Oleyl-branched oligosaccharide phosphate (OA-BOS-P) was prepared by phosphorylation through dry-heating branched oligosaccharide (BOS) from corn starch with metaphosphric acid, and then by oleylating the resulting branched oligosaccharide phosphate (BOS-P) through lipase-catalyzed solid phase synthesis. The multi-functionality of OA-BOS-P was evaluated in respect of its interfacial ability, Ca2+-binding ability, and ability to control the gelatinization and retrogradation behavior of potato starch. OA-BOS-P exhibited markedly lower surface tension and interfacial tension than either BOS-P or oleyl BOS (OA-BOS), and revealed Ca2+-binding ability similar to that of BOS-P. OA-BOS-P offered improved gelatinization behavior, as indicated by the elevated gelatinization temperature, reduced enthalpy, and reduced peak viscosity and breakdown, in comparison with those properties of other related samples. OA-BOS-P also inhibited retrogradation as indicated by the reduced setback viscosity, turbidity and development of an ordered structure depending on the level of addition, whereas BOS-P and OA-BOS elevated the turbidity, in spite of the reduced setback value and development of an ordered structure. OA-BOS-P could therefore be a useful multi-functional food material with interfacial, Ca2+-binding, and starchy food-controlling abilities.
The effects of lysine (Lys), monosodium glutamate (GluNa), glycine (Gly) and alanine (Ala) on the viscosity, turbidity, morphological features, solubility of saccharides and molecular size distribution of the dissolved saccharides in retorted potato starch paste were investigated. Gly and Ala with zero net charge-containing pastes showed a large decrease in the viscosity due to collapse of the swollen starch granules by the retort treatment, whereas GluNa and Lys with a positive or negative net charge could inhibit such viscosity change and collapse of the starch granules. The turbidity of GluNa- and Lys-containing pastes evaluated by the absorbance at 500 nm was higher than that of Gly- and Ala-containing pastes. The dissolved saccharides for each retorted paste mainly consisted of low-molecular-weight molecules below Mr 600. GluNa and Lys could strongly suppress the increased viscosity and turbidity of the retorted paste during storage for 7 days at 5 and 55°C, although Gly and Ala had little effect. The charged amino acids could thus be applied to improving the viscosity and turbidity of the retorted starch paste by maintaining the swollen starch granules.
In this study, the physicochemical properties of starches from transgenic sweetpotato plants modified by RNA interference of the starch synthase II (SSII) gene were examined. The method of genetic manipulation developed during our previous study using White Star cultivar was applied to Konahomare cultivar, which has promising features of high yield and high starch content. SSII gene suppression resulted in the following consistent effects being observed in both cultivars: lowering of gelatinization temperature and gelatinization enthalpy, increase in B-type feature in X-ray diffractograms, decrease in the phosphate content of starch and alteration in chain-length distributions as determined by gel-permeation chromatography and high-performance anion-exchange chromatography. Furthermore, starches from all transgenic lines showed slower retrogradation and higher digestibility by glucoamylase compared to those from control starches. These traits of transgenic starches were similar to those from a starch of Quick Sweet cultivar, suggesting that mutation in the SSII gene could be one of the reasons for the unique property of starch in Quick Sweet. Thus, our RNA interference technique for sweetpotato was successfully applied to starch engineering in the promising cultivar of sweetpotato for starch material, Konahomare. The SSII gene inhibition significantly modified the basic architecture of starch and subsequently altered its functional properties.
Azasugars are known as potent inhibitors of glycoside hydrolases. In this study, we examined the inhibition of Cellvibrio gilvus cellobiose phosphorylase (CBP) by four azasugars (isofagomine, 1-deoxynojirimycin, castanospermine and calystegine B2) and a non-azasugar (glucono-1,5-lactone). Isofagomine strongly inhibited CBP, whereas 1-deoxynojirimycin, castanospermine, and glucono-1,5-lactone exhibited moderate or weak inhibition. Calystegine B2 did not inhibit CBP. Kinetic analysis in the presence of sulfate indicated that it is an extremely weak competitive inhibitor against phosphate. Moreover, crystal structures of CBP complexed with isofagomine or 1-deoxynojirimycin were determined, revealing molecular recognition of the glucosidase inhibitors by the phosphorolytic enzyme. These inhibitors are bound at subsite −1 and form several hydrogen bonds with the protein and anion (phosphate or sulfate). The strong inhibition by isofagomine is probably due to an electrostatic interaction between its endocyclic amino group and phosphate.
This work aims to characterize disproportionating enzyme (DPE1) and its isoform DPE2 in rice. Rice DPE genes (OsDPE1 and OsDPE2) were cloned and expressed in E. coli. The OsDPE1 and OsDPE2 genes encode proteins of 594 and 946 amino acids with a calculated molecular mass of 67 kDa and 108 kDa, respectively. Purified recombinant OsDPE1 and OsDPE2 showed highest activity at around pH 7.0 and pH 6.0-7.0, respectively. The optimum reaction temperature was 30°C for OsDPE1 and 39°C for OsDPE2. Recombinant OsDPE1 disproportionates maltotriose to produce glucose and maltopentaose, and thus shares the defining behavior of D-enzymes. In our experiments, recombinant OsDPE2 catalyzed the glucose transfer reaction from maltose to an acceptor molecule such as glycogen. We also characterized the differences between the diurnal transcription profiles of OsDPE1 and OsDPE2 in rice leaves and seeds, and their temporal expression levels in developing rice seeds.
Xylanases belonging to glycoside hydrolase (GH) family 11 have a wide range of pH optima. A single residue, which is located adjacent to the acid/base catalyst, is primarily responsible for pH optimum determination. This residue is Asp in acidophilic xylanases, whereas it is Asn in neutrophilic and alkaliphilic ones. Aspergillus kawachii produces 2 GH11 xylanases, acidophilic XynC, which has Asp37 at this position, and neutrophilic XynB, which has Asn43. To investigate the mechanism of pH optimum determination in these xylanases, we constructed various mutant enzymes, including mutations of the Asp/Asn residue. Their pH-dependent activities were measured using a natural xylan substrate or a synthetic substrate, o-nitrophenyl β-xylobioside. A D37N mutation raised the pH optimum of XynC from 2.8 to 5.5, whereas an N43D mutation lowered the pH optimum of XynB from 4.2 to 3.6. Crystallographic analysis on the D37N mutant of XynC suggested that a hydrogen bond between Asp(Asn)37 and the acid/base catalyst is weakened by the mutation. Kinetic analysis of the mutants suggested that the ionization states of the ES complex dictate the acidophilicity of XynC. Therefore, mutants of other residues in the active site cleft were also examined, and it was shown that Glu118 and Tyr10 also contribute to the extreme acidophilicity of XynC. Interestingly, an F131W mutation in XynC increased the activity toward the synthetic substrate by 8.4-fold. Crystallographic analysis on the F131W mutant suggested that optimization of the aromatic side chain packing in the substrate-binding cleft increases the catalytic activity.
To investigate the substrate recognition at the minus subsites of glycoside hydrolase family 10 xylanases, the kinetic parameters of four xylanases on all four p-nitrophenyl β-glycosides of β-1,4-gluco/xylo-disaccharides were determined. All four xylanases hydrolyzed all the four substrates examined. The Km values of all the enzymes on the four substrates lined up in the same order, indicating that both the subsites -1 and -2 of all the enzymes prefer xylose to glucose. The comparison of the parameters on the substrates gave detailed information on the substrate recognition at each subsite -1 and -2.
The present paper established new methods for determining the activities of starch branching enzyme (BE) and starch synthase (SS). The methods are based on the calculation of the data on chain-length distribution obtained from the fluorophore-assisted carbohydrate capillary electrophoresis (FACE) method, in which the molar percentage of each linear chain comprising the branched glucan can be precisely determined. The FACE method can give us two important results at the same time, namely the chain-length distribution and the enzyme activity, for characterization of BE and SS. Although the activity of BE has been quantitatively determined by measuring the reducing power of the isoamylorysates of the reaction product and the substrate, this method has difficulties in having a high background value when branched glucan such as amylopectin and glycogen is used as substrate in the in vitro reaction, whereas the FACE method has no such disadvantages.
Quantification of orthophosphate (Pi) in the presence of labile phosphate esters is required for biochemical assays. We developed a method for the enzymatic colorimetric quantification of Pi using pyruvate oxidase and peroxidase. The calibration curve was not affected by the presence of labile phosphate esters. Furthermore, this method allows continuous monitoring of the reaction of Pi-releasing enzymes.
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