Purification and characterization of an extracellular amylase from Lactobacillus plantarum strain A6

Extracellular amylase from Lactobacillus plantarum A6 was purified by fractionated precipitation with ammonium sulphate and by anion exchange chromatography. The homogeneity of the purified fraction was tested by polyacrylamide gel electrophoresis and showed multiple amylase forms. A major form had an estimated molecular weight of 50 kDa. It was identified as an α-amylase, with an optimum pH of 5.5, an optimum temperature of 65°C and K _m value of 2.38 g l^-1 with soluble starch substrate. The enzyme was inhibited by N -bromosuccinimide, iodine and acetic acid. The enzyme activation energy was 30.9 kJ mol^-1.     

Purification and characterization of an extracellular polygalacturonase from Lactobacillus plantarum strain BA 11

Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K _m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release.     

Genome sequence of Lactobacillus pentosus IG1, a strain isolated from Spanish-style green olive fermentations

Lactobacillus pentosus is the most prevalent lactic acid bacterium in Spanish-style green olive fermentations. Here we present the draft genome sequence of L. pentosus IG1, a bacteriocin-producing strain with biotechnological and probiotic properties isolated from this food fermentations.     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Purification and characterisation of mannitol dehydrogenase from Lactobacillus sanfranciscensis

Mannitol dehydrogenase (MDH) was purified and characterised from Lactobacillus sanfranciscensis. Two peptide fragments of MDH were N-terminally sequenced for the first time in the genus Lactobacillus. The purified enzyme had an apparent molecular mass of 44 kDa and catalysed both the reduction of fructose to mannitol and the oxidation of mannitol to fructose. The K(m) value for the reduction reaction was 24 mM fructose and that for the oxidation 78 mM mannitol. The optimum temperature was 35 degrees C, the pH optima for the reduction or oxidation were 5.8 and 8, respectively.     

Structure of the high-molecular weight exopolysaccharide isolated from Lactobacillus pentosus LPS26

The strain Lactobacillus pentosus LPS26 produces a capsular polymer composed of a high- (2.0 × 10⁶ Da) (EPS A) and a low-molecular mass (2.4 × 10⁴ Da) (EPS B) polysaccharide when grown on semi-defined medium containing glucose as the carbon source. The structure of EPS A and its deacetylated form has been determined by monosaccharide and methylation analysis as well as by 1D/2D NMR studies (¹H and ¹³C). We conclude that EPS A is a charged heteropolymer, with a composition of d-glucose, d-glucuronic acid and l-rhamnose in a molar ratio 1:2:2. The repeating unit is a pentasaccharide with two O-acetyl groups at O-4 of the 3-substituted α-d-glucuronic acid and at O-2 of the 3-substituted β-l-rhamnose, respectively.→4)-α-d-Glcp-(1→3)-α-d-GlcpA4Ac-(1→3)-α-l-Rhap-(1→4)-α-d-GlcpA-(1→3)-β-l-Rhap2Ac-(1→This unbranched structure is not common in EPSs produced by Lactobacilli. Moreover, the presence of acetyl groups in the structure is an unusual feature which has only been reported in L. sake 0-1 [Robijn et al. Carbohydr. Res., 1995, 276, 117-136].     

Purification and properties of an extracellular beta-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.     

Mathematical description of the growth of Lactobacillus sake and Lactobacillus pentosus under conditions prevailing in fermented sausages

The growth behaviour of Lactobacillus sake and Lactobacillus pentosus was determined in a model system simulating the conditions of fermenting sausages. Minor effects on the growth were observed by varying the concentrations of glucose, peptone, manganese and sodium nitrite. Temperature and sodium chloride concentration were found to have most effect on the growth. The measured data were used for a mathematical model describing the growth response of L. sake and L. pentosus sufficiently well to estimate the behaviour of the investigated strains. In all combinations relevant to sausage fermentation L. sake proved to be more competitive. It exhibited a shorter lag phase, higher maximal growth rate and higher final cell yield than L. pentosus.     

一株产右旋糖酐酶青霉的分离及酶的纯化和性质

[目的]从土壤中筛选到一株新的产右旋糖酐酶的真菌F1001,为酶法制备药用级右旋糖酐提供新的右旋糖酐酶产生菌株.[方法]通过形态特征和ITS rDNA序列分析方法鉴定菌株.利用硫酸铵盐析、Sepharose 6B凝胶柱纯化,得到纯度较高的酶蛋白.以右旋糖酐70 kDa为底物,对右旋糖酐酶酶学性质及催化机理进行研究.[结...     

Purification and properties of an extracellular glucoamylase from a diastatic strain of Saccharomyces cerevisiae.

The extracellular glucoamylase from certain strains of Saccharomyces cerevisiae can be purified from culture medium by a simple chromatographic procedure. The native enzyme is heavily glycosylated and has an Mr of about 250,000, but gel filtration indicates the existence of oligomers of larger size. Dissociation yields a form of Mr about 70,000. The glucoamylase is rich in serine and threonine and in aspartic acid plus asparagine, and has a pI of 4.62 and a pH optimum of 4.5-6.5. The thermostability and resistance to denaturants of the yeast enzyme is compared with those of two other fungal glucoamylases. Kinetic data for the yeast enzyme and a variety of substrates is presented; the enzyme is particularly ineffective in cleaving alpha-(1----6)-glycosidic bonds.     

Sequencing and expression analysis of the sakacin P bacteriocin produced by a Lactobacillus sakei strain isolated from naturally fermented sausages

A Lactobacillus sakei strain, designated as I151 and isolated from naturally fermented sausages, was found to produce the sakacin P bacteriocin which is active against Listeria monocytogenes. In this study, we performed the sequencing of the gene cluster involved in the production of the sakacin P, and we followed the expression of the sppA gene, encoding for the bacteriocin, in vitro, using Rogosa–Sharpe medium, and in situ, inoculating the strain in fermented sausages as starter culture. The results obtained underlined the high similarity (>99%) of the entire sakacin P gene cluster from the L. sakei studied here with others present in strains of L. sakei already described. Moreover, from the expression experiments, it was shown that the gene is expressed during the exponential phase and that production procedures typical of fermented sausages are not turning off the expression of the gene encoding the bacteriocin. The capability of the strain studied to produce sakacin P during production is considered an advantage for its use as starter culture to improve the safety aspect of traditional fermented sausages produced in Italy.     

口腔乳酸杆菌的分离及其益生特性

[目的]从口腔环境中筛选具有潜在益生特性的乳酸杆菌,用于防治口腔疾病的益生菌疗法.[方法]利用选择性培养基从健康志愿者的唾液和牙菌斑样品中筛选得到乳酸杆菌,然后验证他们对龋齿致病菌变异链球菌生长的抑制作用.同时考察分离得到的微生物是否具有可以定植或在口腔环境中生存的特性.[结果]本研究从牙菌斑样品中分离得到一株发酵乳杆菌Y29.该菌能够抑制变异链球菌的生长,并有自聚集和与其他口腔微生物共聚集形成生物膜的能力.此外,发酵乳杆菌Y29可耐受1.0 mg/mL溶菌酶和140μg/g过氧化氢,有利于其在可能含有多种抑菌物质的口腔动态环境中生存.[结论]发酵乳杆菌Y29在防治龋齿和保证口腔健康方面具有潜在的益生特性.     

Antibacterial activity of Lactobacillus strains isolated from dry fermented sausages

One hundred strains of lactic acid bacteria isolated from dry cured sausages were tested for antagonistic activity against a set of test strains. Nine of 52 strains of Lactobacilus casei and three of 48 strains of Lact. plantarun produced inhibition zones against the indicator species. The substance excreted by Lact. casei CRL 705 was active against Lact. plantarum, Listeria monocytogenes, Staphylococcus aureus and a wide range of Gram-negative bacteria. The activity of the antibacterial compound from Lact. casei CRL 705 was destroyed by papain, trypsin and pepsin, but was resistant to heat (100°C for 20 min), lysozyme and catalase. The agent was produced during the growth cycle and when the concentrated and neutralized supernatant fluid was added to a fresh culture of sensitive cells it produced a rapid inactivation. A decrease in optical density (O.D.) over time, indicative of cell lysis, was also observed. These characteristics allowed us to identify the inhibitory compound as a bacteriocin which we termed Lactocin 705.     

Characterization of beta-fructosidase, an extracellular glycoprotein of carrot cells

Seedlings and suspension-cultured cells of carrot (Daucus carota) contain a cell wall associated as well as a soluble form of beta-fructosidase (beta F). These two forms have different pH optima: 4.6 for cell wall beta F and 5.6 for soluble beta F. Soluble beta F is relatively more abundant in the seedlings and cell wall beta F is relatively much more abundant in the cultured cells. Protoplasts of cultured cells have only the soluble form (pH optimum 5.6) indicating that the cell wall associated form is indeed extracellular in situ. Cell wall beta F was purified to homogeneity and has an Mr = 63,000. Antibodies raised against the deglycosylated enzyme cross-reacted with two soluble enzyme forms: in cultured cells, the soluble enzyme has an Mr = 58,000 and, in seedlings, there are two forms of Mr = 58,000 and 52,000. Treatment of purified cell wall beta F with endoglycosidase H and trifluoromethanesulfonic acid (complete deglycosylation) indicated that the enzyme probably has one high mannose and two complex glycans. This was confirmed by HPLC analysis of [3H]GlcNAc- and [3H]fucose-labeled glycopeptides obtained after trypsin digestion of radioactively-labeled beta F. The amino acid composition shows that cell wall beta F has 18.6% glycine.     

Complete genome sequence analysis of a strain Lactobacillus pentosus ZFM94 and its probiotic characteristics

Lactic acid bacteria have been attracting increased attentions recent years because of harboring probiotic properties. In present study, a Lactobacillus pentosus strain ZFM94 was screened from healthy infant feces and its probiotic characteristics were investigated. We found that ZFM94 was resistant to environmental stresses (temperature, pH and NaCl), tolerant to gastrointestinal juice and bile salts, with inhibitory action against pathogens and capacity of folate production etc. Additionally, complete genome sequence of the strain was analyzed to highlight the probiotic features at genetic level. Genomic characteristics along with the experimental studies is critically important for building an appropriate probiotic profile of novel strains. Genes that correspond to phenotypes mentioned above were identified. Moreover, genes potentially related to its adaptation, such as carbon metabolism and carbohydrate transporter, carbohydrate-active enzymes, and a novel gene cluster RaS-RiPPs, were also revealed. Together, ZFM94 could be considered as a potential probiotic candidate.     

Characterization of an acid phosphatase from Lactobacillus pentosus: regulation and biochemical properties

AIMS: To screen for phosphatase and phytase activities in Lactobacillus isolated from diverse ecosystems and to determine the biochemical properties and the factors that regulate the synthesis of the enzyme responsible for these activities in the selected strain, Lactobacillus pentosus CECT 4023. METHODS AND RESULTS: These activities were determined spectrophotometrically by using p-nitrophenyl phosphate and sodium phytate as substrates. They were maximal at the onset of the stationary phase of growth and repressed in the presence of high glucose concentration and inorganic phosphate. The enzyme responsible for these activities was an acid phosphatase (E.C.3.1.3.2.), with a molecular mass of 69 kDa. The activity was optimum at pH 5.0 and 50 degrees C. It hydrolysed mono-phosphorylated substrates and phytate, albeit at lower rates. It was inhibited by iodoacetic acid, phenyl-methylsulphonyl fluoride, di-sodium pyrophosphate and Ca+2 while activated by Co+2 and low concentrations of L-ascorbic acid and EDTA. CONCLUSIONS: Lactobacillus pentosus CECT 4023 produces a nonspecific acid phosphatase that hydrolyses a number of mono-phosphorylated substrates and phytate. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the phosphatase from L. pentosus CECT 4023 could partly contribute to reduce the phosphorylation degree of phytate and its derivatives and, thereby, their anti-nutrient properties during fermentation processes.     

Purification and characterization of P fimbriae from an Escherichia coli strain isolated from a septicemic turkey

Abstract A pap + Escherichia coli isolate from a turkey with colisepticemia expressed P fimbriae with a major subunit of an apparent molecular mass of 18 kDa which reacted with anti-F11 serum. This fimbriae was purified and polyclonal antiserum was produced in rabbits. The N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of F11. On immunoblotting, the antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae, with F11, and with F165₁ fimbriae. Some antigenic determinants on the major subunits of F13, F7₁, and F7₂ fimbriae, with a stronger reaction against F13 fimbriae, were also recognized. The F11 antiserum reacted similarly to the antiserum against avian P fimbriae although cross-reactions against F13, F7₁, and F7₂ fimbriae were equivalent. In a competitive enzyme-linked immunosorbent assay, serological differences were observed between the purified avian P fimbriae and F11. Thus, the avian P fimbriae is closely related but not identical to F11 fimbriae which are associated with E. coli isolated from human urinary tract infection.     

Purification and characterization of extracellular proteinases excreted by a strain of Bacillus subtilis BS2, isolated from fermented African locust bean 'iru'

Proteinases were excreted by strains of Bacillus subtilis during fermentation of African locust bean cotyledons. Those excreted by one strain were purified and characterized by ammonium sulphate precipitation, ion-exchange chromatography (IEC), gel filtration, inhibition tests and polyacrylamide gel electrophoresis (PAGE). Three proteinases and an esterase without proteolytic activity were identified. A serine proteinase which showed a high degree of hydrophobicity and a neutral proteinase were present. The third proteinase showed both proteolytic and esterolytic activities, and had multiple electrophoretic mobilities on polyacrylamide gel.     

Immunomodulatory activity of Lactobacillus strains isolated from fermented vegetables and infant stool

Four Lactobacillus strains?- Lactobacillus plantarum CJLP133, L. plantarum CJLP243, L. plantarum CJNR26, and Lactobacillus gasseri CJMF3?- were isolated from Korean fermented food or healthy infant feces, and their capacity to modulate cellular and humoral immune responses was studied. Feeding of the tested lactobacilli for 8?weeks did not alter the weight of and cell numbers in the spleen of mice. However, CJLP133 and CJLP243 strains increased the T lymphocyte population in the spleen of mice, while CJNR26 and CJMF3 increased the B lymphocyte population. In splenocytes treated with concanavalin A, ingestion of CJLP133 and CJLP243 promoted T lymphocyte proliferation and secretion of T cell cytokines, whereas feeding of the CJNR26 and CJMF3 strains enhanced B lymphocyte proliferation in splenocytes treated with lipopolysaccharide and plaque formation. These results suggest that CJLP133 and CJLP243 have immunostimulating activity through the enhancement of T cell activation, while CJNR26 and CJMF3 exhibit immunopotentiation through the increment of B cell activation.     

Properties of an extracellular polysaccharide produced by a strain of enterobacter isolated from pond water

A bacterium which was isolated from pond water and identified as Enterobacter cloacae produced a viscous extracellular polysaccharide when it was grown aerobically in a medium containing sucrose as a sole source of carbon. The maximum molecular weight of the polysaccharide was about 9.0 x 10(5). The polysaccharide was composed of fucose, galactose, glucose, and glucuronic acid in a molar ratio of 2:3:2:1, but the molecular weight and the molar ratio of the sugar component were different from those of the polysaccharide produced by the same species reported elsewhere.