• Polymer(Korea)
• /
• v.29 no.6
• /
• pp.613-616
• /
• 2005

In this word as the first step to develop optical biosensors for a single cell level analysis, the preparation method of nano-scale polymer hydrogel spheres containing an enzyme was set up and the feasibility of the spheres as optical biosensors was investigated. The horseradish peroxidase (HRP) was encapsulated in the PEG hydrogel spheres by suspension photopolymerization, yielding spheres of the average size of 305 nm. After the polymerization, the incorporation and activity of HRP within the spheres were determined by the production of fluorescence resulted from the enzymatic reaction between HRP and $\H_{2}O_{2}$. The fluorescence emission response of the HRP-loaded PEG hydrogel spheres increased by nearly 300$\%$ as hydrogen peroxide concentration was changed from 0 to 11 nM in the presence of Amplex Red. The results suggest that the method to prepare the PEG hydrogel nanospheres containing an enzyme could be used for developing optical biosensors to measure various analytes in the very small samples like a single cell.

• Food Science and Biotechnology
• /
• v.18 no.2
• /
• pp.570-573
• /
• 2009

The debranching enzyme of Nostoc punctiforme (NPDE) is a novel enzyme that catalyzes the hydrolysis of $\alpha$-1,6-glycosidic linkages in starch, followed by the sequential hydrolysis of $\alpha$-1,4-glycosidic linkages. The debranching activity of NPDE is highly specific for branched chains with a degree of polymerization (DP)>8. Moreover, the rate of hydrolysis of $\alpha$-1,4-linkages by NPDE is greatly enhanced for maltooligosaccharides (MOs) with a DP>8. An analysis of reaction mixtures containing various starches revealed the accumulation of maltooctaose (G8) with glucose and maltose. Based on the novel enzymatic properties of NPDE, an MO mixture containing more than 60% G8 with yield of 18 g G8 for 100 g starch was prepared by the reaction of NPDE with soluble starch, followed by ethanol precipitation and gel permeation chromatography (GPC). The yield of the G8-rich mixture was significantly improved by the addition of isoamylase. In summary, a 4-step process for the production of a G8-rich mixture was developed involving the enzymatic hydrolysis of starch by NPDE.

• Journal of the Korean Wood Science and Technology
• /
• v.24 no.1
• /
• pp.48-53
• /
• 1996

Brown rot fungus, Tyromyces palustris, has been reported to cause the loss of strength accelerated by oxalate, a non-enzymatic low molecular weight acid, with minute weight loss of decaying wood in early stage. The production of oxalate in relation to wood decaying and the presence of oxaloacetase. an oxalate producing enzyme, were identified during the process. Tyromyces palustris produced the largest amount of oxalate among brown rot fungi. In order to find out the cleavage of pulp fiber, we submerged pulp fiber in oxalate solution and the results showed that the number of short pulp fiber was highly increased, compared with control solution. The pH of decaying wood was decreased to 1.77 which was close to that of saturated oxalate solution, pH 1.2, Thus, the oxalate was thought to be accumulated in the decaying wood, The oxaloacetase which accelerates production of oxalate was derived from fungus, and the production of oxalate by the enzyme was determined by using on UV/Vis spectrophotometer. Therefore, the oxalate was found to be produced by oxaloacetase during decay. The oxalate may cause the acid-hydrolysis of cellulose and hemicellulose. The oxalate was thought to reduce the degree of polymerization and increase the enzyme activity, which resulted in rapid loss of strength in early stage-an identical feature of brown rot fungus.

• Journal of Korean Ophthalmic Optics Society
• /
• v.20 no.2
• /
• pp.237-246
• /
• 2015

Purpose: The present study was conducted to reveal the correlation of structural denaturation and decrease of enzyme activity when the antioxidative enzymes, superoxide dismutase (SOD) and catalase (CAT) were repeatedly exposed to UV-B, and further investigate whether the denaturation and inactivation of those enzymes can be effectively blocked by using UV-inhibitory RGP lens. Methods: Each enzyme solution was prepared from the standardized SOD and CAT, and repeatedly exposed to UV-B of 312 nm for 30 minutes, 1 hour and 2 hours a day over 1, 2, 3, 4 and 5 days. Structural denaturation of SOD and CAT induced by repeat UV-B irradiation was confirmed by the electrophoretic analysis, and their enzyme activity was determined by the colorimetric assay using the proper assay kit. At that time, the change in structure and activity of the antioxidant enzymes directly exposed to UV-B was compared to the case that UV-B was blocked by UV-inhibitory RGP lens. Results: SOD exposed repeatedly to UV-B showed the polymerization pattern in the electrophoretic analysis when it repeatedly exposed for 30 min a day, however, the change of its activity was less than 10%. On the other hand, CAT repeatedly exposed to UV-B reduced size and density of the electrophoretic band which indicated a structure denaturation, and its activity was significantly decreased. In the case that the repeat exposure time was longer, CAT activity was completely lost even though some enzyme band occurred in the electrphoretic analysis. In addition, the degeneration of CAT due to UV-B irradiation was inhibited to some extent by using RGP lens with a UV-B blocking of 63.7%, however, it was not completely inhibited. Conclusions: From these results, it was revealed that the structural denaturation of antioxidative enzymes was not perfectly correlated with the reduction in enzyme activity according to the type of enzyme. It is recommended to minimize the exposure time to UV when wearing contact lens, or wear the contact lenses having UV blocking rate of the FDA Class I blocker or the sunglasses having equivalent UV-blocking rate for reducing the damage of antioxidative enzymes induced by UV.

• Applied Biological Chemistry
• /
• v.38 no.2
• /
• pp.118-122
• /
• 1995

Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of $DH5{\alpha}$, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the s?????? values as those of the wild type enzyme. The $k_{cat}$, of the enzyme was $1.0{\times}10^5min^{-1}$, and $K_ms$ for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.

• Journal of Environmental Science International
• /
• v.18 no.7
• /
• pp.797-802
• /
• 2009

To control degradation rate of biodegradable poly(lactide)s (PLA), the stereochemical PLAs with different ratios of d-lactide and l-lactide units were synthesized by the ring open polymerization and the their degradation kinetics were measured by a Langmuir film balance. The alkaline (pH=11) degradation of poly(l-lactide) (l-PLA) monolayer showed the faster rate at a surface pressure of 4 mN/m in the ranges from to 0 to 7 mN/m. However, the enzymatic degradation of l-PLA with Proteinase K did not occur until 4 mN/m. Above a constant surface pressure of 4 mN/m, the degradation rate was increased with a constant surface pressure. These behaviors might be attributed to the difference in the contacted area with degradation medium: alkaline ions need small contact area with l-PLA while enzymes require much bigger one to be activated due to different medium sizes. The stereochmical PLA monolayers showed that the alkaline degradation was increased with their optical impurities while the enzymatic one was inversed. These results could be explained by the decrease of crystallinity with the optical impurity and the inactivity of enzyme to d-LA unit.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.5
• /
• pp.837-845
• /
• 2016

A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.

• 한국생물공학회:학술대회논문집
• /
• 2002.04a
• /
• pp.528-530
• /
• 2002

We have previously reported recombinant deletion mutant was constructed from cycloinulo-oligosaccharide fructanotransferase(CFTase) gene of Xanthmonas oryzae MGL21. Purification of the mutant protein from E. coli and reation condition for the production of inulo-oligosaccharide(ISO) were studied. The molecular mass of the CFTase deletion mutant protein was estimated to be 90kDa by SDS- Polyacrylamide gel electrophoresis. Optimum reaction pH and temperature were pH6.5 and $40^{\circ}C$, respectively. Under optimal reaction conditions, endo-inulinase activating enzyme was rapidly produced ISO from inulin. Components were DP(degree of polymerization) 3and 4 with trace amount of smaller oligosaccharides.

• Microbiology and Biotechnology Letters
• /
• v.32 no.4
• /
• pp.347-351
• /
• 2004

Hydrolysis of manno-oligosaccharides and galactomannan was studied with the purified Bacillus subtilis WL-7 mannanase from recombinant Eschericoli. The predominant products of hydrolysis were mannose, mannobiose and mannotriose. The enzyme could hydrolyze $\beta$-1 A-linked manno-oligosaccharides larger than mannobiose, but was not active on mannobiose. When the mannanase hydrolyzed manno-oligo saccharides of degree of polymerization(DP) 4-6, it was more active on the substrate of higher DP. Based on analysis of transient reaction products by TLC, the enzyme was found to have a preference for internal $\beta$-IA-mannosidic linkages, which are the central mannosidic bond of mannotetraose and the two middle mannosidic bonds of mannopentaose. The $\beta$-l A-mannosidic bonds situated at the second and fourth positions from the nonreducing end of mannohexaose were preferenhydrolyzed by the mannanase. Locust bean gum(LBG) was enzymatically hydrolyzed with higher efficiency than guar gum, resulting that amount of reducing sugars was liberated more efficiently from LBG than guar gum with same activity of mannanase.

• The Korean Journal of Food And Nutrition
• /
• v.9 no.4
• /
• pp.413-418
• /
• 1996

Two methods were used to extract pectin from apple cell wall by enzyme(Hemicellulase) and by acid. Hemicellulase was used to extract high functional pectin with higher degree of polymerization. The yield of weight of pectin by hemicellulase treatment was slightly higher than that by acid treatment. The optimal condition for extraction was accomplished by providing 1.5 grams of Hemicellulase at 38$^{\circ}C$ for 24 hours. The comparison of the pectin purity and the yield of extracts by the portion of galacturonic acid between two methods showed that the purity of pectin extracted by enzymatic method was lower than that by acid treatment.