scholarly journals The Probiotic Lactobacillus johnsonii NCC 533 Produces High-Molecular-Mass Inulin from Sucrose by Using an Inulosucrase Enzyme

2008 ◽  
Vol 74 (11) ◽  
pp. 3426-3433 ◽  
Author(s):  
Munir A. Anwar ◽  
Slavko Kralj ◽  
Marc J. E. C. van der Maarel ◽  
Lubbert Dijkhuizen
Keyword(s):  
Enzyme Activity ◽  
Probiotic Bacterium ◽  
Reaction Products ◽  
Nmr Analysis ◽  
Lactobacillus Johnsonii ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Thin Layer Chromatographic Analysis

ABSTRACT Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with β(2-6) and β(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, 13C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by 13C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55°C, with a drastic decrease at 60°C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.

scholarly journals Predominant Effect of Host Genetics on Levels of Lactobacillus johnsonii Bacteria in the Mouse Gut

2011 ◽  
Vol 77 (18) ◽  
pp. 6531-6538 ◽  
Author(s):  
Keren Buhnik-Rosenblau ◽  
Yael Danin-Poleg ◽  
Yechezkel Kashi
Keyword(s):  
Genetic Background ◽  
Well Being ◽  
Probiotic Bacterium ◽  
Mouse Genetics ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Lactobacillus Johnsonii ◽  
Content Type ◽  
Isolation And Characterization ◽  
Mouse Lines

ABSTRACTThe gut microbiota is strongly associated with the well-being of the host. Its composition is affected by environmental factors, such as food and maternal inoculation, while the relative impact of the host's genetics have been recently uncovered. Here, we studied the effect of the host genetic background on the composition of intestinal bacteria in a murine model, focusing on lactic acid bacteria (LAB) as an important group that includes many probiotic strains. Based on 16S rRNA gene genotyping, variation was observed in fecal LAB populations of BALB/c and C57BL/6J mouse lines.Lactobacillus johnsonii, a potentially probiotic bacterium, appeared at significantly higher levels in C57BL/6J versus BALB/c mouse feces. In the BALB/c gut, theL. johnsoniilevel decreased rapidly after oral administration, suggesting that some selective force does not allow its persistence at higher levels. The genetic inheritance ofL. johnsoniilevels was further tested in reciprocal crosses between the two mouse lines. The resultant F1 offspring presented similarL. johnsoniilevels, confirming that mouse genetics plays a major role in determining these levels compared to the smaller maternal effect. Our findings suggest that mouse genetics has a major effect on the composition of the LAB population in general and on the persistence ofL. johnsoniiin the gut in particular. Concentrating on a narrow spectrum of culturable LAB enables the isolation and characterization of such potentially probiotic bacterial strains, which might be specifically oriented to the genetic background of the host as part of a personalized-medicine approach.

scholarly journals Structure, expression and duplication of genes which encode phosphoglyceromutase of Drosophila melanogaster.

Genetics ◽  
1994 ◽  
Vol 138 (2) ◽  
pp. 353-363
Author(s):  
P D Currie ◽  
D T Sullivan
Keyword(s):  
Molecular Evolution ◽  
Genomic Sequence ◽  
Glycolytic Enzyme ◽  
Coding Region ◽  
Subsequent Increase ◽  
Rnase Protection ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Genomic Regions

Abstract We report here the isolation and characterization of genes from Drosophila that encode the glycolytic enzyme phosphoglyceromutase (PGLYM). Two genomic regions have been isolated that have potential to encode PGLYM. Their cytogenetic localizations have been determined by in situ hybridization to salivary gland chromosomes. One gene, Pglym78, is found at 78A/B and the other, Pglym87, at 87B4,5 of the Drosophila polytene map. Pglym78 transcription follows a developmental pattern similar to other glycolytic genes in Drosophila, i.e., substantial maternal transcript deposited during oogenesis; a decline in abundance in the first half of embryogenesis; a subsequent increase in the second half of embryogenesis which continues throughout larval life; a decline in pupae and a second increase to a plateau in adults. This transcript has been mapped by cDNA and genomic sequence comparison, RNase protection, and primer extension. Using similar analyses transcripts of Pglym87 could not be detected. Pglym78 has two introns which interrupt the coding region, while the Pglym87 gene lacks introns. This and other features support a model of retrotransposition mediated gene duplication for the origin of Pglym87. The apparent absence of a complete, intact coding frame and transcript suggest that Pglym87 is a pseudogene. However, retention of reading frame and codon bias suggests that Pglym87 may retain coding function, or may have been inactivated recently, substantially after the time of duplication, or that the molecular evolution of Pglym87 is unusual. Similarities of the unusual molecular evolution of Pglym87 and other proposed pseudogenes are discussed.

scholarly journals Subcloning and Expression of a Protease Gene from Bacillus halodurans CM1 in Bacillus subtilis DB104

2019 ◽  
Vol 16 (04) ◽  
pp. 817-826
Author(s):  
Endang Rahmawati ◽  
Abinawanto Abinawanto ◽  
Is Helianti
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Open Reading Frame ◽  
Bacillus Halodurans ◽  
Protease Gene ◽  
Erythromycin Resistance ◽  
Reading Frame ◽  
E Coli ◽  
Protease Enzyme ◽  
Ph Range

ABSTRACT: Proteases are potential enzymes that utilized in various industrial fields, and the demand of these enzymes is increasing. Bacillus halodurans CM1 is Indonesia indigenous bacterium which is detected to be able to produce alkalotermophilic protease enzyme. In this study, we subcloned the protease gene consist of Open Reading Frame of protease gene and its promoter from Bacillus halodurans CM1 in Bacillus subtilis DB104 via conjugation, and analyzed the expression of the recombinant protease. The protease gene is 1 417 bp length including the open reading frame and the promoter, and obtained by PCR and cloned into pGEM T easy. After confirmed by sequencing, the gene was subcloned into vector pBBRE194, then the recombinant plasmid was transformed into E. coli S17-1. This E.coli was then conjugated to Bacillus subtilis DB104. The target recombinant B. subtilis DB104 has been obtained confirmed by plasmid verification and erythromycin resistance. The recombinant protease produced showed the highest enzyme activity at 50oC and pH 9 (with pH range 5-9) which with protease activity 13.66 U/mL.

scholarly journals Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol.

1996 ◽  
Vol 44 (4) ◽  
pp. 389-392 ◽  
Author(s):  
R P van Gijlswijk ◽  
J Wiegant ◽  
A K Raap ◽  
H J Tanke
Keyword(s):  
Enzyme Activity ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dextran Sulfate ◽  
Detection Sensitivity ◽  
Reaction Products ◽  
Free Diffusion ◽  
Enzyme Cytochemistry ◽  
Amplification System

Recently, a peroxidase-mediated amplification system has been described for immunofluorescence and fluorescence in situ hybridization studies. It is based on the deposition of hapten- or fluorochrome-labeled tyramide molecules. Although providing a significantly increased detection sensitivity compared to conventional procedures, its localization properties are inferior because of free diffusion of intermediate reaction products before they are immobilized. In enzyme cytochemistry, it is well established that improved localization of enzyme activity can be achieved through the addition of viscosity-increasing polymers to the incubation media. In this study we analyzed the effect of different polymers on the localization sharpness and sensitivity of the tyramide-peroxidase reaction in FISH applications. Significantly improved localization of the fluorescent endproduct was observed using dextran sulfate or polyvinylalcohol (PVA) with, respectively, no or little loss of sensitivity.

scholarly journals Chitosan Cross-Linking with Acetaldehyde Acetals

Biomimetics ◽  
2022 ◽  
Vol 7 (1) ◽  
pp. 10
Author(s):  
Alexander Pestov ◽  
Yuliya Privar ◽  
Arseny Slobodyuk ◽  
Andrey Boroda ◽  
Svetlana Bratskaya
Keyword(s):  
Chitosan Solution ◽  
Direct Reaction ◽  
Acetate Solution ◽  
Reaction Products ◽  
Acid Type ◽  
Ft Ir ◽  
Low Cytotoxicity ◽  
Ph Range ◽  
Selection Of

Here we demonstrate the possibility of using acyclic diethylacetal of acetaldehyde (ADA) with low cytotoxicity for the fabrication of hydrogels via Schiff bases formation between chitosan and acetaldehyde generated in situ from acetals in chitosan acetate solution. This approach is more convenient than a direct reaction between chitosan and acetaldehyde due to the better commercial availability and higher boiling point of the acetals. Rheological data confirmed the formation of intermolecular bonds in chitosan solution after the addition of acetaldehyde diethyl acetal at an equimolar NH2: acetal ratio. The chemical structure of the reaction products was determined using elemental analysis and 13C NMR and FT-IR spectroscopy. The formed chitosan-acetylimine underwent further irreversible redox transformations yielding a mechanically stable hydrogel insoluble in a broad pH range. The reported reaction is an example of when an inappropriate selection of acid type for chitosan dissolution prevents hydrogel formation.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

DNA-Polymerases α, δ and ε from T-Cell Spontaneous Lymphoblastic Leukemia of Sprague-Dawley Inbred Rat: Isolation and Characterization

1995 ◽  
Vol 60 (9) ◽  
pp. 1555-1572 ◽  
Author(s):  
Pavel Kramata ◽  
Jaroslav Černý ◽  
Gabriel Birkuš ◽  
Ivan Votruba ◽  
Berta Otová ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lymphoblastic Leukemia ◽  
Dna Polymerases ◽  
Sedimentation Coefficient ◽  
Nuclear Antigen ◽  
Exonuclease Activity ◽  
Sprague Dawley ◽  
Dna Primase ◽  
Isolation And Characterization ◽  
Inbred Rat

Using a single isolation procedure and selective assays for the determination of enzyme activity, highly purified DNA-polymerases α, δ and ε were isolated from the lymphoma of Sprague-Dawley inbred rats. For pol α the subunit composition was 170, 70, 57 and 53 kDa with sedimentation coefficient 8.7 S for the native molecule; pol delta consists of two polypeptides (133 and 46 kDa; sedimentation coefficient 8.2 S), while pol ε is a single polypeptide (140 kDa) and its sedimentation coefficient is 7.0 S. Comparison of the interaction of individual enzymes with known inhibitors and proliferating cell nuclear antigen (PCNA) using the template-primer poly dA-oligo dT12-18, gave the following data: (i) pol α is selectively inhibited by N2-(p-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and stimulated by dimethyl sulfoxide; (ii) all the enzymes are inhibited by N-ethylmaleimide and aphidicolin; (iii) PCNA stimulates pol δ approximately 50 times while pol ε is moderately inhibited; (iv) pol α exhibits considerably higher DNA-primase activity with poly dC as template than with poly dT, and negligible 3'-5'-exonuclease activity whereas pol δ and pol ε, which do not exert any DNA-primase activity have approximately the same 3'-5'-exonuclease activity. The ability of individual polymerases to utilize poly dT-oligo dA12-18 as a template-primer at different pH values, ionic strengths and Mg2+-concentrations was also investigated. In comparison to poly dA-oligo dT12-18 template-primer, pol α has 140% of enzyme activity on poly dT-oligo dA12-18 under optimal conditions, whereas the activity of pol ε and pol δ is 4 times and 10 times lower, respectively.

scholarly journals Molecular cloning and analysis of l(1)ogre, a locus of Drosophila melanogaster with prominent effects on the postembryonic development of the central nervous system.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 1033-1044 ◽  
Author(s):  
T Watanabe ◽  
D R Kankel
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Postembryonic Development ◽  
P Element ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
The Central Nervous System ◽  
Highly Hydrophobic ◽  
Conceptual Translation

Abstract Previous genetic studies have shown that wild-type function of the l(1)ogre (lethal (1) optic ganglion reduced) locus is essential for the generation and/or maintenance of the postembryonic neuroblasts including those from which the optic lobe is descended. In the present study molecular isolation and characterization of the l(1)ogre locus was carried out to study the structure and expression of this gene in order to gain information about the nature of l(1)ogre function and its relevance to the development of the central nervous system. About 70 kilobases (kb) of genomic DNA were isolated that spanned the region where l(1)ogre was known to reside. Southern analysis of a l(1)ogre mutation and subsequent P element-mediated DNA transformation mapped the l(1)ogre+ function within a genomic fragment of 12.5 kb. Northern analyses showed that a 2.9-kb message transcribed from this 12.5-kb region represented l(1)ogre. A 2.15-kb portion of a corresponding cDNA clone was sequenced. An open reading frame (ORF) of 1,086 base paris was found, and a protein sequence of 362 amino acids with one highly hydrophobic segment was deduced from conceptual translation of this ORF.

Enzymatic condensation of dopamine and acetaldehyde: a salsolinol synthase from rat brain

Biologia ◽  
2011 ◽  
Vol 66 (6) ◽  
Author(s):  
Xuechai Chen ◽  
Abida Arshad ◽  
Hong Qing ◽  
Rui Wang ◽  
Jianqing Lu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Substantia Nigra ◽  
Human Subjects ◽  
Enzymatic Reaction ◽  
Brain Regions ◽  
Activity Detection ◽  
Reaction Products ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Salsolinol Synthase

AbstractSalsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; Sal) is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is supposed to have a role in the development of Parkinson-like syndrome in both human and non-human subjects. In the human brain, the amount of (R)-enantiomer of Sal is much higher than (S)-enantiomer, suggesting that a putative enzyme may participate in the synthesis of (R)-salsolinol, called (R)-salsolinol synthase. In this study, the (R)-salsolinol synthase activity in the condensation of dopamine and acetaldehyde was investigated in the crude extracts from the brains of Sprague Dawley rats. Identification of the enzymatic reaction products and enzyme activity detection were achieved by HPLC-electrochemical detection. The discovery of this enzyme activity in rat’s brain indicates the natural existence of (R)-salsolinol synthase in the brains of humans and rats, and it is distributed in most brain regions of rat with higher activity in soluble proteins extracted from striatum and substantia nigra.

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