• Title/Summary/Keyword: thermal denaturation temperature

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Trypsins from the Dark Fleshed Fish(Anchovy, Mackerel, Yellowfin Tuna and Albacore) 2. Enzymatic Properties and Thermal Stabilities (혈합육어(멸치, 고등어, 활다랭이 및 날개다랭이)의 Trypsin 2. 성질과 열 안정성)

  • 조득문;허민수;변재형
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.22 no.4
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    • pp.458-464
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    • 1993
  • In the present paper enzymatic properties of the trypsins from the four dark fleshed fish were compared with each other and thermal stabilities of the enzymes were also investigated. The trypsins from the dark fleshed fish showed their activity only in BA-p-NA substrate of the amide substrates such as BA-p-NA and SP-p-NA, and BAEE and TAME of the ester substrate such as ATEE, BAEE, BTEE, and TAME. The enzymes were strongly inhibited by the serine protease inhibitors such as antipain, leupeptin, TLCK, DFP and SBTI, and were also inhibited by such metal ions as Cu$^{2+}$ and Hg$^{2+}$, but fairly activated by $Mg^{2+}$. Denaturation constants of the enzymes were 13.4$\times$10$^{-4}$ sec$^{-1}$ for anchovy trypsin, 47.18$\times$10$^{-4}$ sec$^{-1}$ for mackerel trypsin A, 34.06$\times$10$^{-4}$ sec$^{-1}$ mackerel trypsin B, 42.28$\times$10$^{-4}$ sec$^{-1}$ for yellowfin tuna trypsin and 16.6$\times$10$^{-4}$ sec$^{-1}$ for albacore trypsin at 55$^{\circ}C$. The activation energies of the trypsins at a temperature range of 3$0^{\circ}C$ to 5$0^{\circ}C$ were estimated to be 13.91 ㎉/mole for anchovy trypsin, 11.61㎉/mo1e and 8.43㎉/mole for mackerel trypsin A and for mackerel typsin B, 4.35㎉/mole for yellowfin tuna trypsin, and 3.76㎉/mole for albacore trypsin.

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Study of Interaction of Native DNA with Iron(III)-(2,4-Dihydroxysalophen)chloride (천연 DNA와 2,4-디히드록시살로펜-염화철(III)과 의 상호작용 연구)

  • Azani, Mohammad-Reza;Hassanpour, Azin;Bordbar, Abdol-Khalegh
    • Journal of the Korean Chemical Society
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    • v.54 no.5
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    • pp.573-578
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    • 2010
  • In this study, iron(III)-2,4-dihydroxysalophen chloride (Fe(2,4-DHSalophen)Cl), has been synthesized by combination of 2,4-dihydroxysalophen (2,4-DHSalophen) with $FeCl_2$ in a solvent system. This complex combination was characterized using UV-vis and IR spectroscopies. Subsequently, the interaction between native calf thymus deoxyribonucleic acid (ct-DNA) and Fe(2,4-DHSalophen)Cl, was investigated in 10 mM Tris/HCl buffer solution, pH 7.2, using UV-visible absorption and fluorescence spectroscopies, thermal denaturation technique and viscosity measurements. From spectrophotometric titration experiments, the binding constant of Fe(2,4-DHSalophen)Cl with ct-DNA was found to be $(1.6{\pm}0.2){\times}10^3\;M^{-1}$. The fluorescence study represents the quenching effect of Fe(2,4-DHSalophen)Cl on bound ethidium bromide to DNA. The quenching process obeys linear Stern-Volmer equation in extended range of Fe(2,4-DHSalophen)Cl concentration. Thermal denaturation experiments represent the increasing melting temperature of DNA (about $4.3^{\circ}C$) due to binding of Fe(2,4-DHSalophen)Cl. These results are consistent with a binding mode dominated by interactions with the groove of ct-DNA.

Influence of Thermal Oscillation on Quality of Frozen Foods Stored in Domestic Refrigerator (가정용 냉장고의 제상 주기와 온도 변화가 저장 식품의 품질에 미치는 영향)

  • Kang, Gil-Jin;Auh, Joong-Hyuck;Kim, Myo-Jeong;Cho, Kwang-Yeun;Choi, Young-Hoon;Jung, Dong-Sun;Kook, Seung-Uk;Park, Kwan-Hwa
    • Korean Journal of Food Science and Technology
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    • v.28 no.4
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    • pp.624-631
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    • 1996
  • Effect of fluctuation range and intervals of defrosting temperature on quality of frozen foods stored in a domestic refrigerator equipped with an automatic defrost system was evaluated. As defrost system was operated, temperatures of domestic refrigerators were elevated from $-18^{\circ}C\;to\;-5^{\circ}C\;and\;-15^{\circ}C$, and fluctuation intervals were l6 hrs and 30 hrs, respectively. Quality deterioration such as protein denaturation, vitamin loss, exudate production and changes in appearance of frozen foods was minimized by reducing temperature oscillation during storage. Considerable effects of thermal oscillation on ice crystal sizes were observed for frozen beef tissue and ice cream. TTI (time temperature indicator) system also proved that the temperature control of defrost system in domestic refrigerator can improve the quality of foods during storage.

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Temperature, organic solvent and pH stabilization of the neutral protease from Salinovibrio proteolyticus: significance of the structural calcium

  • Asghari, S. Mohsen;Khajeh, Khosro;Dalfard, Arastoo Badoei;Pazhang, Mohammad;Karbalaei-Heidari, Hamid Reza
    • BMB Reports
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    • v.44 no.10
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    • pp.665-668
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    • 2011
  • In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.

Microbead based micro total analysis system for Hepatitis C detection (마이크로비드를 이용한 초소형 C형 간염 검출 시스템의 제작)

  • Sim, Tae-Seok;Lee, Bo-Rahm;Lee, Sang-Myung;Kim, Min-Soo;Lee, Yoon-Sik;Kim, Byung-Gee;Kim, Yong-Kweon
    • Proceedings of the KIEE Conference
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    • 2006.07c
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    • pp.1629-1630
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    • 2006
  • This paper describes a micro total analysis system ($\mu$ TAS) for detecting and digesting the target protein which includes a bead based temperature controllable microchip and computer based controllers for temperature and valve actuation. We firstly combined the temperature control function with a bead based microchip and realized the on-chip sequential reactions using two kinds of beads. The PEG-grafted bead, on which RNA aptamer was immobilized, was used for capturing and releasing the target protein. The target protein can be chosen by the type of RNA aptamer. In this paper, we used the RNA aptamer of HCV replicase. The trypsin coated bead was used for digesting the released protein prior to the matrix assisted laser desorption ionization time of flight mass spectrometer (MALDI TOF MS). Heat is applied for release of the captured protein binding on the bead, thermal denaturation and trypsin digestion. PDMS microchannel and PDMS micro pneumatic valves were also combined for the small volume liquid handling. The entire procedures for the detection and the digestion of the target protein were successfully carried out on a microchip without any other chemical treatment or off-chip handling using $20\;{\mu}l$ protein mixture within 20 min. We could acquire six matched peaks (7% sequence coverage) of HCV replicase.

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Enzymatic Hydrolysis of Gelatin Layers of X-Ray Films and Release of Silver Particles Using Keratinolytic Serine Proteases from Purpureocillium lilacinum LPS # 876

  • Cavello, Ivana A.;Hours, Roque A.;Cavalitto, Sebastian F.
    • Journal of Microbiology and Biotechnology
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    • v.23 no.8
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    • pp.1133-1139
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    • 2013
  • Enzymatic decomposition of gelatin layers on used X-ray films and repeated utilization of the enzyme for potential application in silver recovery were investigated using keratinolytic serine proteases from Purpureocillium lilacinum LPS # 876. At pH 9.0, the enzymatic reaction was enhanced by the increase of enzyme concentration or by the increase of the temperature up to $60^{\circ}C$. Under the conditions of 6.9 U/ml, $60^{\circ}C$, and pH 9.0, hydrolysis of the gelatin layers and the resulting release of silver particles were achieved within 6 min. The protective effect of polyols against thermal denaturation was investigated. The presence of glycerol and propylene glycol increased enzyme stability. When the reusability of the enzyme for gelatin hydrolysis was tested, it could be seen that it could be effectively reused for more cycles when glycerol was added, compared with the enzyme without protective agents. The results of these repeated treatments suggested that a continuous process of recycling silver from used X-ray is feasible. Keeping in mind that recycling is (at the present time) needed and imperative, it can be remarked that, in this research, three wastes were successfully used: hair waste in order to produce serine proteases; glycerol in order to enhance enzyme thermal stability; and used X-ray films in order to recover silver and PET films.

Conformational Study of Human Serum Albumin in Pre-denaturation Temperatures by Differential Scanning Calorimetry, Circular Dichroism and UV Spectroscopy

  • Rezaei-Tavirani, Mostafa;Moghaddamnia, Seyed Hassan;Ranjbar, Bijan;Amani, Mojtaba;Marashi, Sayed-Amir
    • BMB Reports
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    • v.39 no.5
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    • pp.530-536
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    • 2006
  • Thermal conformational changes of human serum albumin (HSA) in phosphate buffer, 10 mM at pH = 7 are investigated using differential scanning calorimetric (DSC), circular dichroism (CD) and UV spectroscopic methods. The results indicate that temperature increment from $25^{\circ}C$ to $55^{\circ}C$ induces reversible conformational changes in the structure of HSA. Conformational change of HSA are shown to be a three-step process. Interestingly, melting temperature of the last domain is equal to the maximum value of fever in pathological conditions, i.e. $42^{\circ}C$. These conformational alterations are accompanied by a mild alteration of secondary structures. Study of HSA-SDS (sodium dodecyl sulphate) interaction at $45^{\circ}C$ and $35^{\circ}C$ reveals that SDS affects the HSA structure at least in three steps: the first two steps result in more stabilization and compactness of HSA structure, while the last one induces the unfolding of HSA. Since HSA has a more affinity for SDS at $45^{\circ}C$ compared to $35^{\circ}C$, It is suggested that the net negative charge of HSA is decreased in fever, which results in the decrease of HSA-associated cations and plasma osmolarity, and consequently, heat removal via the increase in urine volume.

Kinetics of Thermal Inactivation of Peroxidases and Polyphenol Oxidase in Pineapple (Ananas comosus)

  • Lee, Ting Hun;Chua, Lee Suan;Tan, Eddie Ti Tjih;Yeong, Christina;Lim, Chew Ching;Ooi, Siew Yin;Aziz, Ramlan bin Abdul;Aziz, Azila binti;Sarmidi, Mohd Roji bin
    • Food Science and Biotechnology
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    • v.18 no.3
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    • pp.661-666
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    • 2009
  • The heat tolerance and the inactivation kinetics of peroxidase (POD) and polyphenol oxidase (PPO) in pineapples (Ananas comosus) were studied in the temperature range $45-95^{\circ}C$. The kinetic parameters, such as deactivation rate constant (k), activation energy ($E_a$), and decimal reduction rate (D) of the thermal inactivation process, were determined. POD in pineapples showed biphasic inactivation behavior at temperatures range $45-75^{\circ}C$ but was monophasic at $85-95^{\circ}C$. This indicate that POD has 2 isozymes, namely heat labile and heat resistant, with $E_a$ of 68.79 and 93.23 kJ/mol, respectively. On the other hand, the heat denaturation of pineapple PPO could be described as simple monophasic first-order behavior with $E_a$ of 80.15 kJ/mol. Thus, the results of this study is useful in blanching technology where it shows a shortened time with higher temperature can be applied. The determination of the heat tolerance and inactivation POD and PPO, at different temperature range as done in the present work, was very important to improve the blanching process. This also will help to optimize the pineapple canning process which is one of the most important food industries in many tropical regions.

Immobilization of Fungal Laccase on Keratin-Coated Soil and Glass Matrices

  • Ginalska, G.;Lobarzewski, J.;Cho, Nam-Seok;Choi, T.H.;Ohga, S.;Jaszek, M.;Leonowicz, A.
    • Journal of the Korean Wood Science and Technology
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    • v.29 no.3
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    • pp.112-122
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    • 2001
  • Laccase enzymes from Cerrena unicolor and Trametes versicolor were immobilized on the activated glass beads (CPG), silica gel (SG) and soil (SL). The heterogeneous matrices were activated by ${\gamma}$-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA), and their surfaces were coated by keratin (KER) on activated or non-activated CPG, SG and SL. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. By keratin coating on supports, in the cases of CPG-KER and SL-KER, the immobilization yield was increased from about 80% to 90%. Moreover, much less protein was immobilized in keratin coated matrices than in inorganic ones alone (e.g. on CPG-KER 57.6%, whereas on CPG alone 80.6%). Laccase immobilization on keratin coated inorganic matrices was generally more effective than that of non-coated matrices. Concerned to pH dependency, the optima pH for immobilized laccases generally shifted towards to higher values, 5.5-5.8 and even 5.9 in the case of keratin for C. unicolor and from 5.3 to 5.7 for T. versicolor, respectively, and decreased less gradually both in acidic and alkaline regions. The immobilized laccase was more stable against thermal denaturation. This seems particularly true at $75^{\circ}C$ in the case of C. unicolor, where the activity of immobilized enzyme is > 50% higher than that of the free enzyme. For T. versicolor the respective values were $65^{\circ}C$, and 50%.

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Characterization of Acid- and Pepsin-soluble Collagens from Rockfish Sebastes schlegeli Skin

  • Kim, Hyung-Jun;Jee, Seong-Joon;Yoon, Min-Suck;Youn, Mu-Ho;Kang, Kyung-Tae;Lee, Dong-Ho;Heu, Min-Soo;Kim, Jin-Soo
    • Fisheries and Aquatic Sciences
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    • v.12 no.1
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    • pp.6-15
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    • 2009
  • Biochemical and functional properties of acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from rockfish skin were characterized. Yield of PSC (90.0%) was higher than that of ASC (63.2%). Both ASC and the PSC consisted of ${\alpha}1$ and ${\alpha}2$ chains, and $\alpha$-cross-linked components. According to the results of hydroxylation of proline and lysine, and FT-IR, no difference between the helical structure of ASC and PSC was identified. Thermal denaturation temperature (TDT) of ASC from rockfish skin was $22.8^{\circ}C$, the same as exhibited in PSC. Both ASC and PSC were higher in water absorption capacity (WAC) and oil absorption capacity (OAC) than other vegetable proteins. According to the results of emulsifying activity (EA) and cooking stability (CS), both ASC and PSC from rockfish skin were inferior compared to the commercial emulsifier (Tween-80). The results of FT-IR suggested that the structure of PSC was slightly different when compared to that of ASC. No differences in solubility were established between ASC and PSC from rockfish skin at various pH and NaCl concentrations.