Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans

ABSTRACT A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(−) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and V max for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (β1→4)galactans and (β1→4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (β1→3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

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Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase fromThermoanaerobacterium xylanolyticum

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14025461 ◽

2015 ◽

Vol 71 (1) ◽

pp. 41-44 ◽

Cited By ~ 2

Author(s):

Sompong Sansenya ◽

Risa Mutoh ◽

Ratana Charoenwattanasatien ◽

Genji Kurisu ◽

James R. Ketudat Cairns

Keyword(s):

Amino Acid ◽

Glycoside Hydrolase ◽

Size Exclusion ◽

Glycoside Hydrolase Family ◽

Monoclinic Space Group ◽

X Rays ◽

Amino Acid Residues ◽

Structural Explanation ◽

Exclusion Chromatography ◽

Hydrolase Family

TheThermoanaerobacterium xylanolyticumgene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed inEscherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS–PAGE. The purified TxGH116 enzyme hydrolyzed thep-nitrophenyl (pNP) glycosidespNP-β-D-glucoside,pNP-β-D-galactoside andpNP-N-acetyl-β-D-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 Msodium citrate tribasic, 0.1 MTris–HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120 × 25 × 5 µm. The TxGH116 crystals diffracted X-rays to 3.15 Å resolution and belonged to the monoclinic space groupP21. Structure solution will allow a structural explanation of the effects of human GBA2 mutations.

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Type III CRISPR complexes from Thermus thermophilus.

Acta Biochimica Polonica ◽

10.18388/abp.2016_1261 ◽

2016 ◽

Vol 63 (2) ◽

Cited By ~ 1

Author(s):

Marta Szychowska ◽

Wojciech Siwek ◽

Damian Pawolski ◽

Asgar Abbas Kazrani ◽

Krzysztof Pyrc ◽

...

Keyword(s):

Molecular Mass ◽

Size Exclusion Chromatography ◽

Mass Spectroscopy ◽

Thermus Thermophilus ◽

Size Exclusion ◽

Acquired Immunity ◽

Type I ◽

Type Iii ◽

Exclusion Chromatography

Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.

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Plastics. Determination of average molecular mass and molecular mass distribution of polymers using size-exclusion chromatography

10.3403/bseniso16014 ◽

2015 ◽

Keyword(s):

Molecular Mass ◽

Mass Distribution ◽

Size Exclusion Chromatography ◽

Molecular Mass Distribution ◽

Size Exclusion ◽

Average Molecular Mass ◽

Exclusion Chromatography

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Starch Molecular Mass and Size by Size-Exclusion Chromatography in DMSO-LiBr Coupled with Multiple Angle Laser Light Scattering

Cereal Chemistry ◽

10.1094/cchem.1998.75.4.530 ◽

1998 ◽

Vol 75 (4) ◽

pp. 530-535 ◽

Cited By ~ 59

Author(s):

W. Yokoyama ◽

J. J. Renner-Nantz ◽

C. F. Shoemaker

Keyword(s):

Light Scattering ◽

Molecular Mass ◽

Size Exclusion Chromatography ◽

Laser Light ◽

Laser Light Scattering ◽

Size Exclusion ◽

Exclusion Chromatography

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VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802501115 ◽

2018 ◽

Vol 115 (23) ◽

pp. 5950-5955 ◽

Cited By ~ 6

Author(s):

Bastien Casu ◽

Charline Mary ◽

Aleksandr Sverzhinsky ◽

Aurélien Fouillen ◽

Antonio Nanci ◽

...

Keyword(s):

Molecular Mass ◽

Secretion System ◽

High Molecular Mass ◽

Full Length ◽

Size Exclusion ◽

Cross Linking ◽

X Ray ◽

Exclusion Chromatography ◽

Plasmid Pkm101 ◽

Membrane Bound

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

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Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

International Journal of Molecular Sciences ◽

10.3390/ijms21072400 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2400 ◽

Cited By ~ 1

Author(s):

René Stürmer ◽

Jana Reising ◽

Werner Hoffmann

Keyword(s):

Molecular Mass ◽

Complex I ◽

Size Exclusion ◽

Trefoil Factor ◽

Binding Studies ◽

In Vitro Binding ◽

Vitro Binding ◽

Exclusion Chromatography ◽

Trefoil Factor Family

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

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