• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.644-648
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• 2005

Dextranase (${\alpha}$-1,6-D-glucan-6-glucanogydrolase:E.C. 3.2.1.11) catalyzes the hydrolysis of ${\alpha}$-(1.6) linkages of dextran. A lsd1 gene encoding an extracellular dextranase was isolated from the genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase (lsd1) after codon optimization for gene expression with Pichia pastoris system. A open reading frame of lsd11 gene was 1827 bp and it was inserted into the pPIC3.5K expression vector. The plasmid linearized by Sac I was integrated into the 5'AOX region of the chromosomal DNA of P. pastoris. The lsd11 gene fragment encoding a mature protein of 608 amino acids with a predicted molecular weight of 70 kDa, was expressed in the methylotrophic yeast P. pastoris by controling the alcohol oxidase-1 (AOX1) promoter. The recombinant lds11 was optimized by using the shake-flask expression and upscaled using fermentation technology. More than 9.8 mg/L of active dextranase was obtained after induction by methanol. The optimum pH of LSD11 was found to be 5.5 and the optimum temperature $28^{\circ}C$.

• Korean Journal of Food Science and Technology
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• v.31 no.2
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• pp.391-398
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• 1999

Enzymatic hydrolysis of crushed ginger was carried out to increase the yield of ginger extract, and the quality of the extract was investigated. The first extract was obtained by pressing crushed ginger, and the second extract by pressing ginger pomace hydrolyzed with $\alpha$-amylase at $90^{\circ}C$ for 1 hr. Oleoresin was extracted from the residue of enzymatic hydrolysis with 90% ethyl alcohol. The first extract, second extract and oleoresin were mixed to obtain the final ginger extract. The yield of final extract was increased by 276% on the solid base of the fresh ginger extract. The final ginger extract contained less crude fiber, starch and free amino acids (62, 48 and 40%, respectively), but contained more free sugar (270%) compared to fresh ginger extract.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.925-935
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• 2014

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the ${\alpha}/{\beta}$-class of protein consisted of a central six-stranded ${\beta}$-sheet flanked by eight ${\alpha}$-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were $15^{\circ}C$ and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at $50^{\circ}C$ or $60^{\circ}C$ for 6 h, and 89% of its enzyme activity when preincubated at $70^{\circ}C$ for 1 h. The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by $Na^+$ and $Mg^{2+}$ ions but was strongly inhibited by $Cu^+$ and $Hg^{2+}$ ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a $K_m$ of 0.278 mM and a $k_{cat}$ of $1.9s^{-1}$. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

• Journal of Microbiology and Biotechnology
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• v.23 no.7
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• pp.974-983
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• 2013

Bacillus amyloliquefaciens CB1 was isolated from cheonggukjang, a Korean fermented soy food. B. amyloliquefaciens CB1 secretes proteases with fibrinolytic activities. A gene homologous to aprE of Bacillus subtilis, aprECB1, was cloned from B. amyloliquefaciens CB1, and DNA sequencing showed that aprECB1 can encode a prepro-type serine protease consisting of 382 amino acids. When aprECB1 was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, transformants showed fibrinolytic activity and produced a 28 kDa protein, the size expected for the mature enzyme. The 28 kDa fibrinolytic enzyme was purified from the culture supernatant of B. subtilis WB600 transformant. AprECB1 was completely inhibited by phenylmethylsulfonyl fluoride and almost completely inhibited by EDTA and EGTA, indicating that it is a serine metalloprotease. AprECB1 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin. $A{\alpha}$ and $B{\beta}$ chains of fibrinogen were quickly degraded by AprECB1, but the ${\gamma}$-chain was resistant.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.350-356
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• 2017

We previously reported that the CopA3 peptide (LLCIALRKK, ${\small{D}}-form$) originally isolated from the Korean dung beetle has antimicrobial and immunosuppressive effects. However, the high cost of producing the synthetic peptide, especially the ${\small{D}}-form$, has limited the development of CopA3 for therapeutic purposes. Here, we investigated whether the CopA3 deletion derivative, CopA5, which is composed of only five amino acids (LLCIA) and has the ${\small{L}}-form$ structure, could inhibit the lipopolysaccharide (LPS)-induced activation of macrophages. Peritoneal exudate macrophages (PEM) were isolated from mice and exposed to LPS in the presence or absence of CopA5, and biomarkers of macrophage activation were measured. Our results revealed that LPS-induced nitric oxide (NO) production, tumor necrosis factor $(TNF)-{\alpha}$ secretion, and phagocytic activity of PEM were significantly inhibited by CopA5 treatment. Similar to CopA3, the structurally modified CopA5 peptide had no cell toxicity (as assessed by measurement of cell viability loss and apoptosis) in PEM. Moreover, the LPS-induced upregulation of the activating phosphorylation of signal transducer and activator of transcription 1 (STAT1) was markedly inhibited by CopA5 treatment. These results suggest that, similar to CopA3, CopA5 inhibits macrophage activation by inhibiting STAT1 phosphorylation and blocking the release of NO and $TNF-{\alpha}$. CopA5 may therefore prove therapeutically useful in the realm of immune suppression.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.57-62
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• 2002

Productivity of bacterial cellulose by Acetobacter xylinum GS11 was investigated in the several culture conditions. In various carbon sources, others with the exception of glucose were not found to be effective for cellulose production, and 2% was better in yield than other concentration of glucose. Yeast extract and soytone among several organic nitrogens were effective, but inorganic nitrogen sources tested were not efficient for cellulose production by A. xylinum GS11. The effects of various inorganic salts, amino acids and vitamins were also investigated: $MgSO_4$, phenylalanine and $\alpha$-tocopherol gave the cellulose yield of 1.5, 1.4 and 1.4 fold, respectively, compared with basal medium. In our experiment, cellulose production by A. xylinum GS11 added with 10% coconut milk and 0.5% lignosulfonate in basal medium, was the most efficient among the several material sources employed here, and these were 2.2 and 2.1 fold, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.4
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• pp.465-471
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• 2008

Physicochemical characteristics of black garlic were analyzed. Colorimetry measurement showed that the black garlic, compared with fresh and steamed garlics, was the highest in a value and the lowest in L and b values. Crude lipid, crude protein, and total sugars were the highest in black garlic, which was followed by steamed and fresh garlic. On the other hand, moisture content was the lowest in the black garlic and the highest in the fresh garlic. The pH of garlics was ca. 6.8, 6.5, and 4.4 in fresh, steamed, and black garlic, respectively, which indicated that garlics tended to be acidified with the thermal processing. Total pyruvate and total thiosulfinates were the lowest in steamed garlic ($77{\mu}mol$/g and 0.07 OD/g for each) and the highest in black garlic ($278{\mu}mol$/g and 0.77 OD/g). Arabinose and galactose were detected only in black garlic and their contents were 1.6 and 13 mg/100 g, respectively. Free sugars such as glucose, sucrose and fructose were the highest in the order of fresh, steamed, and black garlic. Potassium was a predominant mineral in all garlics, constituting 76% of total minerals. Glutamic acid, arginine, and aspartic acid were the major composition amino acids in all garlics, regardless of processing conditions. 15 kinds of free amino acids were detected in fresh and steamed garlic, while five more free amino acids, O-phosphoethanolamine, and urea were additionally detected in black garlic.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.21 no.1
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• pp.21-29
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• 1988

This study was carried out to develop the powdered Katsuobushi (a kind of boiled, smoked, and dried fish product which is used for seasoning soup as it is.) using skipjack as a natural flavoring substance. The processing conditions of the powdered Katsuobushi and the changes of taste compounds during processing of the products were examined. In preparation of the powdered Katsuobushl, frozen skipjack was thawed, beheaded, gutted, filleted and then sliced to 1cm of thickness. The silted meats were boiled in skipjack extract for 20 minutes, and then it was smoked for, 3 times to $10\~12\%$ moisture content at $80^{\circ}C$ for 8 hours. The smoked - dried meats were followed to be 50 mesh of particle size. The effect of slicing and boiling in skipjack meat extract on enhancing flavor and on preventing lipid oxidation of product during processing were observed. The moisture content and crude lipid content of the powdered Katsuobushi was in the range of 11 to $12\%$ and 4.3 to $4.8\%$, respectively. The taste compounds of the product were nucleotides and their related compounds, 1135.8mg/100g ; free amino acid and related compounds, 2210.2mg/100g ; non-volatile organic acids, 1148.0mg/100g ; and total creatinine. 592.1mg/100g on dry basis, and small amount of betaine and TMAO. The major elements of mineral in the product were found to be K, Mg, Na, and Ca. The content of IMP was 542.0mg/100g, and major free amino acids were found to be histidine, anserine, taurine, carnosine and alanine of which occupied to $83.6\%$ of total free amino acids. In non -volatile organic acids, major ones were lactic acid, succinic acid, pyroglutamic acid and $\alpha-ketoglutaric$ acid. From the results of the chemical experiments and sensory evaluation, we may conclude that the flavor of the product from present experiment is more desirable than that of conventional products although the processing time used were much shortened than that of conventional method, and it can be commercialized as a seasoning powder.

• Korean Journal of Food Science and Technology
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• v.34 no.1
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• pp.85-90
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• 2002

The changes in taste components of kanjang made with barley bran during fermentation time were examined. The pH was gradually decreased and total nitrogen content reached to 0.7% at 90 days fermentation. Five kinds of free sugars, three kinds of volatile organic acid, and eight kinds of non-volatile organic acid were detected. Lactic acid known as abundant component in kanjang was not detected in kanjang made with barley bran. The content of free amino acid was $422.9{\sim}803.6\;mg%$. Glutamic acid was most abundant component among the amino acids, followed by proline and phenylalanine. Essential amino acid content was revealed $34.3{\sim}37.3%$. Based on result of sensory evaluation, it was most comfortable to eat sample of fermentation $45{\sim}60$ days.

• Analytical Science and Technology
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• v.13 no.4
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• pp.494-503
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• 2000

A 21-residue peptide corresponding to amino acids 84-104 of $p16^{INK4A}$, the tumor suppressor, has been synthesized and its structure was studied by Circular Dichroism, $^1H$ NMR spectroscopy and molecular modeling. A p16-derived peptide (84-104 amino acids) forming stable complex with CDK4 and CDK6 inhibits the ability of CDK4/6 to phosphorylate pRb in vitro, and blocks cell-cycle progression through G1/S phase as shown in the function of the full-length p16. Its NMR spectral data including NOEs, $^3J_{NH-H{\alpha}}$ coupling constants, $C_{\alpha}H$ chemical shift, the average amplitude of amide chemical shift oscillation and temperature coefficients indicate that the secondary structure of a p16-derived peptide is similar to that of the same region of full-length p16, which consists of helix-turn-helix structure. The 3-D distance geometry structure based on NOE-hased distance and torsion angle restraints is characterized by ${\gamma}$-turn conformation between residues $Gly^{89}-Leu^{91}$(${\varphi}_{i+1}=-79.8^{\circ}$, ${\varphi}_{i+1}=60.2^{\circ}$) as evidenced in a single crystal structure for the corresponding region of p18 or p19, but is undefined at both the N and C termini. This compact and rigid ${\gamma}$-turn region is considered to stabilize the structure of p16-derived peptide and serve as a site recognizing cyelin dependent kinase, and this well-defined ${\gamma}$-turn structure could be utilized for the design of anti-cancer drug candidates.