Abstract: Choleragenoid was obtained in pure form by ultra-filteration and fractionation on cationexchange resin-phospho-cellulose column. The choleragenoid was highly pure as judged by the electrophoresis of isoelect...Abstract: Choleragenoid was obtained in pure form by ultra-filteration and fractionation on cationexchange resin-phospho-cellulose column. The choleragenoid was highly pure as judged by the electrophoresis of isoelectric focusing,immunization and SDS-gel electrophoresis.The results of test are thesame as that of the standard choleragenoid.Keywoeds:choleragenoid; vibrio cholerae; purification;ion-exchange; chromatography展开更多
The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent th...The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the Chromatographie condition, several denatured proteins can be refolded and separated simultaneously in a single Chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative scales. That unit for the simultaneous renaturation and purification of proteins (USRPP) had the following four functions: to completely remove denaturant, to renature proteins, to separate renatured proteins from impurities, and to easily recycle waste denaturant. The efficiencies of refolding and purification of proteins by the USRPP are almost comparable to a usual long Chromatographie column in laboratory. In preparative scale, USRPP can be easily, rapidly, and economically applied requiring a low pressure gradient. As an example, recombinant human interferon-γ is employed to elucidate the application of the preparative USRPP.展开更多
Bacteriocins are a large group of chromosome or plasmid-encoded and ribosomally synthesized low-molecular-weight (2 to 6 kDa) antimicrobial and amphiphilous peptides produced by Gr+ or Gr- bacteria [1]. Their low toxi...Bacteriocins are a large group of chromosome or plasmid-encoded and ribosomally synthesized low-molecular-weight (2 to 6 kDa) antimicrobial and amphiphilous peptides produced by Gr+ or Gr- bacteria [1]. Their low toxicity as well as the absence of allergenicity and reactogenicity is confirmed by testing selected bacteriocins [2] [3]. Bacteriocins can be widely used as preservatives and antibiotic alternatives in medicine. Nisin, a Streptococcus lactis-derived bacteriocin, has been in practice in food industry for a long time. A relevant product contains about 2.5% of nisin. For medical use (e.g., when injected into the blood stream), highly purified drugs are required. However, the yield of bacteriocins accounts for no more than a few percents from the total activity in the culture liquid. In this paper, we propose methods (by example of two B. subtilis strains), allowing to increase the yield up to ~80%. It is believed that other bacteriocins may be purified by these methods and with the same yield.展开更多
Acetylcholinesterase （ACHE） plays an important role in enzyme-based detection of pesticides in the environment. In this paper, ACHE from the Triton X-100 extract of brain tissues of Oreochromis aurea was purified by...Acetylcholinesterase （ACHE） plays an important role in enzyme-based detection of pesticides in the environment. In this paper, ACHE from the Triton X-100 extract of brain tissues of Oreochromis aurea was purified by （NH4）2SO4 fractional precipitation, Sephadex G-100 gel filtration, and DEAE-cellulose ion exchange chromatography. Certain biochemical characterizations of the purified enzyme and inhibition of pesticides on the enzyme were also studied. The specific activity of this purified enzyme was 20.628 U/mg protein, fold of purification was 139, and recovery was 22.1%. The optimal temperature of this enzyme was between 35-40 ℃, and optimal pH was between 7.5-8.0. The Michaelis constant （Kin） for acetylthiocholine iodide was 0.183 mmol/L. The enzyme activity was inhibited by excess substrate, and optimal substrate concentration was 6 mmol/L. Four pesticides （dichlorvos, phoxim, triazophos, and methomyl） exhibited strong inhibitions on this enzyme with IC50 less than 5 μg/mL. This study suggests that Oreochromis aurea （tilapia） could be a good enzyme source for pesticide monitoring in water environments.展开更多
疏水层析是分离纯化蛋白质和多肽等生物大分子的常用方法,采用Octyl Se pharose FF疏水层析填料对好食脉孢霉(N.sitophila)发酵产生的纤溶酶进行了初步分离,比较了样品初始盐浓度、缓冲液pH值和洗脱方式对分离效果的影响。实验结果表明...疏水层析是分离纯化蛋白质和多肽等生物大分子的常用方法,采用Octyl Se pharose FF疏水层析填料对好食脉孢霉(N.sitophila)发酵产生的纤溶酶进行了初步分离,比较了样品初始盐浓度、缓冲液pH值和洗脱方式对分离效果的影响。实验结果表明,采用样品初始盐浓度为40%、缓冲液pH值为7.4和步阶式梯度洗脱方式对样品分离效果较好,经测定样品中有3种疏水性不同的纤溶活性组分,其中活性组分Ⅲ经Octyl Sepharose FF疏水层析分离后比活力为855.56U/mg,总纯化倍数为7.12倍。展开更多
文摘Abstract: Choleragenoid was obtained in pure form by ultra-filteration and fractionation on cationexchange resin-phospho-cellulose column. The choleragenoid was highly pure as judged by the electrophoresis of isoelectric focusing,immunization and SDS-gel electrophoresis.The results of test are thesame as that of the standard choleragenoid.Keywoeds:choleragenoid; vibrio cholerae; purification;ion-exchange; chromatography
基金This work was supposed by the National Natural ScienceFoundation of China (Grant Nos.39880003 and 20175016).
文摘The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the Chromatographie condition, several denatured proteins can be refolded and separated simultaneously in a single Chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative scales. That unit for the simultaneous renaturation and purification of proteins (USRPP) had the following four functions: to completely remove denaturant, to renature proteins, to separate renatured proteins from impurities, and to easily recycle waste denaturant. The efficiencies of refolding and purification of proteins by the USRPP are almost comparable to a usual long Chromatographie column in laboratory. In preparative scale, USRPP can be easily, rapidly, and economically applied requiring a low pressure gradient. As an example, recombinant human interferon-γ is employed to elucidate the application of the preparative USRPP.
文摘Bacteriocins are a large group of chromosome or plasmid-encoded and ribosomally synthesized low-molecular-weight (2 to 6 kDa) antimicrobial and amphiphilous peptides produced by Gr+ or Gr- bacteria [1]. Their low toxicity as well as the absence of allergenicity and reactogenicity is confirmed by testing selected bacteriocins [2] [3]. Bacteriocins can be widely used as preservatives and antibiotic alternatives in medicine. Nisin, a Streptococcus lactis-derived bacteriocin, has been in practice in food industry for a long time. A relevant product contains about 2.5% of nisin. For medical use (e.g., when injected into the blood stream), highly purified drugs are required. However, the yield of bacteriocins accounts for no more than a few percents from the total activity in the culture liquid. In this paper, we propose methods (by example of two B. subtilis strains), allowing to increase the yield up to ~80%. It is believed that other bacteriocins may be purified by these methods and with the same yield.
基金supported by Huizhou Science and Technology Planning Project Foundation (Grant No.2008P60)
文摘Acetylcholinesterase （ACHE） plays an important role in enzyme-based detection of pesticides in the environment. In this paper, ACHE from the Triton X-100 extract of brain tissues of Oreochromis aurea was purified by （NH4）2SO4 fractional precipitation, Sephadex G-100 gel filtration, and DEAE-cellulose ion exchange chromatography. Certain biochemical characterizations of the purified enzyme and inhibition of pesticides on the enzyme were also studied. The specific activity of this purified enzyme was 20.628 U/mg protein, fold of purification was 139, and recovery was 22.1%. The optimal temperature of this enzyme was between 35-40 ℃, and optimal pH was between 7.5-8.0. The Michaelis constant （Kin） for acetylthiocholine iodide was 0.183 mmol/L. The enzyme activity was inhibited by excess substrate, and optimal substrate concentration was 6 mmol/L. Four pesticides （dichlorvos, phoxim, triazophos, and methomyl） exhibited strong inhibitions on this enzyme with IC50 less than 5 μg/mL. This study suggests that Oreochromis aurea （tilapia） could be a good enzyme source for pesticide monitoring in water environments.
