털두꺼비하늘소 (Moechotypa diphysis)로부터 Xylanase를 생산하는 Paenibacillus sp. HY-8 균주의 분리 및 특성

허선연,오현우,박두상,김향미,배경숙,박호용,Heo, Sun-Yeon,Oh, Hyun-Woo,Park, Doo-Sang,Kim, Hyang-Mi,Bae, Kyung-Sook,Park, Ho-Yong 한국응용곤충학회 2007 한국응용곤충학회지 Vol.46 No.2

Xylan이 풍부한 식물체를 먹이로 하는 하늘소의 장내에 존재하는 xylanase 생산 미생물의 탐색 과정에서 털두꺼비하늘소 (Moechotypa diphysis) 성충의 장으로부터 우수한 xylanase 생산균주 Paenibacillus sp. HY-8을 분리하였다. 생화학적, 계통학적 분석결과를 바탕으로 이 분리균은 Paenibacillus 속에 속하는 종으로 분석되었다. HY-8 균주에서 xylanase 생산은 제한배지에 xylan을 첨가함으로써 유도되는 특성을 나타내었고 1% 의 yeast extract와 0.5%의 birchwood xylan이 포함된 M9 배지에서 $25^{\circ}C$, 24시간의 배양에 의해 xylanase의 생산이 최대치에 도달하였다. HY-8 균주가 생산하는 xylanase는 pH6.0에서 여러 가지 식물성 사료의 원료에 대하여 대조구로 사용된 Tricoderma sp. 유래의 xylanase에 비해 우수한 분해능을 나타내었다. From the course of screening of useful xylanase producing microorganism from a phytophagous longicorn beetle, we isolated an extra-cellular xylanase producing strain, Paenibacillus sp. HY-8 from the intestine of Moechotypa diphysis adult. On the basis of morphological, biochemical and phylogenetic studies of the new isolate was identified as a Paenibacillus species. Production of xylanase in this strain was strongly induced by adding xylan to the growth medium and repressed by glucose or xylose. The highest xylanase production was attained in the M9 media containing 1% yeast extract and 0.5% birchwood xylan when cultured at $25^{\circ}C$ for 24 hrs. HY-8 producing xylanase showed superior hydrolytic activities against various plant source feedstuff than control xylanase produced by Tricoderma sp. at pH 6.0.

Constitutive overexpression of cyclodextrin glucanotransferase in Bacillus subtilis

허선연,김중균,남수완 한국생명과학회 2003 한국생명과학회 심포지움 Vol.40 No.-

Bacillus subtilis에서 Bacillus stearothermophilus CGTase의 구성적 발현

허선연,김중균,권현주,김병우,김동은,남수완 한국생명과학회 2004 생명과학회지 Vol.14 No.3

B. stearothermophilus NO2의 CGTase 유전자 (cgtS)를 구성적 $P_{JH}$ promoter 하류에 subcloning 하여 재조합 plasmid pIH-CGT1 (8.14 kb)을 구축하고 B. subtilis DB431에 형질 전환하였다. B. subtilis DB431/pJH-CGT1를 5가지 배지(LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG)로 flask 배양하여 균체증식과 CGTase발현량 및 분비국재성을 조사하여 최적 배지를 결정하였다. 그 중 〔5% molasses+2% CSL〕 배지에서 9시간에 1.8 unit/$m\ell$의 CGTase가 발현$.$생산되었다. 이 결과를 토대로 3. subtilis DB431/pJH-CGT1를 〔10% molasses + 5% corn steep liquor〕 배지에서 발효조 회분 배양한 결과, 30시간 배양시 CGTase의 최대 발현량은 4.2 unit/$m\ell$, 90%의 분비 효율, 90% 이상의 plasmid 안정성을 나타내었다. 저렴한 산업용 molasses 배지로 발효조 회분배양시 플라스크 배양보다 균체증식과 CGTase 발현량이 2배 이상의 증가된 값을 얻었다. To overproduce the cyclodextrin glucanotransferase(CGTase) of Bacillus stearothermophilus NO2 in B. subtilis, the pJH-CGTl plasmid (8.14 kb) was constructed, in which the ORF of CGTase gene could be transcribed by strong constitutive promoter, P$\_$JH/. To overproduce CGTase from a recombinant B. subtilis, the effect of media on the cell growth and expression level of CGTase were investigated with various media (LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG) in the flask culture. Among them, [5% molasses+2% CSL] medium revealed the maximum expression level of CGTase with 1.8 unit/$m\ell$ at 9 hr culture. In the batch culture on [10% molasses+5% corn steep liquor] medium the expression level of CGTase, the secretion efficiency, and plasmid stability were about 4.2 unit/$m\ell$, 90% and 90%, respectively, at 30 hr culture. The cell growth and expression level in the fermenter culture with the industrial molasses medium were increased by 2-folds over the flask culture.

재조합 효모 세포의 고농도배양을 통한 섬유소와 자일란 분해효소 유전자의 동시 과발현

김연희,허선연,김군도,남수완 한국미생물·생명공학회 2018 한국미생물·생명공학회지 Vol.46 No.4

For the coexpression of endoxylanase and endoglucanase genes in yeast Saccharomyces cerevisiae, the genes were separately inserted downstream of the yeast ADH1 promoters, resulting the plasmid pAGX3 (9.83 kb). In the batch culture on YPD medium of the yeast transformant, S. cerevisiae SEY2102/pAGX3, the total activities of the enzymes reached about 7.91 units/ml for endoxylanase and 0.43 units/ml for endoglucanase. In the fed-batch culture with intermittent feeding of yeast extract and glucose, the total activities of 24.9 units/ml for endoxylanase and 0.84 units/ml for endoglucanase were produced which were about 3.1- fold and 2.0-fold increased levels, respectively, compared to those of the batch culture. Most of endoxylanase and endoglucanase activities were found in the extracellular media. This recombinant yeast could be useful for the development of simultaneous saccharification bioprocess of the cellulose and xylan mixture. Endoxylanase와 endoglucanase 유전자가 ADH1 프로모터 하류에 따로따로 삽입된 pAGX3 플라스미드를 함유한Saccharomyces cerevisiae에서 endoxylanase와 endoglucanase 유전자는 성공적으로 발현되었으며, YPD 배지에서의 회분배양 결과, endoxylanase는 7.91 units/ml, endoglucanase 는 0.43 units/ml에 달하는 총활성을 보였다. Yeast extract 와 포도당을 간헐적으로 공급하는 유가배양에서 endoxylanase 와 endoglucanase의 총활성은 24.9 units/ml과 0.84 units/ ml을 각각 보였으며, 이는 회분배양에서 발현된 각각 활성의 3.1배와 2배에 해당되었다. 또한, 대부분의 endoxylanase 와 endoglucanase 활성은 세포밖 배지에서 측정되어, 향후이 재조합 효모는 섬유소(cellulose)와 xylan 혼합물의 동시당화 바이오공정 개발에 활용될 가능성이 높다 하겠다.

Characterization of an Extracellular Lipase in Burkholderia sp. HY-10 Isolated from a Longicorn Beetle

박두상,오현우,허선연,정우진,신동하,배경숙,박호용 한국미생물학회 2007 The journal of microbiology Vol.45 No.5

Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60°C. A broad range of lipase substrates, from C4 to C18 ρ-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was ρ-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.

호기적 조건하에서 NH4⁺로부터 N2를 발생시키는 균주의 순수분리 및 동정

조경숙,박경주,허선연,남수완,이민규,김중균 한국생명과학회 2003 한국생명과학회 심포지움 Vol.40 No.-

Bacillus subtilis 유래 재조합 endoxylanase를 이용한 xylooligosaccharide의 최적 생산

김연희(Yeon-Hee Kim),허선연(Sun-Yeon Heo),김미진(Mi-Jin Kim),이재형(Jae Hyung Lee),김영만(Young-Man Kim),남수완(Soo-Wan Nam) 한국생명과학회 2008 생명과학회지 Vol.18 No.1

식물체 구성성분의 하나인 hemicellulose의 대부분을 차지하는 xylan은 농산 폐기물 또는 목재 성분의 약 30%를 차지하는 풍부한 자원물질이다. 이러한 xylan을 효과적으로 분해하기 위해 Bacillus에서 발현시킨 재조합 endoxylanase를 이용하여 기능성 식품소재인 xylooligosaccharide의 최적 생산 조건을 조사하였다. B. subtilis 재조합 균주 DB431/pJHKJ4를 이용한 발효조 회분배양 결과, endoxylanse의 총활성은 857 unit/ml이며, 대부분이 세포밖으로 분비됨을 알 수 있었고, 분비효율은 92%로 나타났다. 재조합 endoxylanase를 이용하여 xylan으로부터 xylooligosaccharide 생성을 위한 최적 반응조건을 검토한 결과, xylooligosaccharide 생산을 위한 기질로는 birchwood xylan이 적합함을 알 수 있었고, 4%의 xylan 농도에서 가장 많은 xylooligosaccharide을 생산하며, xylobiose와 xylotriose가 주생성물임을 알았다. 효소의 농도와 반응시간의 영향은 10 unit의 endoxylanase를 첨가하여 1시간 반응시켰을 때 가장 많은 양의 xylooligosaccharide을 생산할 수 있었고, 반응온도는 40~50℃가 적합함을 알았다. 결론적으로 재조합 endoxylanase을 이용한 xylooligosaccharide생성에는 10 unit endoxylanase과 4% birchwood xylan을 기질로 이용하여 50℃에서 1시간 반응시키는 것이 최적반응 조건임을 알았다. Xylan is a major hemicellulose component of the cell walls of monocots and hardwood, representing up to 30% of the dry weight of these plants. To efficiently hydrolyze xylan, the endoxylanase gene from Bacillus sp. was expressed in B. subtilis DB431 by introducing the plasmid pJHKJ4. The total activity of the recombinant endoxylanase reached about 857 unit/ml by batch fermentation of B. subtilis DB431/pJHKJ4 in LB maltose medium. The majority (>92%) of endoxylanase was efficiently secreted into the culture medium. The recombinant endoxylanase hydrolyzed more the birchwood xylan efficiently than the other xylans. When 4% concentration of xylan was used, the highest production of xylooligosaccharide was observed, and xylobiose and xylotriose were the major products. Optimal amount of enzyme and reaction time for producing xylooligosaccharide were found to be 10 unit and 1 hr, respectively. In addition, the temperature of 40℃~50℃ gave the highest production of xylooligosaccharide. Consequently, the optimized conditions for the production of xylooligosaccharide through the hydrolysis of xylan were determined as follows: 10 unit endoxylanase, 50℃, 4% birch-wood xylan, 1 hr reaction.

Production of Lipid Containing High Levels of Docosahexaenoic Acid by Cultivation of Aurantiochytrium sp. KRS101 Using Jerusalem Artichoke Extract

주정현,오백록,류승규,허선연,김수연,홍원경,김철호,서정우 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.6

In the present study, we evaluated extract of Jerusalem artichoke tubers (JAT) as a substrate for the production of lipid containing high levels of docosahexaenoic acid (DHA) by cultivated Aurantiochytrium sp. KRS101, an oleaginous protist. The optimal conditions for cultivation determined using response surface methods were as follows: pH, 5.9; enzyme loading, 282.6; and temperature, 27.7°C. The maximal levels of lipid (16.4 g/L) and productivity (3.6 g/L/d) were obtained during fed-batch fermentation, which achieved a DHA yield of 7.9 g/L (>48% of total fatty acids by weight). Moreover, JAT was more economical, reducing costs of expensive yeast extract as a nitrogen source by about 40%. These results suggest that JAT may be further explored as a low-cost feedstock for lipid production using microalgae strains.

Tremella fuciformis TFCUV5 Mycelial Culture-derived Exopolysaccharide Production and Its Anti-aging Effects on Skin Cells

조민호,김별,주정현,허선연,안극현,이혜자,염현숙,장한수,김민수,김철호,오백록 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.5

Tremella fuciformis TFC6 mycelia was isolated from its fruiting body. T. fuciformis TFCUV5 was obtained from TFC6 mycelia by ultraviolet mutagenesis to improve extracellular-polysaccharide (EPS) yield. In addition, we investigated the physicochemical properties and functionality of EPS for industrial applications and the effect of EPS on enhancement of the moisturizing factor and antiwrinkle effect in skin cells. The EPS yield was improved upon fermentation by the mutant strain through optimization of the medium composition and culture conditions to a maximum of 8.4 g/L; the productivity was 1.9 g/L·day under optimal culture conditions (initial pH 7.0 at 25°C, 300 rpm, 2 vvm). EPS with a molecular weight of 1.79 × 106 (± 0.721%) comprised mannose, xylose, glucuronic acid, fucose, and glucose. EPS extract increased both moisture and the antiwrinkle factor in skin cells. Therefore, EPS from TFCUV5 might have potential to be used in the cosmetics industry.

곤충 장내미생물로부터 lipase 생산능력이 우수한 Burkholderia sp. HY-10 균주의 분리 및 특성

박두상,오현우,배경숙,김향미,허선연,김남정,설광열,박호용,Park, Doo-Sang,Oh, Hyun-Woo,Bae, Kyung-Sook,Kim, Hyang-Mi,Heo, Sun-Yeon,Kim, Nam-Jung,Seol, Kwang-Youl,Park, Ho-Yong 한국응용곤충학회 2007 한국응용곤충학회지 Vol.46 No.1

곤충으로부터 유용 효소생산 미생물의 탐색 과정에서 우수한 lipase 생산균주 9종을 분리하고 lipase 생산능을 조사하였다 16S rDNA 분석 결과 분리된 균주는 주로 Serratia 속, Pseudomonas 속, Burkholderia 속에 속하는 그람음성균들로 분석되었다. 그 중 lipase 생산능이 가장 우수한 균주를 선별하고 16S rDNA 서열분석 및 생리 생화학적 분석 결과를 바탕으로 Burkholderia sp. HY-10으로 동정하였으며 균주의 lipase생산특성을 조사하였다. 이 균주는 톱하늘소의 장으로부터 분리되었으며 olive oil을 탄소원으로 포함하는 배지에서 배양하였을 때 세포밀도에 의존하여 lipase의 생산이 유도되는 특성을 나타내었고 0.5%의 yeast extract와 0.5%의 olive oil이 포함된 M9배지에서 $30^{\circ}C$, 36-42시간의 배양에 의해 lipase의 생산이 최대치에 도달하였다. From the course of screening of useful enzyme producing microorganism from insect guts, we isolated 9 lipase producing strains and their lipase producing activities were tested. 16S rDNA sequence analysis showed that they were Gram negative bacteria grouped on Serratia sp., Pseudomonas sp., and Burkholderia sp.. Among them, an excellent lipase producing strain, Burkholderia sp. HY-10 identified by 16S rDNA analysis and biochemical methods, was further studied its lipase producing characteristics. It was isolated from a longcorm beetle, Prionus insularis and showed cell density dependent lipase producing activity in the culture media that contained olive oil as a carbon source. Maximum lipase production was achieved in the M9 media containing 0.5% yeast extract and 0.5% olive oil when cultured at $30^{\circ}C$ for 36-42 hrs.