乳链菌肽的分离纯化和部分生物学性质

用乳酸链球菌ＳＭ５２６进行乳链菌肽的发酵生产,产量为４０～５０ｍｇ／Ｌ,经中空纤维超滤器超滤,非极性大孔树脂ＸＡＤ－２层析,ＣＭ－ＳｅｐｈａｄｅｘＣ－２５层析和ＳｅｐｈａｄｅｘＧ－５０层析纯化了该肽。ＳＤＳ－ＰＡＧＥ表达达均一,ＲＰ－ＨＰＬＣ表明其纯度不低于９５％,ＳＤＳ－ＰＡＧＥ测其Ｍｒ约为３６００,用ＩＥＦ测其等电点为９．５,酸性条件下稳定且抗热,胰蛋白酶,胃蛋白酶和木瓜蛋白酶不敏感,但对α－     

与光系统Ⅱ颗粒结合的对DTT敏感的蛋白酶的纯化和性质

菠菜光系统Ⅱ颗粒的１ｍｏｌ／ＬＮａＣｌ抽提液经ｂｕｔｙｌ－Ｔｏｙｏｐｅａｒｌ６５０Ｍ疏水层析和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析分离得一种对ＤＴＴ敏感的蛋白酶。用ＳＤＳ－ＰＡＧＥ和Ｓｕｐｅｒｏｓｅ１２凝胶过滤测得该酶的分子量为３７ｋＤ,其为单体酶。该酶可降解１８ｋＤ和２４ｋＤ水溶性蛋白质,分别产生１３．２ｋＤ、１２ｋＤ、１０．５ｋＤ和２３ｋＤ、２２ｋＤ、２０ｋＤ片段,最适ｐＨ为８．０,对ＮａＣｌ不敏感,对ＤＴＴ和β－Ｍｅ敏感。抑制实验表明该酶不属丝氨酸类蛋白酶。     

湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析的步骤,从湖南产尖吻蝮蛇毒中纯化出两个出血毒素。ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸分别占２３％和２４％。ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具     

番茄叶片胞外β－半乳糖苷酶的纯化和性质

健康或系统感染ＴＭＶ的番茄叶片胞外都存在高比活可溶性β－半乳糖苷酶。系统感染ＴＭＶ的番茄叶胞外提取液经冰冻干燥浓缩、－２０℃丙酮沉淀、ＣＭ－ＳｅｐｈａｄｅｘＣ－２５阳离子交换层析、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５阴离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶层析纯化．获得ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均一的β－半乳糖苷酶。该酶的分子量为７４ｋＤ．酶蛋白带能被过碘酸－Ｓｃｈｉｆｆ试剂染成桃红色,属糖蛋白。β－半乳精苷酶无论在健康或系统感染ＴＭＶ的番茄叶中,均为主要的胞外蛋白组分之一。     

人胎肺细胞的条件化培养液中两种类型纤溶酶原活化物的分离

用正常人胎肺细胞体外培养,从其培养液中分离纤溶酶原活化物（ＰＡ）,通过ＣＭ－ＳｅｐｈａｄｅｘＣ－５０层析,硫酸铵沉淀和Ｆｉｂｒｉｎ－Ｓｅｐｈａｒｏｓｅ,Ｌｙｓｉｎｅ－Ｓｅｐｈａｒｏｓｅ亲和层析及ＳｅｐｈａｄｅｘＧ－５０凝胶过滤等步骤,从１０．５ｌ条件培养液中分离纯化得到两种类型的纤溶酶原活化物,ｔ－ＰＡ９０μｇ,ｕ－ＰＡ８００μｇ．在还原条件下ＳＤＳ－ＰＡＧＥ均显示单带,分子量ｔ－ＰＡ为７２ｋＤ,ｕ－ＰＡ为５４ｋＤ,纤溶比活分别为１５６０００ＩＵ／ｍｇ蛋白和１０６０００ＩＵ／ｍｇ蛋白．     

海枣曲霉β—木糖苷酶的提纯和性质

从海枣曲霉麦麸培养物抽提液中,通过聚乙二醇６０００－磷酸钾缓冲液双水相分离,相继用Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－５０离子交换柱层析、羟基磷石吸附层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－５０离子交换层析、ＳＥ－Ｓｅｐｈａｄｅｘ Ｃ－５０离子交换层析以及最适温度为６５℃,在ｐＨ３．５－６．５之间稳定,酶保温３０分钟时的半失活温度（ｔ１／２）为６８℃。酶的分子量为９     

番茄叶片胞外β—半乳糖苷酶的纯化和性质

健康或系统感染ＴＭＶ的番茄叶片胞外都存在高比活可溶性β－半乳糖苷酶。系统感染ＴＭＶ的番匣叶胞外提取液经冰冻干燥浓缩－２０℃丙酮沉淀、ＣＭ－ＳｅｐｈａｄｅｘＣ－２５阳离子交换层析、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５阴离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶层析纯化,获得ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均一的β－半乳糖苷酶。该酶的分子量为７４ｋＤ,酶蛋白带能被过碘酸－Ｓｃｈｉｆｆ试剂染成档红色,属糖蛋白     

湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－ＳｅｐｈａｄｅｘＣ－２５离子交换层析的步骤,从湖南产尖吻蝮（Ｄｉｅｎａｇｋｉｓｔｒｏｄｏｎａｃｕｔｕｓ）蛇毒中纯化出两个出血毒素（ＤａＨＴ－１和ＤａＨＴ－２）．ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸（Ａｓｘ,Ｇｌｘ）分别占２３％和２４％,ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具蛋白水解酶活性,无对ＴＡＭＥ,ＢＡＥＥ的水解活性和ＰＬＡ２酶活性．两者的蛋白水解酶活力与出血活性并非正相关．ＤａＨＴ－１和ＤａＨＴ－２的最适温度分别为３５℃和４０℃,最适ｐＨ为６－９,对热均不稳定,温度高于６０℃活性完全丧失。金属离子的分析显示每摩尔毒素蛋白约含０．５ｍｏｌ的Ｚｎ,１ｍｏｌ的Ｃａ,较多的Ｎａ、Ｋ、Ｍｇ,不含Ｃｏ。     

系统感染TMV的番茄叶胞外β-1,3-葡聚糖酶

系统感染ＴＭＶ （ｔｏｂａｃｃｏ ｍ ｏｓａｉｃ ｖｉｒｕｓ）的番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）叶胞外蛋白提取液经冰冻干燥浓缩、－ ２０℃丙酮沉淀、ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶层析纯化,获得ＰＡＧＥ均一的β－１,３－葡聚糖酶．ＳＤＳ－ＰＡＧＥ证明,它包含分子量为３６ ｋＤ 和２７ ｋＤ的两个同工酶．以昆布多糖为底物,酶的最适ｐＨ 在４．８—５．２之间,在ｐＨ ４—８稳定;酶的最适温度在３０—４０℃之间,在４０℃保温１ｈ 后酶活性不变;Ｋｍ 值为９．２ ｍ ｇ／ｍ Ｌ．在系统感染ＴＭＶ 的番茄叶胞外蛋白提取液中,有分子量为２２ ｋＤ、２７ ｋＤ和３６ ｋＤ的３个β－１,３－葡聚糖酶同工酶     

甲基单胞菌Methylomonassp.GYJ3中甲烷单加氧酶还原酶组分的…

Ｍｅｙｌｏｍｏｎａｓ ｓｐ．ＧＹＪ３菌的甲烷单加氧酶（ＭＭＯ）粗酶提取液经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ阴离子交换层析,Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤层析和ＤＥＡＥ－ＴＳＫｇｅｌ ＨＰＬＣ分离纯化出ＭＭＯ还原酶组分,经ＨＰＬＣ分析,纯度大于９５％,纯化倍数为４．４,加入至ＭＭＯ羟基化酶和调节蛋白Ｂ的体系中表现比活为２２８ｎｍｏｌ环氧丙烷每分钟毫克蛋白,ＳＤＳ－ＰＡＧＥ电泳表明的酶由     

Quantification of nisin in flow-injection immunoassay systems

A monoclonal-antibody-based, sequential competitive-flow-injection immunoassay system in expanded-bed mode has been developed for the determination of nisin. The system allows the determination of nisin in the presence of suspended particles without any significant interference, illustrating its potential for on-line monitoring of fermentation processes or the analysis of food matrices. The dose response range of the system when operated in expanded-bed mode was 6-90 microM. The detection limit under packed-bed conditions was 3 microM. The results correlated well with the results from conventional ELISA in the analysis of samples of processed cheese. When milk samples, fermentation samples and buffer were spiked with nisin, the mean recoveries were 86% for milk samples, 96% for fermentation samples and 98% for buffer solution.     

乳链菌肽高产菌株的选育及其基因定位

以乳酸乳酸球菌7962为原始菌株,用紫外线、LiCl、(60)~Co及8-MOP+NUV等多种理化诱变剂对其进行诱变处理,获得一株乳链菌肽高产突变株AL2。其效价稳定在2300～2500Iu/ml。经DNA杂交证实,编码乳链菌肽的前体基因位于染色体上,其遗传性状是稳定的。毒理试验表明AL2及其产物属于实际无毒类物质。     

Sensitivities of Germinating Spores and Carvacrol-Adapted Vegetative Cells and Spores of Bacillus cereus to Nisin and Pulsed-Electric-Field Treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

Sensitivities of germinating spores and carvacrol-adapted vegetative cells and spores of Bacillus cereus to nisin and pulsed-electric-field treatment

Treatment of Bacillus cereus spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.     

The lantibiotic nisin, a special case or not?

Nisin is a 34-residue-long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The presence of dehydrated residues and lanthionine rings (thioether bonds) in nisin, imposing structural restrains on the peptide, make it an interesting case for studying the mode of action. In addition, the relatively high activity (nM range) of nisin against Gram-positive bacteria indicates that nisin may be a special case in the large family of pore-forming peptides antibiotics. In this review, we attempted to dissect the mode of action of nisin concentrating on studies that used model membranes or biological membranes. The picture that emerges suggests that in model membrane systems, composed of only phospholipids, nisin behaves similar to the antimicrobial peptide magainin, albeit with an activity that is much lower as compared to its activity towards biological membranes. This difference can be contributed to a missing factor which nisin needs for its high activity. Novel results have identified the factor as Lipid II, a precursor in the bacterial cell wall synthesis. The special high affinity interaction of nisin with Lipid II resulting in high activity and the active role of Lipid II in the pore-formation process make nisin a special case.     

DETECTION AND DEMONSTRATION OF INHIBITORY ACTIVITIES OF BACTERIOCINS BY ISOELECTRIC FOCUSING

A technique utilizing isoelectric focusing (IEF) was developed and compared with a modified Bhunia's sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method for directly detecting antimicrobial activities of inhibitory peptides or proteins (bacteriocins). In IEF, the gel containing separated peptide or protein bands was directly overlaid with indicator bacteria without any fixation or rinsing as done in the SDS-PAGE method. The IEF gave clear zones of inhibition surrounding inhibitory bands. Bhunia's method was modified to reduce the time for fixation and rinsing, and a 15-min fixation followed by 2-h rinsing with dd H₂O was determined to be necessary for a detection similar to the IEF. In addition, results of this study showed that denaturation of bacteriocins and, subsequently, failure to detect the presence of a bacteriocin could occur in the SDS-PAGE analysis while little denaturation was observed in the IEF assay. Therefore, the IEF assay developed in this study was more rapid and less destructive as compared with the SDS-PAGE method.     

Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol

AIMS: The effect of combinations of nisin and ethanol on the survival of Listeria monocytogenes was investigated. METHODS AND RESULTS: Killing by nisin was enhanced during simultaneous exposure to ethanol (2-7% v/v). For example, while 10 IU ml(-1) nisin reduced viability by 1 log unit in 20 min, a combination of this antimicrobial peptide and 5% ethanol, reduced numbers of surviving cells by 3 log units. Increasing the concentrations of either ethanol (2-7%) or nisin (10-50 IU ml(-1)) led to increased cell death with synergy being demonstrated for all combinations tested and at a range of temperatures from 5 to 37 degrees C. CONCLUSIONS: Ethanol can act synergistically with nisin to reduce the survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of ethanol and nisin may be feasible as an effective way of controlling this pathogen in the food processing environment.     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

Carbon dioxide and nisin act synergistically on Listeria monocytogenes

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis(r)) cells grown in broth at 4 degrees C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis(r) cells. Wild-type cells grown in 100% CO(2) were two to five times longer than cells grown in air. Nisin (2.5 microg/ml) did not decrease the viability of Nis(r) cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO(2). There was a quantifiable synergistic action between nisin and CO(2) in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 microg of nisin per ml increased from 3.1 to 12.5 microg/ml over 35 days, but this increase was markedly delayed for cultures in CO(2). This synergism between nisin and CO(2) was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO(2) and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO(2) atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4 degrees C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30 degrees C. Cells grown in the presence of 100% CO(2) and those grown at 4 degrees C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO(2) is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.