Catechol 처리에 의한 Pseudomonas sp. DJ-12의 생화학 및 세포학적 변화

고연자,임재윤,김치경,이기성 한국미생물학회 1999 미생물학회지 Vol.35 No.2

방향족 탄화수소 화학물질들은 자연계에 오염되면 미생물에 의한 분해가 미미하여 장기 축적됨으로써 생명체에 독성을 나타낸다. 이러한 방향족 탄화수소가 준치사 수준의 농도로 미생물에 노출되면 stress-shock 단백질을 합성하거나 세포 구성물질에 생화학적 변화가 일어나 적응현상이 나타나게 된다. 본 연구에서는 Pseudomonas sp. DJ-12를 여러 가지 농도의 catechol 로 처리했을 때 나타나는 stress-shock 단백질의 합성양상과 함께 세포의 형태와 생존을 위한 내성의 변화를 연구하였다. Pseudomonas sp. DJ-12는 0.5~1mM 의 catechol 이 6시간 배양 후 60% 이상이 분해되었으나, 3mM 도는 그 이상의 농도에서는 전혀 분해되지 않앗고 생존 세포수는 30시간 처리 했을 때부터 \10^2$ cell/ml 또는 그 이상이 사멸되었다. DnaK는 1mM 이상의 catechol 로 10분간 처리할 때 유도 생성되었고, GroEL은 0.5 mM 이상에서 생성되었다. 10mM 의 catechol로 처리한 Pseudomonas sp. DJ-12 의 세포는 세포벽에 구멍이 생겼으며 간균의 형태가 일그러지는 변화가 관찰되었다. 준치사 농도인 1mM의 catechol 이나 benzoate 또는 4-chlorobenzoate 로 전처리한 Pseudomonas sp. DJ-12는 stress-shock 단백질이 합성되었을 뿐 아니라, 치사 농도인 10mM 의 catechol에서 tolerance를 나타내었다. Aromatic hydrocarbons which are not easily degraded by microorganisms can be accumulated in the conlaminated environment for a long lime, producing toxic effects on wild lives and humans. However, the sublethal concentrations of the chemicals induce the synthesis of stress-shock proteins in the cells and increase the adaptability of the organisms to the environmental stresses. In this study, therefore, the cells of Psezido~nonus sp. DJ- 12 treated with catechol at various concentrations were inveshgated for their survival, biodegtadability of catechol, production of stress-shock proteins, and cytological changes. The organisms were capable of degrading catechol at the range of 0.5 to 1.0 mM concentration wilhin 6 hours incubation, but they were killed by $10^2$-10$^3$ celllinl at 3 mM or higel- concentration without any catechol degradation. These cells treated with catechol begm lo produce DnaK and GroEL at 1 mM and 0.5 mM. respectively. Pseudumonas sp. DJ-12 treated with 10 mM catechol for I hour exhihiled some punctuated pores on the cell wall and contortion of the rod shape. The cells treated with he sublethal concentration of catechol showed the increased tolerance for suvival when exposed to the lethal concentration, and such tolerant effects were functioned crossly among benzoate, 4-chlorobenzoate, 'and catechol.

바이러스성출혈성패혈증 바이러스 감염에 대한 3-Methyl Catechol의 항바이러스성 활성

조세영,민나래,김영오,김두운 한국수산과학회 2021 한국수산과학회지 Vol.54 No.5

Viral hemorrhagic septicemia virus (VHSV) is a fish pathogen responsible for causing enormous economic loss to the aquaculture industry not only in Korea but worldwide. Thus, it is necessary to identify natural compounds that can be used to control the spread of VHSV. In this study, the anti-VHSV activities of five catechol derivatives, i.e., catechol, pyrogallol, 3-methyl catechol, veratrole, and 3-methyl veratrole-extracted from green tea-were assessed. The antiviral activities of these derivatives were found to be dependent on their structure, i.e., the hydroxyl or methoxyl group and their substituent groups-on the benzene ring. Catechol, pyrogallol, and 3-methyl catechol exhibited relatively high 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities than veratrole, and 3-methyl veratrole. Moreover, 3-methyl catechol harboring a methyl substituent group increased the viability of the virus-infected cells and resulted in a 2.86 log reduction in the gene copies of VHSV N (per mL) in real-time PCR analysis. In conclusion, the catechol derivatives harboring hydroxyl groups in their benzene ring exhibited higher antioxidant activities than those harboring the methoxyl groups. However, catechol derivatives with a methyl group at the 3′-position of the benzene ring exhibited higher antiviral activity than those harboring a hydroxyl group. To our knowledge, this is the first study to evaluate the relationship between the structure and the anti-VHSV activity of catechol derivatives.

Bio-inspired adhesive catechol-conjugated chitosan for biomedical applications: A mini review

Ryu, J.H.,Hong, S.,Lee, H. Elsevier BV 2015 ACTA BIOMATERIALIA Vol.27 No.-

The development of adhesive materials, such as cyanoacrylate derivatives, fibrin glues, and gelatin-based adhesives, has been an emerging topic in biomaterial science because of the many uses of these materials, including in wound healing patches, tissue sealants, and hemostatic materials. However, most bio-adhesives exhibit poor adhesion to tissue and related surfaces due to the presence of body fluid. For a decade, studies have aimed at addressing this issue by developing wet-resistant adhesives. Mussels demonstrate robust wet-resistant adhesion despite the ceaseless waves at seashores, and mussel adhesive proteins play a key role in this adhesion. Adhesive proteins located at the distal end (i.e., those that directly contact surfaces) are composed of nearly 60% of amino acids called 3,4-dihydroxy-l-phenylalanine (DOPA), lysine, and histidine, which contain side chains of catechol, primary amines, and secondary amines, respectively. Inspired by the abundant catecholamine in mussel adhesive proteins, researchers have developed various types of polymeric mimics, such as polyethylenimine-catechol, chitosan-catechol, and other related catecholic polymers. Among them, chitosan-catechol is a promising adhesive polymer for biomedical applications. The conjugation of catechol onto chitosan dramatically increases its solubility from zero to nearly 60mg/mL (i.e., 6% w/v) in pH 7 aqueous solutions. The enhanced solubility maximizes the ability of catecholamine to behave similar to mussel adhesive proteins. Chitosan-catechol is biocompatible and exhibits excellent hemostatic ability and tissue adhesion, and thus, chitosan-catechol will be widely used in a variety of medical settings in the future. This review focuses on the various aspects of chitosan-catechol, including its (1) preparation methods, (2) physicochemical properties, and (3) current applications.

재조합균주 E. coli CNU312가 생산하는 Catechol 2,3-Dioxygenase의 정제 및 특성

임재윤,최경호,최병돈 한국미생물학회 2000 미생물학회지 Vol.36 No.1

Catechol 2,3-dioxygenase was purified from recombinant strain E. coli CNU312 carrying the tomB gene which was cloned from toluene-degrading Burkholderia cepacia G4. The purification of this enzyme was performed by acetone precipitation, Sephadex G-75 chromatography, electrophoresis and electro-elution. The molecular weight of native enzyme was about 140.4 kDa and its subunit was estimated to be 35 kDa by SDS-PAGE. It means that this enzyme consists of four identical subunits. This enzyme was specifically active to catechol, and$K_(m)$ value and $V_(max)$value of this enzyme were 372.6 $\mu$M and 39.27 U/mg. This enzyme was weakly active to 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol, but rarely active to 2,3-DHBP. The optimal pH and temperature of the enzyme were pH 8.0 and $40^{\circ}C$. The enzyme was inhibited by $Co^(2+)$, $Mn^(2+)$, $Zn^(2+)$, $Fe^(2+)$, $Fe^(3+)$, and $Cu^(2+)$ ions, and was inactivated by adding the reagents such as N-bromosuccinimide, and $\rho$-diazobenzene sulfonic acid. The activity of catechol 2,3-dioxygenase was not stabilized by 10% concentration of organic solvents such as acetone, ethanol, isopropyl alcohol, ethyl acetate, and acetic acid, and by reducing agents such as 2-mercaptoethanol, dithiothreitol, and ascorbic acid. The enzyme was inactivated by the oxidizing agent $H_(2)$$O_(2)$, and by chelators such as EDTA, and ο-phenanthroline. Toluene, phenyl 등의 분해균주인 Burkholderia cepacia G4로부터 tomB 유전자를 클로닝하여 얻은 재조합 균주 E. coli CNU312로부터 catechol 2,3-dioxygenase를 정제하여 효소학적 특성을 조사하였다. Catechol 2,3-dioxygenase는 native 분자량이 약 140.4 kDa이었으며 4개의 동일한 35 kDa subunit로 구성된 homotetramer로 생각된다. Catechol의 $K_(m)$값과 $V_(max)$값은 372.6 $\mu$M과 39.27 U/mg이었으며, 1.56 mM 이상의 기질 농도에서는 활성이 감소되었다. 효소 활성의 최적 pH는 8.0이었으며, pH 7.0-8.0 범위에서 안정하였다. 최적 활성온도는 $40^{\circ}C$였으며, $60^{\circ}C$이상에서 완전히 활성을 상실하였다. 또한 $Fe^(2+)$, $Fe^(3+)$ 를 비롯한 대부분의 금속 이온에 의해 활성이 감소되었으며, $Mg^(2+)$, $K^(+)$에는 영향을 받지 않았다. 효소 활성부위를 알아보기 위해 화학변형제를 처리한 결과, tryptophan과 histidine이 효소 활성부위에 존재하는 것으로 추정된다. 그리고 10%의 유기용매에 안정성을 보이지 않았으며, $H_(2)$$O_(2)$, EDTA, ο-phenanthroline에도 활성이 감소되었다. 또한 2-mercaptoethanol, dithiothreitol, 그리고 ascorbic acid와 같은 환원제에 대해서도 안정성을 보이지 않았다. 이 효소는 catechol에 대해 높은 기질 특이성을 보였으며, 3-methylcatechol, 4-methylcatechol, 그리고 4-chlorocatechol에 대해 약간의 활성을 보였다. 그러나 2,3-dihydroxybiphenyl에 대해서는 거의 활성을 보이지 않았다.

PEGylation and HAylation via catechol: α-Amine-specific reaction at N-terminus of peptides and proteins

Song, I.T.,Lee, M.,Lee, H.,Han, J.,Jang, J.H.,Lee, M.S.,Koh, G.Y.,Lee, H. Elsevier BV 2016 ACTA BIOMATERIALIA Vol.43 No.-

The development of chemoselective, site-specific chemistries for proteins/peptides is essential for biochemistry, pharmaceutical chemistry, and other fields. In this work, we found that catechol, which has been extensively utilized as an adhesive molecule for material-independent surface chemistry and as a crosslinker in hydrogel preparation, specifically reacts with N-terminal α-amines, avoiding the ε-amine group in lysine. A conjugate of methoxy-poly(ethylene glycol)-catechol called mPEG-cat chemoselectively reacts with N-terminal amine groups at neutral pH resulting in site-specific PEGylation. To demonstrate the versatility of this catechol chemoselective reaction, we used four proteins (lysozyme, basic-fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), insulin, and erythropoietin (EPO)) as well as two peptides (hinge-3 and laminin-derived peptide (LDP)). All the tested macromolecules showed N-terminal site-specific modifications. Furthermore, we prepared another catechol grafted conjugate called hyaluronic acid-catechol (HA-cat) to demonstrate that this catechol-involved chemoselective chemistry is not specific for PEG conjugates. This new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for a variety of purposes. Statement of Significance: Considering the fact that biological activities of proteins or peptides depend largely on their 3-dimensional conformation, the orientation-controllable reaction is very important for preserving the intrinsic functionality of them. In addition to PEG, many other bio-polymers such as oligonucleotides, antibodies, and oligosaccharides have been conjugated with proteins or peptides for various biomedical applications. Although several chemoselective conjugation chemistries have been reported, conjugation efficiencies are different depending on types of proteins or polymers, and thus there've been strong needs for the development of alternative strategy of chemoselective conjugation that can be applied for a variety of therapeutic proteins towards high biological activities. We are certain this new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for various purposes.

Characterization of an Catechol 2,3-dioxygenase from Pseudomonas putida SU10

Ha, You Mee,Min, Kyung Hee,Jung, Young Hee 숙명여자대학교 자연과학연구소 1998 자연과학논문집 Vol.- No.9

Pseudomonas putida SUl0의 4-methylcatechol 2,3-dioxygenase의 정제를 ammonium sulfate 침전, DEAE 5PW, superdex S-200, Resource-Q의 chromatog chromatography를 통하여 실시하였다. Gel filtration 에 의한 분자량을 약 130 kDa이며 SDS/polyacrylamide gel electrophoresis에 의한 subunit의 분자량은 34 kDa이므로 이 효소는 4개의 동일한 subunit로 되어 있음을 확이하였다. 이 효소의 N-terminal amino acid 서열을 결정한 결과 다음 extradiol dioxygenases와 높은 동질성을 보여주었다. Catechol과 methyl로 치환된 catechol에 대한 활성을 조사한 결과 catechol이나 3-methylcatechol보다 4-methylcatechol에 대하여 높게 나타내었다. 이들 기질에 대한 Km 값은 3.5-5.07μM 이다. A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU1O by a purification procedure consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicates a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit 34 kDa by SDS/ polyacrylamide gel electrophoresis. These results suggest that the native enzyme was composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identify with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. Km values for these substrates with this enzyme are around 3.5-5.7μM.

Biofunctionalization via flow shear stress resistant adhesive polysaccharide, hyaluronic acid-catechol, for enhanced in vitro endothelialization

Joo, H.,Byun, E.,Lee, M.,Hong, Y.,Lee, H.,Kim, P. Korean Society of Industrial and Engineering Chemi 2016 Journal of industrial and engineering chemistry Vol.34 No.-

<P>We report a single-step method for achieving in vitro endothelialization using a water-resistant adhesive polysaccharide, hyaluronic acid (HA). Introduction of adhesive properties is inspired by the catechol chemistry utilized in marine mussels, in which bioactive hyaluronic acid is chemically modified into hyaluronic acid-catechol (HA-catechol). Quantification of cell adhesiveness to HA-catechol coated substrate was performed with microfluidic device-based adhesion assay under shear forces. The HA-catechol exhibited enhanced ability of surface binding under flow shear force condition compared to unmodified HA. HA-catechol was able to retain the adhered endothelial cells under fluid shear stresses up to 150 dyn/cm(2), which is above the physiological condition in human vascular system. This is the first study demonstrating utility of the adhesive force of catechol-tethered polymers under continuous shear force environments such as microfluidic devices. The HA-catechol compound developed herein could provide a straightforward strategy for improving stability in surface retention and preventing substantial tissue damage from the fluctuating blood flow after vascular reconstructive surgery. (C) 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.</P>

Catechol grafted silica particles for enhanced adhesion to metal by coordinate bond

Lee, J.,Ko, J.,Ryu, J.,Shin, J.,Kim, H.,Sohn, D. Elsevier 2016 Colloids and surfaces. A, Physicochemical and engi Vol.511 No.-

Catechol-functionalized materials have been widely studied due to improved adhesion and metal-catechol coordination properties. In this study, silica particles, which are the base material in a multitude of applications, were modified with catechol groups based on a surface grafting reaction via amine- and carboxylic acid-functionalized silica particles. The chemical compositions of the modified particles were analyzed using Fourier transform infrared (FT-IR) and thermal gravimetric analysis (TGA). The surface properties of each particle were investigated using zeta potential, contact angle, and Brunauer-Emmett-Teller (BET) analysis. A series of characterizations verified successful modification with individual catecholic monomers of dopamine onto the silica surface. On the basis of the bioinspired surface modification, catechol-functionalized silica particles (SiO<SUB>2</SUB>?CA) exhibited outstanding adhesion properties with metals. The SiO<SUB>2</SUB>?CA showed enhanced Fe<SUP>3+</SUP>-capturing capacity that was five times that of bare silica particles. Furthermore, SiO<SUB>2</SUB>?CA exhibits outstanding adhesion on the TiO<SUB>2</SUB>-coated layer. The improved adhesion properties of SiO<SUB>2</SUB>?CA were due to high affinity and strong binding as a result of the metal-catechol coordination.

Effect of charge on in vivo adhesion stability of catechol-conjugated polysaccharides

홍상현,류지현,이해신 한국공업화학회 2019 Journal of Industrial and Engineering Chemistry Vol.79 No.-

Mussel-inspired adhesive materials have received much attention for biomedical applications primarilybecause of their underwater/tissue adhesive properties. However, so far, no comparative study focusingon effect of charge differences (i.e., positive vs. negative) in the presence of conjugated catechols has beenconducted. In this study, we investigate the physicochemical characteristics and in vitro/in vivo tissueadhesive properties of hydrogels that use anionic (i.e., hyaluronic acid) and cationic (i.e. chitosan)catechol-conjugated polysaccharides. Fe(III) is utilized to effectively prepare both hydrogels. Hyaluronicacid-catechol (HA-C) hydrogels are immediately formed by iron-catechol coordination bonds, whilechitosan-catechol (CHI-C) hydrogel is formed by both iron-catechol coordination and catechol-aminecovalent reactions. The gelation time and in vitro adhesiveness are similar for both CHI-C and HA-Chydrogels. Differences are found in that the CHI-C hydrogels showed enhanced water-resistance andlong-lasting adhesion in vivo. It is noteworthy to mention that the cationic nature of mussel adhesiveproteins due to lysine and histidine residues in the pads of Mytilus edulis is known to be an importantcontributor to underwater adhesion. Thus, this comparative study provides significant insight for thedesign of effective mussel-inspired adhesive materials.

Formation, Removal, and Reformation of Surface Coatings on Various Metal Oxide Surfaces Inspired by Mussel Adhesives

Kang, Taegon,Oh, Dongyeop X.,Heo, Jinhwa,Lee, Han-Koo,Choy, Seunghwan,Hawker, Craig J.,Hwang, Dong Soo American Chemical Society 2015 ACS APPLIED MATERIALS & INTERFACES Vol.7 No.44

<P>Mussels survive by strongly attaching to a variety of different surfaces, primarily subsurface rocks composed of metal oxides, through the formation of coordinative interactions driven by protein-based catechol repeating units contained within their adhesive secretions. From a chemistry perspective, catechols are known to form strong and reversible complexes with metal ions or metal oxides, with the binding affinity being dependent on the nature of the metal ion. As a result, catechol binding with metal oxides is reversible and can be broken in the presence of a free metal ion with a higher stability constant. It is proposed to exploit this competitive exchange in the design of a new strategy for the formation, removal, and reformation of surface coatings and self-assembled monolayers (SAM) based on catechols as the adhesive unit. In this study, catechol-functionalized tri(ethylene oxide) (TEO) was synthesized as a removable and recoverable self-assembled monolayer (SAM) for use on oxides surfaces. Attachment and detachment of these catechol derivatives on a variety of surfaces was shown to be reversible and controllable by exploiting the high stability constant of catechol to soluble metal ions, such as Fe(III). This tunable assembly based on catechol binding to metal oxides represents a new concept for reformable coatings with applications in fields ranging from friction/wettability control to biomolecular sensing and antifouling.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/aamick/2015/aamick.2015.7.issue-44/acsami.5b06910/production/images/medium/am-2015-06910j_0004.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/am5b06910'>ACS Electronic Supporting Info</A></P>