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You-Jung Lee(이유정),Hye-Jin Kim(김혜진),Wa Gao(고와),Chung-Han Chung(정정한),Jin-Woo Lee(이진우) 한국생명과학회 2011 생명과학회지 Vol.21 No.9
재조합 균주인 E. coli JM109/DL-3를 사용하여 carboxymethylcellulase를 생산하기 위한 배지의 최적 염 농도를 orthogonal array method(OAM)과 response surface method (RSM) 등과 같은 통계학적인 방법으로 확립하고 그 결과를 비교하였다. OAM에 기초를 한 Qualitek-4 Software를 사용하여 실험을 계획하고, 그 결과를 분석한 결과는 K2HPO4가 균체의 생장 및 carboxymethylcellulase의 생산에 미치는 영향이 가장 크다는 사실을 확인 하였다. 균체의 생육에 최적인 K2HPO4, NaCl, MgSO4?7H2O 및 (NH4)2SO4의 농도는 10.0, 1.0, 0.2 및 0.6 g/l이었으나, carboxymethylcellulase의 생산에 최적인 각 염들의 농도는 각각 5.0, 1.0, 0.4 및 0.6 g/l이었다. RSM에 기초를 한 Design-Expert Software를 사용하여 실험을 계획하고, 그 결과를 분석한 결과는 K2HPO4가 균체의 생장 및 carboxymethylcellulase의 생산에 가장 중요한 인자라는 사실을 확인하였다. 균체의 생장에 최적인 K2HPO4, NaCl, MgSO4?7H2O 및(NH4)2SO4의 농도는 7.44, 1.08, 0.22 및 0.88 g/l이었으나, carboxymethylcellulase의 생산에 최적인 각 염들의 농도는 각각 5.84, 0.69, 0.28 및 0.54 g/l이었다. 기본적으로 OAM에 기초한 software를 사용하여 얻은 결과는 RSM에 기초한 software를 사용하여 얻은 결과와 유사하였다. 최적 조건에서 재조합 균주 E. coli JM109/DL-3이 생산하는 carboxymethylcellulase의 생산은 B. amyloliquifacience DL-에 비하여 1.92배 증가하였다. The optimal concentrations of salts in medium for cell growth and the production of carboxymethylcellulase (CMCase) by a recombinant E. coli JM109/DL-3 were established using two statistical methods: orthogonal array method (OAM) and response surface method (RSM). The analysis of variance (ANOVA) of data based on OAM indicated that K2HPO4 gave maximum sum of square (S) and percentage contribution (P) for cell growth as well as production of CMCase. The optimal concentrations of K2HPO4, NaCl, MgSO4?7H2O, and (NH4)2SO4 in medium for cell growth extracted by Qualitek-4 (W32b) Software were 10.0, 1.0, 0.2, and 0.6 g/l, respectively, whereas those for the production of CMCase by E. coli JM109/DL-3 were 5.0, 1.0, 0.4, and 0.6 g/l. The analysis of variance (ANOVA) resulting from RSM indicated that a highly significant salt for cell growth was K2HPO4 ("probe>F" less than 0.0001), whereas K2HPO4 and MgSO4?7H2O were significant for the production of CMCase. The optimal concentrations of K2HPO4, NaCl, MgSO4?7H2O, and (NH4)2SO4 for cell growth extracted by Design Expert Software were 7.44, 1.08, 0.22, and 0.88 g/l, respectively, whereas those for production of CMCase were 5.84, 0.69, 0.28, and 0.54 g/l. The optimal concentrations of salts and their influences on cell growth and production of CMCase extracted by OAM were almost the same as those by RSM. Production of CMCase by a recombinant E. coli JM109/DL-3 under optimized concentration of salts was 1.93 times higher than that by Bacillus amyloliquifaciens DL-3.
Yi-Joon Kim(김이준),Wa Cao(고와),이유정,Sang-Un Lee(이상운),정정한,Jin-Woo Lee(이진우) 한국생명과학회 2012 생명과학회지 Vol.22 No.10
Carboxymethylcellulase를 생산하는 미생물을 해수에서 분리하여 16S rDNA의 염기서열을 분석하고 계통 발생학 방법으로 비교한 결과, Bacillus atrophaeus로 확인되었다. 이 해양 미생물을 B. atrophaeus LBH-18로 명명하였으며 response surface method (RSM)를 사용하여 carboxymethylcellulase의 생산 조건을 최적화하였다. 이 균주의 생육에 최적인 미강, 펩톤 및 배지의 초기 pH는 68.1 g/l, 9.1 g/l 및 7.0이었으나, carboxymethylcellulase의 생산에 최적인 조건은 각각 55.2 g/l, 6.6 g/l 및 7.1이었다. 이 균주의 생육과 carboxymethylcellulase의 생산에 최적인 온도는 30℃이었다. 이 균주의 생육에 최적인 생물배양기의 교반속도 및 통기량은 324 rpm 및 0.9 vvm이었으나, carboxymethylcellulase의 생산에 최적인 조건은 각각 343 rpm 및 0.6 vvm이었다. 파이롯트 규모의 생물배양기를 사용하여 실험한 결과, 이 균주의 생육과 carboxymethylcellulase의 생산에 최적인 내압은 0.06 MPa이었다. 최적 조건의 내압으로 배양한 결과, 이 균주의 carboxymethylcellulase의 생산성은 127.5 U/ml이었으며, 이 결과는 내압을 가하지 않고 배양한 경우에 비하여 1.32배 향상된 것이다. 본 연구를 통하여 쌀 도정 공정의 부산 물인 미강을 기질로 개발하였으며 해양 미생물을 사용하여 carboxymethylcellulase의 생산기간을 7~10일에서 3일로 단축시켰다. A microorganism producing carboxymethylcellulase (CMCase) was isolated from seawater and identified as Bacillus atrophaeus. This species was designated as B. atrophaeus LBH-18 based on its evolutionary distance and the phylogenetic tree resulting from 16S rDNA sequencing and the neighbor-joining method. The optimal conditions for rice bran (68.1 g/l), peptone (9.1 g/l), and initial pH (7.0) of the medium for cell growth was determined by Design Expert Software based on the response surface method; conditions for production of CMCase were 55.2 g/l, 6.6 g/l, and 7.1, respectively. The optimal temperature for cell growth and the production of CMCase by B. atrophaeus LBH-18 was 30℃. The optimal conditions of agitation speed and aeration rate for cell growth in a 7-l bioreactor were 324 rpm and 0.9 vvm, respectively, whereas those for production of CMCase were 343 rpm and 0.6 vvm, respectively. The optimal inner pressure for cell growth and production of CMCase in a 100-l bioreactor was 0.06 MPa. Maximal production of CMCase under optimal conditions in a 100-l bioreactor was 127.5 U/ml, which was 1.32 times higher than that without an inner pressure. In this study, rice bran was developed as a carbon source for industrial scale production of CMCase by B. atrophaeus LBH-18. Reduced time for the production of CMCase from 7 to 10 days to 3 days by using a bacterial strain with submerged fermentation also resulted in increased productivity of CMCase and a decrease in its production cost.
( Wa Gao ),( Eun Jung Lee ),( Sang Un Lee ),( Jian Hong Li ),( Chung Han Chung ),( Jin Woo Lee ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.10
The aim of this work was to establish the optimal conditions for production of carboxymethylcellulase (CMCase) by a newly isolated marine bacterium using response surface methodology (RSM). A microorganism producing CMCase, isolated from seawater, was identified as Cellulophaga lytica based 16S rDNA sequencing and the neighborjoining method. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth were 100.0 g/l, 5.00 g/l, and 7.0, respectively, whereas those for production of CMCase were 79.9 g/l, 8.52 g/l, and 6.1. The optimal concentrations of K2HPO4, NaCl, MgSO4?7H2O, and (NH4)2SO4 for cell growth were 6.25, 0.62, 0.28, and 0.42 g/l, respectively, whereas those for production of CMCase were 3.72, 0.54, 0.70, and 0.34 g/l. The optimal temperature for cell growth and the CMCase production by C. lytica LBH-14 were 35℃ and 25℃, respectively. The maximal production of CMCase under optimized condition for 3 days was 110.8 U/ml, which was 5.3 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen sources for the production of CMCase by C. lytica LBH-14. The time for production of CMCase by a newly isolated marine bacterium with submerged fermentations reduced to 3 days, which resulted in enhanced productivity of CMCase and a decrease in its production cost.
Hye-Jin Kim(김혜진),Wa Gao(고와),You-Jung Lee(이유정),Chung-Han Chung(정정한),Jin-Woo Lee(이진우) 한국생명과학회 2010 생명과학회지 Vol.20 No.4
Carboxymethylcellulose (CMC)를 분해하는 미생물을 해수에서 분리하였으며, 16S rDNA의 염기서열을 분석하여 동정한 결과, Psychrobacter aquinaris로 학인 되어 P. aquinaris LBH-10로 명명하였다. 이 균주는 CMC, 셀로바이오스, 커드란, 여과지, p-nitrophenyl-β-D-glucopyranoside (pNPG), 풀루란 및 자일란을 분해하였으나, avicel 및 섬유소는 분해하지 못하였다. P. aquinaris LBH-10이 생산하는 carboxymethylcellulase (CMCase)의 최적 반응온도는 50℃이었으며, 20℃에서 50℃의 온도 범위에서 24시간이 경과한 후에도 90% 이상의 활성을 유지하였다. 또한, 이 균주가 생산하는 CMCase의 최적 반응 pH는 3.5이었으며 pH 2.5에서 pH 7.0 사이의 산성 조건하에서 24 시간이 경과한 후에도 70% 이상의 활성을 유지하였다. P. aquinaris LBH-10이 생산하는 CMCase의 최적 반응 pH는 지금까지 발견된 섬유소 분해효소 중에서 가장 낮은 pH로 판단된다. 제한된 농도의 CoCl₂, EDTA, 및 PbCl₂은 P. aquinaris LBH-10가 생산하는 CMCase의 활성을 증가시켰으나, HgCl₂, KCl, MnCl₂, NiCl₂ 및 SrCl₂는 이 균주가 생산하는 CMCase의 활성을 감소시켰다. A microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Psychrobacter aquimaris by analysis of 16S rDNA sequences, and named P. aquimari LBH-10. This strain produced an acidic carboxymethylcellulase (CMCase), which hydrolyzed carboxymethylcellulose (CMC), cellobiose, curdlan, filter paper, p-nitrophenyl-β-D-glucopyranoside (pNPG), pullulan, and xylan, but there was no detectable activity on avicel and cellulose. The optimal temperature for CMCase produced by P. aquimari LBH-10 was 50℃ and more than 90% of its original activity was maintained at broad temperatures ranging from 20 to 50℃ after 24 hr. The optimal pH of the CMCase was 3.5, and more than 70% of its original activity was maintained under acidic conditions between pH 2.5 and 7.0 at 50℃ after 24 hr. The optimal pH of CMCase produced by P. aquimaris LBH-10 seems to be lower than those produced by any other bacterial and fungal strain. CoCl₂, EDTA, and PbCl₂ at a concentration of 0.1 M enhanced CMCase-produced P. aquimaris LBH-10, whereas HgCl₂, KCl, MnCl₂, NiCl₂, and SrCl₂ inhibited it.
Kim, Bo-Kyung,Lee, Bo-Hwa,Lee, Yoo-Jung,Jin, Il-Hyuck,Chung, Chung-Han,Lee, Jin-Woo Elsevier 2009 Enzyme and microbial technology Vol.44 No.6
<P><B>Abstract</B></P><P>The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as <I>Bacillus subtilis</I> subsp. <I>subtilis</I> by analyses of 16S rDNA and partial sequences of the <I>gyr</I>A gene, and named as <I>B. subtilis</I> subsp. <I>subtilis</I> A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and <I>p</I>-nitrophenyl-β-<SMALL>D</SMALL>-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50°C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40°C after 24h incubation at 50°C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K<SUP>+</SUP>, Ni<SUP>2+</SUP>, Sr<SUP>2+</SUP>, Pb<SUP>2+</SUP>, and Mn<SUP>2+</SUP>, but inhibited by Co<SUP>2+</SUP> and Hg<SUP>2+</SUP>.</P>