• Korean Journal of Microbiology
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• v.32 no.3
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• pp.222-231
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• 1994

Kinetic analysis was done to elucidate the reaction mechanism of purine nucleoside phosphorylase (PNP) in Saccharomyces cerevisiae. The binary complexes of PNP${\cdot}$phosphate and PNP${\cdot}$ribose 1-phosphate were involved in the reaction mechanism. The initial velocity and product inhibition studies demonstrated were consistent with the predominant mechanism of the reaction being an ordered bi, bi reaction. The phosphate bound to the enzyme first, followed by nucleoside and base were the first product to leave, followed by ribose 1-phosphate. The kinetically suggested mechanism of PNP in S. cerevisiae was in agreement with the results of protection studies against the inactivation of the enzyme by sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and 5,5'-dithiobisnitrobenzoate (DTNB). PNP was protected by ribose 1-phosphate and phosphate, but not by nucleoside or base, supporting the reaction order of ordered bi, bi mechanism. PCMB or DTNB-inactivated PNP was totally reactivated by dithiothreitol (DTT) and the activity was returned to the level of 77% by 2-mercaptoethanol, indicating that inactivation was reversible. The kinetic behavior of the PCMB-inactivated enzyme had been changed with higher $K_m$ value of inosine and lower $V_m$, and was restored by DTT. Inactivation of enzyme by DTNB showed similar pattern of K sub(m) value with that by PCMB, but had not changed the $V_m$ value, significantly. Negative cooperativity was not found with PCMB or DTNB treated PNP at high concentration of phosphate.

• Food Science and Preservation
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• v.21 no.3
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• pp.442-450
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• 2014

In this study, we isolated a cellulase-producing bacterium isolated from traditional Korean fermented soybean paste and investigated the effect of culture conditions on the production of cellulase. This bacterium, which was identified as Bacillus subtilis 4-1 through 16S rRNA gene sequence analysis, showed the highest cellulase activity when the cells were grown at $45^{\circ}C$ for 24 hours in the CMC medium supplemented with 1.0% of soluble starch and 0.1% yeast extract. The initial optimum pH of the medium was observed in the range of 5.0~9.0. The optimal pH and temperature for the production of cellulase from B. subtilis 4-1 were pH 9.0 and $60^{\circ}C$ respectively. In addition, the enzyme showed significant activity in the temperature range of $20{\sim}90^{\circ}C$, which indicates that B. subtilis 4-1 cellulase is an alkaline-resistance and thermo-stable enzyme. This enzyme showed higher activity with CMC as the substrate for endo-type cellulase than avicel or pNPG as the exo-type substrates for exo-type cellulase and ${\beta}$-glucosidase. These results suggest that the cellulase produced from B. subtilis 4-1 is a complex enzyme rather than a mono-enzyme.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.508-513
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• 1997

Thermal characteristics and granular morphology on enzyme-resistant starches (RS) formed during heat-moisture treatment (HMT) and retrogradation were investigated in high amylose corn starches, Hylon V and Hylon VII. With each treatment, both starches showed a similar trend in the increase of RS, but RS yield of Hylon VII is higher than that of Hylon V. Specially, RS was increased remarkably by HMT. It was more than doubled from 11.4% to 26.6% for Hylon V and from 15.9% to 32.8% for Hylon VII. A small increase of RS resulted from retrogradation. HMT on starch increased gelatinization temperature, decreased enthalpy. Retrograded starch exhibited small three endothermic transitions at $94^{\circ}C$, $110^{\circ}C$ and $140^{\circ}C$ in differential scanning calorimetry (DSC) thermogram due to the remained ungelatinized starch granules, dissociation of amylose-lipid complex and melting of recrystallized amylose, respectively. Enzyme-resistant starches isolated from native and heat-moisture treated starches showed a broad endothermic transition at higher temperature than native starch, while retrograded starch exhibited a very sharp peak at ${\sim}150^{\circ}C$ due to the melting of amylose crystallites. Under microscopy, starch granules with HMT was not changed, but retrograded starches showed the aggregates of starch granules because amylose leached out during gelatinization. Iodine stained RS clearly showed the differences in enzyme hydrolysis on the native, heat-moisture treated and retrograded starches.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.100-103
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• 2004

The culture condition and medium composition for the enhanced mycelial growth of Inonotus mikadoi IMSNU 32058 were investigated. The optimal temperature and pH for the mycelial growth were $27^{\circ}C$ and pH 4.5, respectively. Among the complex media tested, the malt extract medium and Phellinus igniarius medium were very good for mycelial growth of I. mikadoi. When Czapek-Dox medium was used as a minimal medium for cultivation of mycelia, xylose, raffinose and carboxymethyl cellulose were very excellent as a carbon and energy source. With respect to carbohydrate, sucrose and glucose were very good carbon sources. In general, organic complex nitrogen sources were better than other inorganic ones. As the organic complex nitrogen sources tested, yeast extract, soytone, proteose peptone and bacto peptone were the best as a source of organic nitrogen. When ammonium sulfate as an inorganic source of nitrogen was used, the enhanced mycelial growth was shown. p-Aminobenzoic acid was proved to be most appropriate source of vitamin. After the mycelia of I. mikadoi was cultivated at $27^{\circ}C$ for 5 days in MEM broth (pH 4.5), the activities of both exomycelial and endo-mycelial enzymes were determined. Among endomycelial enzymes assayed, the specific activity of laccase was much higher than those of other enzymes. When the fungus was grown in MEM broth, exomycelial specific enzyme activity of laccase was comparatively high. However, little or no enzyme activities of protease, chitinase and lipase were found.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1547-1553
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• 2018

Hyaluronidases are a family of enzymes that catalyse the breakdown of hyaluronic acid, which is abundant in the extracellular matrix and cumulus oocyte complex. To investigate the activity of recombinant bovine sperm hyaluronidase 1 (SPAM1) and determine the effect of the Asn-X-Ser/Thr motif on its activity, the bovine SPAM1 open reading frame was cloned into the mammalian expression vector pCXN2 and then transfected to the HEK293 cell line. Expression of recombinant bovine hyaluronidase was estimated using a hyaluronidase activity assay with gel electrophoresis. Recombinant hyaluronidase could resolve highly polymeric hyaluronic acid and also caused dispersal of the cumulus cell layer. Comparative analysis with respect to enzyme activity was carried out for the glycosylated and deglycosylated bovine sperm hyaluronidase by N-glycosidase F treatment. Finally, mutagenesis analysis revealed that among the five potential N-linked glycosylation sites, only three contributed to significant inhibition of hyaluronic activity. Recombinant bovine SPAM1 has hyaluronan degradation and cumulus oocyte complex dispersion ability, and the N-linked oligosaccharides are important for enzyme activity, providing a foundation for the commercialization of hyaluronidase.

• Journal of Animal Science and Technology
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• v.46 no.5
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• pp.763-772
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• 2004

The patterns of fungal growth and fiber digestion under the microscope, and tile productions of fibrolytic enzyme were studied in an in vitro culture with Neocallimastix frontalis SA when either filter paper or rice straw was provided as sole energy source. Under the microscopic observation, active zoospores attachment, sporangium development and complex rhizoidal system were founded on the surface and at the edge of filter paper. After 7 days of incubation, a reduced fiber mass, a decreased fiber cohesion and a weakened fiber structure by fungal digestion were clearly observed. Similar fungal development was observed with rice straw, but fungal growth and digestion took place mostly on the damaged and exposed portion of rice straw. Although there were some differences in absolute concentration and pattern, the concentration of both cellulase and xylanase increased with incubation time with the higher activity being obtained with filter paper. Their differences were large especially after 48 and 96hr of incubation(P< 0.05). The filter paper was more good inducer of cellulolytic and xylanolytic enzymes compared with complex substrate, rice straw. These findings suggest that the filter paper is the better energy source for N frontalis than the complex substrate, and structural disintegration by physical process is able to help rumen fungal growth on the lignified roughage although anaerobic rumen fungi have mechanical and enzymatic functions for fiber digestion.

• Environmental Engineering Research
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• v.23 no.1
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• pp.46-53
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• 2018

This study explores the environmentally innovative and low-impact technology, a zero food waste system (ZFWS) that utilizes food waste and converts it into composts or biofuels and curtails carbon emissions. The ZFWS not just achieves food waste reductions but recycles food waste into fertilizer. Based on a fermentation-extinction technique using bio wood chips, the ZFWS was employed in a field experiment of the system installed in a large-scale apartment complex, and the performance of the system was examined. The on-site ZFWS consisted of three primary parts: 1) a food waste slot into which food waste was injected; 2) a fermentation-extinction reactor where food waste was mixed with bio wood chips made up of complex enzyme and aseptic wood chips; and 3) deodorization equipment in which an ultraviolet and ozone photolysis method was employed. The field experiment showed that food waste injected into the ZFWS was reduced by 94%. Overall microbial activity of the food waste in the fermentation-extinction reactor was measured using adenosine tri-phosphate (ATP), and the degradation rate of organic compounds, referred to as volatile solids, increased with ATP concentration. The by-products generated from ZFWS comply with the national standard for organic fertilizer.

• BMB Reports
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• v.48 no.1
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• pp.25-29
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• 2015

Ubiquitination is a post translational modification which mostly links with proteasome dependent protein degradation. This process has been known to play pivotal roles in the number of biological events including apoptosis, cell signaling, transcription and translation. Although the process of ubiquitination has been studied extensively, the mechanism of polyubiquitination by multi protein E3 ubiquitin ligase, SCF complex remains elusive. In the present study, we identified UbcH5a as a novel stimulating factor for poly-ubiquitination catalyzed by $SCF^{hFBH1}$ using biochemical fractionations and MALDI-TOF. Moreover, we showed that recombinant UbcH5a and Cdc34 synergistically stimulate $SCF^{hFBH1}$ catalyzed polyubiquitination in vitro. These data may provide an important cue to understand the mechanism how the SCF complex efficiently polyubiquitinates target substrates.

• Particle and aerosol research
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• v.9 no.4
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• pp.201-208
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• 2013

Copper (II) and Nickel (II) mimic complexes of enzyme carbonic anhydrase were evaluated under ambient condition for carbon dioxide capture and conversion process. The synthesized complexes were characterized by ATR-FTIR and UV-DR spectroscopy. It was found that all the complexes have biomimetic activity towards $CO_2$ using para-nitrophenyl acetate (p-NPA) hydrolysis as the model reaction. Interestingly, the proper geometry obtained by the restricted orientation of tripodal N atoms in Cu (II) complex of 2,6-bis(2-benzimidazolyl) pyridine showed the highest activity (1.14 au) compared to others. The $CO_2$ bio-mineralization to $CaCO_3$ was carried out via in-vitro crystallization approach. Results indicate that the biomimetic complexes have a role in determining $CaCO_3$ morphology. The present observations establish a qualitative insight for the design of improved small-molecule catalysts for carbon capture.

• BMB Reports
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• v.42 no.12
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• pp.817-822
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• 2009

Type-1 phosphatase (PP-1) was assessed in foot muscle (FM) and hepatopancreas (HP) of estivating (EST) Otala lactea. Snail PP-1 displayed several conserved traits, including sensitivity to inhibitors, substrate affinity, and reduction in size to a 39 kDa catalytic subunit (PP-1c). During EST, PP-1 activity in FM and HP crude extracts was reduced, though kinetics and protein levels of purified PP-1c isoforms were not altered. PP-1c protein levels increased and decreased in nuclear and glycogen-associated fractions, respectively, during EST. Gel filtration determined that a 257 kDa low $K_m$ PP-1$\alpha$ complex decreased during estivation whereas a 76 kDa high $K_m$ complex increased in EST. Western blotting confirmed that the 76 kDa protein consisted of PP-1$\alpha$ and nuclear inhibitor of PP-1 (NIPP-1). A suppression of PP-1 activity factors in the overall metabolic rate depression in estivating snails and the mechanism is mediated through altered cellular localization and interaction with binding partners.