• Journal of Microbiology and Biotechnology
• /
• v.17 no.6
• /
• pp.1031-1035
• /
• 2007

Prostate specific $antigen-{\alpha}_1-antichymotrypsin$ was detected by a double-enhancement strategy involving the exploitation of both colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation. The AuNPs were synthesized and conjugated with horse-radish peroxidase-PSA polyclonal antibody by physisorption. Using the protein-colloid for SPR-based detection of the PSPJACT complex showed their enhancement as being consistent with other previous studies with regard to AuNPs enhancement, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the signal. The limit of detection was found at as low as 0.027 ng/ml of the PSA/ACT complex (or 300 fM), which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1994.04a
• /
• pp.260-260
• /
• 1994

Mammalian pyruvate dehydrogenase complex(PDC) enzyme consists of multiple oopies of three major oligomeric enzymes-El, E2 E3. And protein X is one of the enzymatic constituents which is tightly bound to E2 subunit This complex enzyme is responsible for the oxidative decarboxylation of pyruvate producing of acetyl CoA which is a key intermediate for the entry of carbohydrates into the TCA cycle for its complete metabolic conversion to CO$_2$. And the overall activity of the complex enzyme is regulated via covalent nodification of El subunit by a El specific phosphatase ad kinase. Protein X has lipoyl moiety that undergoes reduction and acetylation during ezymatic reaction and has been known h be involved in the binding of E3 subunit to E2 core and in the regulatory activity of kinase. The purification of protein X has not been achieved majorly because of its tight binding to E2 subunit The E2-protein X subcomplex was obtained by the established methods and the detachment of protein X from E2 was accomplished in the 0.1M borate buffer containing 150mM NaCl. During the storage of the subcomplex in frozen state at -70$^{\circ}C$, the E2 subunit was precipitated and the dissociated protein X was obtained by cntrifegation into the supernatant The verification of protein X was accomplished by (1)the migration on SDS-PAGE, (2)acetylation by 〔2$\^$-l4/C〕 pyruvate, and (3)internal amino acid sequence analysis of tryptic digested enzyme.

• Asian-Australasian Journal of Animal Sciences
• /
• v.27 no.12
• /
• pp.1755-1762
• /
• 2014

The objective of this study was to investigate the effect of maize source and complex enzymes containing amylase, xylanase and protease on performance and nutrient utilization of broilers. The experiment was a $4{\times}3$ factorial design with diets containing four source maize samples (M1, M2, M3, and M4) and without or with two kinds of complex enzyme A (Axtra XAP) and B (Avizyme 1502). Nine hundred and sixty day old Arbor Acres broiler chicks were used in the trial (12 treatments with 8 replicate pens of 10 chicks). Birds fed M1 diet had better body weight gain (BWG) and lower feed/gain ratio compared with those fed M3 diet and M4 diet (p<0.05). Apparent ileal crude protein digestibility coefficient of M2 was higher than that of M3 (p<0.05). Apparent metabolisable energy (AME) and nitrogen corrected AME (AMEn) of M1 were significant higher than those of M4 (p<0.05). Supplementation of the basal diets with enzyme A or B improved the BWG by 8.6% (p<0.05) and 4.1% (p>0.05), respectively. The fresh feces output was significantly decreased by the addition of enzyme B (p<0.05). Maize source affects the nutrients digestibility and performance of broilers, and a combination of amylase, xylanase and protease is effective in improving the growth profiles of broilers fed maize-soybean-rapeseed-cotton mixed diets.

• Food Science and Preservation
• /
• v.7 no.1
• /
• pp.94-98
• /
• 2000

To produce liquor and vinegar using potatoes needs to liquefy and sacchrify potatoes . So selecting the efficient fermenter for proceeding these process successfully is very important . This study was investigated several fermenter and crush types of potatoes for alcohol fermentation. Final sugar contents was high in pottoes saccharificatiion by nuruk or crude enzyme. But pure enzyme and blucoamylase ended liquefaction and saccharificatiion within short ime. So complex type fermenter mixed several fermenters was superior to single type fermenter. Complexfermenter III using crude enzyme and glucoamyulase saccharificed excellently potatoes with 150% of water contents by treatment of 3 hours. Through alcohol fermentation using pressure steamed potatoes (PSP), it could be obtained 6.4% , 150%, of alcohol content and yield. However to perform a series process efficiently , crush steamed pottoes (CSP) was suitable. When it was fermented after saccharification using crush steamed potatoes and complex fermenter III, it could be obtained 6.6% of alcohol and 6.7% of acidity.

• Microbiology and Biotechnology Letters
• /
• v.18 no.1
• /
• pp.39-43
• /
• 1990

The inhibitor had the inhibitory activities against hydrolysis of PNPG, sucrose and ONPG by $\alpha$-Dglucosidase, $\alpha$ - and $\beta$ -galactosidase, but it did not inhibit amylases and other carbohydrases. Kinetic studies exhibited that the inhibitory substance non-competitively inhibited the enzyme reaction with a Ki value of 118 $\mu$g/m$\ell$, and enzyme-inhibitor complex was formed slowly.

• BMB Reports
• /
• v.29 no.3
• /
• pp.230-235
• /
• 1996

The interaction of ${\alpha}-ketoglutarate$ dehydrogenase complex (${\alpha}-KGDC$) with a hydrophobic fluorescent probe [1,1'-bi(4-aniline)naphthalene-5,5'-disulfonic acid] (bis-ANS) was studied. The punfied ${\alpha}-KGDC$ was potently inhibited by bis-ANS with an apparent half maximal inhibitory concentration ($IC_{50}$) of 9.8 ${\mu}m$ at pH 8.0. The catalytic activities of both the E1o and E2o subunits were predominantly inhibited while that of the E3 component was hardly affected. The binding of bis-ANS to the enzyme caused a marked enhancement and blue shift from 523 nm to 482 nm in the fluorescence emission spectrum. The dissociation constant ($K_d$) and the number of binding sites (n) were calculated to be 0.87 mM and 158, respectively. Allosteric regulators such as purine nucleotides and divalent cations further increased the fluorescence intensity of the $bis-ANS-{\alpha}-KGDC$ binary complex. These data suggest that the binding of these allosteric regulators to ${\alpha}-KGDC$ may cause the conformational changes in the enzyme and that bis-ANS could be used as a valuable probe to study the interaction of the multi-enzyme complex and its allosteric regulators.

• Journal of Embryo Transfer
• /
• v.25 no.1
• /
• pp.35-42
• /
• 2010

Many studies propose that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. With a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1) as the gene delivery method, we were able to attain high transduction efficiencies even in the human epithelial cervical cancer cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. Using a rAAV-NDI1, we demonstrated that the Ndi1 enzyme is successfully expressed in HeLa cells. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced HeLa cells were not affected by rotenone which is inhibitor of complex I, but was inhibited by flavone and antimycin A. The NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. In particular, in the NDI1-transduced cells, the yeast enzyme becomes integrated into the human respiratory chain. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

• The Journal of the Korean Society for Microbiology
• /
• v.22 no.4
• /
• pp.445-451
• /
• 1987

Coagglutination method is widely used for the diagnosis of Salmonella infection. This test, however, has a disadvantage of false positive reaction due to the coagglutination of staphylococci with non-specific immune complexes or anti-staphylococci antibody in serum. Salmonell O antigen was detected by enzyme immunoassay with protein A-bearing Staphylococcus aureus as in the solid phase. Horse radish peroxidase was labeled to IgG specific against Salmonella O antigen. This enzyme immunoassay was much more sensitive than conventional coagglutination method without false poitive agglutination. To improve the sensitivity for detection of Salmonella O antigen in samples, we tried to determine the optimal concentration of normal IgG that inhibits non-specific binding of horse radish peroxidase labeled IgG to staphylococci, and to establish the optimal condition of reaction between antigen-antibody complex and staphylococci. Non-specific binding of horse radish peroxidase labeled specific IgG to staphylococci was almost blocked when the enzyme labeled IgG was 500-fold diluted with phosphate buffered saline containing 2mg/ml of normal IgG. When staphylococci coated with antibody to Salmonella O antigen were mixed with antigen-antibody complex and then incubated for 1 hour at room temperature, the minimal detectable concentration of Salmonella O antigen was 1ng/ml. The sensitivity of enzyme immunoassay was 100-fold greater than a conventional coagglutination method. This enzyme immunoassay could be expected as an improved method for detection of other infectious agents.

• BMB Reports
• /
• v.33 no.6
• /
• pp.427-439
• /
• 2000

The activity of the phagocyte respiratory burst oxidase is regulated by complex and dynamic alterations in protein-protein interactions that result in the rapid assembly of an active multicomponent NADPH oxidase enzyme on the plasma membrane. While the enzymatic activity has been studied for the past 20 years, the past decade has seen remarkable progress in our understanding of the enzyme and its activation at the molecular level. This article describes the current state of knowledge, and proposes a model for the mechanism by which protein-protein interactions regulate enzyme activity in this system.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.2
• /
• pp.226-232
• /
• 2016

Dihydrodipicolinate reductase is an enzyme that converts dihydrodipicolinate to tetrahydrodipicolinate using an NAD(P)H cofactor in L-lysine biosynthesis. To increase the understanding of the molecular mechanisms of lysine biosynthesis, we determined the crystal structure of dihydrodipicolinate reductase from Corynebacterium glutamicum (CgDapB). CgDapB functions as a tetramer, and each protomer is composed of two domains, an Nterminal domain and a C-terminal domain. The N-terminal domain mainly contributes to nucleotide binding, whereas the C-terminal domain is involved in substrate binding. We elucidated the mode of cofactor binding to CgDapB by determining the crystal structure of the enzyme in complex with NADP⁺ and found that CgDapB utilizes both NADH and NADPH as cofactors. Moreover, we determined the substrate binding mode of the enzyme based on the coordination mode of two sulfate ions in our structure. Compared with Mycobacterium tuberculosis DapB in complex with its cofactor and inhibitor, we propose that the domain movement for active site constitution occurs when both cofactor and substrate bind to the enzyme.