• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XII)
• /
• pp.34-38
• /
• 2003

For the production of $B^{30}-homoserine$ insulin analog as a novel anti-diabetic drug, the fermentative study was attempted for the maximal gene expression of HTS-fused $B^{30}-homoserine$ insulin precursor in the recombinant Escherichia coli cells. In a batch fermentation, the maximal production of insulin precursor as much as 38.95 mg/L-h, which occupied more than 12.8% of total cell protein. was achieved when the gene expression was induced by 0.5 mM IPTG at the middle logarithmic growth phase. The HTS-fused $B^{30}-homoserine$ insulin precursor was recovered from a batch culture through the processes of cell harvest, collection of insoluble fraction after sonication and purification by nickel affinity column chromatography. The isolated insulin precursor was 14 mg/L with a recovery yield of 35.9% of expressed gene product. The insulin A and B chain mixture was recovered after the insulin precursor was subjected to CNBr cleavage and purified by nickel affinity column chromatography. The isolated insulin chains were then sulfitolyzed with sodium thiosulfat and sodium tetrathionate, and reconstituted to insulin analog with ${\beta}-mercaptoethanol$, followed by purification with CM-Sepharose C-25 column chromatography.

• 한국미생물·생명공학회지
• /
• 제20권1호
• /
• pp.68-72
• /
• 1992

Aeromonas, salmonicida YA7-625가 생산하는 chitinase는 $Hg^{2+}$에 의해 저해를 받았으나, $\beta$-mercaptoethanol 처리에 의해서 활성이 보호되었으며 Na-thiosulfate, ascorbic acid등의 환원제들과 sodium azide, p-CMB 등의 -SH 저해제에대하여 전혀 작용을 받지 않았으므로 본 효소의 활성중심에는 -SH가 없는 것으로 생각되었다. 또한 histidine, cysteine, tyrosine 및 trytophan에 작용하는 iodine에 의하여 저해를 받았으며 tryptophan에 대해 특이적으로 작용하는 N-bromosuccinimide에 의해 실활되는 것으로 보아 활성중심에 tryptophan, histidine 및 tyrosine 가 존재하는 것으로 생각되었다.

• Journal of Microbiology and Biotechnology
• /
• 제14권5호
• /
• pp.938-943
• /
• 2004

A gene responsible for the chlorothalonil-biotransformation was cloned from the chromosomal DNA of Ochrobactrum anthropi SH35B, an isolated bacterium strain from soil. We determined the nucleotide sequences and found an open reading frame for glutathione S-transferase (GST). The drug-hypersensitive Escherichia coli KAM3 cells transformed with a plasmid carrying the GST gene can grow in the presence of chlorothalonil. The GST of O. anthropi SH35B was expressed in E. coli and purified by affinity chromatography. The fungicide chlorothalonil was rapidly transformed by the purified GST in the presence of glutathione. No significant difference in the chlorothalonil-biotransformation effect was observed among the thiol compounds (cysteine, reduced glutathione, and $\beta$-mercaptoethanol). Thus, the result reported here is the first evidence on the chlorothalonil-biotransformation by conjugation with the cellular free thiol groups, especially glutathione, catalyzed by the bacterial GST.

• 한국미생물·생명공학회지
• /
• 제17권5호
• /
• pp.489-493
• /
• 1989

쥐 하이브리도마 Alps 25-3를 이용하여 무혈청 배지 KM3의 각 성분이 세포증식과 항체생성에 미치는 영향을 조사하여 무혈청 배지를 최소화한 KMS를 결정하였다. Alps 25-3에 영향을 미치는 것으로 조사된 transferrin, ethanolamine 그리고 BSA만을 혼합하여 결정한 KMS를 다른 쥐 하이브리도마인 A4W, KW 그리고 HCGK에 적용시켰다. KM5를 KM3와 비교하였을 때 세포증식과 항체생산이 거의 동일한 양상을 보였다. 그러나 혈청배지와 비교하여 보면 최고 세포농도는 50-90%, 최종 단일클론항체의 농도는 85-100%의 수준을 보였다. 본 연구의 결과로 쥐 하이브리도마 세포를 무혈청 배지에서 배양시킬 때 필수적으로 첨가기어야 하는 성분들이 조사되었으므로 대량배양에 응용하거나 대사를 연구하는데 유용하게 이용되어지리라 본다.

• Food Science and Biotechnology
• /
• 제18권4호
• /
• pp.878-883
• /
• 2009

In Brazil canned 'Biuti' peach is a very popular form of this sub-tropical fruit. This production represents an important economic agro-activity in Minas Gerais, Brazil during the summer period, in preparation for the Christmas celebrations. The aim of this work was to characterize the 'Biuti' peach polyphenoloxidase (PPO), since peach products show enzymatic oxidation of the polyphenols by oxidative enzymes, which affects the products during their shelf life. Two different hypothesis for the browning problem in processed peaches were studied: the inadequacy of the blanching treatment and the presence of a latent phenolase in the peaches. The PPO was characterized: pH optimum (5.5) and stability (5.5-6.5); optimum temperature at $20^{\circ}C$ and 80% of the activity retained after 30 min at $15-40^{\circ}C$. The test for the presence of latent PPO in the processed and canned peaches was negative. Ascorbic acid, ${\beta}$-mercaptoethanol, sodium metabisulfite, and cysteine were efficient in inhibiting the PPO.

• Journal of Microbiology and Biotechnology
• /
• 제2권4호
• /
• pp.260-267
• /
• 1992

Pectate lyase from alkalitolerant Bacillus sp. YA-14 is an endo-type pectate lyase which acts randomly at the $\alpha$-1, 4-galacturonan linkage, and requires calcium or strontium ions for its activity. The enzyme is active on low methyl esterified pectin, but the activity toward a high methyl esterified substrate is reduced. The apparent Km's of the enzyme toward sodium polygalacturonic acid, polygalacturonic acid, and various pectins such as apple pectin, citrus pectin, and genu pectin are 0.826 mg/ml, 0.685 mg/ml, and 1.14 mg/ml, respectively. The enzyme activity is inhibited by SDS, urea, and sodium azide, but not by various reducing reagents, such as $\beta$-mercaptoethanol, Na-thiosulfate, Na-sulfate, cystein, and L-ascorbic acid. The enzyme is inactivated by N-bromosuccinimide, $I_2, H_2O_2$. PMSF, and iodoacetate. Judging from the results of their inhibition types, we speculate that tryptophan and serine residues are directly involved in enzyme activity, while tyrosine and methionine residues are indirectly involved in its activity.

• Preventive Nutrition and Food Science
• /
• 제7권4호
• /
• pp.437-441
• /
• 2002

Rice proteins were extracted from brown and milled rice of five varieties: Kwanganbyeo, Daeanbyeo, Daejinbyeo, Surabyeo, Hwaseongbyeo; and their electrophoretic patterns were analyzed by SDS-PAGE. Albumin was extracted with water, globulin with 5% NaCl, prolamin with 70% ethanol, and glutelin with 0.2 M sodium borate buffer (pH 10.0) containing 0.5% SDS, 0.6% $\beta$-mercaptoethanol. The ratios of albumin : globulin : prolamin : glutelin in the brown rice were 10.8~14.1 : 12.4~16.4 : 3.6~5.3 : 68.6~72.8, and in milled rice were 4.4~5.6 : 10.6~12.0 : 3.9~5.4 : 75.7~79.8. In albumin seven major bands were observed with molecular weights ranging from 14.g~96.8 kDa, in globulin four bands with molecular weights in the range of 14.4~56.9 kDa, prolamin had only one band with a molecular weight of 14.4 kDa, and glutelin had four bands with molecular weights of 14.4 ~ 57.4 kDa. There were no differences in electrophoretic patterns between rice varieties or between brown and milled rice.

• Bulletin of the Korean Chemical Society
• /
• 제35권12호
• /
• pp.3542-3546
• /
• 2014

Relationship between molecular freedom of amyloidogenic protein and its self-assembly into amyloid fibrils has been evaluated with ${\alpha}$-synuclein, an intrinsically unfolded protein related to Parkinson's disease, by restricting its structural plasticity through an end-to-end disulfide bond formation between two newly introduced cysteine residues on the N- and C-termini. Although the resulting circular form of ${\alpha}$-synuclein exhibited an impaired fibrillation propensity, the restriction did not completely block the protein's interactive core since co-incubation with wild-type ${\alpha}$-synuclein dramatically facilitated the fibrillation by producing distinctive forms of amyloid fibrils. The suppressed fibrillation propensity was instantly restored as the structural restriction was unleashed with ${\beta}$-mercaptoethanol. Conformational flexibility of the accreting amyloidogenic protein to pre-existing seeds has been demonstrated to be critical for fibrillar extension process by exerting structural adjustment to a complementary structure for the assembly.

• BMB Reports
• /
• 제31권1호
• /
• pp.20-24
• /
• 1998

Thioltransferase, also known as glutaredoxin, is an enzyme that catalyzes the reduction of a variety of disulfides, including protein disulfides, in the presence of reduced glutathione. Thioltransferase was purified from kale through ammonium sulfate fractionation, DE-52 ion-exchange chromatography, Sephadex G-75 gel filtration, and Q-Sepharose ion-exchange chromatography. Its molecular size was estimated to be about 31,000 daltons on SDS-PAGE. The purified enzyme has an optimum pH of about 8.0 with 2-hydroxyethyl disulfide as a substrate. The enzyme also utilizes L-sulfocysteine, L-cystine, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme has $K_m$ values of 0.24-0.67 mM for these substrates. The enzyme was partly inactivated after heating at $80^{\circ}C$ or higher temperature for 30 min. The enzyme was stimulated by various thiol compounds such as reduced glutathione, dithiothreitol, L-cysteine, and $\beta$-mercaptoethanol. This is a second example of a plant thioltransferase which was purified and characterized.

• Asian-Australasian Journal of Animal Sciences
• /
• 제14권2호
• /
• pp.170-173
• /
• 2001

The objective of this study was to investigate the effect of selenium (Se) on in vitro development of bovine embryos. In CR1BSA, FBS-free medium, the bovine embryos could not proceed past the developmental block more efficiently to morula stage than in chemically undefined media. Addition of glutathione precursor, cysteine, with $\beta$-mercaptoethanol did not improve the development in chemically defined medium and neither did glutathione alone. Exogenous selenium improved the embryonic development to the morula and blastocyst stages at 6 days post-insemination (dpi) significantly (67.1% vs 57.5%, p<0.05), and blastocyst stage at 8 dpi (30.1% vs 20.5%, p>0.05). These improvements might be induced by elevated glutathione peroxidase activity due to addition of Se, and a possible mechanism of selenium to elevate the activity of glutathione peroxidase is discussed.