Biotechnological storage and utilization of entrapped solar energy.

Our laboratory has recently developed a device employing immobilized F0F1 adenosine triphosphatase (ATPase) that allows synthesis of adenosine triphosphate (ATP) from adenosine 5'-diphosphate and inorganic phosphate using solar energy. We present estimates of total solar energy received by Earth's land area and demonstrate that its efficient capture may allow conversion of solar energy and storage into bonds of biochemicals using devices harboring either immobilized ATPase or NADH dehydrogenase. Capture and storage of solar energy into biochemicals may also enable fixation of CO2 emanating from polluting units. The cofactors ATP and NADH synthesized using solar energy could be used for regeneration of acceptor D-ribulose-1,5-bisphosphate from 3-phosphoglycerate formed during CO2 fixation.

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

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.

A model of best vitelliform macular dystrophy in rats.

PURPOSE: The VMD2 gene, mutated in Best macular dystrophy (BMD) encodes bestrophin, a 68-kDa basolateral plasma membrane protein expressed in retinal pigment epithelial (RPE) cells. BMD is characterized by a depressed light peak (LP) in the electro-oculogram. Bestrophin is thought to be the Cl channel that generates the LP. The goal was to generate an animal model of BMD and to determine the effects of bestrophin overexpression on the RPE-generated components of the ERG. METHODS: Bestrophin or bestrophin mutants (W93C or R218C) were overexpressed in the RPE of rats by injection of replication-defective adenovirus. Immunofluorescence microscopy and ERG recordings were used to study subsequent effects. RESULTS: Bestrophin was confined to the basolateral plasma membrane of the RPE. Neither wild-type (wt) nor mutant bestrophin affected the a- or b-waves of the ERG. Wt bestrophin, however, increased the c-wave and fast oscillation (FO), but not the LP. In contrast, both mutants had little or no effect on the c-wave and FO, but did reduce LP amplitude. LP amplitudes across a range of stimuli were not altered by wt bestrophin, though the luminance response function was desensitized. LP response functions were unaffected by bestrophin R218C but were significantly altered by bestrophin W93C. CONCLUSIONS: A model of BMD was developed in the present study. Because overexpression of wt bestrophin shifted luminance response but did not alter the range of LP response amplitudes, the authors conclude that the rate-limiting step for generating LP amplitude occurs before activation of bestrophin or that bestrophin does not directly generate the LP conductance.

Potential use of sugar binding proteins in reactors for regeneration of CO2 fixation acceptor D-Ribulose-1,5-bisphosphate.

Sugar binding proteins and binders of intermediate sugar metabolites derived from microbes are increasingly being used as reagents in new and expanding areas of biotechnology. The fixation of carbon dioxide at emission source has recently emerged as a technology with potentially significant implications for environmental biotechnology. Carbon dioxide is fixed onto a five carbon sugar D-ribulose-1,5-bisphosphate. We present a review of enzymatic and non-enzymatic binding proteins, for 3-phosphoglycerate (3PGA), 3-phosphoglyceraldehyde (3PGAL), dihydroxyacetone phosphate (DHAP), xylulose-5-phosphate (X5P) and ribulose-1,5-bisphosphate (RuBP) which could be potentially used in reactors regenerating RuBP from 3PGA. A series of reactors combined in a linear fashion has been previously shown to convert 3-PGA, (the product of fixed CO2 on RuBP as starting material) into RuBP (Bhattacharya et al., 2004; Bhattacharya, 2001). This was the basis for designing reactors harboring enzyme complexes/mixtures instead of linear combination of single-enzyme reactors for conversion of 3PGA into RuBP. Specific sugars in such enzyme-complex harboring reactors requires removal at key steps and fed to different reactors necessitating reversible sugar binders. In this review we present an account of existing microbial sugar binding proteins and their potential utility in these operations.

Cascade of bioreactors in series for conversion of 3-phospho-D-glycerate into D-ribulose-1,5-bisphosphate: kinetic parameters of enzymes and operation variables.

A novel scheme employing enzymatic catalysts is described enabling conversion of D-ribulose-1,5-bisphosphate (RuBP) from 3-phospho-D-glycerate (3-PGA) without loss of carbon. Bioreactors harboring immobilized enzymes namely, phosphoglycerate kinase (PGK), glycerate phosphate dehydrogenase, triose phosphate isomerase (TIM), aldolase, transketolase (TKL), phosphatase (PTASE/FP), epimerase (EMR) and phosphoribulokinase (PRK), in accordance with this novel scheme were employed. These reactors were designed and constructed based on simulations carried out to study their performance under various operational conditions and allowed production of about 56 +/- 3% RuBP from 3-PGA. This method of synthesis of RuBP from 3-PGA employing immobilized enzyme bioreactors may be used for continuous regeneration of RuBP in biocatalytic carbon dioxide fixation processes from emissions where RuBP acts as acceptor of carbon dioxide to produce 3-PGA, rendering the fixation process continuous.

Uniformly oriented bacterial F0F1-ATPase immobilized on a semi-permeable membrane: a step towards biotechnological energy transduction.

The immobilization of F(0)F(1)-ATPase in uniform orientation is reported. The biotinylated and histidine-tagged subunits of the bacterial F(0)F(1)-ATPase complex were used for immobilization of the complex on artificial semi-permeable membranes resulting in 88+/-7.8 and 72+/-5.2% coupling of the enzymes. The immobilized enzymes retained over 90% activity. The immobilized ATPase/synthase was used for generation of ATP from ADP and P(i) at the expense of electrochemical potential energy. The re-usability, ratio of amount of enzyme immobilized to enzymic activity conferred on the membranes, ATP synthesized by assembled system and suitability of ATP generated for use in coupled enzymic reactions were determined.

Solubilization and concentration of carbon dioxide: novel spray reactors with immobilized carbonic anhydrase.

Novel spray reactors are described that employ immobilized biocatalyst (carbonic anhydrase), enabling concentration and solubilization of emitted CO(2) by allowing catalytic contact with water spray. The reactors were fed with simulated emission gas. The performance of the reactors was investigated with respect to operation variable: emission flow rate; gas composition in the emission stream; water flow rate; area-to-volume ratio of immobilized reactor core; and the enzyme load within the core. The reactors were also investigated for pressure drop and extractability of CO(2) from the emission with single vs. multiple reactors (of combined equal volume). The biotechnological process of solubilization and concentration of CO(2) from emission exhausts or streams occurring in the spray reactors could be coupled for further biochemical/chemical conversion of the concentrated CO(2).

CO2 hydration by immobilized carbonic anhydrase.

The enzyme carbonic anhydrase (isoform II) from bovine and human erythrocytes was immobilized using different covalent coupling methods on inert matrices. Immobilized carbonic anhydrase may enable concentration of CO2 for Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase)-catalysed fixation in bioreactors. In the present study the activity of carbonic anhydrase with respect to hydration of CO2 using soluble and immobilized enzymes was determined. The stability of the immobilization matrix, the properties of the immobilized enzymes subjected to a variation in operation variables and the activity profile with respect to storage are reported. Immobilization imparted greater thermal and storage stability and enhanced reusability.