• Microbiology and Biotechnology Letters
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• v.30 no.3
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• pp.235-240
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• 2002

Streptomycetes is a group of Gram-positive soil bacteria that growas a branching vegetative mycelium leading to the formation of spores, and display a physiological differenti-ation related to the synthesis of many secondary metabolites including antibiotics. Their complex life cycle and multicellular differentiation require various levels of regulation and types of signal transduction systems including eukaryotic-type serine/threonine protein kinases and prokaryotic-type histidine/aspartic acid protein kinases. Akt kinase that was found in cells is a sorine/threonine kinase controlling signal pathway for multi-tude of important cellular events. The activation or inactivation of Akt kinase in the cell is one of the critical regulatory points to deliver cell proliferation, differentiation, survival or apoptosis signal. To find the regula-tory protein homologous to Akt in Streptomyces, the fluorescien-labeled synthetic peptide (FITC-TRRSR-TESIT) was designed from the consensus sequence of target proteins for Akt kinase. From the difference of the mobility between the nonphosphorylated and phosphorylated synthetic peptides on Agarose gel electro-phoresis, the Akt-phosphorylating activity was monitored. The cell-free extract prepared from Streptomyces griseus IFO 13350 and the Akt homologous protein was purified by ammonium sulfate fractionation and many steps of column chromatographies such as, DEAE-Sepharose, Mono Q, Resource Phenyl-Soporose and Gel permeation column chromatographies. As a result, the protein phosphorylating the fluorescien-labeled Akt substrate was identified and it's molecular weight was estimated as 39 kDa on SDS-PAGE.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2024-2033
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• 2015

A gene encoding lipolytic enzyme, lip7-3, was isolated from Bacillus sp. W130-35 isolated from a tidal mud flat. The gene encoded a protein of 215 amino acids with a signal peptide composed of 34 amino acid residues. Lip7-3 belonged to the family I.4 lipase and showed its maximal activity at pH 9.0 and 60℃. Its activity increased in the presence of 30% methanol and, remarkably, increased as well to 154.6% in the presence of Ca²⁺. Lip7-3 preferred p-nitrophenyl octanoate (C8) as a substrate and exhibited broad specificity for short- to long- chain fatty acid esters. Additionally, Lip7-3 showed a low degree of enantioselectivity for an S-enantiomer (e.g., (S)-methyl-3-hydroxy-2-methylpropionate). It efficiently hydrolyzed glyceryl tributyrate, but did not hydrolyze glyceryl trioleate, fish oil, or olive oil. Its substrate specificity and activation by the solvent might offer a merit to the biotechnological enzyme applications like transesterification in the production of biodiesel.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.35 no.2
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• pp.39-45
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• 2003

This study was performed to develop the new type enzyme immobilization sheet from carboxymethylated refiner mechanical pulp (CRMP) substrate. Enzyme immobilization was attempted to couple carboxyl groups of CRMP with amino groups of the enzyme, trypsin, through the reaction of carbodiimide reagent, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodimide (EDC ). Immobilization carrier, water insoluble CRMP fraction (CRMP-IS), was successfully reacted with the enzyme, formed peptide linkage like -CONH- at 1680$cm^{-1}$ / and new ester linkage like -COO$CH_3$, methylester at 1735$cm^{-1}$ /, and produced enzyme immobilized substrate (CRMP-IST). The enzyme immobilized handsheet was prepared by mixing the above chelated enzyme immobilized substrate(CRMP-IST) with kraft pulp by paper sheet machine like papermaking process. The sheet weight and strength were increased with increasing dosage of CRMP-IST, and decreased at more than 10％ mixing of CRMP-IST, but higher than the controls. Concerning activities of immobilized trypsin(CRMP-IST) sheet by caseinolysis, the teared-off sheet with shaking was shown higher enzyme activities than sheet shape without shaking. In conclusion, this enzyme immobilized sheet would be expected easy handling for practical application and reutilization.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.40-45
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• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• BMB Reports
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• v.38 no.1
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• pp.58-64
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• 2005

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.751-751
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• 2005

• BMB Reports
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• v.30 no.4
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• pp.280-284
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• 1997

Phenylglyoxal diethyl pyrocarbonate (DEPC), and 1-cyclohexyl-3-[2-morpholinoethyl]-carbodiimide metho-p-toluenesulfonate (CMC) are modifying reagents specific for arginine, histidine, and aspartate or glutamate, respectively. They were found to inactivate S. cerevisiae farnesyl protein transferase (FPTase). The peptide substrate protected the enzyme against inactivation by CMC and the other substrate farnesyl pyrophosphate showed protection against inactivation by phenylglyoxal. while neither of the two substrates protected the enzyme against DEPC inactivation. These results suggest the presence of aspartate/glutamate, arginine and histidine residues at the active site of this enzyme.

• Journal of Microbiology and Biotechnology
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• v.5 no.6
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• pp.319-323
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• 1995

A chromogenic mimic of phenlyalanyl-dipeptide, L-phenylalanyl-L-2-sulfanilylglycine (PSG), was synthesized and examined for its usability in leucine aminopeptidase (LAP) assay. The enzyme activity was easily determined by measuring the amount of diazotized adduct of sulfanilic acid released upon hydrolysis of PSG ($\varepsilon^{420}$=18,000/M/cm). Under the experimental conditions employed, PSG showed a Km of 0.063 mM and a Kcat of 1683/min, assessable less than 0.1 $\mu$ g of LAP per milliliter. And the presence of aminopeptidase M (APM) was suggested to be negligible in LAP assay. This novel assay can circumvent the occasional yellow background in biological systems, i.e., serums, etc..

• Bulletin of the Korean Chemical Society
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• v.20 no.3
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• pp.301-306
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• 1999

Hepatitis C virus (HCV) has been known to be an enveloped virus with a positive strand RNA genome and the major agent of the vast majority of transfusion associated cases of hepatitis. For viral replication, HCV structural proteins are first processed by host cell signal peptidases and NS2/NS3 site of the nonstructural protein is cleaved by a zinc-dependent protease NS2 with N-terminal NS3. The four remaining junctions are cleaved by a separate NS3 protease. The solution conformations of NS4B/5A, NS5A/5B substrates and NS5A/5B inhibitor have been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data suggested that the both NS5A/5B substrate and inhibitor appeared to have a folded tum-like conformation not only between P1 and P6 position but also C-terminal region, whereas the NS4B/5A substrate exhibited mostly extended conformation. In addition, we have found that the conformation of the NS5A/5B inhibitor slightly differs from that of NS5A/5B substrate peptide, suggesting different binding mode for NS3 protease. These findings will be of importance for designing efficient inhibitor to suppress HCV processing.

• Journal of the Korean Magnetic Resonance Society
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• v.28 no.2
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• pp.6-9
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• 2024

HscA is a Hsp70-type chaperone protein that plays an essential role to mediate the iron-sulfur (Fe-S) cluster biogenesis mechanism in Escherichia coli. Like other Hsp70 chaperones, HscA is composed of two domains: the nucleotide binding domain (NBD), which can hydrolyze ATP and use its chemical energy to facilitate the Fe-S cluster transfer process, and the substrate binding domain (SBD), which directly interacts with the substrate, IscU, the scaffold protein of an Fe-S cluster. In the present work, we prepared the isolated SBD construct of HscA (HscA(SBD)) and conducted the solution-state nuclear magnetic resonance (NMR) experiments to have its backbone chemical shift assignment information. Due to low spectral quality of HscA(SBD), we obtained all the NMR data from the sample containing the peptide LPPVKIHC, the HscA-interaction motif of IscU, from which the chemical shift assignment could be done successfully. We expect that this information provides an important basis to execute detailed structural characterization of HscA and appreciate its interaction with IscU.