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Restaino OF, D'ambrosio S, Cassese E, Ferraiuolo SB, Alfano A, Ventriglia R, Marrazzo A, Schiraldi C, Cimini D
Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains
Applied Microbiology and Biotechnology 103(16) (2019)
6771-6782
Escherichia coli O10:K5:H4 (ATCC 23506)
(NCBI TaxID 1246623,
species name lookup)
Escherichia coli O6:K5:H1
(Ancestor NCBI TaxID 562,
species name lookup)
Escherichia coli K5
(Ancestor NCBI TaxID 562,
species name lookup)
Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
NCBI PubMed ID: 31222385Publication DOI: 10.1007/s00253-019-09969-8Journal NLM ID: 8406612Publisher: Springer
Correspondence: Odile Francesca Restaino <odilefrancesca.restaino
unicampania.it>; Donatella Cimini <donatella.cimini
unicampania.it>
Institutions: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania 'Luigi Vanvitelli', Via De Crecchio 7, 80138, Naples, Italy
Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.
Escherichia coli, capsular polysaccharide, experimental, heparin, molecular weight, Chondrotin sulfate, SEC-TDA
Structure type: polymer chemical repeating unit ; 51300-90900
Location inside paper: p.6772, fig.1a, K5 CPS
Trivial name: non-sulfated heparosan-like CPSs (K5 CPS)
Compound class: CPS
Contained glycoepitopes: IEDB_115136,IEDB_137340,IEDB_140630,IEDB_141807,IEDB_151531,IEDB_153764,IEDB_423153
Methods: HPCE, fermentation, anion exchange chromatography, HPGPC, ultrafiltration, SEC-TDA
Comments, role: Both strains E. coli O10:K5:H4 (E. coli K5) and E. coli O6:K5:H1 (E. coli Nissle 1917) produce non-sulfated heparosan-like CPSs (K5 CPS).
Related record ID(s): 2259, 2260, 2261
NCBI Taxonomy refs (TaxIDs): 1246623,
562Reference(s) to other database(s): GTC:G26089XS
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Restaino OF, D'ambrosio S, Cassese E, Ferraiuolo SB, Alfano A, Ventriglia R, Marrazzo A, Schiraldi C, Cimini D
Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains
Applied Microbiology and Biotechnology 103(16) (2019)
6771-6782
Escherichia coli O5:K4:H4
(Ancestor NCBI TaxID 562,
species name lookup)
Escherichia coli K4
(Ancestor NCBI TaxID 562,
species name lookup)
Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
NCBI PubMed ID: 31222385Publication DOI: 10.1007/s00253-019-09969-8Journal NLM ID: 8406612Publisher: Springer
Correspondence: Odile Francesca Restaino <odilefrancesca.restaino
unicampania.it>; Donatella Cimini <donatella.cimini
unicampania.it>
Institutions: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania 'Luigi Vanvitelli', Via De Crecchio 7, 80138, Naples, Italy
Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.
Escherichia coli, capsular polysaccharide, experimental, heparin, molecular weight, Chondrotin sulfate, SEC-TDA
Structure type: polymer chemical repeating unit ; 51300-90900
Location inside paper: p.6772, fig.1a, K4 CPS
Trivial name: non-sulfated chondroitin-like CPS (K4 CPS)
Compound class: CPS
Contained glycoepitopes: IEDB_115136,IEDB_130648,IEDB_137473,IEDB_140630,IEDB_153510,IEDB_423153
Methods: HPCE, fermentation, anion exchange chromatography, HPGPC, ultrafiltration, SEC-TDA
Related record ID(s): 1095, 2260, 2261
NCBI Taxonomy refs (TaxIDs): 562Reference(s) to other database(s): GTC:G09906MP
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There is only one chemically distinct structure:
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Restaino OF, D'ambrosio S, Cassese E, Ferraiuolo SB, Alfano A, Ventriglia R, Marrazzo A, Schiraldi C, Cimini D
Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains
Applied Microbiology and Biotechnology 103(16) (2019)
6771-6782
|
S-2)-+ /Variants 0/-+
| |
-4)-a-L-IdopA-(1-4)-a-D-GlcpN-(1-
|
S-6)-+
/Variants 0/ is:
S-2)-
OR (exclusively)
Ac-2)- |
Show graphically |
Homo sapiens
(NCBI TaxID 9606,
species name lookup)
Taxonomic group: human / Chordata
(Phylum: Chordata)
NCBI PubMed ID: 31222385Publication DOI: 10.1007/s00253-019-09969-8Journal NLM ID: 8406612Publisher: Springer
Correspondence: Odile Francesca Restaino <odilefrancesca.restaino
unicampania.it>; Donatella Cimini <donatella.cimini
unicampania.it>
Institutions: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania 'Luigi Vanvitelli', Via De Crecchio 7, 80138, Naples, Italy
Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.
Escherichia coli, capsular polysaccharide, experimental, heparin, molecular weight, Chondrotin sulfate, SEC-TDA
Structure type: polymer chemical repeating unit ; 12000-15000
Location inside paper: p.6771
Trivial name: heparin (HP)
Contained glycoepitopes: IEDB_137340,IEDB_140959,IEDB_141807,IEDB_142354,IEDB_151531,IEDB_241119,IEDB_241120,IEDB_241121
Methods: HPCE, fermentation, anion exchange chromatography, HPGPC, ultrafiltration, SEC-TDA
Comments, role: major trisulfated disaccharide repeating unit from ref. Linhardt et al., 2003, DOI: 10.1021/jm030176m.
Related record ID(s): 1095, 2259, 2261
NCBI Taxonomy refs (TaxIDs): 9606
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There is only one chemically distinct structure:
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Restaino OF, D'ambrosio S, Cassese E, Ferraiuolo SB, Alfano A, Ventriglia R, Marrazzo A, Schiraldi C, Cimini D
Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains
Applied Microbiology and Biotechnology 103(16) (2019)
6771-6782
Homo sapiens
(NCBI TaxID 9606,
species name lookup)
Taxonomic group: human / Chordata
(Phylum: Chordata)
NCBI PubMed ID: 31222385Publication DOI: 10.1007/s00253-019-09969-8Journal NLM ID: 8406612Publisher: Springer
Correspondence: Odile Francesca Restaino <odilefrancesca.restaino
unicampania.it>; Donatella Cimini <donatella.cimini
unicampania.it>
Institutions: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania 'Luigi Vanvitelli', Via De Crecchio 7, 80138, Naples, Italy
Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.
Escherichia coli, capsular polysaccharide, experimental, heparin, molecular weight, Chondrotin sulfate, SEC-TDA
Structure type: polymer chemical repeating unit ; 14000-70000
Location inside paper: p.6771
Trivial name: chondroitin sulfate (CS)
Contained glycoepitopes: IEDB_115136,IEDB_130648,IEDB_137473,IEDB_140630,IEDB_153510,IEDB_423153
Methods: HPCE, fermentation, anion exchange chromatography, HPGPC, ultrafiltration, SEC-TDA
Comments, role: repeating units could be mono- or di-sulfated.
Related record ID(s): 1095, 2259, 2260
NCBI Taxonomy refs (TaxIDs): 9606Reference(s) to other database(s): GTC:G43702JT
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There is only one chemically distinct structure:
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