Myo-Jeong Kim

Porcine Pancreatic α-Amylase Hydrolysis of Native Starch Granules as a Function of Granule Surface Area

Porcine pancreatic α‐amylase activity on native starch granules is more accurately described as a function of surface area of the granules rather than of substrate concentration. The apparent Km of α‐amylolysis of native starch from potato, maize, and rice expressed as a function of substrate concentration was largest for potato with a single value of Vmax. However, the ratio of the slope of a Lineweaver‐Burk plot to that of rice for enzymatic hydrolysis of native potato and maize starch were 7.78 and 2.58, respectively, which were very close to the ratio of surface area per mass of the two starch granules to that of rice. Therefore, the reciprocal of initial velocity was a linear function of the reciprocal of surface area for each starch granule. Surface area was calculated assuming the starch granules were spherical. The values obtained by this calculation were in good agreement with the value obtained by the photomicrographic method. By comparing enzymatic digestion of native maize granules to that of rice granules, it was concluded that the presence of pores in maize granules appeared to significantly affect overall rate of digestion after sufficient reaction time, but not at the very initial stage of hydrolysis.

Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and beta-cyclodextrin (beta-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and beta-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched beta-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the alpha-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.

Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity

The debranching enzyme Nostoc punctiforme debranching enzyme (NPDE) from the cyanobacterium Nostoc punctiforme (PCC73102) hydrolyzes the α‐1,6 glycosidic linkages of malto‐oligosaccharides. Despite its high homology to cyclodextrin/pullulan (CD/PUL)‐hydrolyzing enzymes from glycosyl hydrolase 13 family (GH‐13), NPDE exhibits a unique catalytic preference for longer malto‐oligosaccharides (>G8), performing hydrolysis without the transgylcosylation or CD‐hydrolyzing activities of other GH‐13 enzymes. To investigate the molecular basis for the property of NPDE, we determined the structure of NPDE at 2.37‐Å resolution. NPDE lacks the typical N‐terminal domain of other CD/PUL‐hydrolyzing enzymes and forms an elongated dimer in a head‐to‐head configuration. The unique orientation of residues 25–55 in NPDE yields an extended substrate binding groove from the catalytic center to the dimeric interface. The substrate binding groove with a lengthy cavity beyond the −1 subsite exhibits a suitable architecture for binding longer malto‐oligosaccharides (>G8). These structural results may provide a molecular basis for the substrate specificity and catalytic function of this cyanobacterial enzyme, distinguishing it from the classical neopullulanases and CD/PUL‐hydrolyzing enzymes. Proteins 2010.

Influence of the Physical Form of Processed Rice Products on the Enzymatic Hydrolysis of Rice Starch in Vitro and on the Postprandial Glucose and Insulin Responses in Patients with Type 2 Diabetes Mellitus

The manufacturing processes used determined the physicochemical properties of the three kinds of rice food, garaeduk, bagsulgi, and cooked rice. The initial rate of hydrolysis by porcine pancreatic α-amylase (PPA) was affected by the food form. The firmer structure of garaeduk was apparently responsible for the difficulty in maceration, resulting in less digestion than with easily digestible food for the same maceration time. The initial rate of hydrolysis of each rice product by PPA increased with increasing maceration time in a Waring Blender for all of the processed rice products. The postprandial glucose and insulin responses to the three processed rice products were also studied in ten patients with type 2 diabetes mellitus (4 men and 6 women aged 56.8±2.3 yr; duration of diabetes, 3.6±1.2 yr; body mass index (BMI), 23.7±2.6 kg/m2; fasting serum glucose, 143.9±5.1 mg/dl; serum insulin, 20.8±2.2 μU/ml). Each subject ingested of the three rice foods after a 12-h overnight fast, and the serum glucose and insulin levels were measured over a 0–240 min period. The postprandial serum glucose and insulin levels at 90 min after ingesting bagsulgi and cooked rice were less than those at 60 min, while the levels at 90 min after ingesting garaeduk were higher than those at 60 min. Garaeduk also significantly decreased the incremental responses of glucose and insulin when compared with bagsulgi and cooked rice. The results suggest that garaeduk would be the most unlikely to increase the postprandial serum glucose and insulin levels among the three rice foods. The food form, which eventually differentiated each food by its specific surface area with the same degree of maceration because of the characteristic physical strength, therefore affected the rate of rice starch hydrolysis both in vitro and in vivo.

Maltosyl-erythritol, a Major Transglycosylation Product of Erythritol by Bacillus stearothermophilus Maltogenic Amylase

This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-α-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol.

Characterization of a novel debranching enzyme from Nostoc punctiforme possessing a high specificity for long branched chains.

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both alpha-(1,6)- and alpha-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with (14)C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-alpha-(1,6)-beta-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

Quality evaluation of fresh tomato juices prepared using high-speed centrifugal and low-speed masticating household juicers

The aim of this study was to evaluate and compare the physicochemical, nutritional, and sensory properties of homemade fresh tomato juices prepared using 2 types of household juicers, a high-speed centrifugal juicer (HSC juicer) and a low-speed masticating juicer (LSM juicer). Juice yield, soluble solids, and the contents of total polyphenol, vitamin C, and lycopene in LSM tomato juice were significantly higher than those in HSC tomato juice. The DPPH radical scavenging activity of LSM tomato juice was also higher than that of HSC tomato juice. HSC tomato juice easily separated into 2 layers with many fine bubbles, while LSM tomato juice was homogeneous. Sensory evaluation revealed that panels significantly preferred LSM tomato juice to HSC tomato juice. In conclusion, the LSM juicer shows several advantages over the HSC juicer for preparing tomato juice of superior quality and taste that is rich in antioxidant phytochemicals at a high yield.

Binding mode analysis between membrane dipeptidase and its substrates

Membrane dipeptidase (MDP) is a membrane-bound glycoprotein involved in the hydrolysis of dipeptides, showing specific activity for dipeptides. Recent study showed that membrane dipeptidase was the receptor for a lung-targeting peptide identified by in vivo phage display and the crystal structure of the cilastatin-liganded human renal dipeptidase was determined. We performed a pharmacophore-based virtual screening and molecular docking in order to characterize MDP binding interactions with its substrates. A ligand-based pharmacophore model represented only a slight enrichment because of a lacked variety and centralization of ligand features. Molecular docking study was used to incorporate ligand conformational changes in the binding sites and the performance was much better than pharmacophore model; only 10% of compound library needed to be screened in order to detect all included active compounds. In addition, we found that one of the crystallographically observed water molecules plays an important role in the binding modes between MDP and its substrate.

Enzymatic browning reaction of apple juices prepared using a blender and a low-speed masticating household juicer

ABSTRACT The aim of this study was to investigate the effect of juicer type (blender or LSM household juicer) on the browning reaction of apple juice and evaluate the remaining antioxidant activity in the juice. The blender apple juice showed a darker brown color and 4.5 times higher PPO activity than LSM apple juice. This result suggested that the blender caused severer damage to plastids in cells leading to leakage of PPO into the juice than the LSM juicer. The total polyphenol and flavonoid content of LSM apple juice was approximately 2 times higher than that of blender apple juice because polyphenols and flavonoids can be used as substrates by PPO. The antioxidant activity of LSM juice was higher than that of blender juice. Together, these results suggested that the LSM juicer is superior to the blender for preparation of fresh apple juices due to the minimization of enzymatic oxidation reactions. Abbreviations: LSM: low-speed masticating; PPO: polyphenol oxidase; ABTS: 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl GRAPHICAL ABSTRACT Graphical abstract: Low-speed masticating household juicer (LSM) minimized the enzymatic oxidation reactions in fresh apple juice compared with blender (BLD).

Kinetic analysis and enzyme concentration effect relevant to dependence of amylolysis of starch granules on specific surface area concentration

The kinetics and enzyme concentration effects on the amylolysis of rice, maize, and potato starch granules by porcine pancreatic α-amylase (PPA) were analyzed. The Km values of the native starches, when expressed as a function of surface area concentration, were similar to each other. Inconsistencies in Vmax values can be attributed to the deviations from real values of the surface area concentration. The deviations were confirmed by that the initial rate differences among the native starches with the same apparent surface area concentration were observed at low enzyme concentrations. Kinetic parameters of dimethyl sulfoxide (DMSO)-solubilized native starches showed no significant difference, demonstrating that structural and compositional differences of starches do not affect amylolysis. The resistance of starch granules against amylolysis predominantly affects the Vmax, and as a result, the kcat is reduced compared with DMSO-solubilized starch. In conclusion, the kinetic analysis and enzyme concentration effect confirmed that surface area concentration mainly affects the initial hydrolysis rate of native starch granules.