• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.58-66
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• 2007

Isolation, expression, and characterization of a novel $endo-{\beta}-1,3(4)-D-glucanase$ with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a ${\beta}-glucanase$ protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other ${\beta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${\beta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{\beta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{\circ}C$. The $K_m,\;V_{max},\;and\;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{\mu}mol/min{\cdot}mg,\;and\;3,131s^{-1}$, respectively. For barley ${\beta}-glucan$ the values are 3.73mg/ml, $8,939{\mu}mol/min{\cdot}mg,\;and\;4,026s^{-1}$, respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1148-1157
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• 2006

A hyperthermophilic bacterium Thernotoga maritima produced thermostable ${\beta}-glucosidase$. The gene encoding ${\beta}-glucosidase$ from T. maritima MSB8 was cloned and expressed in Escherichia coli. The en-zyme (BgIB) hydrolyzed ${\beta}-glucosidase$ linkages between glucose and alkyl, aryl of saccharide groups such as salicin, arbutin, and $_pNPG$. The insert DNA contained ORF with 2,166 bp encodes a 721 amino acids (calculated molecular mass of 80,964 and pl of 4.93). The amino a.id sequence of BglB showed the similarity to family 3 glycosyl hydrolases. The molecular weight of the enzyme was estimated to be approximately 81kDa by MUG-nondenaturing PAGE (4-methylumbelliferyl 13-D-glucoside-nondenaturing polyacrylamide gel electophoresis) and SDS-PACE. The ${\beta}-glucosidase$ exhibited maximal activity at pH 7.0 and $80^{\circ}C$. By exchanging two possible residues (Glu-232 and Asp-242) to Ala by site-directed mutagenesis method, it was found that these were essential for enzymatic activity.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.57 no.2
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• pp.171-181
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• 2012

Lysine is the first limiting essential amino acid in cereals for humans and monogastric animals, although its content is generally low. A chemically induced high-lysine barley mutant, M98, has an agronomically undesirable shrunken endosperm trait. In order to obtain detailed insight into the atypical traits of M98 grains, we characterized amino acid composition and protein profiles of M98 and its parent cultivar Chalssalbori. Among a total of 16 amino acids, the percentage of each of the 7 amino acids, including lysine, was 1.2~1.8 times higher in M98, comparing to Chalssalbori. The percentage of proline and its precursor, glutamic acid, in M98 was about the half of that of the amino acids in Chalssalbori, but arginine synthesized from glutamic acid was 1.8 times higher in M98, compared that in the parent cultivar. Theses results indicated that the mutation in M98 grains might alter the proportion of amino acids linked to each other in a biosynthetic pathway. A comparison of grain proteome profiles between Chalssalbori and M98 revealed 70 differentially expressed protein spots, where 45 protein spots were up-regulated and 25 protein spots down-regulated in M98 compared to those in Chalssalbori. Of these changed protein spots, 53 were identified using nano-electrospray ionization liquid chromatography mass spectrometry. Most of these identified proteins were involved in various biological processes. In particular, 28 protein spots such as ${\beta}$-amylase, serpins and B3-hordein were identified as proteins associated with the atypical traits of M98. It was thought that a genetic study on the unique protein profile of M98 would be needed to develop an agronomically feasible barley cultivar with high-lysine trait.

• Reproductive and Developmental Biology
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• v.41 no.3
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• pp.51-55
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• 2017

The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of ${\beta}$-casein gene and expressed using the gene regulatory DNA sequence of bovine ${\beta}$-casein gene. The knock-in vector consists of 5' arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3' arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5' terminal of bIGF-1 gene and inserted into exon 7 of the ${\beta}$-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the ${\beta}$-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine ${\beta}$-casein gene.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• Journal of Mushroom
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• v.10 no.1
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• pp.29-36
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• 2012

We conducted an analysis by comparing the nutritional contents of Auricularia auricula-judae(black), Auricularia polytricha and Auricularia auricula-judae (brown). In nutritional contents of three strains of Auricularia spp., four free sugars, seven organic acids and 24 amino acids were detected. Auricularia auricula-judae (black) was highly contained free sugar, organic acid and amino acid. There was the most prevalent Vitamin $D_2$ content in Auricularia auricula-judae (black) of Auricularia spp. Dietary fiber of three strains showed contents of about 60% but were not significantly different. ${\beta}$-glucan contents of Auricularia auricula-judae (brown) contained the highest contents with $25.21{\pm}0.37%$ and showed significant differences between Auricularia polytricha and Auricularia auricula-judae (black). Total polyphenol contents of Auricularia polytricha showed the highest contents, followed by Auricularia auricula-judae (brown) and Auricularia auricula-judae (black).

• Journal of the Korea Organic Resources Recycling Association
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• v.18 no.3
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• pp.31-37
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• 2010

Chitinases (EC 3.2.1.14) hydrolyze the ${\beta}$-1,4-linkages in chitin, the second most abundant polymer of N-acetyl-${\beta}$-D-glucosamine which is a structural component of protective biological matrices such as fungal cell walls and insect exoskeletons. The glycosyl hydrolases 18 family including chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Since earthworms live in the soil with a lot of microbial activities and fungi are supposed to be a major component of the diet of earthworm, it has been reported that there would be appropriate immune system to protect themselves from microorganisms attacks. In this study, the novel chitinase, EaChi, from the midgut of earthworm, Eisenia andrei, were identified and characterized. To obtain full-length cDNA sequence of chitinase, RT-PCR and RACE-PCR analyses were carried out by using the previously identified EST sequence amongst cDNA library established from the midgut of E. andrei. EaChi, a partial chitinase gene, was composed of 927 nucleotides encoding 309 amino acids. By the multiple sequence alignments of amino acids with other different species, it was revealed that EaCHI is a member of glycosyl hydrolases 18 family, which has two highly conserved domains, substrate binding and catalytic domain.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.5
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• pp.482-494
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• 1994

Most of carotenoprotein complexes have been extracted by using buffered solutions. However, in this study carotenoprotein from the muscle of Blue mussel(Mytilus edulis) was extracted by a detergent such as Triton X-100. It was purified and characterized by $20\%$ (w/v) $(NH_4)_2SO_4$, DEAE-cellulose ion exchange and Sephacryl S-300 gel filtration. The carotenoprotein(${\lambda}_{max}=462nm$) had an approximate M. W. of 372KDa(gel filtration). SDS-PAGE analysis of the carotenoprotein indicated the presence of four polypeptides of 60KDa($23.70\%$), 46.9KDa($9.14\%$), 26KDa($49.14\%$) and 13KDa($18.02\%$). Carotenoprotein denaturated by treatment with SDS to a final concentration of $0.2\%$ (w/v) caused a hypsochromic shift of ${\lambda}_{max}$ from 462nm to 456nm. The carotenoprotein contained lipids as structure units. The amino acid composition of the carotenoprotein contained large essential amino acid amounts of $62.8\%$, and the content of threonine($35.9\%$) was higher than other amino acids, but histidine, methionine and proline were not present. In the carotenoprotein, the major fatty acids were $C_{16:4},\;C_{16:0},\;C_{20:5}\;and\;C_{22:6}$. The percentages of polyunsaturated fatty acids($62.4\%$) were higher compared to other fatty acids(saturated fatty acids $19.6\%$, monounsaturated fatty acids $18.0\%$). Carotenoid was extracted from the carotenoprotein by acetone and it was separated into five different components by preparative TLC(benzene:petroleum ether:acetone=69:17:14). The major components of carotenoid were mytiloxanthin($74.79\%$) and 3,4,3'- trihydroxy-7',8'-didehydro-${\beta}$-carotene($18.26\%$), and they were at least presented as prosthetic groups of carotenoprotein.

• Preventive Nutrition and Food Science
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• v.10 no.1
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• pp.103-110
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• 2005

Opuntia ficus-indica, commonly known as prickly pear cactus, is commercially grown as a food and medicinal plant in Jeju Island, Korea. The crushed pads and fruits of Opuntia ficus-indica were dried in a freeze-dryer and ground into flour to be used for food materials. The major components of proximate compositions were part of a nitrogen free extract. The major minerals were Ca (4391.2-2086.9㎎%), K (1932.1-2608.7㎎%), and Mg (800.6-1984.8㎎%). The major amino acid was glutamic acid, comprising 16.3% of total amino acids in fruit and 25.2% in pad. Dihydroflavonols were identified as (+)-trans-dihydrokaempferol and (+)-trans-dihydroquercetin. Citric acid methyl esters extracted from fruits showed inhibitory activities against monoamine oxidase-B. The presence of trimethyl citrate has been reported in other plants, but 1,3-dimethyl citrate and 1-monomethyl citrate have not been previously reported. The results of pharmacological efficacy tests, including serum biochemical and hematological parameters, autonomic nervous system, anti-inflammatory, analgestic activity, anti-diabetic activity, antithrombotic, anticoagulant, dopamine beta-hydroxylase, monoamine oxidase activity, hyperlipidemia, the respiratory system, antigastic, and anti-ulcerative actions indicate that the fruit and pad of the Opuntia ficus-indica are rich sources of food and medicinal materials.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.2
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• pp.139-145
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• 2009

We cloned and sequenced the open reading frame (ORF) cDNA encoding myostatin from the muscle of abalone (Haliotis discus hannai). The ORF cDNA of the abalone myostatin is 1134 bp and encoded 377 amino acid residues that were 60-96% homologous with the amino acids of other organism myostatins. In addition, the ORF contained a conserved proteolytic cleavage site (RXRR) and nine conserved cysteine residues in the C-terminus. Semi-quantitative RT-PCR revealed the presence of myostatin mRNA in various tissues. The strongest expression was observed in the mantle of female abalone, and the gills and heart of male abalone.