• 한국동물위생학회지
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• 제42권2호
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• pp.93-112
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• 2019

Calf diarrhea is a common disease in young claves and is still a major cause of productivity and economic loss in livestock farms. Fecal samples from Korean native cattle (n=100) with diarrhea from 64 farms in Gwangju area, Korea from september 2017 to December 2018 were examined for shedding of important protozoan parasitic, viral and bacterial pathogens using culture, rapid test kit and PCR methods. Of 57 (89.1%) of the 64 Korean native cattle farms examined had samples infected with at least one of the investigated pathogens. Among 100 fecal samples, 88 samples were positive for at least one the twelve pathogens and 51 samples were simultaneously positive for two or more pathogens by culture and PCR assay. Bovine group A rotavirus (BRV) was the most common pathogen, found in 43/100 (43.0%) samples on 32/64 (50.0%) farms. Subsequently, kobuvirus (30.0%), pathogenic E. coli (29.0%), bovine parvovirus (17.0%), Giardia spp. (13.0%), Eimeria spp. (10.0%), Clostridium perfringens type A (8.0%), bovine torovirus (8.0%), bovine viral diarrhea virus (6.0%), bovine coronavirus (5.0%), bovine norovirus (2.0%) and Cryptosporidium spp. (2.0%) were detected. Nebovirus, kırklareli virus, bovine adenovirus, Salmonella spp. and intestinal parasites were not detected. Of the 72 calves sampled in this age group, 64 (88.9%) samples were positive for at least one enteropathogen. BRV was identified in 34/72 (47.2%) samples from 27/48 (56.3%) farms. Subsequently, pathogenic E. coli (30.6%), kobuvirus (29.2%), BPaV (22.2%), Giardia spp. (15.3%), Eimeria spp. (9.7%), BVDV (6.9%), Cl. perfringens type A (6.9%), BCoV (4.6%) and Cryptosporidium spp. (2.8%) were detected in fecal samples. A total of ninety-six strains of E. coli were isolated from one hundred fecal samples collected from Korean native cattle with diarrhea. The presence of stx1, stx2, eaeA, LT, STa, STb, ehxA, saa, F4, F5(K99), F6, F17, F18 and F41 genes in the isolates was investigated by PCR. Out of ninety-six E. coli isolates screened for specific genes, 30 strains E. coli were identified to harbor shiga toxin-producing E. coli (STEC) 7 (7.3%), enterohemorrhagic E. coli (EHEC) 8 (8.3%), enteropathogenic E. coli (EPEC) 6 (6.3%), enterotoxigenic E. coli (ETEC) 2 (2.1%) and STEC/ETEC hybrid 7 (7.3%). This study provides epidemiological estimates of the prevalence of Korean native cattle's enteropathogens in Gwangju area, Korea, which would be used for cattle farmers and veterinarians to select appropriate therapeutic method.

• The Plant Pathology Journal
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• 제22권2호
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• pp.125-130
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• 2006

An isolate of Peanut stunt virus (PSV) isolated from black locust tree (Robinia pseudo-acacia L.) showing severe mosaic and malformation symptoms, was designated as PSV-Rp. PSV-Rp was characterized by the tests of host range, physical properties, RNA and coat protein composition and RT-PCR analysis. Nucleotide sequences of the cucumoviruses CP genes were also used for identification and differentiation of PSV-Rp. Six plant species were used in the host range test of PSV-Rp. PSV-Rp could be differentiated from each Cucumovirus strain used as a control by symptoms of the plants. The physical properties of PSV-Rp virus were TIP $65^{\circ}C$, DEP $10^{-3}$, and LIP $2{\sim}3$ days. In dsRNA analysis, PSV-Rp consisted of four dsRNAs, but satellite RNA was not detected. Analysis of the coat proteins by SDS-PAGE showed one major protein band of about 31 kDa. RT-PCR using a part of Cucumovirus RNA3 specific primer amplified ${\sim}950bp$ DNA fragments from the crude sap of virus-infected black locust leaves. RFLP analysis of the RT-PCR product could differential PSV-RP from CMV The nucleotide sequence identity between the PSV-Rp CP and the TAV-P CP genes and the PS-V-RP CP and CMV-Y CP genes were 61.6% and 40.5%, respectively. On the other hand, the nucleotide sequence identity of the PSV-Rp CP gene was $70.9%{\sim}73.4%$ in comparison with those of PSV subgroup I (PSV-ER and PSV-J) and 67.3% with that of PSV subgroup II(PSV-W). Especially, the nucleotide sequence identity of PSV-Rp CP gene and that of PSV-Mi that was proposed recently as the type member of a novel PSV subgroup III was 92.4%.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.534-537
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• 2002

본 연구에서는 기능성 식품 첨가물 소재 또는 의약품으로 사용 가능한 한천올리고당의 대량생산을 위하여 agarase 생산균주인 Bacillus cereus ASK202에 대해 유전자 cloning 방법을 이용하여 한천분해효소 고생산성 균주로의 개발을 시도하였다. 원균주의 chromosomal DNA를 무작위적으로 절단하여 agarase를 생산하는 gene 부위를 선별한 결과, 83,300 Da의 agarase(Eba1)를 생산하는 재조합 균주 E. coli BL21(DE3)/pEBA1를 얻을 수 있었다. E. coli BL21(DE3)/pEBAl에서 유도물질로 IPTG를 첨가한 후 induction 5시간 후에 agarase가 원균주에 비해 8배 증가한 양이 고발현되었다. 발현된 agarase는 Asx(Aspartic acid, Asparagine), Glycine와 같은 산성 아미노산의 함량과 Alanine과 같은 중성 아미노산의 함량이 높았으며, pH 5.6, $40^{\circ}C$에서 최적의 활성을 나타내었다. 최적 기질 agar에 대한 $K_m$과 $V_{max}$값은 0.068 mg/$m{\ell}$과 0.094mg/$m{\ell}{\cdot}min$으로 한천의 ${\beta}$-결합을 자르는 ${\beta}$-agarase로 판명되었다.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1460-1468
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• 2007

In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from $20^{\circ}C$ to $60^{\circ}C$C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's $K_m$ value and $V_{max}$ of the catalase for $H_2O_2$ were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of $A_{406}$ to $A_{280}$ for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1 in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.

• Journal of Microbiology and Biotechnology
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• 제30권3호
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• pp.391-397
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• 2020

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb₂ into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb₂ under optimal conditions (pH 7.0 and 40℃). Kinetic parameters for α-L-arabinopyranosidase showed apparent K_m and V_max values of 0.078 ± 0.0002 μM and 1.4 ± 0.1 μmol/min/mg of protein against p-nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 ㎍/ml), 0.1% of ginsenoside Rb₂ was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb₂.

• Journal of Microbiology and Biotechnology
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• 제26권9호
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• pp.1650-1656
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• 2016

The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40℃ and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters V_max and K_m for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1054-1059
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• 2005

An extracellular protease was identified from Bacillus subtilis KCCM 10257 by N-terminal sequencing and mass spectral analysis. The molecular mass of the extracellular protease was estimated to be 28 kDa by SDS-PAGE. Sequencing of the N-terminal of the protease revealed the sequence of A(G,S,R)QXVPYG(A)V(P,L)SQ. The N-terminal sequence exhibited close similarity to the sequence of other proteases from Bacillus sp. A mass list of the monoisotopic peaks in the MALDI-TOF spectrum was searched after peptide fragmentation of the protease. Six peptide sequences exhibiting monoisotopic masses of 1,276.61, 1,513.67, 1,652.81, 1,661.83, 1,252.61, and 1,033.46 were observed from the fragmented protease. These monisotopic masses corresponded to the lytic enzyme L27 from Bacillus subtilis 168, and the Mowse score was found to be 75. A doubly charged Top product (MS) at a m/z of 517.3 exhibiting a molecular mass of 1034.6 was further analyzed by de novo sequencing using a PE Sciex QSTAR Hybrid Quadropole-TOF (MS/MS) mass spectrometer. MS/MS spectra of the Top product (MS) at a m/z of 517.3 obtained from the fragmented peptide mixture of protease with Q-star contained the b-ion series of 114.2, 171.2, 286.2, 357.2, 504.2, 667.4, 830.1, and 887.1 and y-ion series of 147.5, 204.2, 367.2, 530.3, 677.4, 748.4, 863.4, and 920.5. The sequence of analyzed peptide ion was identified as LGDAFYYG from the b- and y-ion series by de novo sequencing and corresponded to the results from the MALDI-TOF spectrum. From these results the extracellular protease from Bacillus subtilis KCCM 10257 was successfully identified with the lytic enzyme L27 from Bacillus subtilis 168.

• Asian Pacific Journal of Cancer Prevention
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• 제16권3호
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• pp.1011-1018
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• 2015

This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions, signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis). The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4) were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis.

• Molecules and Cells
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• 제19권3호
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• pp.398-401
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• 2005

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to $2.8{\AA}$ resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to $2.2{\AA}$ have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or $I2_12_12_1$) with unit-cell parameters of a = 63.7, b = 124.5, and $c=126.3{\AA}$. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter ($V_M$) is $2.71{\AA}^3Da^{-1}$ and solvent content is 54.6%.

• Journal of Microbiology
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• 제45권4호
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• pp.333-338
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• 2007

Intracellular NADH:quinone reductase involved in degradation of aromatic compounds including lignin was purified and characterized from white rot fungus Trametes versicolor. The activity of quinone reductase was maximal after 3 days of incubation in fungal culture, and the enzyme was purified to homogeneity using ion-exchange, hydrophobic interaction, and gel filtration chromatographies. The purified enzyme has a molecular mass of 41kDa as determined by SDS-PAGE, and exhibits a broad temperature optimum between $20-40^{\circ}C$, with a pH optimum of 6.0. The enzyme preferred FAD as a cofactor and NADH rather than NADPH as an electron donor. Among quinone compounds tested as substrate, menadione showed the highest enzyme activity followed by 1,4-benzoquinone. The enzyme activity was inhibited by $CuSO_4,\;HgCl_2,\;MgSO_4,\;MnSO_4,\;AgNO_3$, dicumarol, KCN, $NaN_3$, and EDTA. Its $K_m\;and\;V_{max}$ with NADH as an electron donor were $23{\mu}M\;and\;101mM/mg$ per min, respectively, and showed a high substrate affinity. Purified quinone reductase could reduce 1,4-benzoquinone to hydroquinone, and induction of this enzyme was higher by 1,4-benzoquinone than those of other quinone compounds.