• Journal of Microbiology and Biotechnology
• /
• 제25권10호
• /
• pp.1742-1750
• /
• 2015

Human insulin is composed of 21 amino acids of an A-chain and 30 amino acids of a B-chain. This is the protein hormone that has the role of blood sugar control. When the recombinant human proinsulin is expressed in Escherichia coli, a serious problem is the formation of an inclusion body. Therefore, the inclusion body must be denatured and refolded under chaotropic agents and suitable reductants. In this study, H27R-proinsulin was refolded from the denatured form with β-mercaptoethanol and urea. The refolding reaction was completed after 15 h at $15^{\circ}C$, whereas the reaction at $25^{\circ}C$ was faster than that at $15^{\circ}C$. The refolding yield at $15^{\circ}C$ was 17% higher than that at $25^{\circ}C$. The refolding reaction could be carried out at a high protein concentration (2 g/l) using direct refolding without sulfonation. The most economical and optimal refolding condition for human preproinsulin was 1.5 g/l protein, 10 mM glycine buffer containing 0.6 M urea, pH 10.6, and 0.3 mM β-mercaptoethanol at $15^{\circ}C$ for 16 h. The maximum refolding yield was 74.8% at $15^{\circ}C$ with 1.5 g/l protein. Moreover, the refolded preproinsulin could be converted into normal mature insulin with two enzymes. The average amount of human insulin was 138.2 g from 200 L of fermentation broth after enzyme reaction with H27R-proinsulin. The direct refolding process for H27R-proinsulin was successfully set up without sulfonation. The step yields for refolding and enzyme reaction were comparatively high. Therefore, our refolding process for production of recombinant insulin may be beneficial to the large-scale production of other biologically active proteins.

• Journal of Microbiology and Biotechnology
• /
• 제15권2호
• /
• pp.254-258
• /
• 2005

We have conducted in vitro reconstitution study of ferritin from its subunits FerH and FerL. For the reconstitution, FerH was produced from an expression vector construct in Escherichia coli and was purified from a heat treated cell extract by using one-step column chromatography. FerL was expressed as inclusion bodies. The denatured form of FerL was obtained by a simple washing step of the inclusion bodies with 3 M urea. The reconstitution experiment was conducted with various molar ratios of urea-denatured FerH and FerL to make the ferritin nanoparticle with a controlled composition of FerH and FerL. SDS-PAGE analysis of the reconstituted ferritins revealed that the reconstitution required the presence of more than 40 molar$\%$ of FerH in the reconstitution mixture. The assembly of the subunits into the ferritin nanoparticle was confmned by the presence of spherical particles with diameter of 10 nm by the atomic force microscopic image. Further analysis of the particles by using a transmission electron microscope revealed that the reconstituted particles exhibited different percentages of population with dense iron core. The reconstituted ferritin nanoparticles made with molar ratios of [FerH]/[FerL]=l00/0 and 60/40 showed that 80 to $90\%$ of the particles were apoferritin, devoid of iron core. On the contrary, all the particles formed with [FerH]/[FerL]=85/ 15 were found to contain the iron core. This suggests that although FerH can uptake iron, a minor portion of FerL, not exceeding $40\%$ at most, is required to deposit iron inside the particle.

• 대한화학회지
• /
• 제40권6호
• /
• pp.394-400
• /
• 1996

결합성 사슬이합체를 형성할 수 있는 올리고펩티드-$(HPPHPPP)_n$-(H: 소수성 아미노산, P: 친수성 아미노산)는 온도, 수소이온 농도, 이온세기, 변성시약 등에 의해 구조적인 변화가 가능하다. 본 연구에서는 변성시약과 온도에 의한 올리고펩티드의 전이 현상을 이론적으로 고찰하였다. 사슬이합체로는 올리고펩티드20R, 변성시약으로는 구아니듐-염산을 사용하였다(20R에는 사슬 내의 정전기적 반발력이 10개가 존재하고, 사슬사이의 정전기적 반발력이 10개가 존재한다). 변성 시약에 의한 올리고펩티드의 나선에서 코일로의 전이는 급격한 것으로 보아, 변성이 일어나는 전이상태에서 올리고펩티드들은 완전한 나선구조와 무질서한 코일구조로만 되어있다. 반면에 온도에 의한 전이는 변성시약에 의한 전이보다 완만하게 일어난다. 낮은 온도에서 긴 나선 구조를 가지는 올리고펩티드가 짧은 나선 구조를 가지는 것보다 다량으로 존재한다. 온도가 증가할수록 부분적으로 변성된 분자들의 몰분율이 증가하여, 전이가 일어나는 온도에서 부분적으로 변성된 올리고펩티드가 널리 분포되어 있다.

• Journal of Dairy Science and Biotechnology
• /
• 제35권4호
• /
• pp.270-285
• /
• 2017

Among milk proteins, caseins are not subjected to chemical changes during heat treatment of milk; however, whey proteins are partially denatured following heat treatment. The degree of whey protein denaturation by heat treatment is decreased in the order of high temperature short time (HTST) > low temperature long time (LTLT) > direct-ultra-high temperature (UHT) > indirect-UHT. As a result of heat treatment, several changes, including variations in milk nitrogen, interactions between beta-lactoglobulin and k-casein, variations in calcium sulfate and casein micelle size, and delay of milk coagulation by chymosin action, were observed. Lysine, an important essential amino acid found in milk, was partially inactivated during heat treatment. Therefore, the available amount of lysine decreased slightly (1~4% decrease) after heat treatment, However, the influence of heat treatment on the nutritional value of milk was negligible. Nutritional value and nitrogen balance did not differ significantly between UHT and LTLT in milk. In conclusion, our results showed that heat treatment of milk did not alter protein quality. Whey proteins denatured to a limited extent during the heat treatment process, and the nutritional value and protein quality were unaffected by heat treatment.

• 한국식품과학회지
• /
• 제16권3호
• /
• pp.279-284
• /
• 1984

분유와 분유로 제조된 cheese의 미세구조(微細構造)가 전자 현미경(顯微鏡)에 의해서 관찰되었다. 동결건조 분유는 사과모양을 나타내었다. 동결건조 분유로 제조된 cheese는 전형적인 용융 cheese와 비교하여 표면의 구조가 비교적 편편하고 백색의 균일한 침적물을 나타내었다. 도입 분유 역시 동결건조 분유와 거의 유사한 모앙을 나타내었고, 도입분유로 제조된 cheese는 균일한 분산을 나타내나 표면이 약간 거칠고 caseins matrix와 침적물 사이에 공간이 있다. 시판 분유는 건조중 변성된 것으로 불규칙한 변성 응고물의 모양을 나타내었다. 시판분유로 제조된 cheese의 모양은 불규칙하고 작은 침적물과 세공성이 큰 것으로 나타나 분유의 열변성도는 cheese의 세공성을 증가시키는 것으로 보인다. 변성된 단백질은 변성되지 않은 단백질보다 polyphosphate에 의한 단백질 분산성이 덜 효과적인 것으로 보인다. 분유로 제조한 cheese의 내부구조에서 지방구막과 casein micelle이 전형적인 용융 cheese에 비해 아주 근접해있고 casein micelle이 아주 치밀한 양상을 나타내었다. 분유의 용융 기작도 전형 적인 cheese의 용융과는 다른 것으로 보인다.

• The Plant Pathology Journal
• /
• 제15권4호
• /
• pp.247-250
• /
• 1999

Wasabies showing mosaic symptoms were collected and extracted for virus purification. Tobacco mosaic virus (TMV) was identified as causal agent by electron microscopy and nucleic acid and coat protein analyses. TMV strains were determined by enzyme-linked immunosorbent assay (ELISA). TMV was identified as W and C strain in wasabi. The results of host reaction indicated that this virus induced local lesions on Nicotiana tabacum cv. Bright Yellow and N. glutinosa, leaf spots on Chenopodium amaranticolor and mosaic symptoms on wasabi. Rot shape virus particles were observed and was about 300 nm in length. About 6.5 kb single RNA molecule was observed from extracted viral RNA sample and 26 KDa coat protein was detected in denatured acrylamide gel. Infection ratio of TMV was 8% for the first cultivation year, but was 22% for the second year when TMV-W antiserum was used. The results of this experiment showed that infection ratios of both TMV-W and TMV-C strains were higher compared to that of TMV-P strain.

• Journal of Biosystems Engineering
• /
• 제15권1호
• /
• pp.33-43
• /
• 1990

Spectral reflectance of paddy, brown rice, milled rice, greenish grain, yellow grain and denatured grain were measured over the range of 340 to 820 nm with "Double Beam Spectrophotometer(Model UV-180)". Variation in the reflectance depending on milling degree of milled rice appeared greatest over the range of 420 to 500 nm, and that between white rice and other rice samples appeared greatest near 500 nm. On the basis of the above results, a whiteness meter to measure milling degree of rice was manufactured using tungsten lamp, photodiodes and amplifier, and its performance was compared with the existing whiteness meters (KETT and Z-II optical sensor). There were very high correlations among those whiteness meters.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제7권4호
• /
• pp.212-215
• /
• 2002

Tyrosinase transcript In the blood Is known as the marker of malignant melanoma and it has been often determined by using reverse transcription-polymerase chain reaction (RT-PCA) . However, after the PCR process, the quantification of amplified CDMA by the gel electrophoresis is not reliable and time-consuming. for this reason, we tried to quantify the PCR product using a cuvette-type biosensor, where the oligonucleotide probe was immobilized on the cuvette surface and the single strand CDMA, the denatured PCH product, was then hybridized onto the immobilized probe to give a response signal. The response was Immediate and takes 15 min to obtain a stable signal. The biosensor was much more sensitive comparing to the gel electrophoresis method. The quantification of PCR product using a cuvette-type biosensor was feasible and rapid.

• Asian-Australasian Journal of Animal Sciences
• /
• 제23권9호
• /
• pp.1127-1136
• /
• 2010

Yogurt gels are a type of soft solid, and these networks are relatively dynamic systems that are prone to structural rearrangements. The physical properties of yogurt gels can be qualitatively explained using a model for casein interactions that emphasizes a balance between attractive (e.g., hydrophobic attractions, casein cross-links contributed by calcium phosphate nanoclusters and covalent disulfide cross-links between caseins and denatured whey proteins) and repulsive (e.g., electrostatic or charge repulsions, mostly negative at the start of fermentation) forces. Various methods are discussed to investigate the physical and structural attributes of yogurts. Various processing variables are discussed which influence the textural properties of yogurts, such as total solids content, heat treatment, and incubation temperatures. A better understanding of factors contributing to the physical and structural attributes may allow manufacturers to improve the quality of yogurt.

• Journal of Microbiology and Biotechnology
• /
• 제8권4호
• /
• pp.305-309
• /
• 1998

The superoxide dismutase (SOD) activity of seven Bifidobacterium spp. strains was examined by an indirect SOD assay method. Some Bifidobacterium spp. showed significant levels of SOD activity. However, we could not observe any significant differences between anaerobic and aerobic cultures. Furthermore, although several Bifidobacterium spp. exhibited some degree of tolerance to paraquat which produces superoxide radicals, the apparent SOD activity of these strains was not correlated with their resistance to paraquat. In addition, when we added increasing amounts of manganese or iron to MRS medium which had been prepared without either of the metal ions, the apparent SOD activity of cell free extracts (CFEs) was increased with increasing concentration of both metal ions. To our surprise, the heat-denatured CFEs also showed nearly identical correlative patterns. Based on these results, the apparent SOD activity was likely due to a nonenzymatic dismutation. These results strongly suggest that high concentration of divalent metal ions ($Mn^{2+}$, $Fe^{2+}$) in MRS medium result in nonenzymatic dismutation which can lead to false positive SOD activities in Bifidobacerium spp.