• Journal of Microbiology and Biotechnology
• /
• v.7 no.2
• /
• pp.151-156
• /
• 1997

Two commercially available lipases, Lipase OF (non-specific lipase from Candida rugosa) and Lipolase 100T (1, 3-specific lipase from Aspergillus niger), were immobilized on insoluble hydrophobic support HDPE (high density polyethylene) by the physical adsorption method. Hydrolysis performance was enhanced by mixing a non-specific Lipase OF and a 1, 3-specific Lipolase 100T at a 2 : 1 ratio. The results also showed that the immobilized lipase maintained its activity at broader temperature ($25~55^{\circ}C$) and pH (4-8) ranges than soluble lipases. In the presence of organic solvent (isooctane), the immobilized lipase retained most of its activity in upto 12 runs of hydrolysis experiment. However, without organic solvent in the reaction mixture, the immobilized lipase maintained most of its activity even after 20 runs of hydrolysis experiment.

• Korean Journal of Agricultural Science
• /
• v.36 no.1
• /
• pp.111-122
• /
• 2009

When the cattail pollen was identified by using fibrinolytic agents, we found that the fibrinolytic activity was controlled by an enzyme. Therefore, for determining the fibrinolytic activity of cattail pollen, the fibrinolytic enzyme in cattail pollen was purified by gel filtration using DEAE-cellulose, Sephadex G-150 and HPLC. Also, its purity was certified by polyacrylamide gel electrophoresis, and its physico-chemical properties, such as pH and temperature stabilities and effects of metal, inhibitors and substrates, were examined. The specific activity, purification fold, and molecular weight of the enzyme were 38U/mg, 86.4,and 75kDa, respectively. The optimum pH for the purified enzyme was at 4.0 and it was stable at pH 4.0-6.0. The optimum temperature was $55^{\circ}C$ and it was stable at $30-60^{\circ}C$. But the enzyme began to be inactivated at $70^{\circ}C$ and its activity was totally lost at temperatures above $80^{\circ}C$. As for substrate specificity, the enzyme was most effective in dissolving fibrin, followed by whole casein, ${\kappa}$-casein, ${\alpha}$-casein, ${\beta}$-casein, and BSA. With casein as the substrate, Km value was found to be 0.44mM and the enzyme showed a high affinity for casein. As for the metal ions affecting enzyme activity, $K^+$, $Na^+$, and $Mg^{2+}$ had no effect on enzyme reaction while $Zn^{2+}$ and $Fe^{2+}$ showed potent inhibitory activity. Judging from the fact that the purified enzyme was also strongly inhibited by PMSF, iodoacetic acid, and SDA, it assumed to be a serine protease.

• Proceedings of the Korean Society for Bioinformatics Conference
• /
• 2005.09a
• /
• pp.249-255
• /
• 2005

Angiotensin-converting enzyme (ACE) is primarily responsible for human hypertension. Current ACE drugs show serious cough and angiodema health problems due to the un-specific activity of the drug to ACE protein. The availability of ACE crystal structure (1UZF) provided the plausible biological orientation of inhibitors to ACE active site (C-domain). Three-dimensional quantitative structure-activity relationship (3D-QSAR) models have been constructed using the comparative molecula. field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) for a series of 28 ACE inhibitors. Alignment for CoMFA obtained by docking ligands to 1UZF protein using FlexX program showed better statistical model as compared to superposition of corresponding atoms. The statistical parameters indicate reasonable models for both CoMFA (q$^2$ = 0.530, r$^2$ = 0.998) and CoMSIA (q$^2$= 0.518, r$^2$ = 0.990). The 3D-QSAR analyses provide valuable information for the design of ACE inhibitors with potent activity towards C-domain of ACE. The group substitutions involving the phenyl ring and carbon chain at the propionyl and sulfonyl moieties of captopril are essential for specific activity to ACE.

• Applied Biological Chemistry
• /
• v.36 no.2
• /
• pp.86-92
• /
• 1993

Myrosinase from radish was purified by DEAE Bio-Gel, Con-A, and Superose-6 column. The purified myrosinase(II) possessed 2 subunits, and their molecular as determined by SDS-polyacrylamide gel electrophoresis were 53 and 39 KD, respectively. The specific activity of purified enzyme was 37,500 units/mg. The enzyme was purified approximately 44-fold compared to the crude enzyme. Optimum pH of the myrosinase was $6.5{\sim}7.0$ in phosphate and Tris-HCl buffer solutions. Optimum temperature of the enzyme was $37{\sim}38^{\circ}C$. The enzyme was stable at pH 7.0, and less than $30^{\circ}C$. Cu or Hg ion significantly inhibited the enzyme activity, but ascorbic acid enhanced, resulting in a maximum activity by 1 mM ascorbic acid. Among the ascorbic acid analogues, dehydroascorbic acid did not affect, whereas others showed a little effect, but less than ascorbic acid itself. Individual 2-mercaptoethanol and dithiothreitol (reducing agents) did not enhance the enzyme activity. but 2-mercaptoethanol effect was enhanced when mixed with ascorbic acid.

• BMB Reports
• /
• v.44 no.2
• /
• pp.77-86
• /
• 2011

Over the years, nanostructures have been developed to enable to support enzyme usability to obtain highly selective and efficient biocatalysts for catalyzing processes under various conditions. This review summarizes recent developments in the nanostructures for enzyme supporters, typically those formed with various inorganic materials. To improve enzyme attachment, the surface of nanomaterials is properly modified to express specific functional groups. Various materials and nanostructures can be applied to improve both enzyme activity and stability. The merits of the incorporation of enzymes in inorganic nanomaterials and unprecedented opportunities for enhanced enzyme properties are discussed. Finally, the limitations encountered with nanomaterial-based enzyme immobilization are discussed together with the future prospects of such systems.

• BMB Reports
• /
• v.30 no.1
• /
• pp.60-65
• /
• 1997

Expression of human Cu.Zn-superoxide dismutase (SOD) with activity comparable to human erythrocyte enzyme was achieved in E. coli B21(DE3) by using the pET-17b expression vector containing a T7 promoter. Recombinant human SOD was found in the cytosol of disrupted bacterial cells and represented > 25% of the total bacterial proteins. The protein produced by the E. coli cells was purified using a combination of ammonium sulfate precipitation, Sephacryl S-100 gel filtration and DEAE-Sephacel ion exchange chromatography. The recombinant Cu,Zn-SOD and human erythrocyte enzyme were compared using dismutation activity, SDS-PAGE and immunoblotting analysis. The mass of the subunits was determined to be 15,809 by using a electrospray mass spectrometer. The copper specific chelator. diethyldithiocarbamate (DOC) reacted with the recombinant Cu,Zn-SOD. At $50{\mu}M$ and $100{\mu}M$ concentrations of DOC, the dismutation activity was not inhibited for one hour but gradually reduced after one hour. This result suggests that the reaction of DOC with the enzyme occurred in two distinct phases (phase I and phase II). During phase I of this reaction, one DOC reacted with the copper center, with retention of the dismutation activity while the second DOC displaced the copper, with a loss of activity in phase II.

• Applied Biological Chemistry
• /
• v.21 no.2
• /
• pp.112-118
• /
• 1978

Xanthine oxidase from bovine thyroid glands was purified to apparent homogeneity when judged by analytical disc gel electrophoresis. The purification procedures include pancreatin digestion, butanol extraction, ammonium sulfate precipitation, calcium phosphate gel adsorption, ultrafiltration, calcium phosphate gel-cellulose column chromatography, gel filtration, preparative Sephadex G-25 column electrophoresis, and preparative polyacrylamide gel electrophoresis. The enzyme was enriched 1,000-fold. However, its specific activity was markedly low as compared with highly purified milk enzyme. Thyroidal xanthine oxidase exhibited a low specificity for substrates and electron acceptors. The kinetic properties of thyroid xanthine oxidase were found to be similar to those of the milk enzyme on the basis of Michaelis constants for common substrates.

• Microbiology and Biotechnology Letters
• /
• v.17 no.6
• /
• pp.552-555
• /
• 1989

A 150-fold purified preparation of NADPH-specific glutamate dehydrogenase of Corynebacterium glutamicum (1) was used for the determination of kinetic parameters of the substrates, NADPH, NH$_4$Cl, and $\alpha$-ketoglutarate in the direction of glutamate synthesis. The kinetic constants determined from this study suggest a biosynthetic role for the enzyme, Based on the analysis of the result derived from initial velocity, the reaction mechanism was postulated to be ordered addition with NADPH as a first substrate to bind in the forward direction. Of the several metabolites tested for a possible function in the regulation of glutamate dehydrogenase activity, only malate and citrate were appeared to have an appreciable influence on the enzyme, Potassium chloride showed to be the most effective for the enzyme activity.

• 한국생물공학회:학술대회논문집
• /
• 2000.11a
• /
• pp.260-263
• /
• 2000

Membranes are widely used to separate target solute molecules such as proteins on the basis of their size in cell broth mixture to minimize the loss of target compounds. In this study, membrane separation system using ultrafilters of MWCO 100 K and 50 K, was operated for concentration and purification of microbial transglutaminase. Fermentation broth containing MTGase was prefiltered by using pore size 1.6 and $0.7\;{\mu}m$ pre-filter made of cellulose fiber and $0.45\;{\mu}m$ microfilter made of cellulose acetate. The prefiltered solution was concentrated by 100 K and 50 K ultrafilter. The final enzyme concentration was 1.29 units/ml and enzyme specific activity was 0.2 units/mg protein. This specific activity were 3.7 times higher than that of initial cell broth mixture. Membrane separation process of microfiltration and ultrafiltration was proved to be very economic, energy efficient and effective separation method used to concentrate MTGase.

• Korean Journal of Clinical Pharmacy
• /
• v.8 no.1
• /
• pp.29-34
• /
• 1998

Murine CD38 is a 42 kDa type II glycoprotein expressed on cell surface of both B and T lymphocytes. CD38 is a multifunctional enzyme that catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD and cADPR hydrolase activity of CD38 catalyzes the hydrolysis of cADPR to ADP-ribose (ADPR). And also, CD38 has the catalytic activity of NAD glycohydrolase (NADase) which catalyzes the hydrolysis of catalyzes the formation and hydrolysis of cyclic adenosine diphosphoribose (cADPR): ADP-ribosyl cyclase activity of CD38 catalyzes the formation of cADPR from NAD to ADPR. In this study, we attempted to purify CD38 from mouse lymphocytes by using the immobilized anti-CD38 monoclonal antibody. The single step immuno-affinity column chromatography resulted in homogeneous purification, showing a single protein of 42 kDa on a SDS polyacrylamide gel. We have investigated the effects of various inhibitors on the enzyme activities of the purified CD38. Cibacron blue (0.5 mM) inhibited all three enzyme activities of CD38, NADase, ADP-ribosyl cyclase and cADPR hydrolase activities. ADPR (2 mM) showed inhibitory effect on both cADPR hydrolase activity and NADase, but not on ADP-ribosyl cyclase activity. However, ATP (2 mM) inhibited only cADPR hydrolase activity. $Zn^{2+}$ (1 mM) showed similar inhibitory effect as that of ADPR, but activated cyclase activity These results suggest that CD38 has three different catalytic activity domains which might be differentially regulated by their specific inhibitors.