• Journal of Applied Biological Chemistry
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• v.44 no.1
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• pp.6-11
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• 2001

An ${\omega}-3$ fatty acid desaturase gene which is involved in de novo synthesis of -Iinolenate was isolated from cDNA library of Perilla frutescens. A cDNA library was constructed with mRNA extracted from perilla seeds of 12 DAF. The cDNA clone consisting of 1317-bp open reading frame encoding 438 amino acids with a relative MW of 50kDa, was isolated and showed 65-83% similarities to other known genes. This cDNA is deduced to encode a plastidal ${\omega}-3$ fatty acid desaturase based on the fact that it has higher homology to plastidal ones than to microsomal ones and its N-terminal sequence shares several characteristics of transit peptides of chloroplast proteins. Southern blot analysis of genomic DNA indicated that more than one gene or alleles for ${\omega}-3$ fatty acid desaturase are present in the genome of perilla. Northern blot analysis showed that the ${\omega}-3$ fatty acid desaturase gene is mainly revealed in early developing seeds and has different expression patterns depending on tissue types compared to the microsomal ones.

• Journal of Microbiology and Biotechnology
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• v.24 no.10
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• pp.1382-1388
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• 2014

Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was $60^{\circ}C$, which was $20^{\circ}C$ higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to $40^{\circ}C$ and in a pH range of 5.0-10.0, whereas soluble K80 maintained its activity up to $35^{\circ}C$ and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

• Preventive Nutrition and Food Science
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• v.9 no.1
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• pp.14-20
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• 2004

Bacillus strains capable of producing fibrinolytic enzyme were isolated from traditional fermented Korean soybean paste and Japanese fermented soybean (Natto). Among the 16 strains, a selected Bacillus sp. was identified as bacillus firmus, with 80.7％ homology, by API kit analysis. Seed starter or B. firmus NA-1 was prepared with 5％ soymilk prepared from micronized soybean powder. To produce fibrinolytic enzyme by B. firmus NA-1 the liquid culture was performed with NB broth (pH 7.0) fortified with 1％ galactose, 0.1% tryptone, and 0.5% $K_2$HPO$_4$, by shaking with 180 rpm at 37$^{\circ}C$. Fibrinolytic enzyme activity reached the highest value at 7.8 unit/mL (plasmin unit) after fermentation for 72 hr. The crude fibrinolytic enzyme showed higher relative activity in the range of pH 7.0∼9.0. The activity of crude fibrinolytic enzyme was well maintained even after concentration by the vacuum evaporation at 5$0^{\circ}C$ for 1 hr.

• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1415-1423
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• 2010

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular mass of the F. pinicola xylanase was determined to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size-exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of $70^{\circ}C$. The enzyme showed a $t_{1/2}$ value of 33 h at $70^{\circ}C$ and catalytic efficiency ($k_{cat}=77.4\;s^{-1}$, $k_{cat}/K_m$=22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.711-718
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• 2011

A highly efficient cellobiohydrolase (CBH)-secreting basidiomycetous fungus, Agaricus arvensis KMJ623, was isolated and identified based on its morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular CBH was purified to homogeneity from A. arvencis culture supernatant using sequential chromatography. The relative molecular mass of A. arvencis CBH was determined to be 65 kDa by SDSPAGE and 130 kDa by size-exclusion chromatography, indicating that the enzyme is a dimer. A. arvencis CBH showed a catalytic efficiency ($k_{cat}/K_m$) of 31.8 $mM^{-1}\;s^{-1}$ for p-nitrophenyl-${\beta}$-D-cellobioside, the highest level seen for CBH-producing microorganisms. Its internal amino acid sequences showed significant homology with CBHs from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, A. arvencis CBH is distinguished from other CBHs by its high catalytic efficiency.

• Journal of the Korea Institute of Information and Communication Engineering
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• v.20 no.9
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• pp.1816-1821
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• 2016

Protein secondary structure is important for the study of protein evolution, structure and function of proteins which play crucial roles in most of biological processes. This paper try to effectively extract protein secondary structure information from the large protein structure database in order to predict the protein secondary structure of a query protein sequence. To find more remote homologous sequences of a query sequence in the protein database, we used PSI-BLAST which can perform gapped iterative searches and use profiles consisting of homologous protein sequences of a query protein. The secondary structures of the homologous sequences are weighed combined to the secondary structure prediction according to their relative degree of similarity to the query sequence. When homologous sequences with a neural network predictor were used, the accuracies were higher than those of current state-of-art techniques, achieving a Q3 accuracy of 92.28% and a Q8 accuracy of 88.79%.

• BMB Reports
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• v.31 no.4
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• pp.370-376
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• 1998

Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

• International Journal of Industrial Entomology and Biomaterials
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• v.27 no.1
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• pp.159-165
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• 2013

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF-${\beta}$) superfamily and are involved in osteoblastic differentiation. The largest TGF-${\beta}$ superfamily subgroup shares genetic homology with human BMPs (hBMPs) and silkworm decapentaplegic (dpp). In addition, hBMPs are functionally interchangeable with Drosophila dpp. Bombyx mori dpp may induce bone formation in mammalian cells. To test this hypothesis, we synthesized the 1,285-base pairs cDNA of full-length B. mori dpp using total RNAs obtained from the fat body of 3-day-old of the $5^{th}$ instar larvae and cloned the cDNA into the pCEP4 mammalian expression vector. Next, B. mori dpp was expressed in C3H10T1/2 cells. The target cells transfected with the pCEP4-Bm dpp plasmid showed biological functions similar to those of osteogenic differentiation induction growth factors such as hBMPs. We determined the relative mRNA expression rates of Runt-related transcription factor 2 (RUNX2), osterix, osteocalcin, and alkaline phosphatase (ALP) to validate the osteoblast-specific differentiation effects of B. mori dpp by performing quantitative real-time RT-PCR. Interestingly, mRNA expression levels of the 3 marker genes except RUNX2, in cells expressing B. mori dpp were much higher than those in control cells and C3H10T1/2 cells transfected with pCEP4. These results suggested that B. mori dpp signaling regulates osterix expression during osteogenic differentiation via RUNX2-independent mechanisms.

• The Plant Pathology Journal
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• v.33 no.6
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• pp.543-553
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• 2017

Sesame (Sesamum indicum) is an important oil seed crop of Asia. Yields can be negatively impacted by various factors, including disease, particularly those caused by fungi which create problems in both production and storage. Foliar diseases of sesame such as Alternaria leaf blight may cause significant yield losses, with reductions in plant health and seed quality. The work reported here determined the incidence of Alternaria species infecting sesame seeds grown in the Punjab, Pakistan. A total of 428 Alternaria isolates were obtained from 105 seed samples and grouped into 36 distinct taxonomic groups based on growth pattern and morphological characters. Isolation frequency and relative density of surface sterilized and non-surface sterilized seeds showed that three isolates (A13, A47 and A215) were the most common morphological groups present. These isolates were further identified using sequencing of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA) and the Alternaria major allergen gene (Alt a 1). Whilst ITS of rDNA did not resolve the isolates into Alternaria species, the Alt a 1 sequences exhibited > 99% homology with Alternaria alternata (KP123850.1) in GenBank accessions. The pathogenicity and virulence of these isolates of Alternaria alternata was confirmed in inoculations of sesame plants resulting in typical symptoms of leaf blight disease. This work confirms the identity of a major source of sesame leaf blight in Pakistan which will aid in formulating effective disease management strategies.

• Applied Biological Chemistry
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• v.45 no.4
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• pp.203-206
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• 2002

${\gamma}-Tocopherol$ methyltransferase (TMT) is an enzyme catalyzing ${\gamma}-tocopherol$ into ${\alpha}-tocopherol$ at the final step of ${\alpha}-tocopherol$ synthesis pathway. Putative TMT cDNA clone specific to Perilla frutescens immature seeds was isolated from cDNA library. The cDNA clone consisted of 1369 bp open reading frame encoding 369 amino acids with a relative Mw of 42 kDa. Results revealed the CDNA has 60% homology to Arabidopsis thaliana TMT, and possesses methyltransferase and S-adenosyl methionine-binding domains, suggesting that cDNA encodes a ${\gamma}-tocopherol$ methyltransferase To characterize the properties of the TMT gene, the cDNA sequences coding for mature TMT were expressed in E. coli and assayed to determine the enzyme activity in vitro.