title: Inactive enzymatic mutant proteins (phosphoglycerate mutase and enolase) as sugar binders for ribulose-1,5-bisphosphate regeneration reactors.
abstract: BACKGROUND: Carbon dioxide fixation bioprocess in reactors necessitates recycling of D-ribulose1,5-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 muM) compared to 3-phosphoglyceraldehyde (822-966 muM) 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.
authors.affiliation: Department of Ophthalmic Research, Cleveland Clinic Foundation, Area I31, 9500 Euclid Avenue, Cleveland, Ohio, 44195, USA. bhattas@ccf.org.
authors.forename: Debojyoti
authors.initials: D
authors.lastname: De
authors.forename: Debajyoti
authors.initials: D
authors.lastname: Dutta
authors.forename: Moloy
authors.initials: M
authors.lastname: Kundu
authors.forename: Sourav
authors.initials: S
authors.lastname: Mahato
authors.forename: Marc T
authors.initials: MT
authors.lastname: Schiavone
authors.forename: Surabhi
authors.initials: S
authors.lastname: Chaudhuri
authors.forename: Ashok
authors.initials: A
authors.lastname: Giri
authors.forename: Vidya
authors.initials: V
authors.lastname: Gupta
authors.forename: Sanjoy K
authors.initials: SK
authors.lastname: Bhattacharya