane juice, and it produced α-1,6-glucan when grown with sugar. The turbid compounds found in sugarcane vinegar were assumed to be α-1,6-glucan produced from sugar by lactic acid bacteria that exist in sugarcane juice.
The structure and properties of starch reserves in rice seeds are strongly affected by deficiencies in specific starch biosynthetic enzymes, which are highly expressed in storage tissues. Rice lines with unique seed starches should be utilized for food and industrial applications in the near future. We are currently developing novel rice mutant lines with distinct starch properties by introducing specific genes from different cultivars into mutant lines and by producing multiple combinations of single mutant lines. Obtaining the homozygous genotypes of the target genes is necessary during the screening process of these materials. In this study, we developed an effective, accurate screening method for identifying rice lines with novel starch composition. Specifically, we produced a novel mutant line in which we introduced genes encoding starch synthase IIa (SSIIa) and granule-bound starch synthase I (GBSSI) from indica cultivar into a starch branching enzyme IIb (BEIIb)-deficient mutant line.