Purification and characterization of an arylamine N-acetyltransferase from Lactobacillus acidophilus.

N-acetyltransferase from Lactobacillus acidophilus was purified by ultrafiltration, DEAE-Sephacel, gel filtration chromatography on Sephadex G-100, and DEAE-5pw on high performance liquid chromatography, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (w/v) slab gel. The purified enzyme was thermostable at 37 degrees C for 1 h with a half-life of 32 min at 37 degrees C, and displayed optimum activity at 37 degrees C and pH 7.0. The K(m) and Vmax values for 2-aminofluorene were 0.842 mM and 2.406 nmol/min/mg protein, respectively. Among a series of divalent cations and salts, Zn2+, Ca2+, Fe2+, Mg2+, and Cu2+ were demonstrated to be the most potent inhibitors. The enzyme had a molecular mass of 44.9 kD. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited dose-, time-, and temperature-dependent inhibition effects. Preincubation of purified N-acetyltransferase with acetyl coenzyme A (AcCoA) provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial activity, and the first two are likely to reside on the AcCoA binding site, but the arginine residue may be located close to the AcCoA binding site. This report is the first demonstration of acetyl CoA:arylamine N-acetyltransferase in L. acidophilus.     

Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26

An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. Methylene-blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino-acid analysis indicated that this photooxidative loss of activity corresponds to the modification of one histidine residue per monomer of protein.     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Screening and selection of novel animal probiotics isolated from bovine chyme

Probiotics, gut-colonizing microorganisms capable of conferring a number of health benefits to their hosts, are highly desirable as animal feed supplements. Members of the Gram-positive genus Bacillus are often utilized as probiotics, since endospores formed by those bacteria render them highly resistant to environmental extremes and therefore capable of surviving gastrointestinal tract conditions. In this study, 84 distinct bacterial colonies were obtained from bovine chyme and 29 isolates were determined as Bacillus species. These isolates were principally screened for their antimicrobial activity against a group of two Gram-positive and four Gram-negative bacteria, including known human and animal pathogens such as Salmonella enterica, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Seven strains displaying strong antimicrobial activity against the test cohort were further evaluated for other properties desirable from animal probiotics, including high spore-forming capacity and adhesiveness, resistance to pH extremes and ability to form biofilms. The isolates were found to resist simulated gastrointestinal conditions and most of the antibiotics tested. In addition, plasmid presence was checked and cytotoxicity tests were performed to evaluate the potential risks of antibiotic resistance transfer and unintended pathogenic effects on host, respectively. We propose that the bacterial isolates are suitable for use as animal probiotics.     

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1_Lp and ram2_Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramA_La). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1_Lp, Ram2_Lp, and RamA_La were able to efficiently hydrolyze rutin and other rutinosides, while RamA_La was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1_Lp and RamA_La enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1_Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.Lactobacilli such as Lactobacillus plantarum have been used for centuries to ferment vegetables such as cabbage, cucumber, and soybean (34). Fruit pulps, for instance, those from tomato, have also been used as a substrate for lactobacilli for the production of probiotic juices (38). Recently, the full genomic sequences of several lactobacilli have become available (1, 22). A number of the plant-based substrates for lactobacilli are rich in rhamnose sugars, which are often conjugated to polyphenols, as in the case of cell wall components and certain flavonoid antioxidants. Utilization of these compounds by lactobacilli would involve α-l-rhamnosidases, which catalyze the hydrolytic release of rhamnose. Plant-pathogenic fungi such as Aspergillus species produce the rhamnosidases when cultured in the presence of naringin, a rhamnosilated flavonoid (24, 26). Bacteria such as Bacillus species have also been shown to use similar enzyme activities for metabolizing bacterial biofilms which contain rhamnose (17, 40).In food processing, rhamnosidases have been applied primarily for debittering of citrus juices. Part of the bitter taste of citrus is caused by naringin (Fig. ​(Fig.1),1), which loses its bitter taste upon removal of the rhamnose (32). More recently, application of rhamnosidases for improving the bioavailability of flavonoids has been described. Human intake of flavonoids has been associated with a reduced risk of coronary heart disease in epidemiological studies (19). Food flavonoids need to be absorbed efficiently from what we eat in order to execute any beneficial function. Absorption occurs primarily in the small intestine (12, 37). Unabsorbed flavonoids will arrive in the colon, where they will be catabolized by the microflora, which is then present in huge quantities. Therefore, it would be desirable for flavonoids to be consumed in a form that is already optimal for absorption in the small intestine prior to their potential degradation. For the flavonoid quercetin, it has been demonstrated that the presence of rhamnoside groups inhibits its absorption about fivefold (20). A number of flavonoids which are present in frequently consumed food commodities, such as tomato and citrus products, often carry rutinoside (6-β-l-rhamnosyl-d-glucose) or neohesperidoside (2-β-l-rhamnosyl-d-glucose) residues (Fig. ​(Fig.1).1). Therefore, removal of the rhamnose groups from such flavonoid rutinosides and neohesperidosides prior to consumption could enhance their intestinal absorption. With this aim, studies were recently carried out toward the application of fungal enzyme preparations as a potential means to selectively remove rhamnoside moieties (16, 30).Open in a separate windowFIG. 1.Chemical structures of rhamnose-containing flavonoids from plants. Relevant carbon atoms in glycoside moieties are numbered. (1) Rutin (quercetin-3-glucoside-1→6-rhamnoside); (2) narirutin (naringenin-7-glucoside-1→6-rhamnoside); (3) naringin (naringenin-7-glucoside-1→2-rhamnoside); (4) p-nitrophenol-rhamnose.In view of the frequent occurrence of lactobacilli on decaying plant material and fermented vegetable substrates, one could anticipate that their genomes carry one or more genes encoding enzymes capable of utilizing rhamnosilated compounds. In the work reported here, we describe the identification of three putative rhamnosidase genes in lactobacillus genomes. We expressed these genes in Escherichia coli and characterized their gene products. The activities of all three lactobacillus rhamnosidases on flavonoids naturally present in tomato pulp were then assessed. One of the L. plantarum genes, which encoded the enzyme with the highest activity and stability in E. coli, was then also expressed in Lactococcus lactis, with the aim of investigating the potential use of such a recombinant organism to improve the bioavailability of fruit flavonoids and thus their efficacy in common foodstuffs.     

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088.

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.     

Isolation and characterization of acidocin A and cloning of the bacteriocin gene from Lactobacillus acidophilus.

Acidocin A, a bacteriocin produced by Lactobacillus acidophilus TK9201, is active against closely related lactic acid bacteria and food-borne pathogens including Listeria monocytogenes. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential ion-exchange and reversed-phase chromatographies. The molecular mass was determined by high-performance liquid chromatography gel filtration to be 6,500 Da. The sequence of the first 16 amino acids of the N terminus was determined, and oligonucleotide probes based on this sequence were constructed to detect the acidocin A structural gene acdA. The probes hybridized to the 4.5-kb EcoRI fragment of a 45-kb plasmid, pLA9201, present in L. acidophilus TK9201, and the hybridizing region was further localized to the 0.9-kb KpnI-XbaI fragment. Analysis of the nucleotide sequence of this fragment revealed that acidocin A was synthesized as an 81-amino-acid precursor including a 23-amino-acid N-terminal extension. An additional open reading frame (ORF2) encoding a 55-amino-acid polypeptide was found downstream of and in the same operon as acdA. Transformants containing this ORF2 became resistant to acidocin A, suggesting that ORF2 encodes an immunity function for acidocin A. The 7.2-kb SacI-XbaI fragment containing the upstream region of acdA of pLA9201 was necessary for acidocin A expression in the acidocin A-deficient mutant, L. acidophilus TK9201-1, and other Lactobacillus strains.     

Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37 degrees C, respectively). The metal ions Cu(2+) and Fe(3+) (at a concentration of 5 mmol liter(-1)) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K(m) of 0.0953 mmol liter(-1) and a V(max) of 86.27 mmol liter(-1) min(-1) mg(-1) of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.     

Purification and partial characterization of ceruloplasmin from chicken serum

The preparation and properties of ceruloplasmin from chicken serum are described. Ethanol-CHCl3 was used to precipitate the crude protein, followed by adsorption and elution from DEAE-Sephadex. Further treatment with Sephadex G-200 and CM-Sephadex yielded an intensely blue protein judged 1572-fold purer than starting serum. epsilon-Aminocaproic acid (0.02 M) was present in all buffers and starting sera. Chicken ceruloplasmin appears to be a single polypeptide, apparent Mr 124,000, with an A610/A280 ratio of 0.07 and an absorption maximum at 602 nm. Hexose, hexosamine, and sialic acid accounted for 7.2% of the weight; copper represented 0.20%, which suggested four or five copper atoms per molecule. Chicken ceruloplasmin catalyzed the azide-sensitive oxidation of p-phenylenediamine (PPD) and N,N'-dimethyl-p-phenylenediamine (DPD), and showed ferroxidase activity similar to that of human ceruloplasmin. Its amino acid composition, although similar in many residues to human ceruloplasmin, was decidedly lower in methionine and tyrosine. The chicken protein had one-third the sialic acid content of human ceruloplasmin and showed immunochemical nonidentity with human ceruloplasmin.     

嗜酸乳酸杆菌ATCC 4356菌株异源二聚体b-半乳糖苷酶的克隆表达

嗜酸乳酸杆菌(Lactobacillus acidophilus)的异源二聚体β-半乳糖苷酶属于糖苷水解酶2家族,由两个部分重叠、协同翻译的基因编码(lacL和lacM).[目的]克隆表达该酶并测定其酶学特性.[方法]参照已全基因组测序的嗜酸乳酸杆菌NCFM菌株,以嗜酸乳酸杆菌ATCC4356菌株基因组为模板,将lacL的RBS到lacM的终止子之间的序列(2834 bp)克隆到pQE31质粒上,并电转化JM109菌株.以下列步骤纯化表达产物:硫酸铵分级沉淀、阴离子交换、亲和层析和凝胶排阻层析.以凝胶排阻层析测定纯化酶的天然分子量,以邻硝基苯基半乳糖为底物测定其酶学特性.[结果]实现了该酶在JM109菌株中的可溶性表达.其氨基酸序列有一处不同于嗜酸乳酸杆菌NCFM菌株,即其大亚基(LacL)的第512位氨基酸不是组氨酸而是精氨酸.纯化酶比活力为226 U/mg蛋白,天然分子量为96.3±4.6 kDa,最适pH为7,最适温度为49℃,Km和Vmax值分别是:2.18±0.12 mmol/L,273±5 U/mg蛋白.     

Molecular cloning and characterization of a bile salt hydrolase from Lactobacillus acidophilus PF01

Phenotypic screening for bile salt hydrolase (BSH) activity was performed on Lactobacillus acidophilus PF01 isolated from piglet feces. A gene encoding BSH was identified and cloned from the genomic library of L. acidophilus PF01. The bsh gene and surrounding regions were characterized by nucleotide sequence analysis and were found to contain a single open reading frame (ORF) of 951 nucleotides encoding a 316 amino acid protein. The potential bsh promoter region was located upstream of the start codon. The protein deduced from the complete ORF had high similarity with other BSHs, and four amino acid motifs located around the active site, FGRNXD, AGLNF, VLTNXP, and GXGXGXXGXPGD, were highly conserved. The bsh gene was cloned into the pET21b expression vector and expressed in Escherichia coli BLR(DE3) by induction with 0.1mM of isopropylthiogalactopyranoside. The BSH enzyme was purified with apparent homogeneity using a Ni2+-NTA agarose column and characterized. The overexpressed recombinant BSH enzyme of L. acidophilus PF01 exhibited hydrolase activity against tauroconjugated bile salts, but not glycoconjugated bile salts. It showed the highest activity against taurocholic acid. The maximum BSH activity occurred at approximately 40oC. The enzyme maintained approximately 70% of its maximum activity even at 60 degrees , whereas its activity rapidly decreased at below 37 degrees . The optimum pH was 6, and BSH activity was rapidly inactivated below pH 5 and above pH 7.     

Isolation and characterization of two bacteriocins of Lactobacillus acidophilus LF221

Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially Clostridium difficile. Received: 2 October 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998     

鸡源和猪源乳杆菌胆盐水解酶的表达及酶学性质比较

【目的】随着抗生素生长促进剂(AGPs)在动物饲料中逐步禁止使用,AGPs替代物的研究成为热点。由于胆盐水解酶(BSH)在脂类代谢中的关键作用,成为AGPs替代物研究的一个重要方向。在原核表达和纯化的基础上鉴定鸡源和猪源乳杆菌BSH在酶学性质方面的差异性。【方法】分别对鸡源胆盐水解酶(BSHc)和猪源胆盐水解酶(BSHp)基因进行原核表达和蛋白纯化,通过测定对6种甘氨结合胆盐和牛磺结合胆盐的水解效率获得两种酶的酶学动力学性质,进而测定了温度、pH和金属离子对酶活力的影响。【结果】BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc对甘氨结合胆盐的水解效率较BSHp稍高;BSHc和BSHp的最适酶解温度分别为45°C和42°C;BSHc和BSHp的最适反应pH分别为6.0和5.4;含有Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)的金属盐对BSHc和BSHp的酶活力均具有不同程度的抑制作用,特别是Cu~(2+)和Fe~(3+)抑制作用比较强;含有Na~+、K~+、Mg~(2+)和Ca2+的金属盐对BSHc和BSHp酶活力的抑制作用相对较弱或无抑制作用,但KIO3对BSHc和BSHp酶活力具有强抑制作用,KI和CaCl_2对BSHp酶活力也具有较强的抑制作用。【结论】原核表达和纯化的BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc的最适酶解温度和pH稍高于BSHp,Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)等金属离子对BSHc和BSHp酶活力具有明显抑制作用,试验结果为鉴定BSH抑制物进而研制AGPs替代物奠定了基础。     

Peptidoglycan Hydrolases as Species-Specific Markers to Differentiate Lactobacillus helveticus from Lactobacillus gallinarum and Other Closely Related Homofermentative Lactobacilli

We propose a new method that allows accurate discrimination of Lactobacillus helveticus from other closely related homofermentative lactobacilli, especially Lactobacillus gallinarum. This method is based on the amplification by PCR of two peptidoglycan hydrolytic genes, Lhv_0190 and Lhv_0191. These genes are ubiquitous and show high homology at the intra-species level. The PCR method gave two specific PCR products, of 542 and 747 bp, for 25 L. helveticus strains coming from various sources. For L. gallinarum, two amplicons were obtained, the specific 542 bp amplicon and another one with a size greater than 1,500 bp. No specific PCR products were obtained for 12 other closely related species of lactobacilli, including the L. acidophilus complex, L. delbrueckii, and L. ultunensis. The developed PCR method provided rapid, precise, and easy identification of L. helveticus. Moreover, it enabled differentiation between the two closely phylogenetically related species L. helveticus and L. gallinarum.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

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

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.     

Characterization of the most abundant Lactobacillus species in chicken gastrointestinal tract and potential use as probiotics for genetic engineering

The count and diffusion of Lactobacilli species in the differ ent gastrointestinal tract （GI） regions of broilers were investigated by quantitative realtime polymerase chain reaction, and the probiotic characteristics of six L. reuteri species isolated from broilers＇ GI tract were also investi gated to obtain the potential target for genetic engineering. Lactobacilli had the highest diversity in the crop and the lowest one in the cecum. Compared with the lower GI tract, more LactobaciUi were found in the upper GI tract. Lactobacillus reuteri, johnsonii, L. acidophilus, L. crispatus, L. salivarius, and L. aviarius were the predominant Lactobacillus species and present throughout the GI tract of chickens. Lactobacillus reuteri was the most abundant Lactobacillus species. Lactobacillus reuteri XC1 had good probiotic characteristics that would be a potential and desirable target for genetic engineering.     

Isolation, partial characterization and mode of action of acidocin J1229, a bacteriocin produced by Lactobacillus acidophilus JCM 1229

Lactobacillus acidophilus JCM 1229 produces a heat-stable bacteriocin, designated as acidocin J1229, that has a narrow inhibitory spectrum. Production of acidocin J1229 in MRS broth was pH dependent, with maximum activity detected in broth culture maintained at pH 5:0. Acidocin J1229 was purified by ammonium sulphate precipitation and sequential cation exchange and reversed-phase chromatographies. The sequence of the first 24 amino acid residues of the N terminus of acidocin J1229 was determined. The molecular mass of acidocin J1229 as determined by mass spectrometry was 6301 Da. Acidocin J1229 showed a bactericidal effect but not a bacteriolytic effect on sensitive cells. Acidocin J1229 dissipated the membrane potential and the pH gradient in sensitive cells, which affected such proton motive force-dependent processes as amino acid transport. Acidocin J1229 also caused an efflux of glutamate, previously taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic a-helix region that could form hydrophilic pores. These results suggest that acidocin J1229 is a pore-forming peptide that creates cell membrane channels through the 'barrel-stave'mechanism.     

产碱性磷酸酶乳杆菌的筛选鉴定、酶的纯化及特性

【背景】碱性磷酸酶(alkaline phosphatase,ALP)是生物体内参与磷酸代谢的调控酶,不同物种的ALP性质与其生理功能有关,提纯后的ALP常用作工具酶,广泛应用于基因工程中,但目前关于乳酸菌中ALP的相关研究甚少。【目的】筛选出一株产ALP且具有潜在益生作用的乳杆菌,对该酶进行分离纯化,并对其性质进行探究,为今后益生菌的开发利用和ALP的工业化生产提供新的微生物资源。【方法】采集蒙古国4个地区的酸马奶样品,通过显色反应初筛和酶活检测复筛对产酶菌株进行筛选,经形态学观察、生理生化鉴定及16S rRNA基因序列同源性比较分析进行菌种鉴定。采用超声破碎法提取ALP,经硫酸铵沉淀、DEAE-52离子交换层析、Sephadex G-200凝胶过滤层析纯化该酶,SDS-PAGE电泳法检测其纯度。【结果】从78株乳酸菌中分离筛选出一株产ALP酶活性最高的乳杆菌(编号为Z23),16S rRNA基因序列长度为1 473 bp,鉴定结果表明为鼠李糖乳杆菌。纯化后的酶比活力为180.27 U/mg,纯化倍数为48.37,酶活回收率为17.05%,该酶亚基相对分子质量为46.7 kD。菌株所产ALP的最适温度为37℃,4℃时酶活最为稳定;最适pH为9.5,在pH 9.0-10.0之间,酶活稳定性可达90%以上;Mg2+和K+对ALP有明显激活作用,Ba2+和Cu2+在低浓度时对ALP有激活作用,高浓度时有抑制作用,Ca~(2+)、Zn~(2+)和EDTA对ALP有强烈的抑制作用。以不同浓度的p-NPP为底物,测得酶的Km值为3.42 mmol/L,Vmax值为1.24 mmol/(L·min)。【结论】本研究对蒙古国地区酸马奶中的益生菌资源有了更为明确的认知,为今后碱性磷酸酶产生菌的筛选和酶的应用开辟了新途径。