Ken’ichi Ohtsubo

Recent development of phosphorylases possessing large potential for oligosaccharide synthesis

Phosphorylases are one group of carbohydrate active enzymes involved in the cleavage and formation of glycosidic linkages together with glycoside hydrolases and sugar nucleotide-dependent glycosyltransferases. Noticeably, the catalyzed phosphorolysis is reversible, making phosphorylases suitable catalysts for efficient synthesis of particular oligosaccharides from a donor sugar 1-phosphate and suitable carbohydrate acceptors with strict regioselectivity. Although utilization of phosphorylases for oligosaccharide synthesis has been limited because only few different enzymes are known, recently the number of reported phosphorylases has gradually increased, providing the variation making these enzymes useful tools for efficient synthesis of diverse oligosaccharides.

Important Sensory Properties Differentiating Premium Rice Varieties

In rice-consuming countries, specific varieties are recognized as premium, “gold standard” varieties, while others are recognized as being superior but second best, despite being identical using the current suite of tools to evaluate quality. The objectives of this study were to determine if there are distinguishable differences in sensory properties of premium and second best varieties and whether these differences are common to premium varieties. Color, an important sensory property, was determined on the raw and cooked rice using a colorimeter. As raw rice, some of the premium varieties were whiter than their second best counterparts while others were not. However, when cooked, with two exceptions, the premium varieties were of the same or greater whiteness than their counterparts. A trained sensory panel employed descriptive sensory analysis, an objective tool, to characterize and analytically measure the flavor (aromatics, taste, mouthfeel) and texture of premium and second best varieties collected from nine rice-consuming countries. Sweet taste, popcorn aroma/flavor, and water-like metallic mouthfeel showed significant differences in intensity between the premium–second best variety pairs. Slickness, roughness, and springiness were the major traits that distinguished the texture of varieties. Quality evaluation programs do not routinely measure these texture and flavor traits, but the fact that they distinguished the varieties in most pairs indicates that their measurement should be added to the suite of grain quality tests in the development of new higher-yielding, stress-tolerant varieties. The incorporation of premium quality will ensure that quality is no impediment to widespread adoption leading to enhanced productivity and food security.

Avidin Expressed in Transgenic Rice Confers Resistance to the Stored-Product Insect Pests Tribolium confusum and Sitotroga cerealella

Rice (Oryza sativa var. Nipponbare) was transformed with an artificial avidin gene. The features of this construct are as follows: (1) a signal peptide sequence derived from barley alpha amylase was added at the N-terminal region, (2) codon usage of the gene was optimized for rice, and (3) the gene was driven by rice glutelin GluB-1, an endosperm-specific promoter. Avidin was produced in the grain of the transgenic rice but not in the leaves. The concentration of avidin in the kernels was about 1,800 ppm. All larvae of the confused flour beetle (Tribolium confusum) and Angoumois grain moth (Sitotroga cerealella) died when fed transgenic avidin rice powder or kernels, respectively, whereas most of the test insects developed into adults when they were fed a nontransgenic rice control diet. Avidin extracted from the transgenic rice kernel lost most biotin-binding activity after 5 min heating at 95 °C.

Palatable and bio-functional wheat/rice products developed from pre-germinated brown rice of super-hard cultivar EM10.

It became possible to produce high-quality and bio-functional wheat/rice bread and wheat/rice noodles by blending, pre-germinated and cooked brown rice of a super-hard cultivar with wheat flour. Super-hard rice (SHR) is not suitable for table rice because of its low palatability. Nevertheless, it was found to be suitable as a blending material for bread-making or noodle-making due to its hard texture and high content of resistant starch. We developed a novel rapid germination method to improve the quality and to save the time needed for germination. By blending pre-germinated and cooked SHR (30% w/w on a dry basis) as a rice gel with wheat flour (70% w/w on a dry basis), the bread became very soft and any hardening after bread-making was markedly retarded. Similarly, blending pre-germinated and cooked SHR as cooked a rice gel with wheat flour gave high-quality noodles with a similar texture to that of durum semolina noodles. The resistant starch of the SHR-blended bread and noodles was also markedly increased. White waxy rice (9%) soaked and cooked with the pre-germinated brown rice of SHR (21%) produced a rice gel that was very useful as a material for bread-making and noodle-making by blending with wheat flour (70%) to prepare soft, tasty and bio-functional wheat/rice bread and wheat/rice noodles.

Grain qualities and their genetic derivation of 7 new rice for Africa (NERICA) varieties.

NERICA are interspecific rice varieties from crossing between the high-yielding Asian rice ( Oryza sativa spp. Japonica) with locally adapted African rice ( Oryza glaberrima). In this study, we analyzed grain qualities of 7 NERICA varieties (NERICA 1 to 7) and genetic derivation of quality-related genes. Quality analyses of NERICA grains showed that 7 NERICA varieties were clearly classified into two groups based on the difference of amylose content, and the difference influenced the pasting and physical properties of grains. Genetic analysis of the gene encoding granule-bound starch synthase I (GBSSI), which is known as a key enzyme on amylose synthesis in rice grain, revealed that varieties with higher amylose content ( approximately 29%) have the gene derived from O. glaberrima parent, and group 2 with lower amylose content ( approximately 22%) have the gene from O. sativa parent. These results indicated that the difference in amylose content as well as grain properties among 7 NERICA varieties is mainly determined by the genetic derivation of GBSSI. Further genetic analysis of starch synthesis-related genes suggested that the genetic derivation of SSIIa also influences the chain length of amylopectin in 7 NERICA varieties.

Characteristics of pregelatinized ae mutant rice flours prepared by boiling after preroasting.

As ae mutant rice, such as EM10, lacks the starch branching enzyme IIb, its amylopectin contains more long-chain glucans than that of ordinary Indica and Japonica rice grains. Although boiled grains of ae rice cultivars are too hard and nonsticky for table rice, they are promising in terms of biofunctionality, such as prevention of diabetes. The present paper investigates the characterization of a novel group of four ae mutant rice cultivars (EM72, EM145, EM174, and EM189). They were subjected to the evaluation for their main chemical components, physical properties, and enzyme activities at different grain conditions (raw milled rice, roasted rice, boiled rice, and rice boiled after preroasting). These mutant rice grains are characterized by high apparent amylose, high protein and high glucose contents, high pasting temperature, high α-amylase activities, high resistant starch, and low degree of gelatinization. A novel method was developed to maintain the high resistant starch contents of gelatinized rice grains. Rice boild after preroasting showed a higher ratio of resistant starch and a lower amount of glucose than ordinary boiled rice. It became possible to produce high-quality and biofunctional pregelatinized rice flours by boiling with frozen fruits, such as tomatoes, after rice grains had been preroasted. These ae mutants were found to be suitable materials for rice/fruit or rice/vegetable products to serve as palatable, low-glucose, and high resistant starch rice products.

Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi

A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4‐O‐β‐d‐glucopyranosyl‐d‐gluconic acid: phosphate α‐d‐glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α‐d‐glucose 1‐phosphate and d‐gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively.

Influence of Physicochemical Properties of Rice Flour on Oil Uptake of Tempura Frying Batter

The physicochemical properties of rice flour and wheat flour influenced the oil uptake of tempura frying batter. Rice flour was better than wheat flour in the overall quality and crispness of the fried tempura batter. Rice flour resisted oil absorption more than wheat flour, and a higher level of apparent starch amylose and higher consistency/breakdown ratio of the pasting properties led to a lower oil uptake of the batter. Super hard EM10 rice showed the highest apparent amylose content and higher consistency/breakdown ratio than the other flour samples, the batter from EM10 revealing the lowest oil content after frying among all the batters examined. The apparent amylose content, consistency/breakdown ratio and oil absorption index are proposed as useful guides for oil absorption when frying from among the physicochemical properties that influence the oil content of fried batter. Our proposal for the “oil absorption index” could be a simple, although not perfect method for estimating the oil content of batter flour.

2-O-α-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on β-D-glucose 1-phosphate.

The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s−1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-d-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a k cat of 2.8 s−1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H2 18O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-d-glucosylglycerol (GG) at the rate of 180 s−1. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s−1. We propose 2-O-α-d-glucopyranosylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-d-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.

Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch.

Not only amylose but also amylopectin greatly affects the gelatinization properties of rice starch and the quality of cooked rice grains. We here characterized the starches of 32 rice cultivars and evaluated the relationship between their iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch (RS) content, and chain length distribution of amylopectin. We found that the iodine absorption curve differed among the various sample rice cultivars. Using the wavelength at which absorbance becomes maximum on iodine staining of starch (λmax), we propose a novel index, “new λmax” (AAC/(λmax of sample rice starches–λmax of glutinous rice starch)). We developed the novel estimation formulae for AAC, RS contents, and amylopectin fractions with the use of λmax and “new λmax.” These formulae would lead to the improved method for estimating starch properties using an easy and rapid iodine colorimetric method. Graphical Abstract We developed the novel estimation formulae for amylose, resistant starch contents, and amylopectin fractions based upon analyses of the iodine absorption curve.