• Journal of agriculture & life science
• /
• v.45 no.6
• /
• pp.201-212
• /
• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.4
• /
• pp.775-784
• /
• 2017

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase ($CcXyn-{\Delta}5C$) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but $CcXyn-{\Delta}5C$ contained less ${\alpha}-helices$ (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature ($45^{\circ}C$) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and $CcXyn-{\Delta}5C$, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, $CcXyn-{\Delta}5C$ exhibited a much lower $K_m$ value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of $CcXyn-{\Delta}5C$ was 2.4-times higher than that of CcXyn. These properties make $CcXyn-{\Delta}5C$ a good model for the structure-function study of $({\alpha}/{\beta})_8$-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

• 한국생물공학회:학술대회논문집
• /
• 2005.04a
• /
• pp.88-92
• /
• 2005

Aptamer is the single-stranded oligonucleotide which binds to various target molecules such as proteins, peptides, lipids and small organic molecules with high affinity and specificity. DNA aptamers specific for the $17{\beta}-estradiol$ were selected by SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random DNA library. These DNA aptamers have a high affinity to $17{\beta}-estradiol$ as an endocrine disrupting chemical. Nanowell and $200{\mu}m$ gold electrode were used as substrate for DNA aptamer immobilization and electrochemical analysis. Especially, nanowell gold electrode was fabricated by e-beam lithography. The size of single nanowell is 130nm and 40,000 nanowells were deposited on one gold electrode. The immobilization method was based on the interaction between the biotinylated aptamer and streptavidin deposited on gold electrode previously. Immobilization procedure was optimized by surface plasma resonance (SPR) and electrochemical analysis. After the immobilization of DNA aptamer on streptavidin modified gold electrode, $17{\beta}-estradiol$ solution was treated on aptamer immobilized gold electrode. The current of gold electrode was decreased by the binding of $17{\beta}-estradiol$ to DNA aptamer immobilized on gold electrode. However, in negative control experiments of 1-aminoanthraquinone and 2-methoxynaphthalene, the current was rarely decreased. And more sensitive data was obtained from nanowell gold electrode comparing with $200{\mu}m$ gold electrode.

• Korean Journal of Food Science and Technology
• /
• v.23 no.4
• /
• pp.414-419
• /
• 1991

Polyphenol oxidase from Jerusalem artichoke(Helianthus tuberosus L.) tubers was partially purified by precipitation with ammonium sulfate, followed by gel filtration on Sephadex G-100. The enzyme showed maximal activity at pH 6.5 and $4^{\circ}C$. Kinetic studies indicated $K_{m}$ value of 3 mM for catechol and activation energy of 72.6 kcal/mole. As for substrate specificity of polyphenol oxidase the enzyme showed high affinity towards diphenol compounds, but not towards monophenols. The enzamatic browning was completely inhibited at 1 mM concentration of L-ascorbic acid, sodium hydrosulfite and L-cystein(HCl). The activity of polyphenol oxidase in 0.1 M potassium phosphate buffer(pH 6.5) was fairly stable for a week at $4^{\circ}C$, while it decreased remarkably at $25^{\circ}C$.

• BMB Reports
• /
• v.31 no.3
• /
• pp.221-226
• /
• 1998

Phosphorylation of phytochrome may play important functional roles to control plant photomorphogenesis. Many attempts have failed to identify the protein kinase that phosphorylates phytochrome in vivo. It has been reported that a polycation-stimulated protein kinase activity was associated with the purified phytochrome. However, it is not known if the kinase activity is an intrinsic property of phytochrome or whether it comes from a contaminant of the purified phytochrome. In the present study, three protein kinases that phosphorylate phytochrome have been identified from etiolated oat seedlings. A polycationstimulated protein kinase that had very similar enzymatic properties with that associated with the purified phytochrome was identified in the cytosolic extract. It phosphorylated several contaminant proteins in the kinase preparation as well as phytochrome and had a broad substrate specificity. A CK II-type protein kinase phosphorylated phytochrome and the exogenously added casein. It is likely that this kinase may not be a feasible candidate for the kinase phosphorylating phytochrome in vivo since the content of the kinase seemed to well exceed the content of phytochrome in the etiolated oat seedlings. Another protein kinase that had unique enzymatic properties phosphorylated phytochrome very specifically and seemed to be present in a small quantity in the etiohlted seedlings. It is expected that one of three kinases may be responsible for the phytochrome phosphorylation in vivo.

• BMB Reports
• /
• v.31 no.3
• /
• pp.274-280
• /
• 1998

An inositol monophosphatase (IMPase) was purified to homogeneity from rat duodenal mucosa for the first time and its enzymatic properties were investigated. Rat duodenal mucosa peculiarly exhibited the highest IMPase activity among various rat tissues examined. By means of ammonium sulfate precipitation, followed by Q-Sepharose, polylysine agarose, reactive-red agarose column chromatography, Uno-Q FPLC, and Bio-Silect FPLC, duodenal IMPase was purified 223-fold to a specific activity of 13.6 U/mg protein. The molecular mass of the native enzyme was estimated to be 48,000 Da on gel filtration. The subunit molecular mass was determined by SDS-PAGE to be 24,000 Da. These results indicate that duodenal IMPase is a dime ric protein made up of identical subunits. Rat duodenal IMPase has distinct properties from brain IMPase. It has a broad spectrum of substrate specificity and is insensitive to $Li^+$. Duodenal IMPase does not absolutely require $Mg^{2+}$ for its catalytic activity. Furthermore, duodenal IMPase is less stable to heat than brain enzyme. It is suggested that the rat duodenal mucosa needs a large amount of IMPase whose properties are quite different from that of the brain enzyme.

• Journal of Plant Biotechnology
• /
• v.2 no.2
• /
• pp.79-82
• /
• 2000

We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which was designated NtCDPK1. Accumulation of the NtCDPK1 mRNA was stimulated by various stimuli, including phytohormones, CaCl$_2$ wounding, fungal elicitors, chitin and methyl jasmonate. The NtCDPK1 gene encodes a functional Ser/Thr protein kinase of which phosphorylation activity is strongly induced by calcium. By analyzing expression of the NtCDPK1-GFP fusion protein and by immunoblotting with antibody which reacts with NtCDPK1, we found that NtCDPK1 is localized in membrane and nucleus in plant cells. Silencing expression of the NtCDPK1 transgene resulted in marked decrease of lateral root development in the transgenic tobacco plants. Yeast two hybrid screening using NtCDPK1 as a bait identified a tobacco homologue of proteasome regulatory subunit 21D7, designated Nt21D7. The 21D7 mRNA has been shown to be predominantly expressed in proliferating tissues in the cell cycledependent manner in carrot. The recombinant NtCDPK1 protein associated with Nt21D7 in vitro, and could phosphorylate the Nt21D7 protein in vitro in the presence of calcium, suggesting that Nt21D7 protein is a natural substrate of NtCDPK1 in tobacco. These results suggest that NtCDPK1 may regulate tell proliferation processes, such as lateral root formation, by regulating specificity and/or activity of proteasome-mediated protein degradation pathway.

• The Korean Journal of Mycology
• /
• v.32 no.2
• /
• pp.119-124
• /
• 2004

Metalloenzyme was purified from the fruiting bodies of Tricholoma sejunctum. MALDI-TOF and ICP/MS analyses revealed that the enzyme had a molecular weight of 18788.25 and includes $Zn^{2+}$ ion. The N-terminal amino acid sequence of the enzyme was Ala-Thr-Tyr-Lys-Ile-X-Ser-Ala-Thr-His-Gln-X-X-Leu-Val. The activity of the enzyme was inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme was a metalloprotease. No inhibition was found with E-64 and pepstatin. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $40^{\circ}C$. The activity of the enzyme was increased by $Zn^{2+}$ and $Co^{2+}$, while it was totally inhibited by $Hg^{2+}$. The enzyme hydrolyzes $A{\alpha}$ subunit of human fibrinogen but did not show any reactivity for $B{\beta}$ and ${\gamma}$ form of human fibrinogen.

• Journal of Dairy Science and Biotechnology
• /
• v.29 no.1
• /
• pp.49-54
• /
• 2011

Bile salt hydrolase (BSH, EC 3.5.1.24) activity, which cleaves amide bond between carboxyl group (bile acid) and amino group (glycine or taurine), is commonly detected in gut-associated species of human and animal. During the screening of BSH active strains from the fecal samples of elderly human volunteers, strain CU30-2 was isolated on the basis of the highly active BSH producing activity. A bsh gene of the isolate was cloned into the pET22b expression vector and overexpressed in Escherichia coli BL21 (DE3) Gold by induction with 1mM IPTG. The overexpressed BSH enzyme with 6x His-tag was purified with apparent homogeneity using a $Ni^+$-NTA agarose column and characterized. The BSH enzyme of E. faecalis CU30-2 exhibited approximately 50 times higher activity against glycol-conjugated bile salts than tauro-conjugated bile salts having the highest activity against glycocholic acid. Considering the prevalence of E. faecalis strains in the human GI tract and glycol-conjugates dominated bile acid composition of human bile, further study is needed to investigate the impact of the BSH activity exerted by E. faecalis strains to the host as well as to the BSH producing strains.

• BMB Reports
• /
• v.33 no.2
• /
• pp.112-119
• /
• 2000

Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitor, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1, 2 and 3 (DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki = $3.6{\times}10^{-9}\;M$), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki = $6.2{\times}10^{-8}\;M$). Pre-treatment of the 33 mg/kg of DI-mixture (active fractions from $C_{18}$ open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.