Naoto Isono

Relationship of Granule Size Distribution and Amylopectin Structure with Pasting, Thermal, and Retrogradation Properties in Wheat Starch

Starches separated from 18 Indian wheat varieties were evaluated to see relationship of granule size distribution and amylopectin structure with pasting, thermal, and retrogradation properties. Average diameter of A-, B-, and C-granules among different starches varied between 23.0 and 28.5, 10.0 and 12.0, and 2.3 and 2.7 mum, respectively. Amylopectin chain length distribution varied significantly, short length chains (DP 6-12) and long length chains (DP > 24) ranged between 44.5 and 52.4% and 3.7 and 6.5%, respectively, whereas amylose content ranged between 18.2 and 28.8%. Short length chains of amylopectin had inverse relationship with starch gelatinization temperatures T(o), T(p), and T(c). Starches with higher crystallinity had higher enthalpy of gelatinization and lower swelling power. Paste characteristics were mainly dependent upon granule type and all pasting parameters except pasting temperature, showed significant positive correlations with A-granules and negative with the proportion of B- and C-granule.

A comparative study of ethanol production by Issatchenkia orientalis strains under stress conditions

The ability of 13 strains of multi-stress-tolerant Issatchenkia orientalis yeast to produce ethanol was examined under different stress conditions, including conditions of elevated Na₂SO₄ and Na₂SO₄ concentrations and increased heat. The MF-121 strain produced a significant amount of ethanol after the incubation in acidic media containing high concentrations of salt, e.g., 50 g/l Na₂SO₄ at pH 2.0, or at high temperatures, e.g., 43°C, when compared with other strains.

Differential characteristics and subcellular localization of two starch-branching enzyme isoforms encoded by a single gene in Phaseolus vulgaris L.

Starch-branching enzymes (SBE) have a dominant role for amylopectin structure as they define chain length and frequency of branch points. We have previously shown that one of the SBE isoforms of kidney bean (Phaseolus vulgaris L.), designated PvSBE2, has a molecular mass (82 kDa) significantly smaller than those reported for isologous SBEs from pea (SBEI), maize (BEIIb), and rice (RBE3). Additionally, in contrast to the dual location of the pea SBEI in both the soluble and starch granule fractions, PvSBE2 was found only in the soluble fraction during seed development. Analysis of a pvsbe2 cDNA suggested that PvSBE2 is generated from a larger precursor with a putative plastid targeting sequence of 156 residues. Here we describe the occurrence of a larger 100-kDa form (LF-PvSBE2) of PvSBE2 found both in the soluble and starch granule fractions of the developing seeds. The determined N-terminal sequence, VKSSHDSD, of LF-PvSBE2 corresponded to a peptide sequence located 111 amino acids upstream from the N terminus of purified PvSBE2, suggesting that LF-PvSBE2 and PvSBE2 are products of the same gene. Analysis of the products by 5′-RACE (rapid amplification of cDNA ends) and reverse transcription PCR indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing. Recombinant LF-PvSBE2 (rLF-PvSBE2) was purified from Escherichia coli and the kinetic properties were compared with those of recombinant PvSBE2 (rPvSBE2). rLF-PvSBE2 had much higher affinity for amylopectin (K m = 4.4 mg/ml) than rPvSBE2 (18.4 mg/ml), whereas theV max of rLF-PvSBE2 (135 units/mg) for this substrate was much lower than that of rPvSBE2 (561 units/mg). These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for localization in starch granules by altering its enzymatic properties.

Diversity in amylopectin structure, thermal and pasting properties of starches from wheat varieties/lines

Structural, thermal and pasting diversity of starches from Indian and exotic lines of wheat was studied. Majority of the starches showed amylose content ranging between 22% and 28%. Endotherm temperatures (T(o), T(p) and T(c)) of the starches showed a range between 56-57, 60 -61 and 65.5-66.5 degrees C, respectively. Exotherms with T(p) between 87.0 and 88.2 degrees C were observed during cooling of heated starches, indicating the presence of amylose-lipid complexes. Exotherm temperatures were negatively correlated to swelling power. Amylopectin unit chains with different degree of polymerization (DP) were observed to be associated with pasting temperature, setback and thermal (endothermic T(o), T(p), and T(c)) parameters. Amylopectin unit chains of DP 13-24 showed positive relationship with endothermic T(o), T(p) and T(c). Pasting temperature showed positive correlation with short chains (DP 6-12) while negative correlation with medium chain (DP 13-24) amylopectins. Setback was positively correlated to DP 16-18 and negatively to DSC amylose-lipid parameters.

Characterization of Starch Synthase I and II Expressed in Early Developing Seeds of Kidney Bean (Phaseolus vulgaris L.)

Plant starch synthase (SS) contributes to the elongation of glucan chains during starch biosynthesis and hence plays an essential role in determining the fine structure of amylopectin. To elucidate the role of SS activity in the formation of amylopectin in kidney bean (Phaseolus vulgaris L.), a study was undertaken to isolate cDNA clones for SS and to characterize the enzymatic properties of the coded recombinant enzymes. Two SS cDNAs, designated pvss1 and pvss21, which were isolated from early developing seeds, encoded SSI and SSII (designated PvSSI and PvSSII-1) that displayed significant identity (more than 65%) with other SSI and SSII members, respectively. RNA gel blot analysis indicated that both transcripts accumulate in leaves and developing seeds at the early stage. Immunoblot analysis with antisera raised against both recombinant proteins (rPvSSI and rPvSSII-1) showed that the accumulation of both proteins parallels the gene expression profiles, although both were detectable only in starch-granule fractions. Recombinant enzymes expressed by Escherichia coli cells showed distinct chain-length specificities for the extension of glucan chains. Our results suggest that these SS isozymes for synthesis of transitory starch are also responsible for synthesis of storage starch in early developing seeds of kidney bean.

Enzymatic characterization of starch synthase III from kidney bean (Phaseolus vulgaris L.)

In plants and green algae, several starch synthase isozymes are responsible for the elongation of glucan chains in the biosynthesis of amylose and amylopectin. Multiple starch synthase isozymes, which are classified into five major classes (granule‐bound starch synthases, SSI, SSII, SSIII, and SSIV) according to their primary sequences, have distinct enzymatic properties. All the starch synthase isozymes consist of a transit peptide, an N‐terminal noncatalytic region (N‐domain), and a C‐terminal catalytic region (C‐domain). To elucidate the enzymatic properties of kidney bean (Phaseolus vulgaris L.) SSIII and the function of the N‐domain of kidney bean SSIII, three recombinant proteins were constructed: putative mature recombinant SSIII, recombinant kidney bean SSIII N‐domain, and recombinant kidney bean SSIII C‐domain. Purified recombinant kidney bean SSIII displayed high specific activities for primers as compared to the other starch synthase isozymes from kidney bean. Kinetic analysis showed that the high specific activities of recombinant kidney bean SSIII are attributable to the high kcat values, and that the Km values of recombinant kidney bean SSIII C‐domain for primers were much higher than those of recombinant kidney bean recombinant SSIII. Recombinant kidney bean SSIII and recombinant kidney bean SSIII C‐domain had similar chain‐length specificities for the extension of glucan chains, indicating that the N‐domain of kidney bean SSIII does not affect the chain‐length specificity. Affinity gel electrophoresis indicated that recombinant kidney bean SSIII and recombinant kidney bean SSIII N‐domain have high affinities for amylose and amylopectin. The data presented in this study provide direct evidence for the function of the N‐domain of kidney bean SSIII as a carbohydrate‐binding module.

Cloning and heterologous expression of a β-fructofuranosidase gene from Arthrobacter globiformis IFO 3062, and site-directed mutagenesis of the essential aspartic acid and glutamic acid of the active site

We have cloned the gene encoding a beta-fructofuranosidase from Arthrobacter globiformis IFO 3062, and subsequently, the gene was heterologously expressed in Escherichia coli. This beta-fructofuranosidase gene encodes a protein of 548 amino acid residues with a calculated molecular mass of 60,519 Da. We have examined the roles of three residues of A. globiformis IFO 3062 beta-fructofuranosidase by site-directed mutagenesis, and found that aspartic acid 130 and glutamic acid 392, which are two of the apparent catalytic residues, are essential for hydrolase activity. This study provides the first experimental evidence showing that these two amino acid residues of beta-fructofuranosidase play a critical role in hydrolyzing sucrose.

Purification and Characterization of 1,3-β-D-Glucan Phosphorylase from Ochromonas danica

1,3-β-D-Glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-β-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C–30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-β-glucans.

A wild and tolerant yeast suitable for ethanol fermentation from lignocellulose.

Ethanol fermentation from food wastes containing mainly starch without carrying out sterilization was investigated by using wild and tolerant yeast, Issatchenkia orientalis MF-121. The MF-121 strain is not a suitable choice for ethanol fermentation from lignocellulosic biomass because it is only capable of fermenting hexoses of glucose, mannose, and fructose to ethanol. Therefore, we first isolated acid- and salt-tolerant yeast that are capable of fermenting various monosaccharides to ethanol, and the isolated yeast that showed the ability to ferment ethanol from glucose, mannose, galactose, fructose, and xylose, was identified as Zygoascus hellenicus LK-5G on the basis of the 26S rRNA sequence analysis.