• Journal of Microbiology and Biotechnology
• /
• v.24 no.11
• /
• pp.1516-1524
• /
• 2014

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) can utilize a variety of external electron acceptors and also has stricter substrate specificity than any other glucose oxidoreductases, which makes it the ideal diagnostic enzyme in the field of glucose biosensors. A gene coding for a hypothetical protein, similar to glucose oxidase and derived from Aspergillus terreus NIH2624, was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 260,000 U/l in the culture supernatant after fed-batch cultivation for 84 h. After a three-step purification protocol that included isopropanol precipitation, affinity chromatography, and a second isopropanol precipitation, recombinant FAD-GDH was purified with a recovery of 65%. This is the first time that isopropanol precipitation has been used to concentrate a fermentation supernatant and exchange buffers after affinity chromatography purification. The purified FAD-GDH exhibited a broad and diffuse band between 83 and 150 kDa. The recombinant FAD-GDH was stable across a wide pH range (3.5 to 9.0) with maximum activity at pH 7.5 and $55^{\circ}C$. In addition, it displayed very high thermal stability, with a half-life of 82 min at $60^{\circ}C$. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.2
• /
• pp.272-279
• /
• 2021

Two genes encoding probable α-ʟ-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45℃ and pH 7.0 in sodium phosphate buffer and at 50℃ and pH 6.0 in sodium acetate buffer, respectively. These exoacting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only ʟ-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)- and α-(1,3)-ʟ-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)- and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

• Korean Journal of Microbiology
• /
• v.45 no.4
• /
• pp.332-338
• /
• 2009

A xylan-decomposing bacterium, HY-20, was isolated from the gut of a honeybee, Apis mellifera, and identified as Bacillus sp. The extracellular GH11 xylanase (XylP) gene (687-bp) of strain HY-20 encoded a protein of 228 amino acids with a deduced molecular mass of 25,522 Da and a calculated pI of 9.33. The primary structure of XylP was 97% identical to that of B. pumilus xylanase (GenBank accession no.: AY526092) that has not been characterized yet. The recombinant His-tagged enzyme (rXylP) overexpressed in Escherichia coli BL21 harboring pET-28a(+)/xylP was purified to electrophoretic homogeneity by cation exchange and gel permeation chromatographies. The purified enzyme exhibited the highest catalytic activity toward birchwood xylan at pH 6.5 and $50^{\circ}C$ and retained approximately 50% of its original activity when pre-incubated at $55^{\circ}C$ for 15 min. The recombinant enzyme was completely inactivated by $Hg^{2+}$ (1 mM) and N-bromosuccinimide (5 mM), while its activity was slightly stimulated by approximately 10% in the presence of $Mn^{2+}$ (1 mM), $Fe^{2+}$ (1 mM), and sodium azide (5 mM). rXylP was able to efficiently degrade various polymeric xylose-based substrates but PNP-sugar derivatives and glucose-based polymers were not susceptible to the enzyme.

• Korean Journal of Plant Resources
• /
• v.9 no.2
• /
• pp.165-170
• /
• 1996

The study was carried out to identify the characteristics of leafy shapes, and to establish the cultural practices such as shading condition, fertilization method, and planting distance of Cirsium nipponicum. Leaf shapes in this plant consist of two kinds, lobation and non-lobation which has two spur type showing large and small spur. Protein band patterns showed that a new protein band in non-lobation with large spur was appeared at the 116.4kDa. For shading condition and fertilization method, number of stems in non-shading and organic matter treatment was higher than that of shading 55% with 3.7. Fresh leaf yield on non-shading and organic matter treatment was higher than that of other treatments. Growth characteristics of leaf number was increased in the $60\times30cm$ treatment, but was redeuced to some extent compared with $60\times45cm$. To increase the fresh leaf yield, the optimum planting distance was $30\times20cm$ with 4,100kg/10a.

• Asian-Australasian Journal of Animal Sciences
• /
• v.29 no.1
• /
• pp.126-133
• /
• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.3
• /
• pp.390-399
• /
• 2012

Endoglucanase gene egIV was cloned from Trichoderma viride AS 3.3711, an important cellulose-producing fungus, by using an RT-PCR protocol. The egIV cDNA is 1,297 bp in length and contains a 1,035 bp open reading frame encoding a 344 amino acid protein with an estimated molecular mass of 35.5 kDa and isoelectronic point (pI) of 5.29. The expression of gene egIV in T. viride AS 3.3711 could be induced by sucrose, corn straw, carboxymethylcellulose (CMC), or microcrystalline cellulose, but especially by CMC. The transcripts of egIV were regulated under these substrates, but the expression level of the egIV gene could be inhibited by glucose and fructose. Three recombinant vectors, pYES2-xegIV, $pYES2M{\alpha}$-egIV, and $pYES2M{\alpha}$-xegIV, were constructed to express the egIV gene in Saccharomyces cerevisiae H158. The CMCase activity of yeast transformants $IpYES2M{\alpha}$-xegIV was higher than that of transformant IpYES2-xegIV or $IpYES2M{\alpha}$-egIV, with the highest activity of 0.13 U/ml at induction for 48 h, illustrating that the modified egIV gene could enhance CMCase activity and that $MF{\alpha}$ signal peptide from S. cerevisiae could regulate exogenous gene expression more effectively in S. cerevisiae. The recombinant EGIV enzyme was stable at pH 3.5 to 7.5 and temperature of $35^{\circ}C$ to $65^{\circ}C$. The optimal reaction condition for EGIV enzyme activity was at the temperature of $55^{\circ}C$, pH of 5.0, 0.75 mM $Ba^{2+}$, and using CMC as substrate. Under these conditions, the highest activity of EGIV enzyme in transformant $IpYES2M{\alpha}$-xegIV was 0.18 U/ml. These properties would provide technical parameters for utilizing cellulose in industrial bioethanol production.

• Parasites, Hosts and Diseases
• /
• v.30 no.4
• /
• pp.289-298
• /
• 1992

Acanthamoeba sap., free-living amoebae inhabited in moist soil, pond, freshwater, sewage, atmosphere and swimming pool, may be causative protozoa of the fatal primary amoebic meningoence-phalitis in experimental animals and humans. In this study, Acar,thamoeba spry. , including Acan. thamoeba sp. YM-4 (isolated strain from Korea) had been compared by the two-dimensional electrophoresis and hybridoma technique as well as the difference of morphological characteristics. Trophozoite of Acenthamoeba sp. YM-4 is usually uninucleate and show the hyaline filamentous projections (acanthopoda) . No aagellate stage observed. Cysts have two walls, the outer wall is nearly circular, but inner wall is oval or some irregular. As results of SDS-PAGE for Iysate of Acanthamoeba sp. VM-4, 16 major protein fractions are similiar to those of A. cuzbertsoni, but different to A. royreba and A. polyphaga. Findings of two-dimensional electrophoretic patterns of Acanthamceba sp. YM-4 are almost same to those of A. culberssoni, The isotope of monoclonal antibodies produced from McAY 6, McAY 7, McAY 8, McAY 13 and McAY 16 clones were IgGl, and McAY 10 and McAY 11 clones were IsM. As results of the cross-reactivity among various amoebae using ELISA with monoclonal antibodies, McAY 7 monoclonal antibody (molecular weight 43 kDa by EITB) was only reacted with Acanthamoeba sp. YM-4, but McAY 6 and McAY 10 monoclonal antibodies were reacted to A. cuzbertsoni as well as Acanthamoeba sp. YM-4.

• Culinary science and hospitality research
• /
• v.23 no.2
• /
• pp.159-168
• /
• 2017

This study was conducted to compare the food quality of domesticated species. Consumers surveyed for safe food intake and proper culture of food distribution. The results of the comparison study are as follows. Muscle moisture content, protein content, and fat content. K, P, and C showed relatively high values in the muscle of the sea bream. Fe showed low contents. As a result of measuring heavy metal component, Cd was not detected in sea bream and mullet, but $0.01{\pm}0.00mg/kg$ was detected in red mine. Other heavy metals were below the reference value or were not detected. Electrophoresis results showed that the band appeared at in red minefish. In the case of sea bream and swordfish, no distinctive features of the band were shown. In the case of sea bream, there was little difference in food science between the similar fish species and the red sea bream fish, but price was different. An environment should be created for consumers to buy the right ingredients at the price they want. It is necessary to educate consumers about food ingredients immediately.

• Journal of Nutrition and Health
• /
• v.39 no.1
• /
• pp.58-73
• /
• 2006

This research has done for 262 people of the aged men and women that are more than 65 years old who are live in 9 areas of Yecheon as target; through twice off ace to face interview for 24 hours recall method, the result of food intake for 2 days is as following. In case of energy, the aged men (women) of sixties ingested 67.7 $(72.0)\%$ of Korean RDA by 1,369 (1264) kcal, for the ages of seventies and eighties, 68.9 $(66.9)\%$ of Korean RDA and 76.3 $(65.8)\%$ by each 1,309 (1104) kcal and 1,368 (1052) kcal. The aged men ingested protein $46.0\~49.6 g$ ($70.6\~82.9\%$ of RDA), and aged women ingested protein $32.7\~40.2 g$ ($59.4\~73.0\%$ of RDA). Calcium intake of aged men was 388.8 mg, 319.8 mg, 284.4 mg by age range, and aged women was 291.9 mg ($41.6\%$ of RDA), 246.5 mg ($35.3\%$ of RDA), 240.1 mg ($34.3\%$ of RDA). Iron intake of aged men was $8.6\~8.9 mg$ ($72\~74%$ of RDA), and aged women ingested 8.6 mg ($71.3\%$ of RDA), 7.5 mg (62.6 of RDA$\%$), 6.6 mg ($55.4\%$ of RDA) for iron by age range. Vitamin $B_1$ intake of aged men was $0.62\~0.71 mg$ ($62\~71\%$ of RDA), and aged women's intake was $0.50\~0.60 mg$ ($50\~60\%$ of RDA). Vitamin $B_2$ intake of aged men was $0.59\~0.60 mg$ ($49\%$ of RDA), and aged women's intake was $0.45\~0.50 mg$ ($37\~42\%$ of RDA). Vitamin C intake by age range, in case of aged men (women) in sixties was 53.1 (48.9) mg, in seventies was 49.9 (33.2) mg and more than eighties was 34.1 (33.4) mg. The average food intake by age range, in aged men (women) of sixties was 828.9 (670.8) g and seventies was 726.8 (568.8) g and more than eighties was 656.0 (525.3) g. Plant food intake of aged men was 490.8-569.5 g and aged women was 417.9-537.7 g. Aged men (women) of MAR by age range, sixties was 0.60 (0.58), seventies was 0.59 (0.50) and more than eighties was 0.56 (0.49), respectively. INQ for protein, phosphorus, iron, vitamin A, vitamin B, niacin, vitamin C was more than 1 in 60's and 70's aged men, but there was no nutrients in eighties of aged women. Aged men and women's KDDS points represent average 3.14 and 3.04 (out of 5 points), and while intake of the milk was the most lacking, but intake of the fruit was the most lacking in DDS.

• The Korean Journal of Food And Nutrition
• /
• v.9 no.4
• /
• pp.452-458
• /
• 1996

A PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-1 primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI1-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPi-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus. The translated protein sequence of PPIase B. stearothermophilus was compared with sequence from periplasmic PPIase from Escherichina coil ; homogies of 16 and 58%, respectively, were found. The clond PPIase gene was over-expressed in E. coil cell using pUC19 as an expression vector. The enzyme was partially purified by heat treatment and colum chromatochraphy on DEAE-Sepharose CL-6B. The molecular weight of the enzyme was dermined to be about 18.0 kDal by SDS-PAGE.