Cheon-Seok Park

Assessment of phenolics-enriched extract and fractions of olive leaves and their antioxidant activities

Recent studies suggest that olive leaf is a significant source of bioactive phenolic compounds comparable to olive oil and fruits. Identifying appropriate extraction methods is thus an important step to increase the yield of such bioactive components from olive leaf, which is otherwise agricultural waste. The present study evaluates phenolic contents and compositions of olive leaf extracted by several solvent methods and to further establish their antioxidant activities using various radical scavenging systems. Total flavonoid and phenolic contents were significantly higher in the 80% ethanol extract, butanol, and ethylacetate fractions than hexane, chloroform and water fractions (p<0.05). Oleuropein was identified as a major phenolic compound with considerable contents in these major three fractions and the extract that correlated with their higher antioxidant and radical scavenging. These results indicate that olive leaf contains significant amounts of oleuropein and phenolics, important factors for antioxidant capacity, which can be substantially modified by different extraction methods.

Development of a RAPD-PCR method for identification of Bacillus species isolated from Cheonggukjang.

A RAPD-PCR (Randomly Amplified Polymorphic DNA-PCR) method was developed for rapid identification of Bacillus species, especially B. subtilis, B. licheniformis, and B. amyloliquefaciens, the most frequently isolated organisms from fermented soy foods such as Cheonggukjang, a Korean traditional food. A RAPD-PCR using a 10-mer (S-30) produced species specific bands reproducibly. All B. subtilis strains tested produced common bands of 0.5 and 0.88 kb in size. All B. amyloliquefaciens strains generated 1.1 and 1.5 kb bands together with 0.5 kb fragment whereas B. licheniformis strains produced 1.25, 1.70, and 1.9 kb bands with an occasional 0.5 kb band. Using the RAPD-PCR protocol, six bacilli strains isolated from Cheonggukjang were identified to the species level, which was difficult by 16S rRNA gene and recA gene sequencing for some isolates. The 0.5 kb fragment, the major band for B. subtilis strains, was an internal part of a ytcP gene encoding a hypothetical ABC-type transporter. A B. subtilis species specific primer pair was designed based on ytcP sequences and PCR using the primer pair produced a 0.46 kb fragment only from B. subtilis strains.

Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea

Amylosucrase (ASase, EC 2.4.1.4) is a member of family 13 of the glycoside hydrolases that catalyze the synthesis of an alpha-(1-->4)-linked glucan polymer from sucrose instead of an expensive activated sugar, such as ADP- or UDP-glucose. Transglycosylation reactions mediated by the ASases of Deinococcus geothermalis (DGAS) and Neisseria polysaccharea (NPAS) were applied to the synthesis of salicin glycosides with sucrose serving as the glucopyranosyl donor and salicin as the acceptor molecule. Two salicin glycoside transfer products were detected by TLC and HPLC analyses. The synthesis of salicin glycosides was very efficient with NPAS with a yield of over 90%. In contrast, DGAS specifically synthesized only one salicin transglycosylation product. The transglycosylation products were identified as alpha-d-glucopyranosyl-(1-->4)-salicin (glucosyl salicin) and alpha-D-glucopyranosyl-(1-->4)-alpha-D-glucopyranosyl-(1-->4)-salicin (maltosyl salicin) by NMR analysis. The ratio between donor and acceptor had a significant effect on the type of product that resulted from the transglycosylation reaction. With more acceptors present in the reaction, more glucosyl salicin and less maltosyl salicin were synthesized.

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.

Molecular Cloning and Functional Expression of a New Amylosucrase from Alteromonas macleodii

The presence of amylosucrase in 12 Alteromonas and Pseudoalteromonas strains was examined. Two Alteromonas species (Alteromonas addita KCTC 12195 and Alteromonas macleodii KCTC 2957) possessed genes that had high sequence homology to known amylosucrases. Genomic clones containing the ASase analogs were obtained from A. addita and A. macleodii, and the deduced amino acid sequences of the corresponding genes (aaas and amas, respectively) revealed that they were highly similar to the ASases of Neisseria polysaccharea, Deinococcus radiodurans, and Deinococcus geothermalis. Functional expression of amas in Escherichia coli was successful, and typical ASase activity was detected in purified recombinant AMAS, whereas the purified recombinant AAAS was nonfunctional. Although maximum total activity of AMAS was observed at 45 °C, the ratio of transglycosylation to total activity increased as the temperature decreased from 55 to 25 °C. These results imply that transglycosylation occurs preferentially at lower temperatures while hydrolysis is predominant at higher temperatures.

Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana

The genes encoding beta-N-acetylglucosaminidase (nagA and cbsA) from Thermotoga maritima and Thermotoga neapolitana were cloned and expressed in Escherichia coli in order to investigate whether Thermotoga sp. is capable of utilizing chitin as a carbon source. NagA and CbsA were purified to homogeneity by HiTrap Q HP and Sephacryl S-200 HR column chromatography. Both enzymes were homodimers containing a family 3 glycoside hydrolase (GH3) catalytic domain, with a monomer molecular mass of 54 kDa. The optimal temperatures and pHs for the activities of the beta-N-acetylglucosaminidases were found to be 65-75 degrees C and 7.0-8.0, respectively. Both enzymes hydrolyzed chitooligomers such as di-N-acetylchitobiose and tri-N-acetylchitotriose, and synthetic substrates such as p-nitrophenyl-beta-D-glucose (pNPGlc), p-nitrophenyl N-acetyl beta-D-glucosamine (pNPGlcNAc), p-nitrophenyl di-N-acetyl beta-D-chitobiose (pNPGlcNAc(2)) and p-nitrophenyl tri-N-acetyl beta-D-chitotriose (pNPGlcNAc(3)). However, the enzymes had no activity against p-nitrophenyl-beta-D-galactose (pNPGal) and p-nitrophenyl N-acetyl beta-D-galactosamine (pNPGalNAc) or highly polymerized chitin. The k(cat) and K(m) values were determined for pNPGlcNAc, pNPGlcNAc(2) and pNPGlcNAc(3). The k(cat)/K(m) value for pNPGlcNAc was the highest among three synthetic substrates. NagA and CbsA initially hydrolyzed p-nitrophenyl substrates to give GlcNAc, suggesting that the enzymes have exo-activity with chitin oligosaccharides from the non-reducing ends, like other beta-N-acetylglucosaminidases. However, NagA and CbsA can be distinguished from other GH3-type beta-N-acetylglucosaminidases in that they are highly active against di-N-acetylchitobiose. Thus, the present results suggest that the physiological role of both enzymes is to degrade the chitooligosaccharides transported through membrane following hydrolysis of chitin into beta-N-acetylglucosamine to be further metabolized in Thermotoga sp.

Molecular cloning and functional characterization of a sucrose isomerase (isomaltulose synthase) gene from Enterobacter sp. FMB-1.

Aims:u2002 Isomaltulose (palatinose) is a slowly digestible sucrose isomer that can reduce both the glycemic and insulinemic response to foods. The aim of this study was to clone and express a sucrose isomerase (SIase) gene and characterize the protein that is responsible for the production of isomaltulose in the micro‐organism Enterobacter sp. FMB‐1.