Doman Kim

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.

Production of antioxidant compounds by culture of Panax ginseng C.A. Meyer hairy roots

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, established by infecting ginseng root discs with Agrobacterium rhizogenes, were used for secondary metabolite production. In this study, several elicitors [salicylic acid (SA), acetylsalicylic acid (ASA), yeast elicitor, and bacterial elicitor] were used to improve the productivity of useful metabolite in P. ginseng hairy root cultures. In SA elicitation, total ginseng saponin content increased slightly at lower elicitor dosages (0.1 to 0.5 mM). Also, the use of ASA as an elicitor resulted in the inhibition of biomass growth and an increase in total ginseng saponin content at every elicitor dosage (0.1 to 1.0 mM) by about 1.1 times. With yeast elicitor addition, hairy root growth was inhibited about 0.8-fold on a dry weight basis compared to the control, but total ginseng saponin content increased by about 1.17 times when compared to the control. The bacterial elicitor showed a slight inhibition of biomass growth, but total ginseng saponin content increased by about 1.23 times upon the addition of 1 mL.

Production of rubusoside from stevioside by using a thermostable lactase from Thermus thermophilus and solubility enhancement of liquiritin and teniposide.

Solubility is an important factor for achieving the desired plasma level of drug for pharmacological response. About 40% of drugs are not soluble in water in practice and therefore are slowly absorbed, which results in insufficient and uneven bioavailability and GI toxicity. Rubusoside (Ru) is a sweetener component in herbal tea and was discovered to enhance the solubility of a number of pharmaceutically and medicinally important compounds, including anticancer compounds. In this study, thirty-one hydrolyzing enzymes were screened for the conversion of stevioside (Ste) to Ru. Recombinant lactase from Thermus thermophiles which was expressed in Escherichia coli converted stevioside to rubusoside as a main product. Immobilized lactase was prepared and used for the production of rubusoside; twelve reaction cycles were repeated with 95.4% of Ste hydrolysis and 49 g L(-1) of Ru was produced. The optimum rubusoside synthesis yield was 86% at 200 g L(-1), 1200 U lactase. The purified 10% rubusoside solution showed increased water solubility of liquiritin from 0.98 mg mL(-1) to 4.70±0.12 mg mL(-1) and 0 mg mL(-1) to 3.42±0.11 mg mL(-1) in the case of teniposide.

Facile preparation of water soluble curcuminoids extracted from turmeric (Curcuma longa L.) powder by using steviol glucosides.

Curcuminoids from rhizomes of Curcuma longa possess various biological activities. However, low aqueous solubility and consequent poor bioavailability of curcuminoids are major limitations to their use. In this study, curcuminoids extracted from turmeric powder using stevioside (Ste), rebaudioside A (RebA), or steviol glucosides (SG) were solubilized in water. The optimum extraction condition by Ste, RebA, or SG resulted in 11.3, 9.7, or 6.7mg/ml water soluble curcuminoids. Curcuminoids solubilized in water showed 80% stability at pH from 6.0 to 10.0 after 1week of storage at 25°C. The particle sizes of curcuminoids prepared with Ste, RebA, and SG were 110.8, 95.7, and 32.7nm, respectively. The water soluble turmeric extracts prepared with Ste, RebA, and SG showed the 2,2-diphenyl-1-picrylhydrazyl radical scavenging (SC50) activities of 127.6, 105.4, and 109.8μg/ml, and the inhibition activities (IC50) against NS2B-NS3(pro) from dengue virus type IV of 14.1, 24.0 and 15.3μg/ml, respectively.

The effect of fermented buckwheat on producing l-carnitine- and γ-aminobutyric acid (GABA)-enriched designer eggs

BACKGROUND The potential of fermented buckwheat as a feed additive was studied to increase l-carnitine and γ-aminobutyric acid (GABA) in designer eggs. Buckwheat contains high levels of lysine, methionine and glutamate, which are precursors for the synthesis of l-carnitine and GABA. Rhizopus oligosporus was used for the fermentation of buckwheat to produce l-carnitine and GABA that exert positive effects such as enhanced metabolism, antioxidant activities, immunity and blood pressure control. RESULTS A novel analytical method for simultaneously detecting l-carnitine and GABA was developed using liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS. The fermented buckwheat extract contained 4 and 34 times more l-carnitine and GABA respectively compared with normal buckwheat. Compared with the control, the fermented buckwheat extract-fed group showed enriched l-carnitine (13.6%) and GABA (8.4%) in the yolk, though only l-carnitine was significantly different (P < 0.05). Egg production (9.4%), albumen weight (2.1%) and shell weight (5.8%) were significantly increased (P < 0.05). There was no significant difference in yolk weight, and total cholesterol (1.9%) and triglyceride (4.9%) in the yolk were lowered (P < 0.05). CONCLUSION Fermented buckwheat as a feed additive has the potential to produce l-carnitine- and GABA-enriched designer eggs with enhanced nutrition and homeostasis. These designer eggs pose significant potential to be utilized in superfood production and supplement industries.

Synthesis of oligosaccharide-containing orange juice using glucansucrase

Orange juice is a well-accepted fruit juice, and is a natural source of various vitamins, especially vitamin C, as well as sugar, potassium, thiamine, folate, flavonoids and antioxidants. The respective fructose, glucose, and sucrose concentrations were 9.3, 22.9, and 48.1 g/L in the original orange juice used in this study, and 183.4, 170.1, and 142.8 g/L after concentration. Over 97% of the sucrose in the juice was enzymatically converted to glucooligosaccharides upon addition of 3 U/mL dextransucrase, prepared from Leuconostoc mesenteroides 512FMCM, at 16°C. The synthesized oligosaccharides comprised 35.0% of the total saccharides in the concentrated juice and 31.7% in the original juice. The optimum conditions for oligosaccharide synthesis using the concentrated juice were 35.2 × 10−1 U/mL dextransucrase and 1% Ca(OH)2. The calories in the original and modified concentrated orange juices were 325.4 and 246.7 kcal/L, respectively. Compared to the original concentrated juice, the enzyme-modified concentrated juice prevented the formation of 62.7% of the insoluble glucan resulting from addition of mutansucrase, produced by Streptococcus mutans.

Synthesis and characterization of glucosyl stevioside using Leuconostoc dextransucrase

Glucosyl stevioside was synthesized via transglucosylation by dextransucrase from Leuconostoc citreum KM20 (LcDexT), forming α-d-glucosyl stevioside. A production yield of 94% was reached after 5days of LcDexT reaction at 30°C. Glucosyl stevioside induced a 2-fold improved quality of taste and sweetness, compared to stevioside. After 15days of storage at 25°C, 98% of glucosyl stevioside in an aqueous solution was present in a soluble form, compared to only 11% for stevioside or rebaudioside A. Furthermore, glucosyl stevioside exhibited a similar or improved stability in commercially available soft drinks, when compared to stevioside and rebaudioside A. These results suggest that glucosyl stevioside could serve as a highly pure and stable sweetener in soft drinks.

Production of steviol from steviol glucosides using β-glycosidase from Sulfolobus solfataricus.

Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer. Through fermentation, SSbgly was expressed as a 61kDa protein with activity of 24.3U/mg and the OD600 of 23 was reached after 18h induction with 10mM lactose. Purified protein was obtained by Ni-Sepharose chromatography with a yield of 92.3%. SSbgly hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50U/ml SSbgly and 90mg/ml Ste at 75°C as determined by the response surface method.

Enhanced Production of Butanol and Isopropanol from Sugarcane Molasses Using Clostridium beijerinckii optinoii

Sugarcane molasses was studied as a substrate for butanol and isopropanol fermentation by Clostridium beijerinckii optinoii in 10 L batch fermentations. Using 3% glucose as a carbon source, the solvent concentrations (butanol and isopropanol) and solvent yields were 10.03 g/L and 0.43 g/g, respectively, with a sugar utilization of 81.7%. However, on 2.5% glucose medium supplemented with an additional 0.5% sugar supplied as sugarcane molasses, sugar consumption was 100% and the solvent concentrations (13.37 g/L) and solvent yields (0.45 g/g) were both higher. Sugarcane molasses (3%) with or without invertase and without P2 vitamin/mineral solution produced a solvent concentration of 12.15 ~ 12.81 g/L and solvent yield of. 0.39 ~ 0.41 g/g with sugar consumption of 94.88 ~ 100%. This work demonstrated the value of sugarcane molasses as a supplementary nutrient for glucose fermentation for butanol and isopropanol production using C. beijerinckii optinoii as well as its value as a low cost carbon and media source.

Molecular cloning and characterization of a novel glucansucrase from Leuconostoc mesenteroides subsp. mesenteroides LM34

Leuconostoc mesenteroides LM34 was isolated from kimchi, a traditional fermented Korean food. L. mesenteroides LM34 produced extracellular glucansucrase (DSRLM34), which is responsible for the synthesis of soluble glucan using sucrose. The DSRLM34 gene consists of a 4,503 bp open reading frame (ORF) and encodes an enzyme of 1,500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (98%) to that of glucansucrase of Lactobacillus lactis. The gene was over-expressed in Escherichia coli strain and the recombinant enzyme (rDSRLM34) was purified. Both DSRLM34 and rDSRLM34 synthesized glucan mainly containing α-1, 6 glucosidic linkage and branched α-1, 3 glucosidic linkages. The enzyme exhibited optimum activity at 30°C and pH 5.0. DSRLM34 has promising potential as a thickening agent in sucrose-supplemented milk.