Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors

Debajyoti De, Debajyoti Dutta, Moloy Kundu, Sourav Mahato, Marc T. Schiavone, Surabhi Chaudhuri, Ashok Giri, Vidya Gupta, Sanjoy K Bhattacharya

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Background: Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose 1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. Results: We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655-796 μM) compared to 3-phosphoglyceraldehyde (822-966 μM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. Conclusions: The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.

Original languageEnglish
Article number5
JournalMicrobial Cell Factories
Volume4
DOIs
StatePublished - Feb 2 2005
Externally publishedYes

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Phosphoglycerate Mutase
Phosphopyruvate Hydratase
Mutant Proteins
Sugars
Binders
Regeneration
Glyceraldehyde 3-Phosphate
Metabolites
Proteins
Enzymes
Carbon Cycle
Manuscripts
Recycling
Fructose
Carbon Dioxide
Edetic Acid
Sucrose
Limit of Detection
Yeasts
Glucose

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Microbiology

Cite this

Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors. / De, Debajyoti; Dutta, Debajyoti; Kundu, Moloy; Mahato, Sourav; Schiavone, Marc T.; Chaudhuri, Surabhi; Giri, Ashok; Gupta, Vidya; Bhattacharya, Sanjoy K.

In: Microbial Cell Factories, Vol. 4, 5, 02.02.2005.

Research output: Contribution to journalArticle

De, Debajyoti ; Dutta, Debajyoti ; Kundu, Moloy ; Mahato, Sourav ; Schiavone, Marc T. ; Chaudhuri, Surabhi ; Giri, Ashok ; Gupta, Vidya ; Bhattacharya, Sanjoy K. / Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors. In: Microbial Cell Factories. 2005 ; Vol. 4.
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abstract = "Background: Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose 1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. Results: We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655-796 μM) compared to 3-phosphoglyceraldehyde (822-966 μM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. Conclusions: The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.",
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AU - De, Debajyoti

AU - Dutta, Debajyoti

AU - Kundu, Moloy

AU - Mahato, Sourav

AU - Schiavone, Marc T.

AU - Chaudhuri, Surabhi

AU - Giri, Ashok

AU - Gupta, Vidya

AU - Bhattacharya, Sanjoy K

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AB - Background: Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose 1,5-bisphosphate (RuBP) for continuous operation. A radically new close loop of RuBP regenerating reactor design has been proposed that will harbor enzyme-complexes instead of purified enzymes. These reactors will need binders enabling selective capture and release of sugar and intermediate metabolites enabling specific conversions during regeneration. In the current manuscript we describe properties of proteins that will act as potential binders in RuBP regeneration reactors. Results: We demonstrate specific binding of 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) from sugar mixtures by inactive mutant of yeast enzymes phosphoglycerate mutase and enolase. The reversibility in binding with respect to pH and EDTA has also been shown. No chemical conversion of incubated sugars or sugar intermediate metabolites were found by the inactive enzymatic proteins. The dissociation constants for sugar metabolites are in the micromolar range, both proteins showed lower dissociation constant (Kd) for 3-phosphoglycerate (655-796 μM) compared to 3-phosphoglyceraldehyde (822-966 μM) indicating higher affinity for 3PGA. The proteins did not show binding to glucose, sucrose or fructose within the sensitivity limits of detection. Phosphoglycerate mutase showed slightly lower stability on repeated use than enolase mutants. Conclusions: The sugar and their intermediate metabolite binders may have a useful role in RuBP regeneration reactors. The reversibility of binding with respect to changes in physicochemical factors and stability when subjected to repeated changes in these conditions are expected to make the mutant proteins candidates for in-situ removal of sugar intermediate metabolites for forward driving of specific reactions in enzyme-complex reactors.

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