• 한국미생물·생명공학회지
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• 제37권1호
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• pp.17-23
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• 2009

PCR법을 이용하여 Thermus thermophilus HJ6 유래 DNA polymerase(Tod) 유전자를 클로닝하고 염기서열을 분석한 결과, ORF는 2,505개의 뉴클레오타이드로 구성되고 834개의 아미노산을 암호화하였다. 아마노산 서열을 바탕으로 상동성을 분석한 결과, Thermus thermophilus HB8 유래 DNA polymerase와 98%, Thermus aquaticus 유래 DNA polymorase와 86%의 identity를 나타내었다. 이 유전자를 박테리오파지 $\lambda$ 유래 온도감수성 프로모터(PR, PL)를 포함하는 pJLA503 벡터를 이용하여 대장균에서 발현하였다. 발현된 효소는 열처리, $HiTrap^{TM}$ Q column과 $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 columun으로 정제하여 94 kDa의 단백질을 얻을 수 있었다. 정제된 효소의 DNA 중합 활성에 대한 최적온도는 $75{\sim}80^{\circ}C$이고 최적 pH가 9.0이었다. $Mg^{2+}$ and $Mn^{2+}$에 대한 최적 농도는 각각 2.5mM과 1mM이었고 효소활성은 2가 양이온의 존재 하에서는 활성화 되지만 1가양이온에서는 저 해되었다. Tod DNA 중합효소를 이용한 PCR 실험결과, Tod DNA 중합효소는 DNA 증폭 및 PCR 관련 기술에 응용 가능할 것으로 생각된다.

• 한국미생물·생명공학회지
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• 제41권2호
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• pp.153-159
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• 2013

Sinorhizobium meliloti 1021 (KCTC 2353) 유전체로부터 mannitol dehydrogenase (SmMDH)로 추정되는 유전자를 클로닝하고, 대장균에서 대량 발현하였다. 이 유전자는 494개의 아미노산(약 54 kDa)을 암호화하는 1,485 bp의 염기로 구성되며, 기존에 보고된 long-chain dehydrogenase/reductase 계열 MDH 효소들과 약 35-55%의 아미노산 서열상동성을 나타내었다. 재조합 SmMDH의 최적 반응온도는 $40^{\circ}C$이며, pH 7.0의 조건에서 최대의 D-fructose 환원활성, 그리고 pH 9.0에서 최대의 D-mannitol 산화활성을 보였다. 특히, 이 효소는 $NAD^+/NADH$ 조효소의 존재 하에서 산화 환원 활성을 나타내며, $NAD^+/NADPH$는 조효소로 이용하지 못하였다. 결론적으로 SmMDH는 전형적인 $NAD^+/NADH$-의존형 mannitol dehydrogenase (EC 1.1.1.67)임을 확인하였다.

• 대한물리치료과학회지
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• 제15권2호
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• pp.41-51
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• 2008

Background: The purpose of this study is to determine how the cognitive state of the elderly influences the activities of daily living(ADL) in order to increase their achievement capacity and their ability to exercise this capacity in daily life. The MMSE-K(Mini-Mental State Examination-Korean) was used to evaluate the daily activities and cognitive state of a group of subjects more than 65 years of age. The goal was to understand (1) How their achievement level in their daily activities changes according to the increase and decrease of their cognition points by analyzing and comparing their achievement points in the mental state examination, and (2) What variables influence that daily achievement level. Methods: The survey was conducted by a group of students from AnSan College who were trained for this method of data collection. The group surveyed 224 senior citizens older than 65 to determine their ADL performance. The subjects were selected from Silver Care Centers in the Gyounggi Province area including centers in Suwon, Anyang, Kwachun, and Sanbon City. The subjects were given enough information to fully understand the purpose of the study and the method by which it would be conducted. The survey method was a direct interview: which involved an interviewer reading the questionnaire to the subject and recording the answer. Result: The following results were calculated using SPSS WIN ver. 13.0: In the lower score of the MMSE-K regarding ADL performance (K-MBI) for activities such as individual hygiene, bathing activity, food activity, toilet management, going up stairs, getting dressed ad undressed, controlling stools and urination, walking, and chair/bed movement, most of the subjects responded that they could not perform these activities by themselves (p<.01). In the higher scores for the same activities, most response that they could perform the tasks by themselves (p<.01). In the results of the multiple regression analysis, there influence of the variables for ADL performance (K-MBI) and MMSE-K score was higher for females than males. Conclusion: This study evaluated the cognitive state of the elderly using the MMSE-K, which is the most convenient method, and examined how the cognitive state influences the achievement capacity of the daily lives with other variables. In conclusion, the higher the score on the MMSE-K, the more independent the elders are in the activities of daily living, especially among women more than men.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1500-1508
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• 2013

Ebosin, a novel exopolysaccharide produced by Streptomyces sp. 139, has obvious antirheumatic arthritis activity in vivo, and its biosynthesis gene cluster (ste), consisting of 27 open reading frames, has been identified. This paper reports our study of the gene functionality of ste8, the predicted protein product of which is homologous to some bacterial chain length determinant Wzz proteins. For characterization of Ste8, ste8 was cloned and expressed in the mutant strain E. coli 086:H2 (${\Delta}wzz$). The functional complementation of wzz by ste8 was demonstrated by the restoration of wild-type lipopolysaccharide biosynthesis and increased levels of serum resistance of E. coli 086:H2 (${\Delta}wzz$) (pET30a-ste8). To examine the function of ste8 in ebosin biosynthesis, the gene was knocked out with a double crossover via homologous recombination. The molecular weight of the ebosin derivative EPS-8m produced by the mutant Streptomyces sp. 139 ($ste8^-$) was much lower than that of ebosin, and the binding activity of EPS-8m for IL-1R decreased significantly compared with ebosin. These results demonstrate that ste8 encodes a chain length determinant (Wzz) that functions in ebosin biosynthesis.

• Molecules and Cells
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• 제26권6호
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• pp.536-547
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• 2008

Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA and genomic DNA sequences of ANS gene (designated as GbANS) were isolated from Ginkgo biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA analysis showed that GbANS gene had three exons and two introns. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbANS at the similar positions like other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The recombinant protein was successfully expressed in E. coli strain with pET-28a vector. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.

• Asian-Australasian Journal of Animal Sciences
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• 제30권5호
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• pp.736-742
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• 2017

Objective: Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1. Methods: The complete sequence of Wild Argali SPLUNC1 cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in Pichia pastoris (P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by $Ni^{2+}$ chelate affinity chromatography. The test of effect of the protein on Mycoplasma ovipneumoniae (MO) was performed with real-time polymerase chain reaction. Results: The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from Ovis aries (accession no. NP_001288334.1), Capra hircus (accession no. XP_005688516.1), Pantholops hodgsonii (accession no. XP_005979709.1), Bos taurus (accession no. NP_776851.1), Felis catus (accession no. XP_006929910.1), Homo sapiens (accession no. NP_001230122.1), Sus scrofa (accession no. NP_001005727.1), Chinchilla lanigera (accession no. NP_001269294.1), Mus musculus (accession no. NP_035256.2), and Rattus norvegicus (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of P. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01). Conclusion: The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.

• BMB Reports
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• 제34권4호
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• pp.334-341
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• 2001

Even though three isotypes of thioredoxins (-f, -m and -h types) have been identified in a variety of plant cells, there are only a few reports on thioredoxin-h that were recently identified. In this study, a cDNA encoding a h-type of thioredoxin was isolated from a cDNA library of Chinese cabbage, and named here CTrx-h. An open reading frame of the gene contained a polypeptide of 133 amino acids with a conserved active center, WCGPC, which appeared in all of the thioredoxin proteins. A deduced amino acid sequence of the CTrx-h showed the highest sequence identity with those of Arabidopsis thioredoxin-h2 (75.2%) and thioredoxin-h5 (46.6%) proteins, but it shared a low sequence homology to other isotypes of plant thioredoxinm and thioredoxin-f. The CTrx-h protein that is expressed in E. coli represented not only an insulin reduction activity, but also electron transferring activity from NADPH to thioredoxin-dependent peroxidase. A genomic Southern blot analysis using the cDNA insert of CTrx-h revealed that the gene consisted of a small multigene family in Chinese cabbage genome. On the contrary to other thioredoxin-h proteins that were widely distributed in most tissues of the plant, the CTrx-h was predominantly expressed in flowers. The expression was very low in other tissues. The data of the Northern blot analysis suggests that the CTrx-h may have other functions in flower development or differentiation, in addition to its defensive role.

• 미생물학회지
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• 제48권4호
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• pp.293-297
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• 2012

극지의 다양한 환경으로부터 분리되어 극지연구소 PAMC(Polar and Alpine Microbial Collection)에 보관중인 169개 균주들을 0.4% colloidal chitin이 첨가된 ZoBell 고체배지에서 배양하여 chitinase 활성을 균주 27개를 선별하였다. 그 중 PAMC 21693 균주는 저온에서 pNP-$(GlcNAc)_1$를 기질로 사용했을 때 가장 큰 활성을 보였고, $4-37^{\circ}C$의 온도 범위 중 $4^{\circ}C$에서 가장 높은 개체수 증가율을 보였다. PAMC 21693의 chitinase 유전자를 클로닝한 결과 2,619 bp의 ORF를 포함하는 총 2,857 bp의 염기서열을 확보하였다. 대장균에서 chitinase 유전자의 재조합 단백질을 발현한 결과 분자량 96 kDa의 재조합 단백질을 확인할 수 있었다. 본 논문에서는 극지 미생물 유래 저온활성 chitinase들의 생물공학 분야에서의 이용가능성을 제시하였다.

• Molecules and Cells
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• 제27권4호
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• pp.423-428
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• 2009

Superoxide dismutases (SODs) constitute the first line of cellular defense against oxidative stress in plants. SODs generally occur in three different forms with Cu/Zn, Fe, or Mn as prosthetic metals. We cloned the full-length cDNA of the Thellungiella halophila Cu/Zn-SOD gene ThCSD using degenerate RT-PCR and rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the ThCSD gene (GenBank accession number EF405867) had an open reading frame of 456 bp. The deduced 152-amino acid polypeptide had a predicted molecular weight of 15.1 kDa, an estimated pI of 5.4, and a putative Cu/Zn-binding site. Recombinant ThCSD protein was expressed in Escherichia coli and assayed for SOD enzymatic activity in a native polyacrylamide gel. The SOD activity of ThCSD was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, confirming that ThCSD is a Cu/Zn-SOD. Northern blotting demonstrated that ThCSD is expressed in roots, stems, and leaves. ThCSD mRNA levels increased by about 30-fold when plants were treated with sodium chloride (NaCl), abscisic acid (ABA), and indole-acetic acid (IAA) and by about 50-fold when treated with UVB light. These results indicate that ThCSD is involved in physiological pathways activated by a variety of environmental conditions.

• Animal Bioscience
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• 제34권5호
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• pp.867-879
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• 2021

Objective: Fibronectin 3 (FN3) and immunoglobulin like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researches have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase belonging to glycosyl hydorlase (GH) family 5 were determined. Methods: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete open reading frames (ORFs). The alkaline, thermostability, tertiary structure of deduced enzymes were predicted by AcalPred, TBI software, Phyre2 and Swiss models. Then, whole and truncated forms of a selected gene were expressed in Escherichia coli and purified by His-tag affinity column for assessment of FN3 ability to enhance enzyme activity, solubility and conformation. Results: From 297 complete ORFs coding for cellulases, 148 sequences containing FN3, Ig were identified. Mostly FN3 appeared in 90.9% beta-glucosidases belonging to glycosyl hydrolase family 3 (GH3) and situated downstream of catalytic domains. The Ig was found upstream of 100% endoglucanase GH9. Rarely FN3 was seen to be situated downstream of X domain and upstream of catalytic domain endoglucanase GH5. Whole enzyme (called XFN3GH5 based on modular structure) and truncate forms FN3, XFN3, FN3GH5, GH5 were cloned in pET22b (+) and pET22SUMO to be expressed in single and fusion forms with a small ubiquitin-related modifier partner (S). The FN3, SFN3 increased GH5 solubility in FN3GH5, SFN3GH5. The SFN3 partly served for GH5 conformation in SFN3GH5, increased modules interaction and enzyme-soluble substrate affinity to enhance SXFN3GH5, SFN3GH5 activities in mixtures. Both SFN3 and SXFN3 did not anchor enzyme on filter paper but exfoliate and separate cellulose chains on filter paper for enzyme hydrolysis. Conclusion: Based on these findings, the presence of FN3 module in certain cellulases was confirmed and it assisted for enzyme conformation and activity in both soluble and insoluble substrate.