• Bulletin of the Korean Chemical Society
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• v.15 no.9
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• pp.719-724
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• 1994

Major cellulase components, such as three endoglucanases (endoglucanases I, II, and III) and one exoglucanase (exoglucanase II), were isolated from a commercial cellulase (Meicelase TP 60) derived from the fungus Trichoderma viride by a series of chromatography procedures. These procedures were the gel filtration on Bio-Gel, the anion exchange on DEAE-Bio-Gel A, the cation exchange on SP-Sephadex C50, and the affinity chromatography on Avicel cellulose. The average molecular weights determined by SDS-polyacrylamide gel electrophoretic analysis were 51,000, 59,000, 41,000 and 62,000 Da for endoglucanases I, II and III and exoglucanase II, respectively. The extinction coefficients, ${\varepsilon}^{1%}$ 280 nm, of these enzymes were 11.7, 3.3, 7.2 and 11.3, respectively. Among them, the endoglucanase II showed the very low value of the coefficient compared with the others. On the other hand, it was found that endoglucanase II and III were of more random hydrolytic mode on carboxymethylcellulose as compared with those of endoglucanase I and exoglucanase II. Especially, endoglucanase I showed less random action than that of exoglucanase II. In the hydrolysis of insoluble cellulose by the enzyme components, cellobiose was the major product, but glucose was the major product by endoglucanase III.

• Journal of Pharmacopuncture
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• v.27 no.1
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• pp.1-13
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• 2024

Background: Diabetic nephropathy (DN) is one of the major complications of chronic hyperglycaemia affecting normal kidney functioning. The ayurvedic medicine curcumin (CUR) is pharmaceutically accepted for its vast biological effects. Objectives: The Curcuma-derived diferuloylmethane compound CUR, loaded on Poly (lactide-co-glycolic) acid (PLGA) nanoparticles was utilized to combat DN-induced renal apoptosis by selectively targeting and modulating Bcl2. Methods: Upon in silico molecular docking and screening study CUR was selected as the core phytocompound for nanoparticle formulation. PLGA-nano-encapsulated-curcumin (NCUR) were synthesized following standard solvent displacement method. The NCUR were characterized for shape, size and other physico-chemical properties by Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and Fourier-Transform Infrared (FTIR) Spectroscopy studies. For in vivo validation of nephro-protective effects, Mus musculus were pre-treated with CUR at a dose of 50 mg/kg b.w. and NCUR at a dose of 25 mg/kg b.w. (dose 1), 12.5 mg/kg b.w (dose 2) followed by alloxan administration (100 mg/kg b.w) and serum glucose levels, histopathology and immunofluorescence study were conducted. Results: The in silico study revealed a strong affinity of CUR towards Bcl2 (dock score -10.94 Kcal/mol). The synthesized NCUR were of even shape, devoid of cracks and holes with mean size of ~80 nm having -7.53 mV zeta potential. Dose 1 efficiently improved serum glucose levels, tissue-specific expression of Bcl2 and reduced glomerular space and glomerular sclerosis in comparison to hyperglycaemic group. Conclusion: This study essentially validates the potential of NCUR to inhibit DN by reducing blood glucose level and mitigating glomerular apoptosis by selectively promoting Bcl2 protein expression in kidney tissue.

• Journal of the Korean Chemical Society
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• v.23 no.4
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• pp.248-258
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• 1979

Glucose 6-phosphate dehydrogenase of Leuconostoc mesenteroides which was purifid by an affinity chromatography was studied on the characterization, kinetics and chemical modification. The apparent molecular weight of the enzyme was 112,000 by the gel filtration method of Sephadex G-200 column. The optimum temperature of $NAD^+$-linked reation was 50$^{circ}C$ and the activation energy and the heat of inactivation were 8.36 kcal/mole and -58.2kcal/mole, respectively. The steady state kinetic study showed KG6P, Kemp, and CX KNADP to be 76.9 PM, 7.46${\mu}M$ and 7.14 ${\mu}M$, respectively, and KGGP, KNAD,and aKNm to be 53.7${\mu}M$, 115.2${\mu}M$ and 702.2${\mu}M$ for the $NAD^+$-linked reaction at pH 7.8, optimum pH. The pH dependent kinetic constants suggested that the two ionizing groups whose pKa is 7.2 .and pKb is 9.0-9.6 were involved in the enzyme-substrate interaction. Evidence by photooxidation and carboxymethylation of the enzyme suggested that the imidazole group of histidine with pKa group may participate in the catalytic site.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.247-268
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• 1996

It has been known for a long time that gonadotropins and steroid hormones play a pivotal role in a series of reproductive biological phenomena including the maturation of ovarian follicles and oocytes, ovulation and implantation, maintenance of pregnancy and fetal growth & development, parturition and mammary development and lactation. Recent investigations, however, have elucidated that in addition to these classic hormones, multiple growth factors also are involved in these phenomena. Most growth factors in reproductive organs mediate the actions of gonadotropins and steroid hormones or synergize with them in an autocrine/paracrine manner. The insulin-like growth factor(IGF) system, which is one of the most actively investigated areas lately in the reproductive organs, has been found to have important roles in a wide gamut of reproductive phenomena. In the present communication, published literature pertaining to the intrauterine IGF system will be reviewed preceded by general information of the IGF system. The IGF family comprises of IGF-I & IGF-II ligands, two types of IGF receptors and six classes of IGF-binding proteins(IGFBPs) that are known to date. IGF-I and IGF-II peptides, which are structurally homologous to proinsulin, possess the insulin-like activity including the stimulatory effect of glucose and amino acid transport. Besides, IGFs as mitogens stimulate cell division, and also play a role in cellular differentiation and functions in a variety of cell lines. IGFs are expressed mainly in the liver and messenchymal cells, and act on almost all types of tissues in an autocrine/paracrine as well as endocrine mode. There are two types of IGF receptors. Type I IGF receptors, which are tyrosine kinase receptors having high-affinity for IGF-I and IGF-II, mediate almost all the IGF actions that are described above. Type II IGF receptors or IGF-II/mannose-6-phosphate receptors have two distinct binding sites; the IGF-II binding site exhibits a high affinity only for IGF-II. The principal role of the type II IGF receptor is to destroy IGF-II by targeting the ligand to the lysosome. IGFs in biological fluids are mostly bound to IGFBP. IGFBPs, in general, are IGF storage/carrier proteins or modulators of IGF actions; however, as for distinct roles for individual IGFBPs, only limited information is available. IGFBPs inhibit IGF actions under most in vitro situations, seemingly because affinities of IGFBPs for IGFs are greater than those of IGF receptors. How IGF is released from IGFBP to reach IGF receptors is not known; however, various IGFBP protease activities that are present in blood and interstitial fluids are believed to play an important role in the process of IGF release from the IGFBP. According to latest reports, there is evidence that under certain in vitro circumstances, IGFBP-1, -3, -5 have their own biological activities independent of the IGF. This may add another dimension of complexity of the already complicated IGF system. Messenger ribonucleic acids and proteins of the IGF family members are expressed in the uterine tissue and conceptus of the primates, rodents and farm animals to play important roles in growth and development of the uterus and fetus. Expression of the uterine IGF system is regulated by gonadal hormones and local regulatory substances with temporal and spatial specificities. Locally expressed IGFs and IGFBPs act on the uterine tissue in an autocrine/paracrine manner, or are secreted into the uterine lumen to participate in conceptus growth and development. Conceptus also expresses the IGF system beginning from the peri-implantation period. When an IGF family member is expressed in the conceptus, however, is determined by the presence or absence of maternally inherited mRNAs, genetic programming of the conceptus itself and an interaction with the maternal tissue. The site of IGF action also follows temporal (physiological status) and spatial specificities. These facts that expression of the IGF system is temporally and spatially regulated support indirectly a hypothesis that IGFs play a role in conceptus growth and development. Uterine and conceptus-derived IGFs stimulate cell division and differentiation, glucose and amino acid transport, general protein synthesis and the biosynthesis of mammotropic hormones including placental lactogen and prolactin, and also play a role in steroidogenesis. The suggested role for IGFs in conceptus growth and development has been proven by the result of IGF-I, IGF-II or IGF receptor gene disruption(targeting) of murine embryos by the homologous recombination technique. Mice carrying a null mutation for IGF-I and/or IGF-II or type I IGF receptor undergo delayed prenatal and postnatal growth and development with 30-60% normal weights at birth. Moreover, mice lacking the type I IGF receptor or IGF-I plus IGF-II die soon after birth. Intrauterine IGFBPs generally are believed to sequester IGF ligands within the uterus or to play a role of negative regulators of IGF actions by inhibiting IGF binding to cognate receptors. However, when it is taken into account that IGFBP-1 is expressed and secreted in primate uteri in amounts assessedly far exceeding those of local IGFs and that IGFBP-1 is one of the major secretory proteins of the primate decidua, the possibility that this IGFBP may have its own biological activity independent of IGF cannot be excluded. Evidently, elucidating the exact role of each IGFBP is an essential step into understanding the whole IGF system. As such, further research in this area is awaited with a lot of anticipation and attention.

• Applied Biological Chemistry
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• v.35 no.5
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• pp.375-381
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• 1992

An exoinulase-producing bacterium was isolated from soil, and identified as Streptomyces sp. The maximum inulase production was achieved when inulin as carbon source and soybean meal as organic nitrogen source were included in the culture. The exoinulase was considered to be a constitutive enzyme produced not only by inulin but also by soluble starch or glucose. The purified enzyme on DEAE-cellulose and Sephadex G-200 column showed the maximal activity at $pH\;5.5{\sim}6.0$ and $50^{\circ}C$, but lost 65% inulase activity at $50^{\circ}C$ after 1 hour incubation. This exoinulase was activated by $Mn^{+2}$, wherease more that 80% inactivation was observed with $Ag^+$, $Hg^{+2}$ and $Fe^{+3}$. The enzyme was possibly a metalloenzyme in that EDTA and 8-hydroxyquinoline inhibited the enzyme. Km values for inulin (16.51 mM) and sucrose (14.62 mM) were in very close range suggesting mostly equal affinity toward the subatrates. However, the maximum velocity for inulin was 10 times greater than for sucrose.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.251-262
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• 1986

High-molecular-weight ${\beta}-glucosidase$ (EC 3.2.1.21) was purified from the culture filtrate of Trichoderma koningii through a four-step procedure including chromatography on Bio-Gel P-150, DEAE-Sephadex A-50 and SP-Sephadex C-50; and chromatofocusing on Polybuffer exchanger PBE 94. The molecular weight of the enzyme was determined to be about 101,000 by SDS-polyacrylamide gel electrophoreses, and the isoelectric point was estimated to be 4.96 by analytical isoelectric focusing. The temperature optimum for activity was about $55^{\circ}C$, and the pH optimumwas 3.5. The enzyme was considerably thermostable, for no loss of activity was observed when the enzyme was preincubated at $60^{\circ}C$ for 5h. Km values for cellobiose, gentiobiose, sophorose, salicin and $p-nitrophenyl-{\betha}-D-glucoside$ were 99.2, 14.7, 7.09, 3.15 and 0.70 mM, respectively, which indicates that the enzyme has much higher affinity towards $p-nitrophenyl-{\betha}-D-glucoside$ than towards the other substrates, especially cellobiose. Substrate inhibition by $p-nitrophenyl-{\betha}-D-glucoside$ and salicin was observed at the conecntrations exceeding 5mM. Gluconolactone was a powerful inhibitor against the action of the enzyme on $p-nitrophenyl-{\betha}-D-glucoside\;(K_i\;37.9\;{\mu}M)$, wherease glucose was much less effective ($K_i$ 1.95 mM). Inhibition was of the competitive type in each case. Transglucosylation activity was detected shen the readtion products formed from $p-nitrophenyl-{\betha}-D-glucoside$ by the enzyme were analysed using high-performance liquid chromatography.

• Journal of Microbiology
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• v.39 no.4
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• pp.279-285
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• 2001

Vibrio parahaemolyticus is a halophilic bacterium associated with seafood gastroenteritis. An unusual strain of Kanagawa-positive urease producing Vibrio parahaemolyticus O1:K1 was isolated from the environment and identified . A polymerase chain reaction assay revealed that this strain harbored both the tdh and the genes. The urease from this strain was studied. Maximum urease production was induced in LB medium containing 0.2% urea, 0.5% glucose, 2% NaCl and pH 5.5 with 6h of culti-vation at 37$\^{C}$ under aeration. Purification of urease was achieved by the process of whole cell lysate, 65% ammonium sulphate precipitation, DEAE-cellulose ion exchange column chromatography, Sepharose CL-6B gel filtration and oxirane activated Sepharose 6B-urea affinity chromatography with 203 fold purification and 2.2% yield. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of the subunits with a molecular weight of 85kDa, 59kDa, 41kDa and the molecular weight for the native enzyme by nondenaturing PAGE and gel filtration chromatography was 255kDa. The purified urease was stable at pH 7.5 and the opeimal pH in HEPES buffer was 8.0 The enzyme was stable at 60$\^{C}$ for 2 h with a residual activity of 32% . The addition of 10$\mu$M if NiCl$_2$maintained stability for 30 min. The Km value of the purified enzyme was 35.6 mM in urea substrate. The TD$\_$50/(median toxic dose) of the purified urease was 2.5$\mu\textrm{g}$/ml on human leukemia cells.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.933-941
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• 2008

An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

• Korean Journal of Food Science and Technology
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• v.32 no.1
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• pp.161-167
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• 2000

The protease produced by a newly isolated Aspergillus wentti from Korean traditional Meju was purified and characterized. The optimal medium composition and culture conditions for maximum protease production were ; bran :1% glucose solution =1 : 1, pH 9.0, $30^{\circ}C$, and 4 days of fermentation. Protease was purified by QAE-Sephadex, SP-Sephadex ion exchange chromatography and Sephadex G-100 chromatography. The specific activity and the purification fold of the purified enzyme were 213 unit/mg protein and 27.3, respectively. The molecular weight of purified protease was found to be 32 kDa by SDS-PAGE. Km and Vmax value's for hammastein milk casein were $3.049{\times}10^{-4}\;M\;and\;151.1\;{\mu}g/min$, respectively. Kinetic parameters showed that the enzyme has higher affinity to casein than isolated soybean protein, hemoglobin and bovine serum albumin. Optimal pH and temperature for reaction of the purified enzyme were 9.0 and $50^{\circ}C$, respectively. The enzyme was stable at pH 4.0-11.0, below $40^{\circ}C$, and the activity was not stimulated by metal ions. 1mM phenylmethylsulfonyl fluoride inhibited the enzyme activity by 98.5%. It means that the enzyme is one of serine protease.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.85-91
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• 1991

.betha.-1,4-D-Glucan glucanohydrolase(EC 3.2.1.4;F-II-IV) purified from Trichoderma koningii was identified as a glycoprotein containing 9% carbohydrate. Isoelectric point of the enzyme was estimated to be 4.9 and molecular weight was determined to be approximately 58,000. The porducts of p-nitrophenyl-cellobioside ($PNPG_{2}$) catalyzed by the enzyme were p-nitrophenol(PNP) and p-nitrophenyl-glucoside($PNPG_{1}$). The Km value for $PNPG_{2}$ was estimated to be 0.97 mM in case of the holoside lindage and 10.4 mM in case of the aglycon linkage and their kcat values were $1.8*10^{5}$$ min^{-1}$ and $7.5*10^{5}$ $min^{-1}$ respectively. The product of p-nitrophenyl cellotriose($PNPG_{3}$) was only $PNPG_{1}$. The Km value for $PNPG_{3}$ was 69.5 .$\mu$M and kcat was $1*10^{8}$ $min^{-1}$ which implicates that the enzyme have higher affinity and higher hydrolysis rate toward $PNPG_{3}$ than toward $PNPG_{2}$. The enzyme showed its optimal activity at pH 4.0-4.5 and at 60.deg.C. The effect of gluconolactone on the activity toward $PNPG_{2}$ showed competitive inhibition pattern but glucose and cellobiose did not. The enzyme contained a high content of acidic and hydroxylated amino acids in contrast to basic amino acids.