• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.160-166
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• 2008

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoding 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with $endo-{\beta}-1,4-D-mannanase$ from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by $Ni^{2+}$ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa ${\beta}$-mannanase having a specific activity of 481.55U/mg. The optimal activity of the purified protein, MANB48, was at $58^{\circ}C$ and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.397-404
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• 2013

Paenibacillus xylanilyticus KJ-03 was isolated from soil samples obtained from a field with Amorphophallus konjac plants. A gene encoding xylanase was isolated from KJ-03 and cloned using a fosmid library. The xynA gene encodes xylanase; it consists of 1,035 bp and encodes 345 amino acids. The amino acid sequence deduced from the P. xylanilyticus KJ-03 xylanase showed 81% and 69% identities with those deduced from the P. polymyxa E681 and Paenibacillus sp. HPL-001 xylanases, respectively. The xynA gene comprises a single domain, consisting of a catalytic domain of the glycosyl hydrolase (GH) 10 family. The xynA gene was expressed in Escherichia coli BL21 (trxB), and the recombinant xylanase was purified by Niaffinity chromatography. The purified xylanase showed optimum activity with birchwood xylan as a substrate at $40^{\circ}C$ and pH 7.4. Treatment with $Mg^{2+}$ and $Li^+$ showed a slight decrease in XynA activity; however, treatment with 5 mM $Cu^{2+}$ completely inhibited its activity. The results of the thin layer chromatography analysis indicated that the major hydrolysis product was xylobiose and small amounts of xylose and xylotriose. XynA showed increased activity with oat spelt xylan and birchwood xylan, but showed only slight activity with locust bean gum.

• YAKHAK HOEJI
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• v.57 no.2
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• pp.125-131
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• 2013

Biologically active peptides, including growth factors and cytokines, participate in various biological processes in human skin. They could provide a great advantage of maintaining healthy skin. Many peptide growth factors like epidermal growth factor (EGF) and human growth hormone (hGH) have been used in cosmetic formulations. The delivery of peptide growth factors across the Stratum corneum, however, seems not sufficient because of their physical properties such as high molecular weight and hydrophilicity. So increasing the penetration of growth factors of interest into skin would be a major concern for ensuring their maximum biological efficacy. In this study, we have identified several skin penetration-enhancing peptides which facilitate delivery of growth factors, when fused at N-terminus of the target protein, into skin. For efficient and rapid screening, we constructed a skin-penetrating assay system using Franz cell and porcine skin. Next, we carried out phage display screening using M-13 bacteriophage with random 12 -amino acid library on its coat protein P3 on that system. After several selection rounds, peptide sequences facilitate the penetration of phages through the porcine skin were identified from a large population of phages. We found that phages with the most potent peptide (S3-2, NGSLNTHLAPIL) could penetrate the porcine skin eight times more than those with control peptide (12 mino acids scrambled peptide). Furthermore, growth factors conjugated with S3-2 peptide penetrate porcine skin three to five times efficiently than non-conjugated growth factors. In conclusion, our data shows that the skin penetration-enhancing peptide we have characterized could increase the delivery of growth factors and is useful for cosmeceutical application.

• Magazine of the Korea Concrete Institute
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• v.8 no.2
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• pp.151-161
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• 1996

This paper presents a series of tests to produce the h~gh quality concrete to be filled Inside the steel tube columns. Thls concrete filled steel tube system requires not only the high strength, but a150 the flowable concrete. Laboratory test has been performed to clarlfy the material characteristics and to produce the optlmal mix design proportion. Full scale site mock up test has been then carried out to slnlulate the actual construct~on conditions including the product~on of concrete at the rermcon batch plant, transportation to the construction site, proper workabil~ ty and man power required , 4ddit1onal mock up test has finally been performec to irivesti gate any unfavorable construction s~tuatioils since the actual concrete placement has been sched uled in cold weather period, so that the high quality concrete construction is convinced to be successfully carried out.

• KSBB Journal
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• v.18 no.4
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• pp.306-311
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• 2003

Solid-phase refolding of immobilized proteins can be an effective way to reuse an immobilized enzyme column. Oriented immobilization methods are known to provide higher activity of the immobilized enzymes. In this study, using recombinant EK (enterokinase) as a model enzyme and a fusion protein, that consisted of recombinant human growth hormone and six His tag that was linked by the peptide of EK-specific recognition sequence, as a model substrate, we evaluated two oriented immobilization methods, i. e., reductive alkylation of N-terminus ${\alpha}$-amine and affinity interaction between poly-histidine tag and Ni-NTA (nickel-nitrilotriacetic acid). The immobilization yield, activity and cleavage of the immobilized enzymes, and the yield of solid-phase refolding were compared. The Ni affinity immobilization and the covalent immobilization yields were about 100％ and 65％, respectively. But the specific activities were the same, about 50％ of that of the soluble enzyme. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. Covalently immobilized EK showed almost 100％ refolding yield but the affinity immobilized EK showed only 70％ yield, which suggested the covalent conjugation provided more rigid ‘reference structure’ for the solid-phase refolding. The monomeric hGH could be easily obtained by capturing the cleaved poly Histidine tag by the Ni affinity column.

• Nuclear Engineering and Technology
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• v.53 no.9
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• pp.3051-3057
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• 2021

The concrete is considered as an important radiation shielding material employed widely in nuclear reactors, particle accelerators, laboratory hot cells and other different radiation sources. The present research is dedicated to the shielding properties study of the ordinary concrete reinforced with different weight fractions of lead oxide micro/nano particles. Lead oxide particles were fabricated by chemical synthesis method and their properties including the average size, morphological structure, functional groups and thermal properties were characterized by XRD, FESEM-EDS, FTIR and TGA analysis. The gamma ray mass attenuation coefficient of concrete composites has been calculated and measured by means of the Monte Carlo simulation and experimental methods. The simulation process was based on the use of MCNP Monte Carlo code where the mass attenuation coefficient (μ/ρ) has been calculated as a function of different particle sizes and filler weight fractions. The simulation results showed that the employment of the lead oxide filler particles enhances the mass attenuation coefficient of the ordinary concrete, drastically. On the other hand, there are approximately no differences between micro and nano sized particles. The mass attenuation coefficient was increased by increasing the weight fraction of nanoparticles. However, a semi-saturation effect was observed at concentrations more than 10 wt%. The experimental process was based on the fabrication of concrete slabs filled by different weight fractions of nano lead oxide particles. The mass attenuation coefficients of these slabs were determined at different gamma ray energies using ²²Na, ¹³⁷Cs and ⁶⁰Co sources and NaI (Tl) scintillation detector. The experimental results showed that the HVL parameter of the ordinary concrete reinforced with 5 wt% of nano PbO particles was reduced by 64% at 511 keV and 48% at 1332 keV. Reasonable agreement was obtained between simulation and experimental results and showed that the employment of nano PbO particles is more efficient at low gamma energies up to 1Mev. The proposed concrete is less toxic and could be prepared in block form instead of toxic lead blocks.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1295-1300
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• 2009

A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.333-337
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• 1996

For continuous production of galactooligosaccharides(GOS), $\beta-galactosidase$ with h1gh transgalactosylation activity from Bacillus sp. A 4442 was Immobilized onto $Diaion^{TM}$ HPA 75(styrene-divinylbenzene resin). The parameters influencing enzyme immobilization were scrutinized in order to maximize immobilization yield while minimizing enzyme inactivation. The optimum conditions turned out to be: Tris buffer concentration 30 mM, pH 8.0, contact time at room temperature 3 hr, and enzyme loading 25 mg protein/g resin. Both the thermal stability and the operational stability of immobilized enzyme were markedly enchanced by the treatment with 0.5% glutaraldehyde as a cross-linker. Under the experimental conditions established, the yield of ${\beta}-galactosidase$ immobilization was 40% or more and the activity of the immobilized enzyme ca. 200 U/g resin. When a packed-bed reactor was employed to continuously convert lactose to GOS, the specific production, which refers to as the amount of commercially valuable GOS produced by a unit amount of immobilized ${\beta}-galactosidase$, was found to be ca. 300 g GOS/g carrier.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.332-338
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• 2009

A xylan-decomposing bacterium, HY-20, was isolated from the gut of a honeybee, Apis mellifera, and identified as Bacillus sp. The extracellular GH11 xylanase (XylP) gene (687-bp) of strain HY-20 encoded a protein of 228 amino acids with a deduced molecular mass of 25,522 Da and a calculated pI of 9.33. The primary structure of XylP was 97% identical to that of B. pumilus xylanase (GenBank accession no.: AY526092) that has not been characterized yet. The recombinant His-tagged enzyme (rXylP) overexpressed in Escherichia coli BL21 harboring pET-28a(+)/xylP was purified to electrophoretic homogeneity by cation exchange and gel permeation chromatographies. The purified enzyme exhibited the highest catalytic activity toward birchwood xylan at pH 6.5 and $50^{\circ}C$ and retained approximately 50% of its original activity when pre-incubated at $55^{\circ}C$ for 15 min. The recombinant enzyme was completely inactivated by $Hg^{2+}$ (1 mM) and N-bromosuccinimide (5 mM), while its activity was slightly stimulated by approximately 10% in the presence of $Mn^{2+}$ (1 mM), $Fe^{2+}$ (1 mM), and sodium azide (5 mM). rXylP was able to efficiently degrade various polymeric xylose-based substrates but PNP-sugar derivatives and glucose-based polymers were not susceptible to the enzyme.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.10
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• pp.1411-1415
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• 2008

The purpose of the present study was to determine the onset of breeding season, the occurrence of silent and true heats and the duration of estrus in female Markhoz goats of the Kurdistan province in Iran. Ten, 3 years-old Markhoz does with an average weight of $34.05{\pm}2.62kg$ and with one kidding record, were used. The goats were maintained in an open barn under constant nutritional levels and natural photoperiod. One aproned buck was used twice a day every 12 h to detect estrus from mid August to early January. For the determination of the onset of reproductive activity as well as occurrence of silent heat, blood samples were collected every 10 days, from the beginning of the experiment. After $2^{nd}$ standing heat, blood samples were obtained twice a week in order to assess luteal activity and the length of estrous cycles. In this study, estrous behavior was observed including sniffing, vocal exchange, following courtship, flehman, standing heat and mounting. The results of the progesterone assay indicated that in this goat silent heats occur mostly in the early breeding season. The first standing heat was observed in mid-October which was considered as the onset of the breeding season. Duration of the estrous cycle and estrus was recorded as being $20.93{\pm}1.56days$ and $38.86{\pm}15.19h$, respectively. The correlation coefficient between length of daylight and occurrence of estrus was negative (r = -0.470) but not significant (p>0.05). The data showed that there was no significant effect of body weight on estrous cycles (first, second and third) and estrous periods (first, second, third and fourth). Progesterone levels were not significantly different in the first, second and third estrous cycles at days 0, 4, 10, 12, 14 and 19. The results of progesterone assay during the estrous cycle indicate that follicular and luteal phases last 4-5 and 14-15 days, respectively and the concentration of serum progesterone in these phases was $0.88{\pm}0.08$ and $7.44{\pm}0.26ng/ml$, respectively. The study concluded that Markhoz does could be considered as a breed with a short breeding season and an optimal estrous activity in autumn.