Hyun-Dong Shin

Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon.

NADPH, a major reducing power in microorganisms, is mostly generated from the pentose phosphate (PP) pathway by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) expressed by the zwf and gnd genes, respectively. The characteristics of these two genes in Escherichia coli were compared after their re-introduction into the parent strain for over-expression. zwf encoding G6PDH increased the level of NADPH 3 folds compared to gnd encoding 6PGDH. An excess of NADPH depressed cell growth mainly due to the inhibition of citrate synthase in the TCA cycle. Recombinant plasmids containing zwf or gnd co-integrated with the phbCAB operon from Ralstonia eutropha were constructed, and introduced into E. coli for the oddball biosynthesis of PHB. The amount of PHB increased after enforcing the genes; especially the zwf gene an increase of around 41%, due to the rise in NADPH and the depressed TCA cycle, leading to the metabolic flux of intermediates to the pathway for the biosynthesis of PHB.

Effects of ß-glucan fromAureobasidium pullulans on acute inflammation in mice

The effects of ß-glucan isolated fromAureobasidium pullulans were observed on acute xylene-induced inflammation, ß-glucan at a dose of 62.5, 125 or 250 mg/kg were administered once orally to xylene-treated mice (0.03 mL of xylene was applied on the anterior surface of the right ear to induce inflammation), and the body weight change, ear weight, histological profiles and histomorphometrical analyses of ear were conducted upon sacrifice. The xylene was topically applied 30min after dosing with ß-glucan. The results were compared to those of diclofenac, indomethacin and dexamethasone (15 mg/kg injected once intraperitoneally). All animals were sacrificed 2 h after xylene application. Xylene application resulted in marked increases in induced ear weights compared to that of intact control ear; hence, the differences between intact and induced ear were also significantly increased. The histological characteristics of acute inflammation, such as severe vasodilation, edematous changes of skin and infiltration of inflammatory cells, were detected in xylene-treated control ears with marked increase in the thickness of the ear tissues. However, these xylene-induced acute inflammatory changes were significantly and dose-dependently decreased by ß-glucan treatment. We conclude that ß-glu-can fromA. pullulans has a somewhat favorable effect in the reduction of the acute inflammatory responses induced by xylene application in mice.

Metabolic engineering of pentose phosphate pathway in Ralstonia eutropha for enhanced biosynthesis of poly-β-hydroxybutyrate

Poly‐β‐hydroxybutyrate (PHB) biosynthesis in Ralstonia eutrophafrom gluconate as a carbon source is carried out through the Entner‐Doudoroff (ED) pathway and the pentose‐phosphate (PP) pathway generating NADPH and glyceraldehyde‐3‐phosphate that flows to acetyl‐CoA, actively in the unbalanced PHB accumulation phase. The gnd gene encoding 6‐phosphogluconate dehydrogenase (6PGDH) and the tktA gene encoding the transketolase (TK) in PP pathway of E. coli were transformed into R. eutropha H16 to modify the metabolic flux of gluconate to the PHB biosynthesis. Over‐generated NADPH by the amplified gnd gene tended to depress the cell growth and PHB concentration. Meanwhile, the amplified tktA gene significantly increased both PHB biosynthesis and cell growth as a result of the effective flow of glyceraldehyde‐3‐phosphate into acetyl‐CoA along with the concomitant supplementation of NADPH. The amplified tktA gene also activated the enzyme activities directly associated with PHB biosynthesis. The transformant R. eutropha harboring tktA gene was cultivated using pH‐stat‐fed‐batch to achieve the overproduction of PHB.

Enantioselective hydrolysis of insoluble (R, S)-ketoprofen ethyl ester in dispersed aqueous reaction system induced by chiral cyclodextrin

The enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester to the optically active (S)-ketoprofen was carried out in a dispersed aqueous lipase reaction system induced by the inclusion of chiral cyclodextrins for complexation of the substrate. Hydroxypropyl-β-cyclodextrin was the most effective chiral selector and disperser giving an enantiomeric excess and conversion yield of 0.99 and 0.49, respectively.

Role of tktA gene in pentose phosphate pathway on odd-ball biosynthesis of poly-β-hydroxybutyrate in transformant Escherichia coli harboring phbCAB operon

A cloned tktA gene encoding transketolase (TK), the most critical enzyme in the nonoxidative pentose phosphate (PP) pathway, was reinforced into the parent Escherichia coli for metabolic flux control of carbohydrate. It was also transformed into the transformant E. coli harboring phbCAB operon for the odd-ball biosynthesis of PHB. The biosynthesis of PHB significantly increased up to 1.7-fold after the cotransformation of the phbCAB operon and tktA gene, due to the active supplementation of the precursor molecules, acetyl-CoA and NADPH.

Immobilization of Cyclodextrin Glucanotransferase on Amberlite IRA-900 for Biosynthesis of Transglycosylated Xylitol

Cyclodextrin glucanotransferase (CGTase) fromThermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L−1·h−1, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.

Esterification of hydrophobic substrates by lipase in the cyclodextrin induced emulsion reaction system

Cyclodextrin (CD), a doughnut-shaped molecule having a hydrophilic surface and hydrophobic interior, can form inclusion complexes with various hydrophobic guest molecules inducing them into the emulsion state. The CD-induced emulsion reaction system was applied to the esterification between the hydrophobic oleic acid as the carboxylic acid and n-butanol as the alcohol catalyzed by lipase. The rate and yield were substantially increased in the CD-induced emulsion reaction system. The optimal reaction condition and the kinetic constants of the esterification reaction were determined. The enhancing mechanism was investigated through comparing the microscopic observation of the reaction mixtures, the inclusion complex formability, and the distribution of substrates, product and lipase at aqueous phase and CD-induced water immiscible phase in the CD-induced emulsion reaction system. The increase seems to have been caused by three reasons, the increased accessibility of lipase due to the emulsion formation, the shift of the equilibrium to the product formation side through the decrement of the affinity of butyl oleate with lipase, and the activation of lipase adsorbed on interface of oil-droplets in CD-induced water immiscible phase.

Isolation and characterization of Acinetobacter sp. ES-1 excreting a lipase with high enantioselectivity for (S)-ketoprofen ethyl ester

A new Acinetobacter sp. ES-1, grown on triolein, tryptone and Triton X-100, excreted a lipase that hydrolyzed 10m M (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10Uml-1 and, at 30°C and pH7 over 48h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.

In vivo blending of medium chain length polyhydroxy-alkanoates and polyhydroxybutyrate using recombinant Pseudomonas putida harboring phbCAB operon of Ralstonia eutropha

The in vivo blending of medium chain length polyhydroxyalkanoates (mcl-PHA) and polyhydroxybutyrate (PHB) was carried out using recombinant Pseudomonas putida after transforming the phbCAB operon of Ralstonia eutropha. The most suitable carbon sources for the production of mcl-PHA and PHB blends were identified to be octanoate and gluconate. The molar fractions of 3-hydroxyoctanoate and 3-hydroxybutyrate in the polymer blends were effectively modulated by controlling the mixing ratio of octanoate and gluconate, thereby producing a composition ranging from 95% mcl-PHA to 78% PHB.

Enzymatic hydrolysis of hydrophobic triolein by lipase in a mono-phase reaction system containing cyclodextrin; Reaction characteristics

A hydrophobic substrate triolein was hydrolyzed by lipase in a mono-phase reaction system containing cyclodextrin(CD) as emulsifier. The triolein was transformed to an emulsion-like state in the CD containing reaction system in contrast to the oil-droplet like state without CD due to the formation of an inclusion complex between the lipids and CDs. The hydrolysis reaction increased substantially in the CD containing reaction system, and the optimum reaction conditions including the amount of lipase, β-CD concentration, and mixing ratio of triolein and β-CD, were determined. The performance of the enzyme reaction in a mono-phase reaction system was compared with that of a two-phase reaction system which used water immiscible hexane as the organic solvent. The role of a CD in the mono-phase reaction system was elucidated by comparing the degree of the inclusion complex formation with triolein and oleic acid, Km and Vmax values, and product inhibition by oleic acid in aqueous and CD containing reaction systems. The resulting enhanced reaction seems to be caused by two phenomena; the increased accessibility of lipase to triolein and reduced product inhibition by oleic acid through the formation of an inclusion complex.