• BMB Reports
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• v.36 no.6
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• pp.572-579
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• 2003

An expressed sequence tag (EST) library was established from the hypopharyngeal glands of Apis cerana. Sixty-six recombinant clones, possessing inserts >500 bp, were randomly selected and unidirectional sequenced. Forty-two of these (63.6%) were identified as homologues of Major Royal Jelly Proteins families 1, 2, 3, and 4 of A. mellifera (AmMRJP) for which MRJP1 was the most abundant family. The open-reading frame of the MRJP1 homologue (AcMRJP1) was 1299 nucleotides that encoded 433 deduced amino acids with three predicted N-linked glycosylation sites. The AcMRJP1 sequence showed 93% and 90% homologies with nucleotide and deduced amino acid sequences of AmMRJP1, respectively. Two complete transcripts of apisimin, and one and two partial transcripts of $\alpha$-glucosidase and glucose oxidase, were also isolated. In addition, the royal jelly proteins of A. cerana were purified and characterized using Q-Sepharose and Sephadex G-200 column chromatography. The native forms of protein peaks A1, A2, B1, and C1 were 115, 55, 50, and 300 kDa, respectively. SDS-PAGE analysis indicated that A1 and C1 were dimeric and oligomeric forms of the 80 kDa and 50 kDa subunits, respectively. The ratio of the total protein quantities of A1 : A2 : B1 : C1 were 2.52 : 4.72 : 1 : 12.21. Further characterization of each protein, using N-terminal and internal peptide sequencing, revealed that the respective proteins were homologues of MRJP3, MRJP2, MRJP1, and MRJP1 of A. mellifera.

• The Journal of Korean Medicine
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• v.28 no.2 s.70
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• pp.54-65
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• 2007

Objectives : In the present study, the author investigated whether Naenghyo-hwan(NHH) significantly affect mucin secretion from airway epithelial cells. Methods : Confluent hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled with 3H-glucosamine for 24 hrs and chased for 30 min in the presence of NHH to assess the effect of the agent on 3H-mucin secretion. Total elutionprofiles of control spent media and treatment sample through Sepharose CL-4B column were analysed. Effect of NHH on contractility of isolated tracheal smooth muscle was investigated. Also, effect of the agent on MUC5AC gene expression in cultured NCI-H292cells was investigated. Possible cytotoxicities of the agent were assessed by measuring both lactate dehydrogenase (LDH) release from HTSE cells and examining the rate of survival and proliferation of NCI-H292 cells. Results : NHH significantly increased mucin secretion from cultured HTSE cells, with significant cytotoxicity. NHH chiefly affected the 'mucin' secretion. NHH inhibited ACh-induced contraction of isolated tracheal smooth muscle. NHH disturbed both the extraction of total RNA from NCI-H292 cells and polymerase chain reaction, nonspecifically. Therefore, in this experiment, theeffect of NHH on the expression levels of MUC 5AC gene in cultured NCI-H292 cells could not be elucidated. Conclusions : The author suggests that the effect of NHH with their components should be further investigated and it is valuable to find, from oriental medical prescriptions, novel agents which might regulate mucin secretion from airway epithelial cells.

• Microbiology and Biotechnology Letters
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• v.18 no.4
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• pp.376-382
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• 1990

An extracellular cellulase producing bacterium YE-5 was isolated from soil, and identified as a Cellulomonas sp. by its taxonomical characteristics. The maximal activities of avicelase (0.35 units/ml), CMCase (3.18 units/ml), FPase (0.315 units/ml) and $\beta$-glucosidase (0.882 units/ml) were obtained when this strain was cultured for 48 hrs at $30^{\circ}C$ in a medium containing 0.8% (w/v) Solka floc, 0.06010 (wlv) urea, 0.1% (w/v) $K_2HP0_4$, 0.1% (w/v) $MgS0_4.7H2_0$, 0.2% (w/v) bacto peptone, 0.2% (w/v) yeast extract and pH 6.5. The cellulase was purified by ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex 6-100 column chromatography from culture filtrate of Cellulomonus sp. YE-5. The molecular weights of purified avieelase, CMCase I, and CMCase II were estimated to be about 95,000 ~ 105,000, 46,000 ~ 47,000 and 120,000 ~ 125,000, respectively.

• BMB Reports
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• v.37 no.6
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• pp.684-690
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• 2004

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and $50^{\circ}C$. It was rather stable within the range of 25 to $50^{\circ}C$ but lost activity rapidly above $50^{\circ}C$. The enzyme was activated by $Co^{2+}$ or EDTA and stabilized by dithiothreitol. The kinetic constants, $K_m$ and $V_{max}$ for heparin were $1.3{\times}10^{-5}\;M$ and $8.8\;{\mu}mol/min{\cdot}mg$. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.252-259
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• 1992

Extracellular laccase excreted from Lentinus edodes ATCC 48085 was purified through a series of DEAF, Sephadex A-50. Con A-Sepharosc and Sephadex G-150 chromatography. Extracellular enzyme. which consists of a single polypeptide, has a n~olecular mass of 87.000 daltons and contains 12.0'%, carbohydrate. The N-terminal amino acid sequence (I5 residues) of the puritied enzyme was similar to that of laccases of PIeurotus ostreatus and Coriolus hirsutus. The enzyme showed optimal activity at near pH 4.8 and $40^{\circ}C$. The enzyme was stable at pH 7-9 and below $30^{\circ}C$. $K_{M}$ and $k_{cat}$ values for syringaldazine were estimated to be $0.4\mu\textrm{M}$ and 77 sec, respectively. The developed patterns of reaction products of thevenzyme on thin layer chromatography were similar to those of laccase of Pleurotus ostreatus.

• The Journal of Pediatrics of Korean Medicine
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• v.20 no.1
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• pp.109-135
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• 2006

Objective : In the present study, the author tried to investigate whether six oriental medical prescriptions named gamisingitang (SGT), gamijungtang (IJT), gamicheongpyetang (CPT), galhwengchihyosan (CHS), chwiyeontong (CYT), sigyoungcheongpyetang (SCPT) significantly affect mucin release from cultured hamster tracheal surface epithelial (HTSE) cells. Methode : Confluent HTSE cells were inetabolically radiolabeled with $^{3}H-glucosamine$ for 24 hrs and chased for 30 min in the presence of drugs aforementioned, respectively, to assess the effect of each drug on $^{3}H-mucin$ release. Possible cytotoxicities of effective drugs were assessed by measuring lactate dehydrogenase(LDH) release. Additionally, total elution profiles of control spent media and treatment sample (CPT, CHS, SCPT and CYT) through Sepharose CL-4B column were analysed and effect of CPT, CHS and CYT on MUC5AC mRNA expression in cultured HTSE cells were invsetigated. Results : (1) SGT and IJT did not affect mucin release without cytotoxicity; (2) CPT, SCPT and CHS significantly stimulated mucin release from cultured HTSE cells, with significant cytotoxicity; (4) CPT, CHS, SCPT and CYT chiefly affected the 'mucin' release and did not affect significantly the release of the releasable glycoproteins with less molecular weight than mucin. This result suggests that the four herbal prescriptions specifically affect the release of mucin ; (5) CTP and CHS did not significantly affect the expression levels of MUC 5AC mRNA, however, CYT significantly inhibit the expression levels of MUC 5AC mRNA. Conclusion : CYT can decrease the synthesis of mucin at gene level in cultured HTSE cells.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.604-610
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• 2007

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at $30^{\circ}C$, and was unstable at temperatures higher than $30^{\circ}C$, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24h incubation at $4^{\circ}C$. The addition of $Ca^{2+}\;and\;Mg^{2+}$ enhanced the enzyme activity of LipA1, whereas the $Cd^{2+},\;Zn^{2+},\;CO^{2+},\;Fe^{3+},\;Hg^{2+},\;Fe^{2+},\;Rb^{2+}$, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate $(C_{14}\;acyl\; groups)$.

• Korean Journal of Animal Reproduction
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• v.14 no.2
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• pp.93-100
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• 1990

These experiments were carried out to investigate the effect of rabbit anti-bovine cumulus cell antibodies on in vitro maturation of bovine follicular oocytes. Antisera to bovine cumulus cell were produced Japanese Ginat rabbit by repeated immunization of intact or solubilized bovine cumulus cell and purified by ammonium sulfate precipitation and Sepharose CL-4B protein-A affinity chromatography. The bovine cumulus cell-specific antibodies were confirmed by indirect ELISA. The results obtained in these experiments were summarized as follows : 1. The titer of the antibodies to cumulus cell determined by indirect ELISA using intact or solubilized bovine cumulus cell coated plates was very high in both intact and solubilized cumulus cells. Namely, the optical density at 1:12,800 dilution of antibodies was still significantly higher than that of non-immunized control serum. 2. When the follicular oocytes were treated with antibody to intact cumulus cells, the maturation rate of cumulus compacted and removed oocytes was ranged 47.6 to 59.1%. These value is significantly lower(p<0.05) than that(78.8%) of follicular oocytes cultured without the antibody. 3. the maturation rate of cumulus compacted and removed oocytes treated with antibody to solubilized cumulus cells was ranged 46.7 to 59.1%, significantly lower(p<0.05) than that(82.1%) of ooyctes cultured in antibody free medium. From above mentioned results, it could be said that cumulus cells promote nuclear maturation of follicular oocytes and that the beneficial effect of cumulus cells to the oocyte maturation is inhibited by the action of antibody to cumulus cells.

• Microbiology and Biotechnology Letters
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• v.22 no.5
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• pp.507-514
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• 1994

Acetyl xylan esterase was produced by E. coli HB101 harboring a recombinant plasmid pKMG6 which contained the estI gene of Bacillus stearothermophilus. The maximum production was observed when the E. coli strain was grown at 37$\circC for 12 hours in the medium containing 0.5% acetyl xylan, 1.0% tryptons, 1.0% sodium chloride, and 0.5% yeast extract. The esterase produced was purified to homogeneity using a combination of ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration. The native enzyme had an apparent molecular mass of 60 kd and was composed of two identical subunits of 29 kd. The N-terminal amino acid sequence of the polypeptide was Ala-X-Leu-Gln- Ile-Gln-Phe-X-X-Gln. The acetyl esterase displayed a pH optimum of 6.5 and a temperature opti- mum of 45$\circC. The heavy metal ions such as Ag$^{++}$, Hg$^{++}$ and Cu$^{++}$ inhibited nearly completely the activity of the esterase, and no specific metal ion was found to be required for the enzyme activity. The enzyme readily cleaved MAS, $\beta$-D-glucose pentaacetate, $\alpha$-naphthyl acetate, $\rho$-nitrophenyl acetate as well as acetyl xylan, but had no activity on $\rho$-nitrophenyl propionate, $\beta$-nitrophenyl butyrate or $\beta$-nitrophenyl valerate. The Km and Vmax values for MAS were 2.87 mM and 11.55 $\mu$mole/min, respectively. Synergistic behavior was demonstrated with a combination of xylanase and esterase from B. stearothermophilus in hydrolyzing acetyl xylan.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.256-261
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• 2004

In order to detect chitooligosaccharides (COS) in soybean paste, tandem immunoaffinity chromatography and enzyme-linked immunosorbent assay (ELISA) were developed. Polyclonal anti-chitooligosaccharides mixture (CaSM) antibody specific to COSM was attached to Sepharose gel for initial sample cleanup and concentration of COS in soybean paste. COS was eluted and quantified by competitive direct ELISA (cdELISA). Average ELISA recoveries from the column using binding buffer spiked with COSM at levels of 0.5, 2.0, 5.0, and $10.0\mu$g/ml were 79.8, 72.0, 77.7, and 60.6%, respectively, with a mean recovery of 72.5%. Mean inter-well and inter-assay coefficients of variation (CV) were 7.7% and 10.3%, respectively. Average recoveries from soybean paste spiked with COSM at levels of 2, 6, 20, and $60\mu$g/g were 115, 91.7, 91, and 73.3%, respectively, with a mean recovery of 92.8%. Mean inter-well and inter-assay CV were 12.9% and 16%, respectively. The COS was detected from 24 out of 25 homemade Korean soybean paste samples at an average of $14.0\mu$g/g (n, 25; range, $0-51.2 \mu$g/g) and from 13 out of 14 commercially made soybean paste samples at an average of $4.1\mu$g/g(n, 14; range, $0-18.4\mu$g/g). The tandem immunoaffinity chromatography-cdELISA that was developed in this study showed that the level of COS eluted from homemade soybean paste was higher than that of the commercially made ones. In addition, the level of COS eluted from commercially available soybean paste in Korea was higher than that of the ones in Japan.