아교버섯(Phlebia tremellosa)의 리그닌 분해효소 형질전환체를 이용한 염료의 탈색

금현우,이성숙,유선화,최형태,Kum, Hyun-Woo,Ryu, Sun-Hwa,Lee, Sung-Suk,Choi, Hyoung-T. 한국미생물학회 2010 미생물학회지 Vol.46 No.1

리그닌 분해효소 군을 가진 백색부후균들은 다핵방향족 화합물을 포함하여 염료와 폭약 및 내분비장애 물질의 분해 등 다양한 난분해성 물질을 분해할 수 있다. 리그닌 분해효소 중 laccase와 manganese peroxidase (MnP)를 각각 발현하는 벡터를 국내에서 분리한 백색부후균류의 하나인 아교버섯(Phlebia tremellosa)에 형질전환 방법으로 도입시킨 형질전환체를 사용하여 염료의 탈색능력을 분석하였다. Methyl green의 경우 3일 후 약 50%의 탈색을 보인 야생형에 비하여 laccase 형질전환체(TF2-1)와 MnP 형질전환체(T5) 모두 90% 이상의 탈색을 보였다. Remazol brilliant blue R (RBBR)에서는 야생형이 약 67%의 탈색을 보인 반면 두 가지 형질전환체들은 약 85%의 탈색을 보였다. 각각의 형질전환체들은 laccase와 MnP의 활성 및 유전자 발현도 활발한 것으로 확인되었다. White rot fungi which have lignin degrading enzymes show high degrading activity to diverse recalcitrant compounds such as polycyclic aromatic compounds, dyes, explosives and endocrine disrupting chemicals. We have examined decolorizing activity of dyes by Phlebia tremellosa and two transformants which had genetically transformed using laccase or manganese peroxidase (MnP) gene. In case of methyl green, wild type strain showed 50% decolorization while laccase transformant (TF2-1) and MnP transformant (T5) showed more than 90% decolorization on day 3. Remazol brilliant blue R(RBBR) was decolorized up to 85% by two transformants while the wild type showed 67% decolorization on day 3. Transformants TF2-1 and T5 both showed increased laccase and MnP activity respectively during the whole growing phase.

구름버섯 망간 과산화효소를 도입한 아교버섯 형질전환체에 의한 내분비장애 물질의 생분해

금현우,김명길,최형태,Kum, Hyun-Woo,Kim, Myung-Kil,Choi, Hyoung-T. 한국미생물학회 2009 미생물학회지 Vol.45 No.1

내분비장애물질은 분해가 매우 어려워 자연계에서 먹이그물을 통하여 사람에게 농축 전달된다. 이들은 정상적인 내분비계에 혼란을 일으키며, 특히 성호르몬의 작용에 많은 피해를 준다. 이를 효율적으로 분해하고 이들의 에스트로겐 활성을 제거하고자 백색부후균의 하나인 아교버섯(Phlebia tremellosa)을 활용하여 4가지 내분비계 장애물질의 분해에 대한 실험을 수행하였다. 아교버섯의 manganese peroxidase (MnP) 활성을 높이기 위하여 구름버섯의 MnP 유전자를 아교버섯에 도입하여 형질전환체를 확보하였으며 이들은 유전적으로 MnP 활성을 안정되게 나타냈다. 내분비 장애물질을 분해하는 조건에서 내분비장애 물질에 따라 30${\sim}$45%의 분해율을 보인 야생형에 비하여 이 형질전환체들 중 T5는 70${\sim}$88%의 분해율을 보였으며 에스트로겐 활성의 제거에도 약 2배 향상된 능력을 보였다. Endocrine disrupting chemicals (EDCs) disturb animal hormonal system even at very low concentrations, and finally give harmful effects to human through the food web. A white rot fungus Phlebia tremellosa isolated in Korea, was reported to have good degrading activity against the endocrine disrupting phthalates. However, this fungus has very low manganese peroxidase (MnP) activity under various culture conditions while laccase and lignin peroxidase activities were high. We have isolated an MnP cDNA from Trametes versicolor which was involved in the degradation of EDCs, and constructed an MnP expression vector to use in the genetic transformation of P. tremellosa in order to get higher MnP producing strains. Many transformants had integrated expression vector in their chromosomal DNAs, and showed increased MnP activity. One of two transformants showed increased degradation of 4 EDCs (70${\sim}$88%) than the wild type (30${\sim}$45% degradation rates), and showed twice better removal of estrogenic activities generated by the EDCs than the wild type.

Generation of a Transformant Showing Higher Manganese Peroxidase (Mnp) Activity by Overexpression of Mnp Gene in Trametes versicolor

Yeo, Su-Min,Park, Nam-Mee,Song, Hong-Gyu,Choi, Hyoung-T. The Microbiological Society of Korea 2007 The journal of microbiology Vol.45 No.3

Trametes versicolor has a lignin degrading enzyme system, which is also involved in the degradation of diverse recalcitrant compounds. Manganese-dependent peroxidase (MnP) is one of the lignin degrading enzymes in T. versicolor. In this study, a cDNA clone of a putative MnP-coding gene was cloned and transferred into an expression vector (pBARGPE1) carrying a phosphinothricin resistance gene (bar) as a selectable marker to yield the expression vector, pBARTvMnP2. Transformants were generated through genetic transformation using pBARTvMnP2. The genomic integration of the MnP clone was confirmed by PCR with bar-specific primers. One transformant showed higher enzyme activity than the recipient strain did, and was genetically stable even after 10 consecutive transfers on non-selective medium.

The HA-incorporated nanostructure of a peptide–drug amphiphile for targeted anticancer drug delivery

Choi, Huyeon,Jeena, M. T.,Palanikumar, L.,Jeong, Yoojeong,Park, Sooham,Lee, Eunji,Ryu, Ja-Hyoung The Royal Society of Chemistry 2016 Chemical communications Vol.52 No.32

<P>A simple peptide based prodrug of camptothecin (CPT) has been synthesised in which the CPT is conjugated to a tripeptide (KCK) via a disulfide linkage (KCK-CPT) and self-assembled into well-defined nanostructures in water depending on the concentration. The hyaluronic acid (HA) complex of KCK-CPT exhibited target specific toxicity with excellent antitumour efficiency.</P>

Intra-mitochondrial assembly of peptide amphiphiles for cancer therapy

M T Jeena,L. Palanikumar,Sooham Park,Huyeon Choi,Eun Min Go,Hye-kyeong Lee,Lee Seon Ik,In-Hae Kim,Chaekyu Kim,Sung Cheol Bae,Hyun Woo Rhee,Eunji Lee,Sang-Kyu Kwak,Ja Hyoung Ryu 한국고분자학회 2016 한국고분자학회 학술대회 연구논문 초록집 Vol.41 No.2

먹물버섯의 키틴질 분해효소에 의한 식물병원균류의 생장억제

강유리,최형태,Kang, Yuri,Choi, Hyoung T. 한국미생물학회 2012 미생물학회지 Vol.48 No.4

먹물버섯 C. congregatus의 자가분해되는 자실체 조직에서 발현되는 chitinase (Chi2)가 식물병원성 균류인 A. alternata와 F. graminearum를 대상으로 고체배지와 액체배지에서 포자의 발아 및 균사 생장에 대하여 보이는 억제효과 실험을 수행하였다. 액체배지에서 $50{\mu}g/ml$의 Chi2 농도에서 A. alternata의 포자 발아가 90% 이상 억제되었고, $70{\mu}g/ml$의 농도에서 실험균류 모두 생장이 억제되었다. 고체배지에서 하루에 $6{\mu}g$씩 2회 더한 Chi2에 의하여 F. graminearum 균사 생장이 억제됨을 확인하였고, 이때 균사의 branching이 상대적으로 감소하였다. Experiments for growth inhibition of two different plant pathogenic fungal species, Alternaria alternata (KCTC26781) and Fusarium graminearum ZO3639 (type culture) and ASR1R1 (isolate from rice), by a chitinase (Chi2) expressed in the autolysing tissue of Coprinellus congregatus were carried out. In liquid media, Chi2 ($50{\mu}g/ml$) inhibited more than 90% of germination of A. alternata spore, and the growth of each fungal strain was totally inhibited by the addition of Chi2 at the concentrations of $70{\mu}g/ml$. When $6{\mu}g$ of Chi2 was added twice a day at the hyphal tip zone, both strains of F. graminearum showed growth inhibition as well as decreased hyphal branching.

Intra-mitochondrial biomineralization for inducing apoptosis of cancer cells

Kim, Sangpil,Palanikumar, L.,Choi, Huyeon,Jeena, M. T.,Kim, Chaekyu,Ryu, Ja-Hyoung Royal Society of Chemistry 2018 Chemical Science Vol.9 No.9

<▼1><P>Mitochondria targeting mineralization can form biominerals inside cancerous mitochondria through concentration dependent silicification, resulting in dysfunction of mitochondria leading to apoptosis. These results suggest potential therapeutics for cancer treatment.</P></▼1><▼2><P>The use of biomineralization that regulates cellular functions has emerged as a potential therapeutic tool. However, the lack of selectivity still limits its therapeutic efficacy. Here, we report a subcellular-targeting biomineralization system featuring a triphenylphosphonium cation (TPP) (the mitochondria-targeting moiety) and trialkoxysilane (the biomineralization moiety <I>via</I> silicification). The TPP-containing trialkoxysilane exhibited approximately seven times greater cellular uptake into cancer cells (SCC7) than into normal cells (HEK293T) due to the more negative mitochondrial membrane potentials of the cancer cells. In turn, its accumulation inside mitochondria (pH 8) induces specific silicification, leading to the formation of silica particles in the mitochondrial matrix and further activation of apoptosis. <I>In vivo</I> assessment confirmed that the biomineralization system efficiently inhibits tumor growth in a mouse xenograft cancer model. Exploiting both the subcellular specificity and the targeting strategy provides new insight into the use of intracellular biomineralization for targeted cancer therapy.</P></▼2>

A numerical experiment on the stability and convergence characteristics of some splitting mixed-finite element methods for solving the incompressible Navier-Stokes equations

Myung H. Cho,Sang T. Ha,Jung Y. Yoo,Hyoung Gwon Choi 대한기계학회 2023 JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY Vol.37 No.9

In the present study, a fractional three-step P2P1 finite element method (FEM) for solving the unsteady incompressible Navier-Stokes equations, which is a variation of P1P1 four-step splitting FEM [1], was compared with conventional one-step time-integration schemes in terms of the CPU time and convergence characteristics of an iterative solver by the solution of some benchmark problems. One-step time-integration schemes were temporarily discretized by either the Crank-Nicolson or the Adams-Bashforth method. Fractional three-step P2P1 FEM consists of three steps: a non-linear momentum equation with the pressure in the previous time step is solved to obtain an intermediate velocity field by the Crank-Nicolson method in the first step and another intermediate velocity field is calculated using the pressure in the previous time step in the second step, and a divergence-free constraint is imposed in the last step to update the pressure field, in which a symmetric saddle-point type matrix (SPTM) is solved. It was shown that the fractional three-step method is more efficient than one-step time-integration schemes because a symmetric SPTM is assembled only once during the entire computation and solved once at each time-step; further, the cost of solving the nonlinear momentum equation in a fully-implicit manner is relatively low. Furthermore, the proposed method was found to be more stable than one-step time-integration schemes as it provided stable solutions at higher CFL numbers.

먹물버섯 키틴질 분해효소에 의한 인체유해성 Cryptococcus neoformans 세포벽 생성억제 및 Alternaria alternata에 기인한 생쥐의 피부알레르기의 감소

강유리,최형태,Kang, Yuri,Choi, Hyoung T. 한국미생물학회 2016 미생물학회지 Vol.52 No.2

먹물버섯 Coprinellus congregatus의 버섯조직이 자가분해되는 시기에 발현되는 chitinase (Chi2)는 사람에게 질병을 일으키는 Cryptococcus neoformans의 세포벽 두께를 정상 세포벽의 32% 수준까지 감소시킬 뿐만 아니라, 세포 외 섬유상 물질도 제거하였다. Chi2 처리에 의하여 사람에게 알레르기를 유발하는 Alternaria alternata의 생장은 물론 생쥐 모델에서 이 균에 의한 피부알레르기 증상도 감소시켰다. The growth of two different fungal species, human pathogenic Cryptococcus neoformans and plant pathogenic Alternaria alternata were inhibited by a chitinase (Chi2) expressed in the autolysing tissue of Coprinellus congregatus. The cell wall thickness was reduced (up to 32%) in C. neoformans compared with that of normal cell, and polysaccharide fibers located outside of the cell wall were also severely removed. The hyphal growth of A. alternata on agar plate was stopped by the enzyme. The allergic inflammation induced by A. alternata was reduced by the enzyme reaction when compared with untreated control in a mouse model.

이형 재조합한 먹물버섯 Coprinellus congregatus Chitinase 1 (Chi1)의 발현과 생화학적 특성 분석

유예은,최형태,Yoo, Yeeun,Choi, Hyoung T. 한국미생물학회 2013 미생물학회지 Vol.49 No.4

Fungal cell walls consist of various glucans and chitin. Fungi produce chitinases for their growth and development. The inky cap, Coprinellus congregatus, produces at least two different chitinases during its life cycle. Chitinase 1 (chi1) is expresses throughout its life cycle while chitinase 2 (chi2) is expressed at the mushroom autolysing phase. The cloned cDNA of chi1 is successfully expressed as a fusion protein with c-myc in Pichia pastoris, and purified by the affinity chromatography. The optimum pH and temperature of Chi1 was pH 8.0 and $35^{\circ}C$, respectively when 4-nitrophenyl N,N',N"-triacetyl-${\beta}$-D-chitotrioside was used as the substrate. The $K_m$ value and $V_{max}$ for the substrate was 0.780 mM and 0.10 OD $min^{-1}unit^{-1}$, respectively. The addition of purified Chi1 resulted in total growth inhibition against several plant pathogenic fungi such as Alternaria alternata, Fusarium graminearum and Trichoderma harzianum at the concentration of 60 ${\mu}g/ml$. 진균류의 세포벽은 다양한 결합의 글루칸과 키틴질로 구성되었으며 이들의 생활사 전체에 해당하는 균사생장과 분화과정에서 키틴질 분해효소(chitinase)를 생성한다. 먹물버섯의 하나인 Coprinellus congregatus는 전 생활사 동안 최소 2종 이상의 chitinase를 생성한다. Chi1은 균사시기부터 버섯의 자가분해 시기까지 고르게 생성되며, chitinase 2는 버섯 자가분해 시기에 다량 생성된다. Chi1의 cDNA 유전자를 Pichia pastoris의 발현벡터의 하나인 pPICZB 벡터의 c-myc과 6x Histidine tag을 포함하는 Chi1 재조합 단백질 발현벡터를 구축하고 이를 발현시킨 후, Histidine tag에 의한 affinity chromatography 방법으로 Chi1 단백질을 정제하였다. 정제된 Chi1은 endochitinase의 기질인 4-nitrophenyl N,N',N"-triacetyl-${\beta}$-D-chitotrioside에 대하여 활성을 보였다. 이를 기질로 사용했을 때 최적 pH는 8.0이었고, 최적 온도는 $35^{\circ}C$로 확인되었다. $K_m$ 값과 $V_{max}$ 값은 각각 0.780 mM과 0.10 (OD $min^{-1}unit^{-1}$)로 나타났다. 이러한 생화학적 특성은 버섯 자가분해 시기에 작용할 것으로 판단되는 Chi2가 exochitinase 활성을 보이며, pH 4.0에서 최대 활성을 보이는 것을 종합하면 상호 보완작용을 할 것이라고 판단된다. 정제된 Chi1은 Alternaria alternata, Fusarium graminearum 및 Trichoderma harzianum 등과 같은 식물 병원성 균류에 처리한 결과 60 ${\mu}g/ml$ 농도에서 이들의 생장을 완전하게 억제하였다.