Identification of the upstream 4-chlorophenol biodegradation pathway using a recombinant monooxygenase from <i>Arthrobacter chlorophenolicus</i> A6

Cho, Su Yeon,Kwean, Oh Sung,Yang, Jun Won,Cho, Wooyoun,Kwak, Seonyeong,Park, Sungyoon,Lim, Yejee,Kim, Han S. Elsevier 2017 Bioresource technology Vol.245 No.2

<P><B>Abstract</B></P> <P>This study aimed to clarify the initial 4-chlorophenol (4-CP) biodegradation pathway promoted by a two-component flavin-diffusible monooxygenase (TC-FDM) consisting of CphC-I and CphB contained in <I>Arthrobacter chlorophenolicus</I> A6 and the decomposition function of CphC-I. The TC-FDM genes were cloned from <I>A. chlorophenolicus</I> A6, and the corresponding enzymes were overexpressed. Since CphB was expressed in an insoluble form, Fre, a flavin reductase obtained from <I>Escherichia coli</I>, was used. These enzymes were purified using Ni<SUP>2+</SUP>-NTA resin. It was confirmed that TC-FDM catalyzes the oxidation of 4-CP and the sequential conversion of 4-CP to benzoquinone (BQN)→hydroquinone (HQN)→HQL. This indicated that CphC-I exhibits substrate specificity for 4-CP, BQN, and HQN. The activity of CphC-I for 4-CP was 63.22U/mg-protein, and the Michaelis-Menten kinetic parameters were <I>v<SUB>max</SUB> </I> =0.21mM/min, <I>K<SUB>M</SUB> </I> =0.19mM, and <I>k<SUB>cat</SUB> </I>/<I>K<SUB>M</SUB> </I> =0.04mM<SUP>−1</SUP> min<SUP>−1</SUP>. These results would be useful for the development of a novel biochemical treatment technology for 4-CP and phenolic hydrocarbons.</P> <P><B>Highlights</B></P> <P> <UL> <LI> TC-FDM catalyzes initial 4-chlorophenol biodegradation pathway. </LI> <LI> 4-CP is converted sequentially to BQN→HQN→HQL by CphC-I. </LI> <LI> The activity of CphC-I for 4-CP was 63.22U/mg-protein. </LI> <LI> <I>v<SUB>max</SUB> </I> =0.21mM/min, <I>K<SUB>M</SUB> </I> =0.19mM, and <I>k<SUB>cat</SUB> </I>/<I>K<SUB>M</SUB> </I> =0.04mM<SUP>−1</SUP> min<SUP>−1</SUP>. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

북태평양 조업선박의 운영 효율성 및 생산성 분석

조우연(Wooyoun Cho),조건식(Geonsik Jo),여기태(Gitae Yeo) 한국항만경제학회 2014 韓國港灣經濟學會誌 Vol.30 No.2

지구온난화와 더불어 조업이 가능한 수역이 넓어지고 있다. 특히 수온상승으로 풍부한 어족자원이 이동하고 있는 북극해의 경우 최근 관심이 높아지고 있다. 북태평양지역은 북극해에 가장 가깝고 진입하기 용이한 지역이다. 현재 북태평양에서 조업하고 있는 한국수산업은 미래 북극해 조업에 대비하여 효율성분석 등을 통하여 미래를 대비하여야 한다. 이러한 측면에서 본 연구는 자료포락분석(DEA)방법 및 Malmquist지수를 활용하여 북태평양 수역에 조업하는 선박 중 2009년부터 2013년까지 운항한 16척의 선박의 효율성을 분석하는 것을 연구의 목적으로 하였다. 효율성 분석을 위해 투입변수로는 선박톤수, 선박마력수, 조업일수를, 산출변수로는 연간어획량을 선정하였다. 측정결과에 의하면, 지난 5년간(2009년~2013년) 평균 효율성은 CCR효율성 0.8405, BCC효율성 0.9484, 규모효율성 0.8858로 나타나, 운영하고 있는 선박들은 전반적으로 선박톤수를 38%, 선박마력수를 36%, 조업일수를 29% 줄여야 현재보다 효율적인 운영이 가능할 것으로 나타났다. With the global warming phenomenon, the deep sea water area that fishing vessels can enter and operate is ever widening. For example, the Arctic Ocean recently has overall competitive advantages due to having many deep-sea fish stocks. The North Pacific region is a strategic coastal district, the closest access point of Arctic Ocean. For Korean fishing vessels which now operate in North Pacific region, and want to entry the Arctic Ocean, the analysis of technical efficiency is needed for preparing the better industry"s future. This paper aims to analyze the relative efficiency, and select the low effective deep-sea fishing vessels in the North Pacific, and to suggest their desirables strategies. As a research methodology, Data Envelopment Analysis (DEA) and Malmquist Index are applied to 16 fishing vessels for the periods(2009 to 2013). To draw out the efficiency of targeted deep-sea fishing vessels, gross tons, horsepowers, and operating days are used as input variables while total catch stands for an output variable. As a result, CCR efficiency, BCC efficiency and scalability efficiency are measured to be 0.8405, 0.9484 and 0.8858 respectively for 5 years (2009 to 2013). In conclusion, 38% of total tons, 36% of horsepowers and 29% of operating days each fishing vessel should be reduced to keep their competitive powers.

4-Chlorophenol biodegradation facilitator composed of recombinant multi-biocatalysts immobilized onto montmorillonite

Kwean, Oh Sung,Cho, Su Yeon,Yang, Jun Won,Cho, Wooyoun,Park, Sungyoon,Lim, Yejee,Shin, Min Chul,Kim, Han-Suk,Park, Joonhong,Kim, Han S. Elsevier 2018 Bioresource technology Vol.259 No.-

<P><B>Abstract</B></P> <P>A biodegradation facilitator which catalyzes the initial steps of 4-chlorophenol (4-CP) oxidation was prepared by immobilizing multiple enzymes (monooxygenase, CphC-I and dioxygenase, CphA-I) onto a natural inorganic support. The enzymes were obtained via overexpression and purification after cloning the corresponding genes (<I>cphC-I</I> and <I>cphA-I</I>) from <I>Arthrobacter chlorophenolicus</I> A6. Then, the recombinant CphC-I was immobilized onto fulvic acid-activated montmorillonite. The immobilization yield was 60%, and the high enzyme activity (82.6%) was retained after immobilization. Kinetic analysis indicated that the Michaelis-Menten model parameters for the immobilized CphC-I were similar to those for the free enzyme. The enzyme stability was markedly enhanced after immobilization. The immobilized enzyme exhibited a high level of activity even after repetitive use (84.7%) and powdering (65.8%). 4-CP was sequentially oxidized by a multiple enzyme complex, comprising the immobilized CphC-I and CphA-I, via the hydroquinone pathway: oxidative transformation of 4-CP to hydroxyquinol followed by ring fission of hydroxyquinol.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Enzymatic biodegradation facilitator was developed. </LI> <LI> Recombinant 4-CP mono- and di-oxygenases were immobilized together. </LI> <LI> Immobilization improved enzyme stability and handling efficiency. </LI> <LI> 4-CP is converted to hydroxyquinol and then ring-fissioned by the facilitator. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Oxidative biodegradation of 4-chlorophenol by using recombinant monooxygenase cloned and overexpressed from <i>Arthrobacter chlorophenolicus</i> A6

Kang, Christina,Yang, Jun Won,Cho, Wooyoun,Kwak, Seonyeong,Park, Sungyoon,Lim, Yejee,Choe, Jae Wan,Kim, Han S. Elsevier 2017 Bioresource technology Vol.240 No.-

<P><B>Abstract</B></P> <P>In this study, <I>cphC-I</I> and <I>cphB,</I> encoding a putative two-component flavin-diffusible monooxygenase (TC-FDM) complex, were cloned from <I>Arthrobacter chlorophenolicus</I> A6. The corresponding enzymes were overexpressed to assess the feasibility of their utilization for the oxidative decomposition of 4-chlorophenol (4-CP). Soluble CphC-I was produced at a high level (∼50%), and subsequently purified. Since CphB was expressed in an insoluble form, a flavin reductase, Fre, cloned from <I>Escherichia coli</I> was used as an alternative reductase. CphC-I utilized cofactor FADH<SUB>2</SUB>, which was reduced by Fre for the hydroxylation of 4-CP. This recombinant enzyme complex exhibited a higher specific activity for the oxidation of 4-CP (45.34U/mg-protein) than that exhibited by CphC-I contained in cells (0.18U/mg-protein). The Michaelis-Menten kinetic parameters were determined as: <I>v<SUB>max</SUB> </I> =223.3μM·min<SUP>−1</SUP>, <I>K<SUB>M</SUB> </I> =249.4μM, and <I>k<SUB>cat</SUB>/K<SUB>M</SUB> </I> =0.052min<SUP>−1</SUP>·μM<SUP>−1</SUP>. These results could be useful for the development of a new biochemical remediation technique based on enzymatic agents catalyzing the degradation of phenolic contaminants.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Arthrobacter chlorophenolicus</I> A6 possesses DNA genes for 4-chlorophenol degradation. </LI> <LI> Soluble form CphC-I was expressed, whereas CphB expressed in an insoluble form. </LI> <LI> CphC-I exhibited high specific activity when coupled with a flavin reductase, Fre. </LI> <LI> <I>v</I> <SUB>max</SUB> =223.3μM·min<SUP>−1</SUP>, <I>K<SUB>M</SUB> </I> =249.4μM, and <I>k<SUB>cat</SUB>/K<SUB>M</SUB> </I> =0.052min<SUP>−1</SUP>·μM<SUP>−1</SUP>. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>Color change from yellow to orange-red after 4-CP degradation by CphC-I. CphC-I concentration from 0.1 to 25μM from left to right.</P> <P>[DISPLAY OMISSION]</P>

Remediation of heavy metal-contaminated soils using eco-friendly nano-scale chelators

Lim, Heejun,Park, Sungyoon,Yang, Jun Won,Cho, Wooyoun,Lim, Yejee,Park, Young Goo,Kwon, Dohyeong,Kim, Han S. Techno-Press 2018 Membrane water treatment Vol.9 No.3

Soil washing is one of the most frequently used remediation technologies for heavy metal-contaminated soils. Inorganic and organic acids and chelating agents that can enhance the removal of heavy metals from contaminated soils have been employed as soil washing agents. However, the toxicity, low removal efficiency and high cost of these chemicals limit their use. Given that humic substance (HS) can effectively chelate heavy metals, the development of an eco-friendly, performance-efficient and cost-effective soil washing agent using a nano-scale chelator composed of HS was examined in this study. Copper (Cu) and lead (Pb) were selected as target heavy metals. In soil washing experiments, HS concentration, pH, soil:washing solution ratio and extraction time were evaluated with regard to washing efficiency and the chelation effect. The highest removal rates by soil washing (69% for Cu and 56% for Pb) were achieved at an HS concentration of 1,000 mg/L and soil:washing solution ratio of 1:25. Washing with HS was found to be effective when the pH value was higher than 8, which can be attributed to the increased chelation effect between HS and heavy metals at the high pH range. In contrast, the washing efficiency decreased markedly in the low pH range due to HS precipitation. The chelation capacities for Cu and Pb in the aqueous phase were determined to be 0.547mmol-Cu/g-HS and 0.192mmol-Pb/g-HS, respectively.

Immobilization of enzymes onto clay minerals for the biochemical decomposition of 4-chlorophenol

( Oh Oh Sung Kwean ),( Su Yeon Cho ),( Jun Won Yang ),( Wooyoun Cho ),( Seonyeong Kwak ),( Sungyoon Park ),( Yejee Lim ),( Han S. Kima ) 한국물환경학회 2017 한국물환경학회·대한상하수도학회 공동 춘계학술발표회 Vol.2017 No.-

In this study an oxidative enzyme was immobilized onto inorganic backbone materials to stimulate the detoxification of toxic aromatic hydrocarbon compounds. Smectite clay minerals and soil organic matter were screened as an enzyme support and a binding agent, respectively. Montmorillonite of which inner pores are layered with nano-scale spacing planar was activated by humic acid. A dioxygenase obtained by cloning of its corresponding gene from Arthrobacter chlorophenolicus A6 was immobilized onto the humic acid-activated montmorillonite. Oxygenated metabolites such as catechol and 4-chlorocatechol were selected as target aromatic contaminants (primary substrates of enzyme). The enzyme immobilization yield was as high as 63% and the reductions in enzyme activity for the decomposition of substrate compounds during enzyme immobilization were minimal: 15% for catechol and 24% for 4-chlorocatechol, respectively. The kinetic analysis of the free and immobilized enzymes demonstrated a slight decrease of vmax and a marginal increase of KM as compared with those for the free enzyme, indicating the changes in enzyme activity perhaps due to the changes in enzyme conformation associated with its immobilization were minimal. The results for the effects of environmental factors including pH, temperature, and ionic strength on the activity of free and immobilized enzymes showed that the activity of free enzyme changed significantly in response to the changes of the environmental factors whereas that of immobilized enzyme was pretty much consistent. This indicated that the stability of enzyme against the abrupt changes in environmental factors can be greatly improved by enzyme immobilization. The results of this study support the feasibility of a new environmental fusion technology based on bio-technology and nano-technology for the development of biochemical treatment processes.

톨루엔 산화 및 탈수소효소의 복제, 발현, 정제와 효소기능분석

양준원 ( Jun Won Yang ),조우연 ( Wooyoun Cho ),임예지 ( Yejee Lim ),박성윤 ( Soungyoon Park ),김한승 ( Han S. Kim ) 한국물환경학회(구 한국수질보전학회) 2018 한국물환경학회·대한상하수도학회 공동 춘계학술발표회 Vol.2018 No.-