• 대한한방내과학회지
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• 제25권2호
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• pp.277-287
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• 2004

Objective : Diabetes is a disease in which the body does not produce or properly use insulin. Etiological studies of diabetes and its complications showed that oxidative stress might playa major role. Therefore, many efforts have been made to regulate oxygen free radicals for treating diabetes and its complications. Because Jindangwon has been known to be effective in treatment of diabetes, the methanol extract of Jindangwon was tested for its effectiveness in reducing the oxidative stress induced by Streptozotocin. Methods : Jindangwon was washed, dried in the shade and crushed. The crushed Jindangwon was extracted 3 times, each time with 3 volumes of methyl alcohol at $60^{\circ}C$ for 24 hours. The extract was filtered and evaporated under reduced pressure using a rotary evaporator to yield 30.6 g. Jindangwon extract was oral-administered to the diabetic rats induced by streptozotocin 50 mg per 1 kg of body weight for 15 days. The efficacy of the Jindangwon extract was examined with regard to the enzymatic pathways involved in the oxygen free radical production and the glutathione balance. Results : he effects of the methanol extract of Jindangwon in streptozotocin-induced diabetics rats with regard to body weight, blood glucose level, hepatic lipid peroxide level, hepatic xanthine oxidase activity and type conversion rate, hepatic glutathione level, hepatic glutathione peroxidase activity, hepatic glutathione reductase activity, hepatic aldose reductase activity, and hepatic sorbitol dehydrogenase activity were favorable enough to suggest that it is a cure for diabetes and its complications. Conclusions :These results support Jindangwon as an effective reducing agent for oxidative stress in the tissues and organs by regulating the production of oxygen free radicals. Jindangwon, in particular, shows promising results for its use as a cure, or preventative medicine for diabetes and its complications by reducing oxidative stress in beta-cells of the pancreas.

• 한국미생물·생명공학회지
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• 제20권6호
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• pp.681-686
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• 1992

D,L-ATC의 L-cysteine으로의 생물학적 전환에 있어 균체를 이용할 때, 계면활성제의 영향, 효소의 안정성 및 연속생산공정중의 고정화 균체 반응기의 안정성에 대하여 분석하였다. 계면활성제의 첨가없이 균체만을 이용할 때 반응은 매우 미미하게 이루어졌으나 SDS와 Triton X-100을 첨가할 때 cell-free 조효소액을 이용하는 경우와 비슷한 정도의 결과가 얻어졌다. 효소 활성은 $30^{\circ}C$에서 7시간 저장후 50 저하되었으며 질소가스하의 혐기적인 조건에서는 활성저하가 거의 일어나지 않았다. 이와 같은 효소의 불활성화는 효소에 대한 산소의 작용으로 사료되었다. 그러나, alginate로 고정화한 균체를 이용한 연속반응공정에서 혐기적으로 조건하에서 150시간 내에 대부분의 활성을 잃어버렸으며, L-cysteine 저해제인 hydroxylamine을 첨가할 때 효소활성이 급속히 감소되었다.

• Journal of Microbiology and Biotechnology
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• 제26권9호
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• pp.1586-1592
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• 2016

Klebsiella pneumoniae is a gram-negative, non-motile, rod-shaped, and encapsulated bacterium in the normal flora of the intestines, mouth, skin, and food, and has decarboxylation activity, which results in generation of diamines (cadaverine, agmatine, and putrescine). However, there is no specific information on the exact mechanism of decarboxylation in K. pnuemoniae. Specifically lysine decarboxylases that generate cadaverine with a wide range of applications has not been shown. Therefore, we performed a functional study of lysine decarboxylases. Enzymatic characteristics such as optimal pH, temperature, and substrates were examined by overexpressing and purifying CadA and LdcC. CadA and LdcC from K. pneumoniae had a preference for L-lysine, and an optimal reaction temperature of 37℃ and an optimal pH of 7. Although the activity of purified CadA from K. pneumoniae was lower than that of CadA from E. coli, the activity of K. pneumoniae CadA in whole cell bioconversion was comparable to that of E. coli CadA, resulting in 90% lysine conversion to cadaverine with pyridoxal 5'-phosphate L-lysine.

• Journal of Microbiology and Biotechnology
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• 제26권10호
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• pp.1781-1789
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• 2016

Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into Escherichia coli JM109. The transformed E. coli cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1244-1251
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• 2020

Phospholipase A₂ (PLA₂) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA₂ in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA₂ was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA₂ (P-PLA₂), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA₂. Finally, we observed that the extracellular PLA₂ from the recombinant E. coli P-PLA₂ culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA₂ expression system led to extracellular production of PLA₂ to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA₂.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.856-862
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• 2015

The objective of this study was to optimize the slurry contents and salt concentrations for ethanol production from hydrolysates of the seaweed Eucheuma spinosum. A monosaccharide concentration of 44.2 g/l as 49.6% conversion of total carbohydrate of 89.1 g/l was obtained from 120 g dw/l seaweed slurry. Monosaccharides from E. spinosum slurry were obtained by thermal acid hydrolysis and enzymatic hydrolysis. Addition of activated carbon at 2.5% (w/v) and the adsorption time of 2 min were used in subsequent adsorption treatments to prevent the inhibitory effect of HMF. The adsorption surface area of the activated carbon powder was 1,400-1,600 m²/g and showed selectivity to 5-hydroxymethyl furfural (HMF) from monosaccharides. Candida tropicalis KCTC 7212 was cultured in yeast extract, peptone, glucose, and high-salt medium, and exposed to 80, 90, 100, and 110 practical salinity unit (psu) salt concentrations in the lysates. The 100 psu salt concentration showed maximum cell growth and ethanol production. The ethanol fermentations with activated carbon treatment and use of C. tropicalis acclimated to a high salt concentration of 100 psu produced 17.9 g/l of ethanol with a yield (Y_EtOH) of 0.40 from E. spinosum seaweed.

• Journal of Microbiology and Biotechnology
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• 제23권10호
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• pp.1403-1412
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• 2013

Ethanol fuel production from lignocellulosic biomass is emerging as one of the most important technologies for sustainable development. To use this biomass, it is necessary to circumvent the physical and chemical barriers presented by the cohesive combination of the main biomass components, which hinders the hydrolysis of cellulose and hemicellulose into fermentable sugars. This study evaluated the hydrolytic capacity of enzymes produced by yeasts, isolated from the soils of the Brazilian Cerrado biome (savannah) and the Amazon region, on sugarcane bagasse pre-treated with $H_2SO_4$. Among the 103 and 214 yeast isolates from the Minas Gerais Cerrado and the Amazon regions, 18 (17.47%) and 11 (5.14%) isolates, respectively, were cellulase-producing. Cryptococcus laurentii was prevalent and produced significant ${\beta}$-glucosidase levels, which were higher than the endo- and exoglucanase activities. In natura sugarcane bagasse was pre-treated with 2% $H_2SO_4$ for 30 min at $150^{\circ}C$. Subsequently, the obtained fibrous residue was subjected to hydrolysis using the Cryptococcus laurentii yeast enzyme extract for 72 h. This enzyme extract promoted the conversion of approximately 32% of the cellulose, of which 2.4% was glucose, after the enzymatic hydrolysis reaction, suggesting that C. laurentii is a good ${\beta}$-glucosidase producer. The results presented in this study highlight the importance of isolating microbial strains that produce enzymes of biotechnological interest, given their extensive application in biofuel production.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1472-1482
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• 2017

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with BmCYP106A2 (Bacillus megaterium ATCC 13368). In this study, soluble BaCYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity ($K_d$ value) to 4-androstenedione was $387{\pm}37{\mu}M$. Moreover, the crystal structure of BaCYP106A2 was determined at $2.7{\AA}$ resolution. Structural analysis suggested that the ${\alpha}8-{\alpha}9$ loop region of BaCYP106A2 is intrinsically mobile and might be important for initial ligand binding. The hydroxyl activity of BaCYP106A2 was identified using in vitro enzyme assays. Its activity was confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using chromatography and mass spectrometry methods. The main products were mono-hydroxylated compounds with high conversion yields. This is the second study on the structure of CYP106A steroid hydroxylases, and should contribute new insight into the interactions of bacterial CYP106A with steroid substrates, providing baseline data for studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

• International Journal of Industrial Entomology and Biomaterials
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• 제7권1호
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• pp.37-44
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• 2003

Alcohol dehydrogenases (AHDs) are enzymes responsible for the catalysis of the reversible conversion of various alcohols to their corresponding aldehydes and ketonesis. Until now cDNA sequences of ADH gene is informed exclusively from several diptean species. We describe here the cDNA sequence and mRNA expression of a putative ADH gene from the mole cricket, Gryllotalpa orientalis, and phylogenetic relationships among known insect ADHs. The G. orientalis ADH cDNA sequences comprised of 798 bp encoding 266 amino acid residues. The multiple sequence alignment of G. orientalis ADH gene and known dipteran ADHs shared 100％ identity in the nine amino acid residues that are important for the enzymatic activity in Drosophila melanogaster. Percent sequence identity ranged from 25％ to 32％ among all insect ADHs including both types of ADHs. G. orientalis ADH gene showed no clear resemblance to any dipteran species and type. Phylogenetic analysis of the deduced amino acid sequences of G. orientalis ADH gene with available dipteran ADH genes including both types of ADHs further confirmed that the G. orientalis ADH gene is not clearly assigned to either type of ADHs. Northern blot analysis revealed a stronger signal in the fat body than midgut and epidermis, indicating that the fat body possibly is a main site for the synthesis of the G. orientalis ADH protein.

• 대한한의학회지
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• 제27권1호
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• pp.91-103
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• 2006

Objectives : Etiological studies of diabetes and its complications showed that oxidative stress might play a major role. Therefore, many effects have been fried to regulate oxygen free radicals for treating diabetes and its complications. Because Holotrichia has been known to be effective for the treatment of diabetes, the methanol extract of Holotrichia was tested for its effectiveness in reducing the oxidative stress induced by streptozotocin. Methods : Holotrichia was washed, dried in the shade and crushed. The crushed Holotrichia was extracted 3 times, each time with 3 volumes of methyl alcohol at $60^{\circ}C$ for 24h. The extract was filtered and evaporated under a reduced pressure using a rotary evaporator to yield 17 g. Holotrichia extract was oral-administed to the diabetic rats induced by streptozotocin 50 mg per 1 kg of body weight for 20 days. The efficacy of the Holotrichia extract was examined with regard to the enzymatic pathways involved in the oxygen free radical production and the glutathione balance. Results : The Effects of the methanol extract of Holotrichia in streptozotocin-induced diabolic rats with regards to body weight, blood glucose level, hepatic lipid peroxide level, hepatic superoxide anion radical content. hepatic xanthine oxidase activity and type conversion rate, hepatic glutathione level, hepatic aldose reductase activity, and hepatic sorbitol dehydrogenase activity were shown to be good enough to cure and prevent the diabetes and its complications. Conclusions : These results indicated that Holotrichia might reduce the oxidative stress in the tissues and organs by regulating the production of oxygen free radicals. Especially, Holotrichia might prevent and cure the diabetes and its complications by reducing the oxidative stress in the ${\beta}$-cells of pancreas. Some suggestions on biophoton experiments were made.