• Title/Summary/Keyword: protein immobilization

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Nanoscale Fabrication of Biomolecular Layer and Its Application to Biodevices

  • Park, Jeong-Woo;Nam, Yun-Suk;Masamichi Fujihira
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.9 no.2
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    • pp.76-85
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    • 2004
  • Biodevices composed of biomolecular layer have been developed in various fields such as medical diagnosis, pharmaceutical screening, electronic device, photonic device, environmental pollution detection device, and etc. The biomolecules such as protein, DNA and pigment, and cells have been used to construct the biodevices such as biomolecular diode, biostorage device, bioelectroluminescence device, protein chip, DNA chip, and cell chip. Substantial interest has focused upon thin film fabrication or the formation of biomaterials mono- or multi-layers on the solid surfaces to construct the biodevices. Based on the development of nanotechnology, nanoscale fabrication technology for biofilm has been emerged and applied to biodevices due to the various advantages such as high density immobilization and orientation control of immoblized biomolecules. This review described the nanoscale fabrication of biomolecular film and its application to bioelectronic devices and biochips.

Effects of Different Types of Dietary Fat on Muscle Atrophy According to Muscle Fiber Types and PPAR${\delta}$ Expression in Hindlimb-Immobilized Rats (지방의 종류가 다른 식이의 섭취가 하지고정 흰 쥐의 근 섬유별 근 위축과 PPAR${\delta}$ 활성에 미치는 영향)

  • Lee, Ho-Uk;Park, Mi-Na;Lee, Yeon-Sook
    • Journal of Nutrition and Health
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    • v.44 no.5
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    • pp.355-365
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    • 2011
  • This study investigated how dietary fat affects muscle atrophy and lipid metabolism in various muscles during hindlimb immobilization in rats. Twenty-four male Sprague?Dawley rats had their left hindlimb immobilized and were divided into four groups by dietary fat content and composition. The contralateral hindlimb (control) was compared with the immobilized limb in all dietary groups. Rats (n = 6/group) were fed a 4% corn oil diet (CO), 2.6% corn oil + 1.4% fish oil diet (FO), 30% corn oil diet (HCO), or a 30% beef tallow diet (HBT)after their hind limbs were immobilized for 10 days. Data were collected for the gastrocnemius, plantaris and soleus muscles. Muscle atrophy was induced significantly after 10 days of hindlimb immobilization, resulting in significantly decreased muscle mass and total muscle protein content. The protein levels of peroxisome proliferator activated receptor ${\delta}$ (PPAR${\delta}$) in the plantaris, gastrocnemius, and soleus increased following hindlimb immobilization irrespective of dietary fat intake. Interestingly, the PPAR${\delta}$ mRNA level in the plantaris decreased significantly in all groups and that in the FO group was lower than that in the other groups. The soleus PPAR${\delta}$ mRNA level decreased significantly following hindlimb immobilization in the FO group only. Muscle carnitine palmitoyl transferase 1 (mCPT1) mRNA level was not affected by hindlimb immobilization. However, the mCPT1 mRNA level in the FO group was significantly lower in the plantaris but higher in the soleus than that in the other groups. The pyruvate dehydrogenase kinase 4 (PDK4) mRNA level in the plantaris decreased significantly, whereas that in the soleus increased significantly following hindlimb immobilization. The plantaris, but not soleus, PDK4 mRNA level was significantly higher in the FO group than that in the CO group. The increased PPAR${\delta}$ protein level following hindlimb immobilization may have suppressed triglyceride accumulation in muscles and different types of dietary fat may have differentially affected muscle atrophy according to muscle type. Our results suggest that ${\omega}$-3 polyunsaturated fatty acids may suppress muscle atrophy and lipid accumulation by positively affecting the expression level and activity of PPAR${\delta}$ and PPAR${\delta}$-related enzymes, which are supposed to play an important role in muscle lipid metabolism.

Electrostatic Immobilization of D-Xylose Isomerase to a Cation Exchanger for the Conversion of D-Xylose to D-Xylulose (D-xylose에서 D-xylulose로의 전환을 위한 D-xylose Isomerase의 정전기적 고정화)

  • Hang, Nguyen Thi;Kim, Sung-Gun;Kweon, Dae-Hyuk
    • Microbiology and Biotechnology Letters
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    • v.40 no.2
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    • pp.163-167
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    • 2012
  • Since D-xylose is not fermentable in Saccharomyces cerevisiae, its conversion to D-xylulose is required for its application in biotechnological industries using S. cerevisiae. In order to convert D-xylose to D-xylulose by way of an enzyme immobilized system, D-xylose isomerase (XI) of Escherichia coli was fused with 10-arginine tag (R10) at its C-terminus for the simple purification and immobilization process using a cation exchanger. The fusion protein XIR10 was overexpressed in recombinant E. coli and purified to a high purity by a single step of cation exchange chromatography. The purified XIR10 was immobilized to a cation exchanger via the electrostatic interaction with the C-terminal 10-arginine tag. Both the free and immobilized XIR10 exhibited similar XI activities at various pH values and temperatures, indicating that the immobilization to the cation exchanger has a small effect on the enzymatic function of XIR10. Under optimized conditions for the immobilized XIR10, D-xylose was isomerized to D-xylulose with a conversion yield of 25%. Therefore, the results of this study clearly demonstrate that the electrostatic immobilization of XIR10 via the interaction between the 10-arginine tag and a cation exchanger is an applicable form of the conversion of D-xylose to D-xylulose.

Immobilization of Glucose Oxidase using Branched Polyethyleneimines of Various Molecular Weights for Glucose Based Biofuel Cell (글루코스 기반 바이오연료전지를 위한 다양한 분자량의 폴리에틸렌이민을 이용한 글루코스 산화효소 고정화)

  • Ahn, Yeonjoo;Chung, Yongjin;Kwon, Yongchai
    • Korean Chemical Engineering Research
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    • v.54 no.5
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    • pp.693-697
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    • 2016
  • In this study, we fabricated the catalysts for enzymatic biofuel cell anode with carbon nanotube (CNT), glucose oxidase (GOx) and various molecular weights branched poly(ethyleneimine)(bPEI) and terephthalaldehyde (TPA) as cross-linker. In case of GOx/bPEI/CNT using only physical entrapments for immobilization, the molecular weights of bPEI didn't affect to electrochemical performances and long term stability. but that of the catalysts cross linked via TPA (TPA[GOx/bPEI/CNT]) improved and the mass transfer of glucose to FAD was interrupted as increasing of the bPEI's molecular weights. Furthermore, it was confirmed that the optimum molecular weight of PEI for TPA [GOx/bPEI/CNT]) structure is 750k that showed marvelous high performance (maximum power density of $0.995mW{\cdot}cm^{-2}$).

Comparative study on Hsp25 expression in Mongolian gerbil and mouse cerebellum

  • Lee, Heang-Yeon;Kim, Seong-Hwan;Lee, Jae-Bong;Shin, Chang-Ho
    • Korean Journal of Veterinary Service
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    • v.29 no.4
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    • pp.469-482
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    • 2006
  • The term 'heat shock protein (Hsps)' was derived from the fact that these proteins were initially discovered to be induced by hyperthermic conditions. In response to a range of stressful stimuli, including hyperthermia, immobilization, UV radiation, amino acid analogues, arsenite, various chemicals, and drugs the mammalian brain demonstrates a rapid and intense induction of the heat shock protein. Moreover, Hsps were expressed on the various pathological conditions including trauma, focal or global ischemia, hypoxia, infarction, infections, starvation, and anoxia. Especially, Hsp25 has a protective activity, facilitated by the ability of the protein to decrease the intracellular levels of reactive oxygen species (ROS) as well as its chaperone activity, which favors the degradation of oxidized proteins. Recently, it has clearly demonstrated that Hsp25 is constitutively expressed in the adult mouse cerebellum by parasagittal bands of purkinje cells in three distinct regions, the central zone (lobule VI-VII) and nodular zone (lobule IX-X), and paraflocculus. The Mongolian gerbil has been introduced into stroke study model because of its unique brain vasculature. There are no significant connections between the basilarvertebral system and the carotid system. This anatomy feature renders the mongolian gerbil susceptible to forebrain ischemia-induced seizure. The present study is designed to examine the pattern of Hsp25 expression in the cerebellum of this animal in comparison with that in mouse.

Stability Analysis of Bacillus stearothermopilus L1 Lipase Fused with a Cellulose-binding Domain

  • Hwang Sangpill;Ahn Ik-Sung
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.10 no.4
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    • pp.329-333
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    • 2005
  • This study was designed to investigate the stability of a lipase fused with a cellulose­binding domain (CBD) to cellulase. The fusion protein was derived from a gene cluster of a CBD fragment of a cellulase gene in Trichoderma hazianum and a lipase gene in Bacillus stearother­mophilus L1. Due to the CBD, this lipase can be immobilized to a cellulose material. Factors affecting the lipase stability were divided into the reaction-independent factors (RIF), and the re­action-dependent factors (RDF). RIF includes the reaction conditions such as pH and tempera­ture, whereas substrate limitation and product inhibition are examples of RDF. As pH 10 and $50^{\circ}C$ were found to be optimum reaction conditions for oil hydrolysis by this lipase, the stability of the free and the immobilized lipase was studied under these conditions. Avicel (microcrystal­line cellulose) was used as a support for lipase immobilization. The effects of both RIF and RDF on the enzyme activity were less for the immobilized lipase than for the free lipase. Due to the irreversible binding of CBD to Avicel and the high stability of the immobilized lipase, the enzyme activity after five times of use was over $70\%$ of the initial activity.

Immobilization and Characterization of a Liposome-Mediated Reconstituted Nicotinic Acetylcholine Receptor

  • Suh, Jeong-Ihn;Palk, Bo-Hyun;Oh, Se-Zu;Suh, Jung-Hun;Cho, Key-Seung;Palk, Young-Ki
    • BMB Reports
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    • v.28 no.2
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    • pp.155-161
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    • 1995
  • A nicotinic acetylcholine receptor (nAchR) isolated from the electric tissues of Torpedo californica has been reconstituted into a vesicle comprising a bifunctional azo-ligand (Bae 1) compound, and a liposome containing phospholipids and cholesterol (1 : 1, w/w). The liposome-mediated reconstituted receptor showed a concentration-dependent response to cholinergic drugs in a lithium ion flux assay. This liposome-mediated reconstituted nAchR was immobilized onto an electrode using various synthetic polymers which were tested for their response to the cholinergic ligands. The immobilized nAchR not only exhibited a linear response to a wide range of cholinergic ligand concentrations but also retained an operational stability which lasted for longer than 6 days. Thus, this result provides a basis for application of the immobilized nAchR-based biosensor in detecting cholinergic ligands in vitro.

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Characterization of Proteus vulgaris K80 Lipase Immobilized on Amine-Terminated Magnetic Microparticles

  • Natalia, Agnes;Kristiani, Lidya;Kim, Hyung Kwoun
    • Journal of Microbiology and Biotechnology
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    • v.24 no.10
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    • pp.1382-1388
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    • 2014
  • Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was $60^{\circ}C$, which was $20^{\circ}C$ higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to $40^{\circ}C$ and in a pH range of 5.0-10.0, whereas soluble K80 maintained its activity up to $35^{\circ}C$ and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

Performance Improvement of Glucose Sensor Adopting Enzymatic Catalyst bonded by Glutaraldehyde (글루타알데하이드에 의해 결합된 효소촉매를 이용한 글루코스 센서의 성능향상)

  • AHN, YEONJOO;CHUNG, YONGJIN;LEE, KYUBIN;KWON, YONGCHAI
    • Transactions of the Korean hydrogen and new energy society
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    • v.27 no.4
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    • pp.378-385
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    • 2016
  • In this study, we synthesized a biocatalyst consisting of glucose oxidase (GOx), polyethyleneimine (PEI) and carbon nanotube (CNT) with addition of glutaraldehyde (GA)(GA/[GOx/PEI/CNT])for fabrication of glucose sensor. Main bonding of the GA/[GOx/PEI/CNT] catalyst was formed by crosslinking of functional end groups between GOx/PEI and GA. Catalytic activity of GA/[GOx/PEI/CNT] was quantified by UV-Vis and electrochemical measurements. As a result of that, high immobilization ratio of 199% than other catalyst (with only physical adsorption) and large sensitivity value of $13.4{\mu}A/cm^2/mM$ was gained. With estimation of the biosensor stability, it was found that the GA/[GOx/PEI/CNT] kept about 88% of its initial activity even after three weeks. It shows GA minimized the loss of GOx and improved sensing ability and stability compared with that using other biocatalysts.