• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.838-845
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• 2011

The present work describes the identification, purification, and characterization of bile salt hydrolase (BSH) from Bifidobacterium animalis subsp. lactis. The enzyme was purified to electrophoretic homogeneity by hydrophobic chromatography, ion-exchange chromatography and ultrafiltration. SDS-PAGE analysis of putative BSH and gel filtration revealed that the analyzed protein is presumably a tetramer composed of four monomers each of about 35 kDa. The purified enzyme was analyzed by liquid chromatography coupled to LTQ FT ICR mass spectrometry and unambiguously identified as a bile salt hydrolase from B. animalis. The isoelectric point of the studied protein was estimated to be around pH 4.9. The pH optimum of the purified BSH is between 4.7 to 6.5, and the temperature optimum is around 50oC. The BSH of B. animalis could deconjugate all tested bile salts, with clear preference for glycine-conjugated bile salts over taurine-conjugated forms. Genetic analysis of the bsh showed high similarity to the previously sequenced bsh gene from B. animalis and confirmed the usefulness of bile salt hydrolase as a genetic marker for B. animalis identification.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권5호
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• pp.362-368
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• 2004

A new one-column chromatography process, analogous to a four-zone simulated moving bed (SMB), was presented. The basic principle of the process was identical to that of a four-zone SMB. The process consisted of one chromatographic column and four tanks, instead of the four columns in the four-zone SMB (1-1-1-1), and has been used for the separation of two amino acids, phenylalanine and tryptophan, using an ion exchange resin. The operating parameters for the one-column process and four-zone SMB were obtained from equilibrium theory. Computer simulations were used to compare the performances of the new one column process to that of the general four-zone SMB, using Aspen $Chromatography^{TM}$ v 11.1. The differences between the one-column and SMB processes in terms of the purities and yields of phenylalanine and tryptophan were less than 4 and about $6\%$, respectively. The lower purities of the one-column process were due to the loss of the developed concentration profiles in the column when the liquid was stored in tanks. The one-column process gave great flexibility, and would be useful for reconstructing an existing conventional chromatography process to one of a SMB.

• Journal of Microbiology
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• 제34권1호
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• pp.90-94
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• 1996

The carboxymethyl cellulase (CMCase) was purified from the induced culture filtrate of hybrid TAPW15703 between Aspergillus niger and penicillium verruculosum made by nuclear transfer. The enzyme was purified 80 fold with an overall yield 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and DEAE-ion exchange column chromatography. The molecular weight of the CMCase has estimated to be 32,000 daltons on SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel permeation chromatography. The purified enzyme functions optimally at pH 4.0 and 4$0^{\circ}C$ The Km value for carbosymethyl cellulose was 68 mM. The enzyme activity was increased by the presence of $Mg^{2+}$and Mn$^{2+}$.

• Journal of Applied Biological Chemistry
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• 제55권1호
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• pp.13-17
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• 2012

In order to enzymatically produce chitooligosaccharide using the crude enzyme preparation from Bacillus cereus D-11, we first studied the optimal reaction conditions. It was found that the optimal temperature for hydrolysis of chitosan was $55^{\circ}C$. The ratio of enzyme/substrate should not be lower than 0.13 U/mg in the reaction mixture. The enzyme activity was stable below $50^{\circ}C$. The products of enzymatic reaction were analyzed by both thin layer chromatography and high performance liquid chromatography. Under the appropriate condition, chitosan was hydrolyzed using the enzyme preparation. The resulting chitooligosaccharides were purified and separated by Dowex ($H^+$) ion exchange chromatography. From 4 g soluble chitosan, 0.95 g $(GlcN)_2$, 1.43 g $(GlcN)_3$, and 1.18 g $(GlcN)_4$ were recovered.

• 미생물학회지
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• 제26권4호
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• pp.324-331
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• 1988

Pleuratus ostreatus로부터 ascorbate oxidizing enzyme을 황산암모늄 침전, preparative polyacrylamide gel 전기영동, DEAE Sepharose CL-6B 이온교환크로마토그라피, Sephadex G-150 gel 여과크로마토그라피의 단계를 거쳐 순수분리 하였다. 이 효소의 분자량은 gel 여과크로마토그라피에 의하여 140,000 정도로, 효소의 소단위 분자량은 SDS-polyacrylamide gel 전기영동에 의하여 66,000 정도로 추정되었다. Isoelectric focusing에 의하여 이 효소는 6.0의 등전점을 갖는 것으로 밝혀졌고, 최적반응온도는 $85^{\circ}C$ 정도, 최적반응 pH는 5.2 정도인 것으로 나타났다. 본 효소는 L-ascorbic acid와 D-isoascorbic acid에 대하여 동일한 친화도를 갖는 것으로 보이며, Km값은 두가지 기질에 대해 모두 2.2µM 이였다.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.854-860
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• 1999

Superoxide dismutase (SOD) was purified to homogeneity from fruiting bodies of edible mushroom, Lentinus edodes, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose FF ion-exchange chromatography, Sephacryl S-200 gel filtration chromatography, and preparative PAGE. The molecular weight of the purified enzyme was estimated to be approximately 54 kDa by gel filtration chromatography, and the enzyme was shown to be consisted of two identical subunits of molecular weight 27 kDa by SDS-PAGE. The isoelectric point of the enzyme was 4.9 as determined by isoelectric focusing. The enzyme had optimal pH and temperature of pH 8.0 and $20^{\circ}C$, respectively. The activity of the enzyme was inhibited by hydrogen peroxide, but inhibited less by cyanide and azide. The native enzyme was found to contain 0.89g-atom of iron, 0.75g-atom of zinc, and 0.46g-atom of copper per mol of enzyme. Analysis of amino acids composition revealed that the SOD from L. edodes contained a relatively large amount of glutamic acid/glutamine, proline, cysteine, isoleucine, and leucine, but only a small amount of aspartic acid/asparagine, tyrosine, and tryptophan when compared to the other iron-containing SODs.

• KSBB Journal
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• 제13권1호
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• pp.77-82
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• 1998

미생물 효소에 의해 생산된 생산모액 내의 글루타치온(L-${\gamma}$-glutamyl-L-cysteinylglycine, GSH)을 액체크로마토그래피를 이용하여 분리하였다. GSH와 결합하는 수지를 선택하기 위해 여러 수지와 GSH 수용액을 사용하여 회분식 흡착실험을 한 결과, pH 8.0에서 음이온 교환수지인 Q-sepharose와 QAE- sephadex에 GSH가 결합하였으나, QAE-sephadex는 수지와 결합된 GSH를 이탈시키기 위해 사용된 salt에 의해 부피가 줄어들어 부적합하였다. GSH 분리를 위한 기초실험을 위하여 GSH, cysteine, glutamate, glycine, $\gamma$-glutamylcysteine, ATP, glucose의 혼합액에서 GSH와 $\gamma$-glutamylcysteine를 다른 물질로부터 1차 분리할 수 있었다. NaCl의 농도를 조절하여 두 물질이 중첩되는 현상을 제거하여 분리하고자 하였으며, GSH의 tailing현상을 줄이도록 노력하였다. NaCl(35mM)을 용해시킨 Tris buffer를 사용함으로써 두 물질의 분리가 가능하였고, 생산모액을 사용하여 실험한 결과, 혼합 시료와 유사한 분리결과를 얻을 수 있었다. Standard solution에서의 GSH 분리결과 72.6%의 회수율을 보였으며, 생산 모액에서는 84.4%의 회수율을 각각 보였다.

• 생명과학회지
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• 제12권5호
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• pp.528-534
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• 2002

우리나라에서 식용으로 뿐만아니라 한방재료로 널리 사용되고 있는 녹두(vigna radiata L. wilczek) 로부터 trypsin inhibitor (Mung bean trypsin inhibitor, MBTI)를 분리정제하여 그 특성을 조사하였다 또한 병태동물모델 즉, septic shock induced guinea pig model을 이용하여 MBTI의 약물학적 효과를 평가하였다. MBTI의 분리 및 정제과정은 Sephadex C-50 chromatography, DEAE-celluloseion exchange chromatography 및 trypsin affinity column 을 차례로 이용하였다. 정제한 MBTI는 전기영동 및 아미노산 서열분석결과 분자량 약 8,000 Da 의 BBI-type (Bowman-birk inhibitor type)임을 알 수 있었으며 이들의 생화학적 특성을 구명하였다. 또한 pseudomonal elastase로 유도된 septic shock guinea pig model에서 MBTI 10 mg/kg를 전처치한 결과 hypotention shock 유발이 억제됨을 알 수 있었다.

• FOCUS: LIFE SCIENCE
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• 제1호
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• pp.1.1-1.4
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• 2024

This article provides comprehensive guidance on the maintenance, cleaning, regeneration, and storage of silica-based HPLC (High-Performance Liquid Chromatography) columns. The general considerations emphasize the importance of using in-line filters and guard cartridges to protect columns from blockage and irreversible sample adsorption. While these measures help, contamination by strongly adsorbed sample components can still occur over time, leading to an increase in back pressure, loss of efficiency, and other issues. To maximize column lifetime, especially with UHPLC (Ultra-High Performance Liquid Chromatography) columns, it is advisable to use ultra-pure solvents, freshly prepared aqueous mobile phases, and to filter all samples, standards, and mobile phases. Additionally, an in-line filter system and sample clean-up on dirty samples are recommended. However, in cases of irreversible compound adsorption or column voiding, regeneration may not be possible. The document also provides specific recommendations for column cleaning procedures, including the flushing procedures for various types of columns such as reversed phase, unbonded silica, bonded normal phase, anion exchange, cation exchange, and size exclusion columns for proteins. The flushing procedures involve using specific solvents in a series to clean and regenerate the columns. It is emphasized that the flow rate during flushing should not exceed the specified limit for the particular column, and the last solvent used should be compatible with the mobile phase. Furthermore, the article outlines the storage conditions for silica based HPLC columns, highlighting the impact of storage conditions on the column's lifetime. It is recommended to flush all buffers, salts, and ion-pairing reagents from the column before storage. The storage solvent should ideally match the one used in the initial column test chromatogram provided by the manufacturer, and column end plugs should be fitted to prevent solvent evaporation and drying out of the packing bed.

• 한국식품영양과학회지
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• 제25권4호
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• pp.701-707
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• 1996

Aspergillus niger SFN-416으로 부터 생성한 xylanase I를 분리.정제하여 특성을 조사하였다. Aspergillus niger SFN-416의 배양액을 ethanol(70%) 침전, $(NH_4)_2-SO_4$(30~90%) 침전, Sephadex G-100 chromatography 및 DEAE-Sephacel ion chromatography 등의 정제과정을 거친 결과, 10.2배 정제되었고, 정제효소의 최적 활성온도는 $50^{\circ}C$ 였다.최적 pH는 3.5이었고, pH 안정성은 6.0 이상에서 활성이 급격히 감소하였다. 또한 금속이온에 대한 효소의 활성은 대부분 억제를 보였고, 특히 $Hg^{2+}$는 18.5%로 가장 낮은 상대활성을 보였지만, $Fe^{2+}$는 117.0%, $Mn^{2+}$는 129.9%로 오히려 효소활성이 증가되었다. 정제 효소의 분자량은 SDS-PAGE에 의하여 31,000 daltons이 었으며, 유기용매에 대한 활성과 안정성은 10%의 methanol, ethanol, isopropanol 및 1-butanol 대하여 모두 낮은 활성을 나타내어 유기용매에는 안정하지 않는 것으로 생각된다.