• Korean Journal of Plant Tissue Culture
• /
• v.28 no.1
• /
• pp.7-13
• /
• 2001

Calli were induced from diploid and haploid tobacco after 4 weeks and maintained on MS medium with combination of 2.0 mg/L 2,4-D,0.1 mg/L BAP and 2.0 mg/L kinetin. Suspension cells were screened through 65 $\mu$m-nylon mesh and 100 $\mu$m-mesh, then they were smeared on selection medium combined with cadmium and PFP by using the low melting agarose of 0.8%. After 30days smeared cultures of the medium the cell was treated with 500 $\mu$M and 1000 $\mu$M to select the resistant cell line were selected. Plant regeneration was induced from the selected cell lines on medium with 0.5, 1.5, 2.0 mg/L BAP and on media with combination of auxin and BAP under 500 $\mu$M and 1000 $\mu$M cadmium. At this time, plant regeneration was achived on cadmium free medium. In case of haploid, occurred from the cell line which is selected in medium with cadmium and PFP. In case of diploid regeneration occurred is in the medium with cadmium alone. The plantlet regenerated from cadmium resistant calli grew well in cadmium 500 $\mu$M. Protein pattern of leaf, root, stem of regenerated plants was analyzed. The quantum was 6.5188 ug/mg.fr.wt in the leaf of plant, 5.3611 ug/mg.fr.wt in the stem, 3.0213 ug/mg.fr.wt in the root. On the other hand, 5.9652 ug/mg.fr.wt. in the leaf of control, 3.5974 ug/mg.fr.wt in the stem of the control, 4.3766 ug/mg.fr.wt. in the root of the control. The one dimension bends regenerated from cadmium resistant calli resistant to cadmium in leaf were 49 involving 198.7KD etc. Disappeared were 4 involving 160.5KD etc, The protein bends were combinized were 3 involving 83.4KD etc. The bends resistant to cadmium stress in stem were 41 involving 4.3KD etc. Disappeared were 5 involving 114.8KD etc. The protein bends combinized were 6 involving 128.7KD etc. The bends which had the resistance to cadmium stress in root is 27 in volving 166,9KD etc. The bends which disappeared were 198.7KD etc. There were 5 involving 83.4KD etc.

• Korean Journal of Poultry Science
• /
• v.48 no.3
• /
• pp.133-142
• /
• 2021

The purpose of this study was to investigate whether enzyme-hydrolyzed poultry by-product meal (EHPBM) is more effective as a protein source than poultry by-product meal (PBM) and soybean meal (SBM) for broiler chickens. A group of 300 one-day-old broiler chicks was randomly allocated to three treatments with five replicates (20 birds/replicate) for five weeks. The treatments consisted of basal diets containing 1) SBM, 2) PBM, and 3) EHPBM. The EHPBM-fed group (1,853 g±125.60) showed the highest final body weight (P<0.05) when compared to the PBM-fed group (1,723 g±76.81) and SBM-fed group (1,545 g±62.31). The feed conversion ratio of the EHPBM treatment group (1.740±0.104) was significantly higher (P<0.05) than those of the SBM (1.653±0.056) and PBM groups (1.674±0.072). It can be speculated that the increased feed intake in the EHPBM group led to higher body weight gain and FCR. There was no significant effect of treatments on internal organ weight except for the bursa of Fabricius. Blood biochemical characteristic analysis showed that aspartate aminotransferase and alkaline phosphatase levels were higher in the EHPBM and PBM groups (P<0.05), probably due to the strained liver caused by the rapid growth of birds. In conclusion, EHPBM may partly replace conventional dietary protein sources such as soybean meal or poultry by-product meal and can be used to improve the productivity of broilers.

• Journal of Microbiology
• /
• v.44 no.6
• /
• pp.622-631
• /
• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.

• Korean Journal of Food Science and Technology
• /
• v.40 no.4
• /
• pp.394-399
• /
• 2008

In this study, peptides were isolated from Crassostrea gigas using an ultrasonification process at $40^{\circ}C$. The yield of the peptides was greater than 34%, and their cytotoxicity was found to be less than 22.8% against several cell lines that were treated with the extracts at a dose of 1.0 mg/mL. In addition, the tyrosinase inhibitory and melanin synthesis of the peptides isolated from Crassostrea gigas were also evaluated to determine if they could be used as a potential cosmetic agent. The peptides were found to significantly inhibit the melanin synthesis of the clone M-3 cell line by up to 62.7%. The inhibitory activities of the tyrosinase were observed 34.51% in ascorbic acid, 42.49% in extract with the ultrasonification at $40^{\circ}C$ and 35.37% in $40^{\circ}C$ extract at 1.0 mg/mL concentration, respectively. Finally, when samples were treated with the peptide extracts at a concentration of 0.6 mg/mL, PGE2 expression was significantly decreased. Taken together, these results indicate that Crassostrea gigas may be a source of cosmetic agents capable of improving physiological hyperpigmenting and immuno-modulating skin disorders.

• Korean Journal of Food Science and Technology
• /
• v.44 no.5
• /
• pp.600-606
• /
• 2012

A Bacillus sp. that produces fibrinolytic enzyme was isolated from Cheonggukjang, a traditional Korean soybean-fermented food. According to 16S rRNA gene base sequencing, the bacillus was identified as a variety of Bacillus subtilis, and named Bacillus subtilis IDCC 9204. Fibrinolytic enzyme NK-IL9204 was stable up to $60^{\circ}C$ and within pH range of 5-10. Purified NK-IL9204 was detected through fibrin zymography. The molecular weight and isoelectric point of the enzyme were estimated to be 27.7 kDa and 6.7 by SDS-PAGE and 2D electrophoresis, respectively. Its amino acid sequence was similar to that of nattokinase (identities 99.5%) and different from that of nattokinase BPN (identities 86.4%). The plasma fibrinolytic activity of NK-IL9204 was measured by euglobulin clot lysis times (ECLT). The NK-IL9204 was orally administered to SD rats for 3 weeks (1,000 FU/rat/day). The ECLT was significantly shortened by supplementation of NK-IL9204.

• Journal of Life Science
• /
• v.19 no.10
• /
• pp.1424-1431
• /
• 2009

Shrimp Jeot-Gal is a popular traditional Korean fermented seafood and has been used for seasoning. We isolated a bacterium showing strong extra-cellular fibrinolysis and chitinase activity from shrimp Jeot-Gal and the strain was designated SC082. SC082 was identified as Bacillus licheniformis by 16S rRNA sequence homology search. B. licheniformis SC082 exhibited optimum temperature, pH, and salt concentration at $37^{\circ}C$, pH 7.0, and 6%, respectively. Substrate specificity of the culture supernatant from B. licheniformis SC082 was detected in fibrin, skim milk, and chitin plate. The fibrinolytic activity was highly maintained up to $50^{\circ}C$ at a pH of 7.0 for 3 hr and was stable up to pH 9.0 at $37^{\circ}C$ for 3 hr. The chitinase activity was remarkably induced by addition of 1.0% colloidal chitin and the pH and temperature optima of the enzyme were 5.0 and $45^{\circ}C$, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis, this strain produced three fibrinolytic isozymes and two chitinase isozymes. The approximate molecular weights of the putative fibrinolytic enzymes were 23.0, 62.0, and 72.0 kDa and those of the chitinases were 62.0 and 55.0 kDa, respectively. The antioxidant activity of SC082 was also measured by using 2,2-diphenyl-l-picryl-hydrazyl (DPPH) free radical. The DPPH radical scavenging was slightly increased in a dose-dependent manner.

• BMB Reports
• /
• v.35 no.3
• /
• pp.313-319
• /
• 2002

N-Acetyl-$\beta$-D-hexosaminidase ($\beta$-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sative L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions($F_1-F_7$) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S-300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-$\beta$-D-hexosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-$\beta$-D-hexosaminide (pNP-GalNAc) as substrates, which are typical properties of $\beta$-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-$\beta$-glucopyranoside, or pNP-$\beta$-glucopyranoside. The enzyme showed $K_m$, $V_{max}$ and $K_{cat}$ for pNP-GlcNAc of 1.65 mM, $79.49\;mM\;min^{-1}$, and $4.79{\times}10^6\;min^{-1}$, respectively. The comparison of kinetic values for pNP-GlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNP-GlcNAc of 5.0 and $50^{\circ}C$, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and $20-40^{\circ}C$. The enzyme activity was completely inhibited at a concentration of 0.1 mM $HgCl_$ and $AgNO_3$, suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.

• The Journal of Korean Medicine
• /
• v.18 no.1
• /
• pp.357-374
• /
• 1997

In order to investigate the effects of Sakunjatang(四君子湯) and Samultang(四物湯) aqua-acupuncture on immune response, Sprague-Dawley male rats were used and randomly divided into four group. Normal group was normal control, Control group was injected i.v. with 2mg/kg MTX on 9th and 11th day after sensitization with SRBC on 5th day, Sakunjatang aqua-acupunctured group(Sample A) was aqua-acupunctured daily for 18 days into the locus corresponding Bisu(B20) locus of the human body to Rat and MTX was administered 9th and 11th experimental day, Samultang aqua-acupunctured group(Sample B) was aqua-acupunctured daily for 18 days into the locus coresponding Bisu(B20) locus of the human body to Rat and MTX was administered 9th and 11th experimental day. In the 9th day and the 11th day after aqua-acupuncture, MTX was injected to reduce immune function in the tail of rat. leukocyte count, lymphocyte ratio, lymphocyte count of spleen, lymphocyte count of bonemarrow, contact hypersensitivity to DNFB, morphologic change of thymus cell, and electropherogram of serum protein. The result were summarized as follows: 1. Before MTX injection, leukocyte count had no significant difference in Sakunjatang aqua-acupunctured group and Samultang aqua-acupunctured group compared to control group and after MTX injection, leukocyte count had no significant difference Sakunjatang aqua-acupunctured group and Samultang aqua-acupunctured group. 2. Before MTX injection, lymphocyte ratio was decreased significantly Sakunjatang aqua-acupunctured group and Samultang aqua-acupunctured group compared to control group. 3. After MTX injection, lymphocyte ratio was increased significantly Sakunjatang aqua-acupunctured group, Samultang aqua-acupunctured group had no significant difference compared to control group. 4. The lymphocyte count of spleen was increased significantly Sakunjatang aqua-acupunctured group and Samultang aqua-acupunctured group compared to control group. 5. The lymphocyte count of bonemarrow was increased significantly Sakunjatang aqua-acupunctured group, Samultang aqua-acupunctured group had no significant difference compared to control group. 6. Contact hypersensitivity was no significant difference in Sakunjatang aqua-acupunctured group and Samultang aqua-acupunctured group compared to control group. 7. In the morphologic change of thymus cell, control group compared to normal group had a indistinct boundary between cortex and medulla, and lymphocyte cell density of thymus was low, Sakunjatang aqua-acupuryctured group had a distinct boundary between cortex and medulla, and lymphocyte cell density of thymus was high. 8. In the SDS-PAGE electrophorogram of serum protein, Sakunjatang aqua-acupunctured group and Samultang aqua-acupunctured group had a wide band of nearby 50,000 and 25,000 Dalton which meant IgG generate more activity.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.3
• /
• pp.292-300
• /
• 2012

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters $K_m$, $V_{max}$, and $k_{cat}$ for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at $50^{\circ}C$, but the optimal temperature of activity was $37^{\circ}C$. It also showed maximal and optimal activities at pH 9.0. The values of $K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$ were $8.9{\pm}0.967{\times}10^{-3}$ M, $128{\pm}2.8$ U/mg protein, $106{\pm}2s^{-1}$, and $1.2{\pm}0.105{\times}10^4M^{-1}s^{-1}$, respectively. The L-asparaginase activity was reduced in the presence of $Mn^{2+}$, $Zn^{2+}$, $Ca^{2+}$, and $Mg^{2+}$ metal ions for about 52% to 31%. In addition, we found that $NH_4{^+}$, L-Asp, D-Asn, and ${\beta}$-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobial-type asparaginase II family.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.10
• /
• pp.1529-1536
• /
• 2006

We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-t1 (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -t1, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their $M_rs$ were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some $50{\mu}g$ of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at $10{\mu}l$ of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose ($10{\mu}l$) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-t1, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.