• 한국자원식물학회:학술대회논문집
• /
• 한국자원식물학회 2010년도 정기총회 및 춘계학술발표회
• /
• pp.17-17
• /
• 2010

Ethylene, known as a stress hormone regulate wide developmental processes including germination, root hair initiation, root and shoot primordial formation and elongation, leaf and flower senescence and abscission, fruit ripening. The acceleration of ethylene biosynthesis in plant associated with environmental and biological stresses. 1-Aminocycloprophane-1-carboxlyate deaminase(ACCD) is an enzyme that cleaves ACC into and ammonia, a precursor of the plant hormone ethylene. Plant growth-promoting rhizobacteria (PGPR) having ACCD can decrease endogenous ACC level of tissue, resulting in reduced production of ethylene in plants. ACC deaminse was a key enzyme for protect stressed plants from injurious effects of ethylene. ACCD gene was encoded from Pseudomonas flourescens, PGPR and was cloned in Escherichia coli. We expressed the recombinant ACCD(rACCD) containing 357 amino acids with molecular weight 39 kDa that revealed by SDS-PAGE and western blot. The rACCD was purified by Ni-NTA purification system. The active form of rACCD having enzyme activity converted ACC to a-ketobutyrate. The optimal pH for ACC deaminase activity was pH 8.5, but no activity below pH 7.0 and a less severe tapering activity at base condition resulting in loss of activity at over pH 11. The optimal temperature of the enzyme was $30^{\circ}$ and a slightly less severe tapering activity at 15 - 30$^{\circ}$, but no activity over $35^{\circ}$. P. flourescens ACC deaminase has a highly conserved residue that plays in allowing substrate accessibility to the active sites. The enzymatic properties of this rACCD will provide an important reference for analysis of newly isolated ACCD and identification of newly isolated PGPR containing ACCD.

• 미생물학회지
• /
• 제45권4호
• /
• pp.291-299
• /
• 2009

본 연구에서는 Human HtrA3 (HtrA3)의 효소활성을 분자수준에서 연구하기 위해 HtrA간의 상동성과 기존에 알려진 maturation site들을 비교 분석하여 예상 mature HtrA3인 M1-HtrA3와 M2-HtrA3를 발현하는 construct를 제작하였다. pGEX system을 통해 Top10 균주에서 발현, 정제한 M1-HtrA3 단백질은 $10{\mu}g$/L를 정제할 수 있었으며 발현량 대비 1%를 회수할 수 있었다. M2-HtrA3는 M1보다 5배 가량 많은 양을 정제할 수 있었으며 발현량 대비 회수율은 3배 정도 더 높았다. $\beta$-Casein을 이용한 in vitro cleavage test를 통해 M1, M2 form 모두 protease 활성을 갖는 것을 확인하였다. 또한, $\beta$-casein cleavage를 통해 HtrA serein protease들 간의 상대적인 활성을 비교한 결과, HtrA3와 HtrA2는 HtrA1보다 약 2배 더 높은 proteolytic cleavage 활성을 보였다. 본 연구에서 정립한 protease 활성을 지닌 HtrA3의 제작과 정제 조건은 HtrA3의 substrate를 탐색을 용이하게 할 수 있을 것이며, HtrA3 연관된 질환의 발병기전과 세포 신호전달을 이해하는 연구에 활용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
• /
• 제19권11호
• /
• pp.1295-1300
• /
• 2009

A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.

• Journal of Nutrition and Health
• /
• 제19권6호
• /
• pp.363-373
• /
• 1986

This study was devised to elucidate whether soused shrimp exhibits a digestive action on boiled pork meats. and the mechanism by which sousing with a high concentration of sodium chloride preserves nutrients in foods for a prolonged pe\ulcornerriod. Protease was isolated from soused shrimp using a combination of ammonium sulfate fractionation. DEAE - cellulose ion exchange chromatography and gel filtra\ulcornertion. The isolated protease had specific activity of 1.560 units. 210 purification fo\ulcornerld with an yield of 38%. Its optimum pH and temperature were 8.0 and $43^{\circ}C$ respectively. The molecular weight of the enzyme was 35.000. The Km value of the enzyme for casein was 1.6 x $10^{-6}$ M The e=yme required the presence of cu\ulcornerpric ion to exhibit its full activity. Eighty eight percent of the enzyme activity was in\ulcornerhibited by 3.5M NaCI showing a reversibly linear decrease of the enzyme activity as NaCI concentration increased. The nature of the inhibition by NaCl was rever\ulcornersible and noncompetitive. The protease activity in soused shrimp was well preser\ulcornerved with the elapse of time at least in part due to NaCI induced suppression of autodigestion. The enzyme was denatured by acid easily. i.e. 1% of the original activity remained after staying at pH 2 for 10 minutes. which is within the norm\ulcorneral range of pH of the human stomach. Soused shrimp was observed to be one of those containing the highest protease activity compared with the other soused foo\ulcornerds such as soused oyster. squid. clam. and Pollack intestine with respect to spec\ulcornerific activities of dialized 1:4 whole homogenates(w/v) in 5 mM sodium phospha\ulcornerte - 2.4 mM j3 - mercaptoethanol buffer. pH 8.0. Casein and boiled meats including pork, beef, and chicken appeared to be the good substrates for the protease. Casein was the best. Therefore. the ingestion of boiled meats including pork together with soused sh\ulcornerrimp would help digestion of boiled pork in human not only by increasing appe\ulcornertite also by the direct proteolytic digestion of boiled meats by soused shrimp to\ulcorner some extent. And a high concentration of sodium chloride inhibited the protease activity reversibly in a remarkable degree, which ensued in a significant retardat\ulcornerion of autodigestion of protein in foods by proteases, and hereby contributed to the preservation of foods for an extended period.

• Journal of Microbiology and Biotechnology
• /
• 제21권10호
• /
• pp.1012-1020
• /
• 2011

A gene coding for an endoglucanase (EglA), of the glycosyl hydrolase family 12 and derived from Aspergillus niger VTCC-F021, was cloned and sequenced. The cDNA sequence, 717 bp, and its putative endoglucanase, a 238 aa protein with a predicted molecular mass of 26 kDa and a pI of 4.35, exhibited 98.3-98.7% and 98.3-98.6% identities, respectively, with cDNA sequences and their corresponding endoglucanases from Aspergillus niger strains from the GenBank. The cDNA was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 1.59 U/ml culture supernatant, after 72 h of growth in a YP medium induced with 1% (v/v) of methanol. The molecular mass of the purified EglA, determined by SDS-PAGE, was 33 kDa, with a specific activity of 100.16 and 19.91 U/mg toward 1% (w/v) of ${\beta}$-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of $55^{\circ}C$ and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of $30-37^{\circ}C$ and at pH range of 3.5-4.5. Metal ions, detergents, and solvents tested indicated a slightly inhibitory effect on rEglA activity. Kinetic constants ($K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$) determined for rEglA with ${\beta}$-glucan as a substrate were 4.04 mg/ml, 102.04 U/mg, 2,040.82 $min^{-1}$, and 505.05, whereas they were 10.17 mg/ml, 28.99 U/mg, 571.71 $min^{-1}$, and 57.01 with CMC as a substrate, respectively. The results thus indicate that the rEglA obtained in this study is highly specific toward ${\beta}$-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme.

• 한국식품영양과학회지
• /
• 제39권11호
• /
• pp.1647-1653
• /
• 2010

군소에서 추출한 다당류로부터 DEAE-Sepharose 상에서 glycosaminoglycan(GAG)을 정제하여 기능기의 분포, 구성당의 분포, 이당류의 조성과 당 구조를 조사하였다. 정제한 GAG는 기본 형태를 구성하는 이당류 단위가 전체 구성물 중 55% 이상을 차지하고 있는 다당 복합체였다. 정제한 GAG는 1648 $cm^{-1}$에서 amide I의 특징적인 띠와 1457 $cm^{-1}$에서 C-O stretch, 탄수화물 및 아미노산의 특징, 866 $cm^{-1}$에서 단당류의 특징을 보이는 것으로 나타났다. 정제한 GAG는 fucose, N-acetylgalactosamine, N-acetylglucosamine, glucose, galactose, 미량의 mannose와 xylose로 구성되어 있는 것으로 나타났고, 이중에서 N-acetylgalactosamine, N-acetylglucosamine이 70% 이상을 차지하는 다당 복합체인 heparan sulfate인 것으로 추정되었다. 군소 GAG는 단백질핵의 threonine 잔기에 O-연결된 GlyUA(2S)-GlcNS와 GlyUA-GlcNS(6S) 구조를 가지고 있는 것으로 나타났다.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• 제19권3호
• /
• pp.265-286
• /
• 1997

Bone morphogenetic proteins(BMPs) are a group of transforming growth factor beta(TGF-${\beta}$)-related factors and multifunctional proteins, especially the only known biologic factors capable of inducing endochondral bone formation at an extraskeletal site. This study was performed to investigate the effect of the partially purified porcine BMP(pBMP) at an ectopic site. PBMP was partially purified from porcine bone matrix and its activity was monitored by an in vivo bioassay. The purification method utilized extraction of the bone-inducing activity with 4M guanidine, followed by chromatography on heparin-Sepharose. Active fractions were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. And the fractions were reconstituted with inactive insoluble collagenous bone matrix from rats, acid soluble type I collagen from rat tail and chondroitin-6-sulfate sodium salt and implanted into the pectroralis muscle pouches of Sprague-Dawley rats. And the carrier complex was implanted on the opposite side as control. The rats were sacrificed at the day of 1st, 3rd, 5th, 7th, 11th, 14th and 21st after implantation and examined histologically, radiologically and biochemically. And alkaline phosphatase activity and calcium content were used as indices of bone formation. The results were as follows ; 1. Active fractions were localized in a zone between 31 and 40 KDa on SDS-PAGE. 2. The implanted 3.0mg of the partially purified pBMP induced cartilage and bone in the muscle tissue of rats through an endochondral ossification process. 3. Inactive insoluble bone matrix, type I collagen and chondroitin-6-sulfate have functioned as carriers for pBMP, but revealed some foreign body reactions. 4. Soft X-ray didn't reveal significant change between the experimental and the control group. 5. The alkaline phosphatase activities in the experimental group of 5th, 7th, 11th, 14th and 21st were increased significantly compared with control (p<0.01) with the peak in the group of 11th day. 6. With time, the calcium content of the experimental group increased. And the calcium contents in the experimental group of 11th, 14th and 21st were increased significantly compared with control (p<0.01).

• The Korean Journal of Physiology
• /
• 제24권2호
• /
• pp.363-375
• /
• 1990

The present investigation was performed to purify bombesin-like immunoreactivity (BBS-LI) from the skin of frogs, B. orientalis inhabiting Korea. For extraction of BBS-LI, the fresh skin of 360 g from frogs was immersed in 1,800 ml of 100% methanol and then kept at $4^{\circ}C$ for 5 days. BBS-LI was partially purified by liquid chromatography using an alkaline alumina column followed by a Sephadex G-10 column. BBS-LI was further purified by using sequential HPLC of reversed phase C18 preparation, gel permeation, SP-ion exchange and reversed phase C18 analysis. BBS-LI in fractions of each step was monitored by radioimmunoassay for which bombesin antiserum with a titer of 1 : 188,800 was raised in a guinea pig. Eventually, two different BBS-LI were successfully purified and each BBS-LI showed the following character. 1) BBS-LI was well separated into two peaks in SP-ion exchange HPLC. One (BBS-LI-K1) bound to the column while the other (BBS-LI-K2) did not. 2) BBS-LI-K1, 73.8% of total BBS-LI, was not differentiated from synthetic bombesin in reversed phase C18 analytical and gel permeation HPLC. 3) BBS-LI-K2, 26.2% of total BBS-LI, eluted later than synthetic bombesin in reversed phase C18 analytical HPLC, but it eluted with a retention time identical to that of synthetic bombesin in gel permeation HPLC. 4) The two forms of BBS-LI and synthetic bombesin identically stimulated gastrin release and pancreatic exocrine secretion including volume, protein output and amylase output in anesthetized rats. It is concluded from the above results that the skin of B. orientalis contains two different forms of BBS-LI which are very identical to bombesin immunologically and biologically. In comparison with synthetic bombesin containing 14 amino acid residues, the major form shows quite similar pattern in all HPLC used in the present study, but the minor form exhibits quite different pattern in SP-ion exchange and reversed phase C18 analytical HPLG.

• 한국잠사곤충학회지
• /
• 제50권2호
• /
• pp.145-149
• /
• 2012

세크로핀(cecropin)은 곤충의 체액성 면역에 있어서 효과적인 방어인자로 작용하는 항균 펩타이드로 잘 알려져 있다. 본 연구에서는 면역 유도된 천잠, Antheraea yamamai 유충 혈림프로부터 세크로핀 항균 펩타이드 분리 및 정제를 실시하였다. 먼저 항균 펩타이드를 분리하기 위해서, 면역 유도된 유충 및 정상 유충으로부터 추출된 혈림프 단백질 시료에 대한 단백질 전기영동(SDS-PAGE)를 통하여 비교분석하였다. 정상누에 혈림프 시료에 비해 면역 유도된 혈림프 추출물에서만 특이적으로 발현되는 분자량 4,223.01 Da의 펩타이드 밴드를 검출하였다. 선발된 면역유도 특이적 발현 펩타이드의 특성 분석을 위해서 이온교환 크로마토그래피 및 gel permeation 크래마토그래피을 수행하여 특이적으로 발현되는 펩타이드를 성공적으로 순수 정제하였다. 정제된 펩타이드는 Edman degradation법으로 N말단 아미노산 서열을 결정하였고 다른 나비목곤충의 세크로핀과 매우 높은 상동성을 나타내어 세크로핀으로 동정하였다. 또한 정제된 천잠 세크로핀 항균 펩타이드는 그람음성세균, 그람양성세균 및 곰팡이에 대해 폭 넓은 항균 스펙트럼을 나타냈었다.

• 한국수산과학회지
• /
• 제30권4호
• /
• pp.556-561
• /
• 1997

해수로부터 분리한 V. cholerae non-O1 FM-3 이 생산하는 hemolysin의 특성에 관하여 연구한 결과를 요약하면 다음과 같다. 1. V. cholerae non-O1 FM-3이 생산하는 hemolysin은 대수증식기일 때 가장 높은 활성을 나타내었으며, Sephadex G-100 gel column chromatography로 부분정제한 후 hemolysin의 특성을 실험한 결과, pH 7.0, $4^{\circ}C$에서 가장 안정하였으며, $60^{\circ}C$, 30분 가열에 의해 완전히 실활되었다. 2. 적혈구와 hemolysin이 반응할 때, $Na^+,\;K^+$과 같은 1가 양이온을 첨가하면 약간의 활성증가를 보였으며, $Ca^{2+},\;Mg^{2+},\;Mn^{2+}$과 같은 2가 양이온을 첨가하였을 때 급격한 활성증가를 보였고, 반면 $Zn^{2+}$ 첨가시에는 급격히 활성이 저하되었다. 3. 적혈구 현탁액과 hemolysin을 각각 다른 온도대에서 반응시킨 결과, $4^{\circ}C$에서도 온도와 관계없이 적혈구막과 hemolysin의 결합이 가능한 온도 비의존적인 제1단계와, 용혈황성의 최적온도대인 $37^{\circ}C$에서만 용혈작용이 일어나는 온도 의존적인 제2단계로 나누어졌다.