Kayoko Kawakami

Production of dipeptidyl peptidase IV inhibitory peptides from defatted rice bran

The insulinotropic hormone glucagon-like peptide-1 is metabolised extremely rapidly by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, human DPP-IV is a key regulator involved in the prevention and treatment of type 2 diabetes. To simplify the method of producing an inhibitory peptide against DPP-IV, we focused on rice bran (RB) as a source and subjected proteins from defatted RB to enzymatic proteolysis using 2 commercial enzymes. The RB peptides produced with Umamizyme G exhibited 10 times the inhibitory activity as those produced with Bioprase SP. The half-maximal inhibitory concentration (IC(50)) value of the RB peptides was 2.3 ± 0.1mg/ml. Leu-Pro and Ile-Pro were identified as the inhibitory peptides among the RB peptides produced with Umamizyme G. Ile-Pro was the strongest DPP-IV inhibitor among the 15 Xaa-Pro dipeptides and Pro-Ile tested. Ile-Pro competitively inhibited DPP-IV (K(i)=0.11 mM). Mass spectrometry indicated that the contents of Leu-Pro and Ile-Pro in the RB peptides were 2.91 ± 0.52 μg/mg.

Enzymatic Production of Ferulic Acid from Defatted Rice Bran by Using a Combination of Bacterial Enzymes

Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an α-l-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.

The structural analysis and the role of calcium binding site for thermal stability in mannanase

Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K(a) of 3.02 × 10(4) M(-1) and 1.52 × 10(4) M(-1), respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.

Inhibitory effect of collagen-derived tripeptides on dipeptidylpeptidase-IV activity

Abstract The collagen tripeptide fragments Gly-Ala-Hyp, Gly-Pro-Ala and Gly-Pro-Hyp were generated by hydrolyzing collagen from pig-skin, cattle-skin, fish-scales and chicken-feet, respectively, with Streptomyces collagenase. Collagenase treatment increased the concentration of tripeptides in the hydrolysates by 13–15% (w/w). Of the three peptides, Gly-Pro-Hyp was a true peptidic inhibitor of dipeptidylpeptidase-IV (DPP-IV), because DPP-IV could not hydrolyze the bond between Pro-Hyp. This tripeptide was a moderately competitive inhibitor (Ki = 4.5 mM) of DPP-IV, and its level in the collagen hydrolysates could be greatly increased (4–9% [w/w]) using Streptomyces collagenase.

Binding of bivalent ions to actinomycete mannanase is accompanied by conformational change and is a key factor in its thermal stability.

The study aimed to define the key factors involved in the modulation of actinomycete mannanases. We focused on the roles of carbohydrate-binding modules (CBMs) and bivalent ions. To investigate the effects of these factors, two actinomycete mannanase genes were cloned from Streptomyces thermoluteus (StManII) and Streptomyces lividans (SlMan). CBMs fused to mannanase catalytic domains do not affect the thermal stability of the proteins. CBM2 of StManII increased the catalytic efficiency toward soluble-mannan and insoluble-mannan by 25%-36%, and CBM10 of SlMan increased the catalytic efficiency toward soluble-mannan by 40%-50%. Thermal stability of wild-type and mutant enzymes was enhanced by calcium and manganese. Thermal stability of SlMandC was also slightly enhanced by magnesium. These results indicated that bivalent ion-binding site responsible for thermal stability was in the catalytic domains. Thermal stability of mannanase differed in the kinds of bivalent ions. Isothermal titration calorimetry revealed that the catalytic domain of StManII bound bivalent ions with a K(a) of 5.39±0.45×10(3)-7.56±1.47×10(3)M(-1), and the catalytic domain of SlMan bound bivalent ions with a K(a) of 1.06±0.34×10(3)-3.86±0.94×10(3)M(-1). The stoichiometry of these bindings was consistent with one bivalent ion-binding site per molecule of enzyme. Circular dichroism spectrum revealed that the presence of bivalent ions induced changes in the secondary structures of the enzymes. The binding of certain bivalent ion responsible for thermal stability was accompanied by a different conformational change by each bivalent ion. Actinomycete mannanases belong to GHF5 which contained various hemicellulases; therefore, the information obtained from mannanases applies to the other enzymes.

Hepatoprotective effects of rice-derived peptides against acetaminophen-induced damage in mice

Glutathione, the most abundant intracellular antioxidant, protects cells against reactive oxygen species induced oxidative stress and regulates intracellular redox status. We found that rice peptides increased intracellular glutathione levels in human hepatoblastoma HepG2 cells. Acetaminophen is a commonly used analgesic. However, an overdose of acetaminophen causes severe hepatotoxicity via depletion of hepatic glutathione. Here, we investigated the protective effects of rice peptides on acetaminophen-induced hepatotoxicity in mice. ICR mice were orally administered rice peptides (0, 100 or 500 mg/kg) for seven days, followed by the induction of hepatotoxicity via intraperitoneal injection of acetaminophen (700 mg/kg). Pretreatment with rice peptides significantly prevented increases in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels and protected against hepatic glutathione depletion. The expression of γ-glutamylcysteine synthetase, a key regulatory enzyme in the synthesis of glutathione, was decreased by treatment with acetaminophen, albeit rice peptides treatment recovered its expression compared to that achieved treatment with acetaminophen. In addition, histopathological evaluation of the livers also revealed that rice peptides prevented acetaminophen-induced centrilobular necrosis. These results suggest that rice peptides increased intracellular glutathione levels and could protect against acetaminophen-induced hepatotoxicity in mice.

Inhibitory Effects of Pomegranate Extracts on Recombinant Human Maltase–Glucoamylase

α-Glucosidase inhibitors are currently used in the treatment of type 2 diabetes. In this study, we investigated the inhibitory activities of aril and pericarp extracts from pomegranates obtained various regions against recombinant human maltase-glucoamylase (MGAM). The inhibitory activities of the aril extracts tended to be stronger than those of the pericarp extracts. The Iranian aril extract was the most effective inhibitor. We investigated the polyphenol content of the pomegranate extracts using the Folin-Ciocalteu method. Among the aril extracts, the Iranian aril extract showed the highest polyphenol content. We further evaluated inhibitory activity against α-glucosidase from the rat small intestine. Pomegranate extract used in this study showed slightly different inhibitory activities according to α-glucosidase origin. Iranian aril extract was the most effective inhibitor of α-glucosidases, especially recombinant human MGAM. Bioassay-guided fractionation of the pomegranate arils led to identification of punicalagin and oenothein B as potent inhibitors of α-glucosidase. Oenothein B showed inhibitory activity with a half-maximal inhibitory concentration (IC(50)) value of 174 μM. Its potency was comparable to that of the α-glucosidase inhibitor acarbose with an IC(50) value of 170 μM. Dixon plot kinetic analysis of oenothein B showed a noncompetitive inhibition with a K(i) value of 102 μM. These results suggest that pomegranate arils would be useful for suppressing postprandial hyperglycemia.

Sake lees hydrolysate protects against acetaminophen-induced hepatotoxicity via activation of the Nrf2 antioxidant pathway

Acetaminophen is a commonly used analgesic. However, an overdose of acetaminophen causes severe hepatotoxicity via depletion of hepatic glutathione. Here, we investigated the protective effects of sake lees hydrolysate against acetaminophen-induced hepatotoxicity in mice. Sake lees hydrolysate was administered orally to ICR mice for seven days. Six hours after acetaminophen treatment, the mice were sacrificed, and blood and liver samples were collected for analysis. Treatment with acetaminophen markedly increased the levels of serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase. Pretreatment with sake lees hydrolysate significantly prevented the increases in the serum levels of these enzymes and inhibited acetaminophen-mediated glutathione depletion. In addition, histopathological evaluation of the livers also revealed that sake lees hydrolysate prevented acetaminophen-induced centrilobular necrosis. The expression of γ-glutamylcysteine synthetase (γ-GCS), hemeoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the liver were decreased after acetaminophen treatment, whereas pretreatment with sake lees hydrolysate led to an increased expression of all three proteins. Furthermore, sake lees hydrolysate induced the expression of these proteins in HepG2. These results suggested that sake lees hydrolysate could induces HO-1 and γ-GCS expression via activation of the Nrf2 antioxidant pathway, and protects against acetaminophen-induced hepatotoxicity in mice.