Hiroki Hamada

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.

Glycosylation of capsaicin Derivatives and phenylpropanoid Derivatives Using cultured plant cells

Biotransformations of capsaicinoids such as capsaicin and 8-nordihydrocapsaicin and phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid have been investigated using cultured plant cells. Capsain and 8-nordihydrocapsaicin were converted into the corresponding glycosides which are three glycosides respectively using the cultured cells of Catharanthus roseus. In a time-course study under sterile conditions, the changes in amounts of their reaction products were determined. Furthermore phenypropanoid, such as cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid have been biotransformed using the cultured cells of the Eucalyptus perriniana, and then cinnamic acid was converted into two glycosides. In addition, p-coumaric acid, caffeic acid and ferulic acid were converted into four, four and three glycosides respectively. Then in time-course study under sterile conditions, the change in amounts of their reaction products were determined. Finally it was found that the cultured plant cells have the ability to glycosylate the phenolic group of capsacinoids and phenylpropanoids regioselectively.

Enzymatic Synthesis and Anti-Allergic Activities of Curcumin Oligosaccharides

Curcumin 4‘-O-glucooligosaccharides were synthesized by a two step-enzymatic method using almond β-glucosidase and cyclodextrin glucanotransferase (CGTase). Curcumin was glucosylated to curcumin 4‘-O-β-D-glucopyranoside by almond β-glucosidase in 19% yield. Curcumin 4‘-O-β-D-glucopyranoside was converted into curcumin 4‘-O-β-glucooligosaccharides, i.e. 4‘-O-β-maltoside (51%) and 4‘-O-β-maltotrioside (25%), by further CGTase-catalyzed glycosylation. Curcumin 4‘-O-β-glycosides showed suppressive action on IgE antibody formation and inhibitory effects on histamine release from rat peritoneal mast cells.

Chemo-Enzymatic Synthesis of Ester-Linked 2-Phenylindole-3-Carboxaldehyde-Monosaccharide Conjugate as Potential Prodrug

Chemo-enzymatic synthesis of ester-linked 2-phenylindole-3-carboxaldehyde-glucose conjugate (2-phenylindole-3-carboxyl-10″-O-β-D-glucosyl ester) was achieved by using plant cell cultures as biocatalysts. The anticancer agent, 2-phenylindole-3-carboxaldehyde, induced apoptosis in cells, whereas 2-phenylindole-3-carboxyl-10″-O-β-D-glucosyl ester showed no cytotoxicity and induced no apoptosis.

Synthesis of Capsaicin Glycosides and 8-Nordihydrocapsaicin Glycosides as Potential Weight-Loss Formulations:

The enzymatic synthesis of capsaicin glycosides and 8-nordihydrocapsaicin glycosides was investigated using almond β-glucosidase and cyclodextrin glucanotransferase (CGTase). Capsaicin and 8-nordihydrocapsaicin were converted into their β-glucoside and β-maltooligosaccharide (amylose conjugate), i.e. β-maltoside and β-maltotrioside, by sequencial glycosylation with almond β-glucosidase and CGTase. The β-glucoside and β-maltoside of capsaicin and β-glucoside of 8-nordihydrocapsaicin showed inhibitory effects on high-fat-diet-induced elevations in body weight of mice.

Chemo-Enzymatic Synthesis of Glycolyl-Ester-Linked Taxol-Monosaccharide Conjugate and Its Drug Delivery System Using Hepatitis B Virus Envelope L Bio-Nanocapsules

Chemo-enzymatic synthesis of glycolyl-ester-linked taxol-glucose conjugate, ie, 7-glycolyltaxol 2′-O-α-D-glucoside, was achieved by using α-glucosidase as a biocatalyst. The water-solubility of 7-glycolyltaxol 2′-O-α-D-glucoside (21 μM) was 53 fold higher than that of taxol. The hepatitis B virus envelope L particles (bio-nanocapsules) are effective for delivering 7-glycolyltaxol 2′-O-α-D-glucoside to human hepatocellular carcinoma NuE cells.

Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world.

Bioremediation of Benzophenone by Glycosylation with Immobilized Marine Microalga Chrysocampanulla spinifera and Amphidinium crassum

Reduction and glycosylation of benzophenone, which is an endocrine disrupting chemical, were investigated using immobilized marine microalga and plant cells from the viewpoint of bioremediation of benzophenone. Immobilized marine microalga of Chrysocampanulla spinifera reduced benzophenone to diphenylmethanol. Immobilized marine microalga of Amphidinium crassum glucosylated diphenylmethanol to the corresponding glucoside. The sequential biotransformation with C. spinifera and A. crassum effectively converted benzophenone into diphenylmethyl glucoside. On the other hand, immobilized plant cells of Catharanthus roseus transformed benzophenone to diphenylmethanol, diphenylmethyl glucoside, and diphenylmethyl primeveroside, which was a new compound, by one-step biotransformation.