• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.445-448
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• 2002

The Pseudomonas fluorescens KCTC 1767, a selected and identified as potential candidate for stereo-specific resolution of rac-ketoprofen ethyl ester, was systematically investigated in order to induce the high level expression and detailed characterization of the expressing enzyme esterase. We cloned the esterase gene from chromosomal DNA of Pseudomonas fluorescens KCTC 1767 by PCR with two synthetic primers that desinged for simple purification. The recombinant esterase from Pseudomonas fluorescens KCTC 1767 exibited a high conversion rate and enantioselectivity to the (S)-ketoprofen ethyl ester as expected. The enzyme was easily purified to homogeniety by using a metal chelating affinity chromatography as a protein with poly histidine taq, and thus obtained 0.6 mg of protein from a 100 mL culture broth in a single step. The purified enzyme was steadily stable at the pH range from 7.0 to 10. The activity was also retained to be about 70% after the preincubation at $40^{\circ}C$ but over $50^{\circ}C$ lost the activity completely. The molecular mass of the esterase was estimated to be about 43 kDa on SDS-PAGE, and an identical result was also shown in gel filteration chromatography. The specific activity was calculated 27 mM/mg-protein/min by using the rac-ketoprofen ethly ester as a substrate.

• BMB Reports
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• v.39 no.1
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

• Journal of Microbiology and Biotechnology
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• v.28 no.4
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• pp.638-644
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• 2018

In this study, the immobilization of xylanase using a protein-inorganic hybrid nanoflower system was assessed to improve the enzyme properties. The synthesis of hybrid xylanase nanoflowers was very effective at $4^{\circ}C$ for 72 h, using 0.25 mg/ml protein, and efficient immobilization of xylanase was observed, with a maximum encapsulation yield and relative activity of 78.5% and 148%, respectively. Immobilized xylanase showed high residual activity at broad pH and temperature ranges. Using birchwood xylan as a substrate, the $V_{max}$ and $K_m$ values of xylanase nanoflowers were 1.60 mg/ml and $455{\mu}mol/min/mg$ protein, compared with 1.42 mg/ml and $300{\mu}mol/min/mg$ protein, respectively, for the free enzyme. After 5 and 10 cycles of reuse, the xylanase nanoflowers retained 87.5% and 75.8% residual activity, respectively. These results demonstrate that xylanase immobilization using a proteininorganic hybrid nanoflower system is an effective approach for its potential biotechnological applications.

• Molecules and Cells
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• v.21 no.1
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• pp.104-111
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• 2006

Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex virus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory bystander effect; (b) short-lived expression of the protein. To study the capacity of the protein transduction domain (PTD) of HIV-1 TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much of it is translocated to the nucleus. The transduced HepG2 cells are killed by exogenously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the introduced recombinant protein is stable and remains functional for several days at least, probably because nuclear localization protects it from the cytoplasmic degradation machinery and provides access to the nuclear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.

• International Journal of Industrial Entomology and Biomaterials
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• v.48 no.3
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• pp.107-113
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• 2024

The efficiency of protein extraction from Hongjam, a steamed mature silkworm, was quantitatively evaluated using various chemical buffers and physical methods. This study considers the difficulty of protein extraction yield due to the high content of hydrophobic amino acids in Hongjam compared to 5^th instar-3^rd day silkworm larvae. Results indicated that urea buffer enhanced protein yield more effectively than RIPA buffer. Additionally, the application of physical methods such as microwave treatment to samples treated with RIPA buffer increased yields by up to 22%, achieving concentrations as high as 3.9 mg/mL. Circular dichroism (CD) analysis showed that proteins extracted with urea buffer retained their structural integrity, exhibiting deeper and more prominent peaks associated with random coil structure. In addition, physical methods such as vortexing, sonication, microwave and homogenization increased the extraction yield of larger molecules without altering protein structures, suggesting their potential scalability for industrial applications. These results demonstrate the critical role of selecting appropriate extraction methods to optimize the yield and functionality of proteins from Hongjam, with implications for its use in biotechnological applications and nutraceuticals.

• Journal of Nutrition and Health
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• v.18 no.2
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• pp.139-146
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• 1985

This study was attempted to investigate the effect of fermentation of soybean on protein and mineral bioavailability. The traditional, oriental fermented soybean foods, Meju and Tempeh, and isolated soybean protein( ISP ) and cooked soybean were prepared and fed to rats as sources of protein. The C-PERs of ISP, cooked soybean, Meju and Tempeh were 1.82, 1.98, 2.11, and 2.36, respectively C-PERs of fermented soybean products, Meju and Tempeh were higher than ISP and cooked soybeans. However, they were not significantly different The percent retention of protein of rats fed with casein, ISP, cooked soybean, Meju and Tempeh were 47.44%, 51.83%, 47.67%, 50.90% and 45.97%, respectively, showing no significant differences among the diets. The rates of calcium retention percent were 62.26%, 59.22%, 61.59%, 55.78% and 67.09 % in rats fed with casein, ISP, cooked soybean, Meju and Tempeh, respectively, demonstrating no significant differences. Rats fed with cooked soybeans did show significantly higher iron retention rate than other samples. However, this study failed to any significant increase in iron availability. The percent of fine retained in rats fed with ISP was significantly lower than those of rats fed with other diets, however, significantly high amounts of zinc were retained in rats fed with Tempeh compared with other diets. The percents of zinc, iron, calcium retained in the bones of rats were not significantly different among the diets.

• Journal of Life Science
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• v.17 no.6 s.86
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• pp.847-858
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• 2007

The endoplasmic reticulum (ER) is an important intracellular organelle for folding and maturation of newly synthesized transmembrane and secretory proteins. The ER provides stringent quality control systems to ensure that only correctly folded proteins exit the ER and unfolded or misfolded proteins are retained and ultimately degraded. The ER has evolved stress response both signaling pathways the unfolded protein response (UPR) to cope with the accmulation of unfolded or misfolded proteins and ER overload response (EOR). Accumulating evidence suggests that, in addition to responsibility for protein processing, ER is also an important signaling compartment and a sensor of cellular stress. In this respect, production of bio-functional recombinant-proteins requires efficient functioning of the ER secretory pathway in host cells. This review briefly summarizes our understanding of the ER signaling developed in the recent years to help of the secretion capacities of recombinant cells.

• Animal Bioscience
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• v.34 no.4
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• pp.558-566
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• 2021

Objective: Objectives were to estimate energy and protein requirements of dairy crossbred steers, as well as to evaluate equations previously described in the literature (HH46 and CS16) to predict the carcass and empty body chemical composition of crossbred dairy cattle. Methods: Thirty-three Holstein×Zebu steers, aged 19±1 months old, with an initial shrunk body weight (BW) of 324±7.7 kg, were randomly divided into three groups: reference group (n = 5), maintenance level (1.17% BW; n = 4), and the remaining 24 steers were randomly allocated to 1 of 4 treatments. Treatments were: intake restricted to 85% of ad libitum feed intake for either 0, 28, 42, or 84 d of an 84-d finishing period. Results: The net energy and the metabolizable protein requirements for maintenance were 0.083 Mcal/EBW0.75/d and 4.40 g/EBW0.75, respectively. The net energy (NEG) and protein (NPG) requirements for growth can be estimated with the following equations: NEG (Mcal/kg EBG) = $0.2973_{({\pm}0.1212)}{\times}EBW^{0.4336_{({\pm}0.1002)}$ and NPG (g/d) = 183.6(±22.5333)×EBG-2.0693(±4.7254)×RE, where EBW, empty BW; EBG, empty body gain; and RE, retained energy. Crude protein (CP) and ether extract (EE) chemical contents in carcass, and all the chemical components in the empty body were precisely and accurately estimated by CS16 equations. However, water content in carcass was better predicted by HH46 equation. Conclusion: The equations proposed in this study can be used for estimating the energy and protein requirements of crossbred dairy steers. The CS16 equations were the best estimator for CP and EE chemical contents in carcass, and all chemical components in the empty body of crossbred dairy steers, whereas water in carcass was better estimated using the HH46 equations.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.12
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• pp.1681-1690
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• 2000

A study on energy and protein utilization, and milk production of Bali cows on grass-legume diets was carried out using 12 first lactation cows (initial BW $263.79{\pm}21.66kg$) during a period of 16 weeks starting immediately post calving. The animals were randomly allotted into 4 dietary treatment groups R1, R2, R3 and R4, receiving from the last 2 months of pregnancy onwards, graded improved rations based on a mixture of locally available grass and legume feed ad libitum. R1 contained on a DM basis 70% elephant grass (PP, Penisetum purpureum) plus 30% Gliricidia sepia leaves (GS), R2 was 30% PP plus 55% GS supplemented with 15% Hibiscus tilliactus leaves (HT, defaunating effect), R3 and R4 were 22.5% PP+41.25% GS+11.25% HT+25% concentrate, where R3 was not and R4 supplemented with zinc di-acetate. TDN, CP and zinc contents of the diets were 58.2%, 12.05% and 18.3 mg/kg respectively for R1, 65.05%, 16.9% and 25.6 mg/kg respectively for R2, 66.03%, 16.71% and 29.02 mg/kg respectively for R3 and 66.03%, 16.71% and 60.47 mg/kg respectively for R4. Milk production and body weight were monitored throughout the experimental period. In vivo body composition by the urea space technique validated by the body density method and supported by carcass data was estimated at the start and termination of the experiment. Nutrient balance and rumen performance characteristics were measured during a balance trial of 7 days during the 3rd and 4th week of the lactation period. Results indicated that quality of ration caused improvement of ruminal total VFA concentration, increments being 52 to 65% for R2, R3 and R4 above R1, with increments of acetate being less (31 to 48%) and propionate being proportionally more in comparison to total VFA increments. Similarly, ammonia concentrations increased to 5.24 to 7.07 mM, equivalent to 7.34 to 9.90 mg $NH_3-N/100ml$ rumen fluid. Results also indicated that feed quality did not affect DE and ME intakes, and heat production (HP), but increased GE, UE, energy in milk and total retained energy (RE total) in body tissues and milk. Intake-, digestible- and catabolized-protein, and retained-protein in body tissues and milk (Rprot) were all elevated increasing the quality of ration. Similar results were obtained for milk yield and components with mean values reaching 2.085 kg/d (R4) versus 0.92 kg/d (R1) for milk yield, and 170.22 g/d (R4) vs 71.69 g/d (R1), 105.74 g/d (R4) vs 45.35 g/d (R1), 101.34 g/d (R4) vs 46.36 g/d (R1) for milk-fat, -protein, and -lactose, respectively. Relatively high yields of milk production was maintained longer for R4 as compared to the other treatment groups. There were no significant effects on body mass and components due to lactation. From the relationship $RE_{total}$ (MJ/d)=12.79-0.373 ME (MJ/d); (r=0.73), it was found that $ME_{m}=0.53MJ/kgW^{0.75}.d$. Requirement of energy to support the production of milk, ranging from 0.5 to 3.0 kg/d, follows the equation: Milk Prod. ($Q_{mp}$, kg/d)=[-2.48+4.31 ME($MJ/kg^{0.75}.d$)]; (r=0.6) or $Q_{mp}$=-3.4+[0.08($ME-RE_{body\;tissue}$)]MJ/d]; (r=0.94). The requirement for protein intake for maintenance ($IP_m$) equals $6.19 g/kg^{0.75}.d$ derived from the relationship RP=-47.4+0.12 IP; (r=0.74, n=9). Equation for protein requirement for lactation is $Q_{nl}$=[($Q_{mp}$)(% protein in milk)($I_{mp}$)]/100, where $Q_{nl}$ is g protein required for lactation, $Q_{mp}$ is daily milk yield, Bali cow's milk-protein content av. 5.04%, and $I_{mp}$ is metabolic increment for milk production ($ME_{lakt}/ME_{m}=1.46$).

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.7
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• pp.948-956
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• 2000

The effect of high and low level of feed intakes on nutrient digestibility, nutrient losses through methane, energy and protein utilization by goats fed on alfalfa (Medicago sativa L.) pellets based diets was investigated in this study. Twelve castrated Japanese goats were employed in two subsequent digestion and metabolism trials. The goats were divided into three groups, offered three diets. Diet 1 consisted of 100% alfalfa pellet, Diet 2 was 70% alfalfa pellet and 30% corn, and Diet 3 was 40% alfalfa pellet and 60% corn. The two intake levels were high (1.6 times) and low (0.9 times) the maintenance requirement of total digestible nutrients (TON). Rumen ammonia nitrogen ($NH_3$-N) level of Diet 1 was lower (p<0.001) compared to Diets 2 and 3, but the values were always above the critical level (I50 mg/liter), The pH values of rumen liquor ranged from 6.02 to 7.30. Apparent digestibility of nutrient components did not show differences (p>0.05) between the two intake levels but inclusion of corn significantly altered the nutrient digestibility. Diet 3 had highest (p<0.001) dry matter (DM), organic matter (OM), ether extract (EE) and nitrogen fee extract (NFE) digestibility followed by the Diet 2 and Diet 1. The crude protein (CP) digestibility values among the three diets were in a narrow range (70.1 to 70.8%). Crude fiber (CF) digestibility for Diet 3 was slight higher (p>0.05) than that for other two diets. When alfalfa was replaced by corn, there were highly significant (p<0.001) increases in DM, OM, EE and NFE apparent digestibility and a slight increase in the CF digestibility (p>0.05). There were no differences (p>0.05) in energy losses as methane ($CH_4$) and heat production among the diets but energy loss through urine was higher for the Diet 1. The total energy loss as $CH_4$ and heat production were higher for the high intake level but the energy loss as $CH_4$ per gram DM intake were same (0.305 kcal/g) between the high and low intake level. Retained energy (RE) was higher for Diet 3 and Diet 2. Nitrogen (N) losses through feces and urine were higher (p<0.001) for Diet 1. Consequently, N retention was lower (p>0.05) for Diet 1 and higher in Diets 3 and 2. It is concluded that inclusion of corn with alfalfa increased the metabolizable energy (ME) and RE, and retained N through reducing the energy and N losses. The high level of intake reduced the rate of nutrient losses through feces and urine.