Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.     

Purification, characterization and partial amino acid sequences of carnitine palmitoyl-transferase from human liver

Carnitine palmitoyl-transferase has been extracted with 0.5% Tween-20 from human liver homogenate and purified to homogeneity. The purified enzyme has a native Mr of 274 kDa. The subunit Mr is of 66 kDa, as shown by SDS-PAGE and immunoblots obtained with antibodies raised against human CPT. Purified CPT shows high affinity for palmitoyl-CoA and palmitoyl-carnitine and is not inhibited by malonyl-CoA. Seven tryptic peptides and the N-terminal of purified human CPT have been sequenced, and found homologous to rat CPT sequence. Both antibodies and peptide sequences are important tools for the investigation of the molecular basis of CPT deficiency in man.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

Purification,characterization and partial amino acid sequences of a novel cephalosporin-C deacetylase from Bacillus subtilis

Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretically to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yield of 9 % and a specific activity of 187.4 U/mg protein. The native enzyme (molecular weight, 280,000) was composed of eight identical subunits with apparent molecular weights of 35,000. The cephalosporin-C deacetylase was stable up to 60°C for 30 min at pH 7.0. The enzyme exhibited Michaelis-Menten kinetics with the substrates cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and p-nitrophenyl acetate; the K_m values were 24.0, 7.9 and 1.0 mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak non-competitive inhibitor and other product, acetate, was a weak competitive inhibitor; the K_i values were 171 and 290 mM, respectively. However, these weak product inhibitors did not prevent the completion of the deacylation of 7-ACA. The pI value of the enzyme was determined to be 5.3 using isoelectric focusing. The observed data indicate that the enzyme is different from known cephalosporin-C deacetylases. In addition, amino acid sequencing of the N-terminus and Achromobacter proteinase I-digested peptides yielded no sequences with similarities to other known proteins by a computer search.     

1,2 Propanediol utilization by Lactobacillus reuteri DSM 20016, role in bioconversion of glycerol to 1,3 propanediol, 3-hydroxypropionaldehyde and 3-hydroxypropionic acid

The objective of the presented work is to demonstrate the metabolism of 1,2 propandiol by Lactobacillus reuteri and to elucidate the metabolites produced during the process. This Metabolic pathway is crucial for biotechnological applications using L. reuteri in bioconversion of glycerol to industrially important plate-form chemicals. L. reuteri grown on minimal media containing 1,2 propanediol was able to utilize the compound as a sole carbon and energy source. The growth of the bacteria was linear with time; however the specific growth rate was significantly low compared to bacteria grown on the same media in the presence of glucose.The fermentation of 1,2 propanediol by L. reuteri in presence and absence of glucose was followed for 72 h and the metabolites produced during the process were detected using HPLC. 1,2 Propanediol was completely converted to propionaldhyde in a time dependent fashion, this process had a higher rate in presence of glucose. Consequently the produced propionaldhyde was converted to propionic acid and propanol in a skewed equimolar manner. In presence of glucose: acetic acid, lactic acid, succinic acid and ethanol were detected while in absence of glucose only minute amounts of acetic acid and lactic acid were detected which indicates presence of different metabolic pathways for glucose and 1,2 propanediol metabolism. Resting cells of L. reuteri induced in presence of 1,2 propanediol have shown significant capabilities to convert aqueous glycerol to 1,3 propanediol, 3-hydroxypropionaldhyde and a compound proposed to be 3-hydroxypropionic acid as detected by gas chromatographic technique.     

Purification, characterization and partial amino acid sequences of a xylanase produced by Penicillium chrysogenum.

An extracellular xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8, endo 1,4-beta-xylanase) was found to be the major protein in the culture filtrate of Penicillium chrysogenum when grown on 1% xylan. In contrast to other microorganism no xylanase multiplicity was found in P. chrysogenum under the conditions used. This enzyme was purified to homogeneity by high performance anion-exchange and size-exclusion chromatography. It had an M(r) of 35,000 as estimated by SDS-PAGE and was shown to be active as a monomer. No glycosylation of the protein could be detected neither by a sensitive glycostain nor by enzymatic deglycosylation studies. The enzyme hydrolyzed oat spelt and birchwood xylan randomly, yielding xylose and xylobiose as major end products. It had no cellulase, CMCase, beta-xylosidase or arabinogalactanase activity but acted on p-nitrophenylcellobioside. The pH and temperature optima for its activity were pH 6.0 and 40 degrees C, respectively. Eight peptides obtained after endoproteinase LysC digestion of xylanase have been sequenced, six of them showed considerable amino acid similarity to glucanases and high M(r)/acidic xylanases from different bacteria, yeasts and fungi.     

Purification and Characterization of Glycerol Dehydratase from Lactobacillus reuteri

A coenzyme B₁₂-dependent glycerol dehydratase from Lactobacillus reuteri has been purified and characterized. The dehydratase has a molecular weight of approximately 200,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single major band with a molecular weight of 52,000. K_m values for substrates and coenzyme B₁₂ were in the millimolar and the submicromolar range, respectively.     

Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP gamma S, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4,5-bisphosphate hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.     

Purification and characterization of acid urease from Lactobacillus fermentum

Summary Acid urease was purified to an electrophoretically homogeneous state, and the molecular weight was estimated to be 220 000. The enzyme consisted of three kinds of subunits, designated , and , with molecular weights of 67 000, 16 800 and 8600, respectively, in a (₁ \₂₁)₂ structure. The isoelectric point of the enzyme was 4.8. The nickel content was found to be 1.9 atoms of nickel per ₁₂₁ unit. The amino acid profile was different from those of known bacterial neutral ureases. The enzyme was most active at pH 2 and around 65° C. It was stable between pH 3 and 9, and below 50° C. The K _m for urea was 2.7 mM at pH 2. The enzyme activity was inhibited by Ag⁺, Hg²⁺, Cu²⁺, p-chloromercuribenzoate and acetohydroxamate. The enzyme was separated into three subunits by reverse phase HPLC. The amino terminal amino acid sequences of the subunits , and were Ser-Phe-Asp-Met-, Met-Val-Pro-Gly- and Met-Arg-Leu-Thr-, respectively.Offprint requests to: S. Kakimoto     

Purification and Characterization of Invertase from Lactobacillus reuteri CRL 1100

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited by bivalent metal ions (Ca⁺⁺, Cu⁺⁺, Cd⁺⁺, and Hg⁺⁺), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K _m and V_max values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a β-fructofuranosidase. Received: 13 August 1999 / Accepted: 15 September 1999     

Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938

The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of beta-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known beta-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The beta-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.     

Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced

Gene disruptions in the diploid opportunistic human fungal pathogen Candida albicans are usually created using multiple rounds of targeted integration called the 'ura-blaster' method. Resulting heterozygous and homozygous null mutants can be auxotrophic (Ura(-)) or prototrophic (Ura(+)) for uracil biosynthesis. Here we demonstrate that the Ura-status of otherwise isogenic mutants affected the adhesion of C. albicans. Moreover the effect of Ura-status on adhesion was also dependent on the null mutant background, the nature of the underlying surface and the carbon source for growth. Therefore the Ura-status is not neutral in determining adhesive properties of C. albicans mutants that are generated via the ura-blaster protocol.     

Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp. paracasei CHCC 2115

AIM: Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. METHODS AND RESULTS: The purification protocol consisted of anion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa. The AT converted isoleucine, leucine and valine at a similar rate with alpha-ketoglutarate as the amino group acceptor; minor activity was shown for methionine. The enzyme had pH and temperature optima of 7.3 and 43 degrees C, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5.2, 4% NaCl). Hg2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5'-phosphate-dependent enzymes, when studying the effect of other metal ions, thiol- and carbonyl-binding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei CHCC 2115 may contribute to development of flavour in cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of transamination performed by cheese-related bacteria, and in the understanding and control of amino acid catabolism and the production of aroma compounds.     

新生期大鼠补充罗伊乳杆菌DSM 17938 对肠道菌群及肠道发育的影响

目的 探讨新生期应用罗伊乳杆菌DSM 17938对大鼠肠道菌群的影响。 方法 24只自然分娩出生的新生SD大鼠随机分为对照组和益生菌组(每组12只),益生菌组大鼠在生后2 d(PND2)开始予罗伊乳杆菌DSM 17938[1×106 CFU/(g·bw),1次/d]灌胃,至出生后第6天(PND6),对照组同期予等量生理盐水灌胃。PND7及PND42时,两组分别处死6只SD大鼠,留取空肠、结肠黏膜及其肠内容物标本,采用16S rDNA V4区二代测序法检测空肠、结肠菌群构成;采用Image Pro Plus 6.0测量空肠、结肠绒毛长度及隐窝深度。 结果 益生菌组和对照组大鼠的体质量增量差异无统计学意义(t值分别为0.410、0.856,均P>0.05),PND42时两组大鼠空肠、结肠黏膜绒毛长度及隐窝深度差异均无统计学意义(t值分别为1.796、0.115、1.122和0.715,均P>0.05),两组大鼠空肠和结肠菌群的α多样性及β多样性差异在PND7及PND42时差异均无统计学意义(均P>0.05),不同组间的肠道菌群LEfSe分析显示PND7及PND42时益生菌组和对照组大鼠肠道菌群组成无差异。 结论 自然分娩出生的健康SD大鼠生命早期添加罗伊乳杆菌DSM 17938不影响生命早期、远期肠道菌群的总体组成及远期肠道上皮组织结构的发育。     

Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520.

Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).     

Purification, properties, and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520.

Two types of xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) were isolated from the culture filtrate of a thermophilic actinomycete, Streptomyces thermoviolaceus OPC-520. The enzymes (STX-I and STX-II) were purified by chromatography with DEAE-Toyopearl 650 M, CM-Toyopearl 650 M, Sephadex G-75, Phenyl-Toyopearl 650 M, and Mono Q HR. The purified enzymes showed single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of STX-I and STX-II were 54,000 and 33,000, respectively. The pIs were 4.2 (STX-I) and 8.0 (STX-II). The optimum pH levels for the activity of STX-I and STX-II were pH 7.0. The optimum temperature for the activity of STX-I was 70 degrees C, and that for the activity of STX-II was 60 degrees C. The enzymes were completely inhibited by N-bromosuccinimide. The enzymes degraded xylan, producing xylose and xylobiose as the predominant products, indicating that they were endoxylanases. STX-I showed high sequence homology with the exoglucanase from Cellulomonas fimi (47% homology), and STX-II showed high sequence homology with the xylanase from Bacillus pumilus (46% homology).     

Localization and primary characterization of bile salt hydrolase from Lactobacillus reuteri

The bile salt hydrolase (BSH) of Lactobacillus reuteri CRL 1098 is a single, constitutive, intracellular enzyme which is only detectable in stationary phase cells. It has optimal activity at pH 4.5–5.5 and 37–45 °C. The enzyme (80 kDa apparent mass) has sulphydryl groups in the catalytic active site and hydrolyzes both glycine and taurine conjugated bile acids with higher affinity for glyco-conjugates.     

Biochemical and crystallographic characterization of a glucansucrase from Lactobacillus reuteri 180

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742–1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2₁2₁2₁). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.