• KSBB Journal
• /
• v.16 no.2
• /
• pp.188-199
• /
• 2001

A gene encoding the dextransucrase(dsCB) that synthesizes mostly $\alpha-(1\rightarrow6)$ linked dextran with low amount(10%) of $\alpha-(1\rightarrow3)$ branching was cloned and sequenced from Leuconostoc mesenteroides B-742CB. The 6.1 kbp DNA fragment carrying dsCB showed one open reading frame(ORF) composed of 4,536bp. The deduced amino acid sequence shows that it begins from the start codon(ATG) at position 698 of the cloned DNA fragment and extends to the termination condon(TAA) at position 5,223. The enzyme is consisted of 1,508 amino acids and has an calculated molecular mass of 168.6kDa. This calculated Mw was in good agreement with an activity band of 170kDa on non-denaturing SDS-PAGE. A recombinant E. coli DH5 $alpha$ harboring pDSCB produced extracellular dextransucrase in 2% sucrose medium, and synthesized both soluble and insoluble dextran. To compare the properties of enzyme with B-742CB dextransucrase, the acceptor reaction, hydrolysis of dextran and methylation were performed. The expressed enzyme showed the same properties as B-742CB dextransucrease, but its ability to synthesize $\alpha-(1\rightarrow3)$ branching was lower than that of B-742CB dextransucrase. In order to identify the critical amino acid residues known as conserved regions related to catalytic activity, Asp-492 was replaced with Asn. D492N resulted in a 1.6 fold decrease in specific activity.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.10
• /
• pp.1589-1603
• /
• 2018

Twenty analogs of $[Orn^6,D-Ala^9]{\alpha}-factor$ were synthesized and assayed for their biological activities: seven analogs of $[Orn^6,X^9]{\alpha}-factor$, seven analogs of $[X^6,D-Ala^9]{\alpha}-factor$, five analogs of $[X^5,X^6,D-Ala^9]{\alpha}-factor$, and native ${\alpha}-factor$ (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native ${\alpha}-factor$ (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU-STE2 vector. $[Dap^6,D-Ala^9]{\alpha}-factor$ with weak halo activity (10%) showed the highest receptor affinity (> 230%) and the highest gene induction activity (167%). $[Arg^6,D-Ala^9]{\alpha}-factor$ showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newly-designed fluorescence-based detector, $[Arg^6,D-Ala^9]{\alpha}-factor-Edan$, with high sensitivity (12,500-fold higher than the absorption-based detector $[Orn^6]{\alpha}-factor-[Cys]_3$).

• Food Science and Preservation
• /
• v.24 no.1
• /
• pp.134-144
• /
• 2017

This study evaluated the changes of physiochemical properties, phytochemical compounds (isoflavones and phenolic acids), and biological activity during the fermentation of Doenjang without and with bitter melon powder (BMP). The pH decreased from 6.41-5.83 to 5.81-5.24, during the fermentation of Doenjang, while the acidity increased from 0.42-0.65% to 1.28-1.48%. The viable cell numbers of Bacillus and Yeast, salinity, and total amino acid contents increased at the end fermentation (60 day). Also, the fermented Doenjang (FD) with 10% BMP showed the highest ${\gamma}$-aminobutyric acid (GABA, 129.87 mg/100 g) contents, among all the Doenjang samples. The FD exhibited significantly higher inhibitory activities than unfermented Doenjang (UFD) on radicals and ${\alpha}$-glucosidase. The phytochemical compounds including isoflavone-aglycones and phenolic acids increased, whereas isoflavoneglycosides decreased in the BM following fermentative processing. Moreover, the total phenolic, isoflavone-aglycone, and phenolic acid contents were markedly increased, leading to a general increase in antioxidant and ${\alpha}$-glucosidase inhibition activities after fermentation. These results suggest that BMP may be used to prepare a new type of fermented Doenjang with improved antioxidant and antidiabetic activities.

• 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.

• BMB Reports
• /
• v.44 no.11
• /
• pp.719-724
• /
• 2011

The $Sec61{\alpha}$ subunit is the core subunit of the protein conducting channel which is required for protein translocation in eukaryotes and prokaryotes. In this study, we cloned a $Sec61{\alpha}$ subunit from Penicillium ochrochloron ($PoSec61{\alpha}$). Sequence and 3D structural model analysis showed that $PoSec61{\alpha}$ conserved the typical characteristics of eukaryotic and prokaryotic $Sec61{\alpha}$ subunit homologues. The pore ring known as the constriction point of the channel is formed by seven hydrophobic amino acids. Two methionine residues from transmembrane ${\alpha}$-helice 7 (TM7) contribute to the pore ring formation and projected notably to the pore area and narrowed the pore compared with the superposed residues at the corresponding positions in the crystal structures or the 3D models of the $Sec61{\alpha}$ subunit homologues in archaea or other eukaryotes, respectively. Results reported herein indicate that the pore ring residues differ among $Sec61{\alpha}$ subunit homologues and two hydrophobic residues in the TM7 contribute to the pore ring formation.

• Korean Journal of Food Science and Technology
• /
• v.27 no.5
• /
• pp.762-767
• /
• 1995

The ${\alpha}-Amylase$ inhibitor from black bean(Phaseolus vulgaris) was purified to homogeneity using 70% saturated ammonium sulfate, DEAF-cellulose, Concanavalin-A sepharose chromatography and gel filtration with Superose 6. The purified α-amylase inhibitor showed a single band of 25 KD in molecular weight on the SDS-PAGE. The specific activity of the inhibitor was 544.0 units/mg and the purity was enhanced about 18-fold. The amino acids of ${\alpha}-Amylase$ inhibitor from black bean was mainly glutamic acid, aspartic acid and lysine. The inhibitor was glycoproteins and its carbohydrate contents was 3.2%.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.23 no.1
• /
• pp.12-24
• /
• 1990

Two trypsin-like enzymes, designated trypsin A and 3, purified from the intestine of menhaden by $(NH_4)_2SO_4$ fractionation, Benzamidine-Sepharose 6B affinity chromatography, DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The two trypsins were subjected to compare the enzymatic properties of the trypsin-like enzymes from the other dark fleshed fishes. Both trypsins catalysed the hydrolysis of N$\alpha$-benzoyl-DL-arginine-p-nitroanilide and they were remarkably inhibited by several well known trypsin-inhibitors, tosyllysyl chloromethyl ketone, soybean trypsin inhibitor, be-nzamidine, leupeptin and antipain, etc. Therefore, it was ascertained that the two enzymes are serine-type trypsins. The molecular weights of these enzymes were about 25,000 and 26,200, respectively, ;Is determined by SDS-PAG electrophoresis and by Sephadex G-100 gel filtration, and the molecular weights of these two enzymes are somewhat fewer than those from the other dark fleshed fishes. Both enzymes had less basic amino acids such as arginine and Iysine, whereas they had slightly high contents of neutral amino acids, glycine, alanine and tryptophane. The enzymes showed a pH optimum of $8\~11$ at $60^{\circ}C$ against the $N\alpha$-benzoyl-DL-argi-nine-p-nitroanilide substrate and they were quite unstable above $40^{\circ}C$ and under the atidic pH region. The Km constant of the two enzymes against the $N\alpha$-benzoyl-DL-arginine-p-nitroanilide was $1.4\times10^{-4}M$ for trypsin A and $4.3\times10^{-5}M$ for trypsin B, respectively.

• Natural Product Sciences
• /
• v.20 no.3
• /
• pp.160-169
• /
• 2014

44 compounds and 9 minerals were isolated from and detected in the New Zealand deer velvet antler Cervus elaphus var. scoticus L$\ddot{o}$nnberg. The chemical structures of (1 - 26) were identified on the basis of the spectroscopic methods and comparisons with literature, respectively. The structures were identified as cholesterol (CS, 6), 7-keto-CS (7), $7{\beta}$-hydroxy-CS (8), and $7{\alpha}$-hydroxy-CS (9), and included 12 steroid $3{\beta}$-O-(palmitic/stearic/myristic acid esters; PM/SA/MS) [CS-$3{\beta}$-O-PM (1 - 1), CS-$3{\beta}$-O-SA (1 - 2), CS-$3{\beta}$-O-MR (1 - 3), 7-keto-CS-$3{\beta}$-O-PM (2 - 1), 7-keto-CS-$3{\beta}$-O-SA (2 - 2), 7-keto-CS-$3{\beta}$-O-MR (2 - 3), $7{\beta}$-hydroxy-CS-$3{\beta}$-O-SA (3 -1), $7{\beta}$-hydroxy-CS-$3{\beta}$-O-PM (3 - 2), $7{\beta}$-hydroxy-CS-$3{\beta}$-O-MR (3 - 3), $7{\alpha}$-hydroxy-CS-$3{\beta}$-O-SA (4 - 1), $7{\alpha}$-hydroxy-CS-$3{\beta}$-O-PM (4 - 2), and $7{\alpha}$-hydroxy-CS-$3{\beta}$-O-MR (4 - 3)], dinonyl phthalate (5), 8 nucleic acids analogues [uracil (10), deoxyguanosine (11), deoxyuridine (12), uridine (13), deoxyadenosine (14), adenosine (15), inosine (16), and guanosine (17)], and the 9 free amino acids [L-phenylalanine (18), L-isoleucine (19), L-leucine (20), L-tyrosine (21), L-valine (22), L-proline (23), L-threonine (24), L-alanine (25), and L-hydroxyproline (26)]. Also, there are 8 kinds of amino acids [asparagine, serine, glutamine, glycine, histidine, arginine, methionine, and lysine], 2 sialic acids [N-acetylneuraminic acid (27), ketodeoxynonulosonic acid (28)], and 9 minerals [Na > K > Ca > Mg > Fe > Zn > B > Al > Cu] were detected from the autoaminoacid analyzer and ICP spectrometer, HPAEC-PAD/HPLC-FLD, respectively. 9 kinds of oxycholesterol-$3{\beta}$-O-fatty acid ester (2 - 1, 2 - 2, 2 - 3, 3 - 1, 3 - 2, 3 - 3, 4 - 1, 4 - 2, and 4 - 3) and 3 nucleic acids (12, 14, and 15) were isolated from the velvet antler for the first time. 6 kinds of steroids (7, 8, 9, 2 - 1, 3 - 1, and 4 - 1) were examined for their anti-proliferative effects against L1210, P388D1, K562, MEG-01, KG-1, MOLT-4, A549, HepG2, MCF-7, SK-OV-3, and SW-620 cancer cell lines. They showed anti-proliferative effects with $IC_{50}$ values of 0.06, 2.16, 2.42, > 50.0, 1.66 and $8.31{\mu}M$ against L1210, while the values were 24.05, 9.44, 5.22, 0.25. 9.48 and $49.77{\mu}M$ against P388D1, respectively. The others were inactive.

• Journal of Life Science
• /
• v.11 no.1
• /
• pp.57-64
• /
• 2001

The cDNA encoding human NeuAc ${\alpha}$2,3Gal$\beta$ 1,3GalNAc GalNac ${\alpha}$2,6-Sialyltransferase (hST6GalNac IV) was isolated by screening of human fetal liver cDNA library with a DNA probe generated from the cDNA sequence of mouse ST6Gal NAc IV (mkST6GalNAc IV). The cDNA sequence included an open reading frame coding for 302 amino acids, and comparative analysis of this cDNA with mST6GalNAc IV showed that each sequence of the predicted coding region contains 88% and 85% identifies in nucleotide and amino acid levels, respecively. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short N-terminal cytoplamic domain, a transmembrane domain and a large C-terminal active domain. This enzyme expressed in COS-7 cells echibited transferase activity toward NeuAc ${\alpha}$2,3Gal$\beta$ 1,3GalNAc, fetuin and GM1b, although the activity toward the later is very low, no significant activity being detected toward Gal${\beta}$ 1,3Gal NAc or asialofetuin, the other glycoprotein substrates tested. The $^{14}$ C-sialylated residue of fetuin sialylated by this enzyem with CMP-[$^{14}$C]NeuAc was sensitive to treatment with ${\alpha}$2,8-specific sialidase of Vibrio cholerae but resistant to treatment with ${\alpha}$2,3-specific sialidase (NaNase I), and ${\alpha}$2,3- and ${\alpha}$2,8-specific sialidase of Newcastle disease virus. These results clearly indicated that the expressed enzyme is a type of GalNAc ${\alpha}$2,6-sialyltransferase like mST6GalNAc IV, which requires sialic acid residues linked to Gal${\beta}$1,3GalNAc-residues for its activity.

• Journal of Animal Science and Technology
• /
• v.46 no.6
• /
• pp.909-916
• /
• 2004

CCAAT/enhancer binding proteins(C/EBP) are a group of transcription factors expressed during preadipocyte differentiation. In the C/EBPs, C/EBPa plays an important role in lipid deposition and adipocyte differentiation. In this studies, we report the identification, characterization, and expression of a Hanwoo CIEBP$\alpha$ The Hanwoo C/EBP$\alpha$DNA includes a 1059 bp open reading frame encoding a protein of 353 amino acids. The CIEBPa amino acid sequences of the Hanwoo show strong conservation with the corresponding sequences reported in other species. The distribution of C/EBP$\alpha$ mRNA in various tissues of Hanwoo aged 12 months were investigated using Northern blotting analysis. The highest expression was detected in adipose tissue and more lower expression was detected in colon and lung. We also identified expression of C/EBPa mRNA in Hanwoo sirloin and adipose tissue aged 12, 26, and 30 months by real-time RT-PCR. The higest expression were detected at 26 months in the sirloin and at 12 and 26 months in the adipose tissue.