Tae-Jip Kim

Biotechnological production of human milk oligosaccharides.

Human milk contains a large variety of oligosaccharides (HMOs) that have the potential to modulate the gut flora, affect different gastrointestinal functions, and influence inflammatory processes. This review introduces the recent advances in the microbial and coupled enzymatic methods to produce HMOs with grouping them into trisaccharides (sialyllactose and fucosyllactose) and complex oligosaccharides (lacto-N-biose derivatives). The high purity and low cost of HMOs should make their use possible in new fields such as the food or pharmaceutical industries.

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.

A novel amylolytic enzyme from Thermotoga maritima, resembling cyclodextrinase and α-glucosidase, that liberates glucose from the reducing end of the substrates☆

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.

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.

Production of L-lactate in Leuconostoc citreum via heterologous expression of L-lactate dehydrogenase gene.

D-form lactate is often found in fermented foods and excessive dietary intake of D-lactate may cause metabolic stress in both infants and patients. Leuconostoc citreum is a major lactic acid bacterium that produces D-lactate in fermented foods. The aim of this study was to change the pyruvate carbon flux in L. citreum from D-lactate into L-lactate by heterologous expression of L-lactate dehydrogenase (ldhL) gene. For this, ldhL from Lactobacillus plantarum was cloned and introduced into L. citreum using a shuttle vector pLeuCM. In the transformant, ldhL was successfully transcribed and L-lactate dehydrogenase was expressed. As a consequence of transformation, the ratio between D- and L-isomers was changed due to the increment of L-lactate and the decrement of D-lactate, but no significant differences were found in total lactate concentration between the host and transformant cells. This is the first report of metabolic engineering in Leuconostoc by modulating the central carbon flux into health-favored way.

Characterization of the major dehydrogenase related to d-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293

Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate mainly to d-(-)-lactic acid by using d-(-)-lactate dehydrogenase (ldhD). The aim of this study was to identify the gene responsible for d-lactic acid formation in this organism and to characterize the enzyme to facilitate the production of optically pure d-lactic acid. A genomic analysis of L. mesenteroides ATCC 8293 revealed that 7 genes encode lactate-related dehydrogenase. According to transcriptomic, proteomic, and phylogenetic analyses, LEUM_1756 was the major gene responsible for the production of d-lactic acid. The LEUM_1756 gene, of 996bp and encoding 332 amino acids (36.5kDa), was cloned and overexpressed in Escherichia coli BL21(DE3) Star from an inducible pET-21a(+) vector. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 4450U/mg, significantly higher than those of other previously reported ldhDs. The gel permeation chromatography analysis showed that the purified enzyme exists as tetramers in solution and this was the first report among lactic acid bacteria. The pH and temperature optima were pH 8.0 and 30°C, respectively, for the pyruvate reduction reaction, and pH 11.0 and 20°C, respectively, for the lactate oxidation reaction. The K(m) kinetic parameters for pyruvate and lactate were 0.58mM and 260mM, respectively. In addition, the k(cat) values for pyruvate and lactate were 2900s(-1) and 2280s(-1), respectively. The enzyme was not inhibited by Ca(2+), Co(2+), Cu(2+), Mg(2+), Mn(2+), Na(+), or urea, but was inhibited by 1mM Zn(2+) and 1mM SDS.

Detailed modes of action and biochemical characterization of endo-arabinanase from Bacillus licheniformis DSM13

An endo-arabinanase (BLABNase) gene from Bacillus licheniformis DSM13 was cloned and expressed in Escherichiacoli, and the biochemical properties of its encoded enzyme were characterized. The BLABNase gene consists of a single open reading frame of 987 nucleotides that encodes 328 amino acids with a predicted molecular mass of about 36 kDa. BLABNase exhibited the highest activity against debranched α-(1,5)-arabinan in 50 mM sodium acetate buffer (pH 6.0) at 55°C. Enzymatic characterization revealed that BLABNase hydrolyzes debranched or linear arabinans with a much higher activity than branched arabinan from sugar beet. Enzymatic hydrolysis pattern analyses demonstrated BLABNase to be a typical endo-(1,5)-α-s-arabinanase (EC 3.2.1.99) that randomly cleaves the internal α-(1,5)-linked L-arabinofuranosyl residues of a branchless arabinan backbone to release arabinotriose mainly, although a small amount of arabino-oligosaccharide intermediates is also liberated. Our results indicated that BLABNase acts preferentially along with the oligosaccharides longer than arabinopentaose, thus enabling the enzymatic production of various arabino-oligosaccharides.

Isolation and characterization of human intestinal Enterococcus avium EFEL009 converting rutin to quercetin.

Quercetin is a flavonol believed to have beneficial effects on human health. Rutin, found in many plants, fruits and vegetables, is metabolized by human intestinal bacteria and converted to quercetin, where it is absorbed through the intestinal epithelium. This study aimed to isolate and characterize human intestinal bacteria capable of converting rutin to quercetin. A bacterium that can metabolize rutin was isolated from human faecal samples and identified by 16S rRNA gene sequencing. The whole‐cell enzymatic activities on flavonoid glycoside and the conversion profiles of the isolate were also analysed. The bacterium was identified as Enterococcus avium EFEL009 and was shown to convert rutin to isoquercetin and then to quercetin under anaerobic conditions. Microscopic analysis revealed short chains of cocci with diameters of approx. 1 μm. β‐Glucosidase was shown to be constitutively expressed in Ent. avium, while α‐rhamnosidase was expressed following induction by rutin. Both enzymes were mainly localized to the cell surface. This study is the first report on the isolation of a quercetin‐producing Ent. avium FEEL009, which could be a potential industrial starter bacterium.

A Double-Blind, Placebo Controlled-Trial of a Probiotic Strain Lactobacillus sakei Probio-65 for the Prevention of Canine Atopic Dermatitis.

Canine atopic dermatitis (CAD) is a ubiquitous, chronic inflammatory skin disorder prevalent in dogs, which results in production of abnormal levels of IgE antibodies in reciprocation to an allergen challenge. In this study, administration of the probiotic strain Lactobacillus sakei probio-65 for 2 months significantly reduced the disease severity index in experimental dogs diagnosed with CAD. In addition, one month pre-medication of L. sakei probio-65 revealed significant difference in the PVAS score in experimental dogs for both probio-65 and placebo groups. However, post 2 months treatment resulted in a significant decrease in the CASESI score values in the probio-65-treated group (p < .0.06).

Effect of temperature and ph on interconversion between fructose and mannose catalyzed by Thermotoga neapolitana mannose-6-phosphate isomerase

The gene encoding a putative mannose-6-phosphate isomerase (TnMPI) from Thermotoga neapolitana DSM4359 was cloned and expressed in Escherichia coli. TnMPI showed the highest isomerization activity between d-fructose and d-mannose at 75°oC in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM of Cu2+. TnMPI can be activated by some divalent metal ions, such as Cu2+, Mn2+, and Co2+. In the presence of 1 mM Cu2+, TnMPI activity on conversion from d-fructose to d-mannose was significantly enhanced up to 271% of that without Cu2+. In addition, its isomerization equilibrium between d-fructose and Dmannose was strongly affected by reaction temperature and pH. As reaction temperature decreased from 95 to 55°C, the equilibrium ratio of d-fructose to d-mannose was gradually shifted from 73:27 to 55:45. As reaction pH decreased from pH 8.5 to 5.5, the equilibrium ratio of Dfructose to d-mannose was shifted from 68:32 to 49:51.