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生物技术通报 1994年第5期 104-105页

作者：田桂英

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Purification and some characteristics of enterocin ON-157, a bacteriocin produced by Enterococcus faecium NIAI 157

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第1期88卷 81-89页

作者： Ohmomo, S Murata, S Katayama, N Nitisinprasart, S Kobayashi, M Nakajima, T Yajima, M Nakanishi, K Minist Agr Forestry & Fisheries Natl Inst Anim Ind Tsukuba Ibaraki 3050901 Japan Shizuoka Prefecture Toubu Livestock Hlth & Hyg Of Shizuoka Japan Tokyo Univ Agr Tokyo Japan Kasetsart Univ Fac Agroind Bangkok Thailand Asahi Chem Ind Co Ltd Chuo Ku Tokyo Japan

Bacteriocin-like activity (BLA) was screened in 690 strains of lactic acid bacteria isolated from plant materials such as silages and fermented vegetables. Among them, a strain identified as Enterococcus faecium NIAI 157 showed a clear BLA against the indicator strain, Ent. faecium IFO 13712. The proteinaceous nature and antimicrobial activity against closely related species strongly indicated that this BLA was a bacteriocin and was designated enterocin ON-157. The bacteriocin activity of this strain was extracellularly produced in the logarithmic growth phase in MRS broth and purified by ultrafiltration, ammonium sulphate precipitation and cation-exchange chromatography. Purified enterocin ON-157 had a molecular weight of approximately 2500 Da in SDS-PAGE analysis and was easily inhibited by treatment with alpha-amylase and proteolytic enzymes. Enterocin ON-157 had a bactericidal mode of action and inhibited the growth of the enterococci, Lactobacillus sake and Listeria monocytogenes. Enterococcus faecium NIAI 157 harboured two plasmids, 49.0 kb and 43.7 kb, and a variant missing a larger plasmid by curing with novobiocin lost the bactriocin activity.

关键词：抗菌药/药理学细菌素类/分离和提纯细菌素类/药理学电泳琼脂凝胶电泳聚丙烯酰氨凝胶屎肠球菌/化学屎肠球菌/药物作用屎肠球菌/代谢乳杆菌属/药物作用乳杆菌属/生长和发育单核细胞增生利斯特菌/药物作用单核细胞增生利斯特菌/生长和发育微生物敏感性试验乳链球菌素/药理学质粒

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Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第2期88卷 349-357页

作者： Jennes, W Dicks, LMT Verwoerd, DJ Univ Stellenbosch Dept Microbiol ZA-7600 Stellenbosch South Africa Onderstepoort Vet Inst Onderstepoort South Africa

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml(-1), but increased further to 1600 AU ml(-1) in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.

关键词：细菌素类/生物合成细菌素类/分离和提纯细菌素类/药理学肠球菌属/生长和发育肠球菌属/分离和提纯肠球菌属/代谢革兰氏阳性菌/药物作用肠/微生物学微生物敏感性试验鸵形目/微生物学动物

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Triton X-114 phase partitioning for the isolation of a pediocin-like bacteriocin from Carnobacterium divergens

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LETTERS IN APPLIED MICROBIOLOGY 2000年第1期30卷 42-46页

作者： Métivier, A Boyaval, P Duffes, F Dousset, X Compoint, JP Marion, D INRA Lab Biochim & Technol Prot Nantes 03 France INRA Rech Technol Laitiere Lab F-35042 Rennes France ENITIAA Microbiol Lab Nantes France

A new procedure combining Triton X-114 phase partitioning and cation exchange chromatography was developed to purify a bacteriocin from a complex culture medium. This pediocin-like bacteriocin, secreted by Carnobacterium divergens and named divercin V41, was entirely recovered in the lower detergent-rich phase whereas all other substances (compounds from culture medium, bacterial metabolites) remained in the upper detergent-poor phase. Subsequent cation-exchange chromatography of the TX-114-rich phase allowed recovery of the pure active bacteriocin and also detergent removing. This new purification method is versatile, fast (only two steps) and call be carried out on whole broth.

关键词：细菌/代谢细菌蛋白质类/分离和提纯细菌素类/分离和提纯色谱法高压液相色谱法离子交换培养基/化学洗涤剂聚乙烯二醇类

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Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第5期88卷 877-886页

作者： Zhu, WM Liu, W Wu, DQ Shandong Med Univ Dept Microbiol & Immunol Jinan 250012 Peoples R China Shandong Univ Dept Microbiol Jinan 250100 Peoples R China Peking Univ Dept Biochem & Mol Biol Beijing 100871 Peoples R China

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.

关键词：氨基酸类/分析细菌素类/拮抗剂和抑制剂细菌素类/分离和提纯细菌素类/药理学色谱法高压液相梭菌属/药物作用电泳聚丙烯酰氨凝胶肠球菌属/药物作用粪便/微生物学热温度氢离子浓度乳杆菌属/遗传学乳杆菌属/代谢利斯特菌属/药物作用分子量人类婴儿

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Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第9期65卷 3880-3887页

作者： Qi, FX Chen, P Caufield, PW Univ Alabama Birmingham Sch Dent Dept Oral Biol Birmingham AL 35294 USA

Previously, members of our group reported the isolation and characterization of mutacin II from Streptococcus mutans T8 and the genetic analyses of the mutacin II biosynthesis genes (J. Novak, P. W. Caufield, and E. J. Miller, J. Bacteriol. 176:4316-4320, 1994;F;Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:652-658, 1999;P. Chen, F. Qi, J. Novak, and P.W. Caufield, Appl. Environ. Microbiol. 65:1356-1360, 1999). In this study, we cloned and sequenced the mutacin III biosynthesis gene locus from a group III strain of S. mutans, UA787. DNA sequence analysis revealed eight open reading frames,which we designated mutR, -A, -A', -B, -C, -D, -P, and -T. MutR bears strong homology with MutR of mutacin II, while MutA, -B,-C, -D, -P, and -T are counterparts of proteins in the lantibiotic epidermin group. MutA' has 60% amino acid identity with MutA and therefore appears to be a duplicate of MutA. Insertional inactivation demonstrated that mutA is an essential gene for mutacin III production, while mutA' is not required. Mutacin III was purified to homogeneity by using reverse-phase high-pressure liquid chromatography. N-terminal peptide sequencing of the purified mutacin III determined mutA to be the structural gene for prepromutacin III. The molecular mass of the purified peptide was measured by laser disorption mass spectrophotometry and found to be 2,266.43 Da, consistent with our supposition that mutacin III has posttranslational modifications similar to those of the lantibiotic epidermin.

关键词：氨基酸序列细菌/药物作用细菌素类/生物合成细菌素类/遗传学细菌素类/分离和提纯细菌素类/药理学克隆分子培养基抗药性微生物基因细菌微生物敏感性试验分子序列数据序列分析 DNA 链球菌变异/遗传学链球菌变异/生长和发育链球菌变异/代谢

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Production of a nisin-like bacteriocin by Lactococcus lactis subsp lactis A164 isolated from Kimchi

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第4期88卷 563-571页

作者： Choi, HJ Cheigh, CI Kim, SB Pyun, YR Yonsei Univ Dept Biotechnol Seoul 120749 South Korea Yonsei Univ Bioprod Res Ctr Seoul 120749 South Korea

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha,beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.

关键词：细菌素类/生物合成细菌素类/分离和提纯细菌素类/药理学革兰氏阳性菌/药物作用氢离子浓度乳酸/代谢乳乳球菌/分离和提纯乳乳球菌/代谢乳链球菌素/生物合成乳链球菌素/分离和提纯乳链球菌素/药理学温度蔬菜/微生物学

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Purification and characterization of a bacteriocin produced by Lactobacillus acidolphilus IBB 801

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JOURNAL OF APPLIED MICROBIOLOGY 1999年第6期87卷 923-931页

作者： Zamfir, M Callewaert, R Cornea, PC Savu, L Vatafu, I De Vuyst, L Inst Biol Genet Engn Lab Bucharest 79651 Romania Free Univ Brussels Dept Appl Biol Sci Res Grp Ind Microbiol Fermentat Technol & Downstr Brussels Belgium

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6.5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 degrees C), and maintained in a wide pH range, The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v), Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.

关键词：细菌素类/化学细菌素类/分离和提纯细菌素类/药理学电泳聚丙烯酰氨凝胶大肠杆菌/药物作用氢离子浓度半数抑制浓度嗜酸乳杆菌/化学嗜酸乳杆菌/代谢微生物敏感性试验胃蛋白酶A/药理学沙门菌属/药物作用胰蛋白酶/药理学

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Efficient method for the detection of microbially-produced antibacterial substances from food systems

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第1期89卷 56-62页

作者： Morgan, SM Hickey, R Ross, RP Hill, C TEAGASC Dairy Prod Res Ctr Fermoy Cork Ireland Natl Univ Ireland Univ Coll Cork Dept Microbiol Cork Ireland Natl Univ Ireland Univ Coll Cork Natl Food Biotechnol Ctr Cork Ireland

A novel method for the isolation of microbially-derived inhibitory substances from food sources was developed. The method involves an enrichment step coupled to a killing assay which is initially carried out in multi-well plates. The technique has advantages in that large numbers of samples can be tested in parallel. The assay can be completed in less than 60 h and is more sensitive than direct plating due to the enrichment step. This novel screening approach was compared with the standard direct plating approach in an effort to identify the antimicrobial potential of a number of Kefir grains. Kefir grains were incubated in 10% reconstituted skim milk for 20 h at 32 degrees C to enable production of any potential biopreservatives. Following overnight incubation, fermentates were aliquoted into multi-well plates and a known number of indicator cells was added to each well. The fermentates were incubated for a further 20 h and counts were carried out to determine whether a reduction in indicator cell numbers had occurred. A reduction in cell-forming units indicated the presence of an inhibitory substance and these inhibitory fermentates were selected for further investigation. Using the protocol outlined, Kefir fermentates capable of inhibiting Listeria innocua DPC1770 and Escherichia roll O157:H45 were identified.

关键词：酸类/药理学细菌素类/分离和提纯细菌素类/药理学大肠杆菌O157/生长和发育发酵过氧化氢/药理学利斯特菌属/生长和发育微生物敏感性试验乳/微生物学动物

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Characterization and mechanism of action of cerein 7, a bacteriocin produced by Bacillus cereus Bc7

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第2期89卷 361-369页

作者： Oscáriz, JC Pisabarro, AG Univ Publ Navarra Dept Agr Prod Pamplona 31006 Spain

Cerein 7 is a peptidic antibiotic produced by Bacillus cereus Bc7 (CECT 5148) at the end of exponential growth but before sporulation onset. Cerein 7 has a broad spectrum of antibacterial activity against Gram-positive bacteria, but it is inactive against Gram-negative bacteria. The sequence of its amino-terminal end and its characteristics of hydrophobicity and molecular mass make cerein 7 unique among the bacteriocins produced by the soil bacterium B. cereus. In this paper a further characterization of cerein 7 is presented, it is shown that it can be classified as a Klaenhammer's class II bacteriocin and that its mode of action corresponds to that of a membrane-active compound.

关键词：蜡状芽孢杆菌/生长和发育蜡状芽孢杆菌/代谢细菌素类/分类细菌素类/分离和提纯细菌素类/代谢细菌素类/药理学溶菌作用集落计数微生物剂量效应关系药物革兰氏阳性菌/药物作用革兰氏阳性菌/生长和发育微生物敏感性试验

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