Cascade of bioreactors in series for conversion of 3 ...

Journal of Biotechnology 111 (2004) 203–217

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 Sumana Bhattacharya a , Marc Schiavone a , James Gomes b , Sanjoy K. Bhattacharya c,∗ b

a Environmental Biotechnology Division, ABRD Company LLC, 1555 Wood Road, Cleveland, OH 44121, USA Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India c Department of Ophthalmic Research/I31, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

Received 4 September 2003; received in revised form 31 March 2004; accepted 1 April 2004

Abstract 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. © 2004 Elsevier B.V. All rights reserved. Keywords: Enzymes; RuBP; 3-PGA

1. Introduction Enzymes are potentially useful catalysts for preparation of chemical substances especially biologically relevant substances. Attempts to use the enzymes in sugar conversions required in pharmaceutical, confectionery and cosmetic industries have been reported (Schmid et al., 2001). Anthropogenic carbon dioxide emission is one of the major sources of pollution and has unwarranted consequences for the environment ∗ Corresponding author. Tel.: +1-216-445-0676; fax: +1-216-297-9892.

(Joos et al., 1999; Schnur, 2002). However, despite widespread concern, there is a lack of technological development enabling fixation of emitted carbon dioxide from stationary or mobile sources that may hold the carbon in fixed state for a long period in the global carbon cycle. Based on engineering considerations it appears that the biocatalytic carbon fixation has potential to abate this pollution for stationary emission sources (Bhattacharya et al., 2002). In biocatalytic fixation carbon dioxide gas is fixed on a five-carbon acceptor (RuBP) resulting in formation of two molar equivalents of a three-carbon sugar 3-PGA (Bhattacharya, 2001; Chakrabarti et al., 2003a). The

0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2004.04.002

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S. Bhattacharya et al. / Journal of Biotechnology 111 (2004) 203–217

regeneration of RuBP from 3-PGA (RuBP recycling) is essential if the process of biocatalytic carbon dioxide fixation is to be continuous. The regeneration of RuBP from 3-PGA is necessary for continuous fixation of CO2 and is completely different than batch processes for RuBP synthesis with d-glucose or adenosine monophosphate as starting material, which occurs with a loss of carbon in gas form (Chakrabarti et al., 2003b). For reliable and industrial level recycling of RuBP it would be essential to develop a continuous, efficient and stable conversion process for conversion of 3-PGA into RuBP. The process is described here that employs the enzymes: phosphoglycerate kinase (PGK), glycerophosphate-3-dehydrogenase (GAPDH), triose phosphate isomerase (TIM), aldolase (ADH/FBP), phosphatase (PTASE/FP), transketolase (TKL), aldolase (ADH/SuBP), phosphatase (PTASE/SBPase), transketolase (TKL), epimerase (EMR), ribulose-5-phosphate kinase (RPK) in series, enabling the conversion of 3-PGA into RuBP. 2. Materials and methods The media ingredients including LB media were procured from Life Technologies, The Sepharose CL-4B and other column chromatography matrix were procured from Amersham Pharmacia Biotech. The Ni-NTA resin was procured from Qiagen. All other chemicals were procured from Sigma unless stated otherwise. 3. Purification of enzymes 3.1. PGK purification PGK was purified from Z. mobilis cell extracts using Dye adsorbent column (Thomas and Scopes, 1998), followed by Sephacryl 200 gel filtration and finally by Q-Sepharose ion-exchange chromatography (Pawluk et al., 1986). The purified enzyme analyzed by SDS–PAGE was over 90% pure and retained a specific activity of about 380 units/mg at 25 ◦ C. 3.2. Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was procured from Worthington Biochemical Corporation and was used without further purification.

3.3. Purification of recombinant triose phosphate isomerase The recombinant Thermotoga maritima TIM was expressed in Escherichia coli and purified as described elsewhere (Beaucamp et al., 1995, 1997). 3.4. Purification of recombinant aldolase The recombinant his-tagged aldolase was expressed in E. coli cells harboring plasmid pET16b (Novagen, Madison, MI) containing aldolase gene form Streptomyces galbus DSM40480 (Wehmeier, 2001). The three his-tagged recombinant enzymes: aldolase, SBPase and RPEase used in this study were subjected to similar purification steps following protein expression. The cell lysate prepared by sonication was centrifuged and supernatant purified by Ni-NTA chromatography (Qiagen) following protocols recommended by the resin supplier. 3.5. Purification of recombinant FBPase/SBPase The wheat SBPase was overexpressed using vector pET19b-SBPase in E. coli host BL21 DE3 pLysS (Novagen) and purified using published protocols (Dunford et al., 1998). 3.6. Purification of recombinant RPEase The recombinant RPEase from Rice (Oryza sativa) was expressed in E. coli BL21DE3 pLysS host harboring plasmid pET19b using suitable modification of published protocols (Teige et al., 1995; Kopriva et al., 2000). 3.7. Purification of recombinant transketolase The transketolase gene of Ascidia sydneiensis samea cDNA cloned in pMAL-c2 expression vector (New England Biolabs, MA) was expressed in E. coli BL21DE3 pLysS host harboring plasmid pTKT-MBP (Ueki et al., 2000). The supernatant containing transketolase-MBP fusion protein from cell extract was purified using amylose resin. The purification was achieved following protocol recommended by the supplier of resin (New England Biolabs, MA).


3.8. Purification of wild-type PRK Spinach PRK was purified using suitable modification of the published protocols (Porter et al., 1986), briefly, after successive (NH4 )2 SO4 precipitation and centrifugation steps, the supernatant was subjected to gel filtration (Ultragel AcA 34 column; Amersham Pharmacia Biotech), ion exchange (DE52 cellulose column; Whatman) and reactive red 120 agarose-CL column chromatography.

4. Enzyme assays and kinetic parameters estimation 4.1. Assay of 3-phosphoglycerate kinase (PGK) PGK was assayed for its activity related to production of 1,3-bisphosphoglycerate using a GAPDH coupled assay. The GAPDH-coupling enzyme (from muscle) was procured from Worthington Biochemical Corporation, traces of PGK was removed using gel-filtration. One unit of enzyme is defined as 1 ␮mol product formed per minute, kinetic parameters were determined with modification of published protocol (Thomas and Scopes, 1998). 4.2. Glyceraldehyde-3-phosphate dehydrogenase activity The glyceraldehyde-3-phosphate dehydrogenase activity was measured spectrophotometrically determining absorbance at 340 nm using a protocol provided by Worthington Biochemical Corporation.

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fructose-1,6-bisphosphate (FBP) as a substrate (Rutter et al., 1966). The assay is based on conversion of FBP by aldolase into glyceraldehyde-3-phosphate and DHAP. Oxidation of 2 ␮mol of ␤-NADH (12.44 OD units at 340 nm) reflects the cleavage of 1 ␮mol of fructose bisphosphate under assay conditions. The activity is determined by measuring absorbance at 340 nm. 4.5. SBPase/FBPase activity determination The assay is based on colorimetric detection of inorganic phosphate (Pi) released from sedoheptulose bisphosphate (SBP). Reactions were carried out in a total volume of 100 ␮l at room temperature for 10 min initiated by addition of 1 ␮g of purified enzyme. Detection of released Pi was carried out 100 ␮l of ammonium molybdate solution (0.3%, w/v in 0.56 M H2 SO4 ). Color development was allowed to proceed for 20 min after addition of 20 ␮l malachite green solution (0.035%, w/v in water) subsequently absorbance was measured at 630 nm. 4.6. Transketolase activity Transketolase activity was determined spectrophotometrically by the rate of reduction of ␤-NAD with coupled enzymatic assay of GAPDH. The reaction in a volume of 1 ml was initiated by addition of one unit of transketolase at 25 ◦ C. The progress of reaction was monitored spectrophotometrically at 340 nm (Kovina et al., 1997). 4.7. Ru5PEpimerase assays

The assays were carried out either with glycerate-3phosphate (G-3-P) or with dihydroxyacetone phosphate (DHAP) as substrate in 1 ml volume. The initial rates were determined at a constant 25 ◦ C and monitored at 340 nm with a spectrophotometer (Beaucamp et al., 1995, 1997).

Ribulose-5-phosphate epimerase activity was determined spectrophotometrically at 340 nm, 25 ◦ C, and pH 8.0 using a coupled enzyme assay in the presence of transketolase, triose-phosphate isomerase, glycerol-3-phosphate dehydrogenase, and ribulose-5-phosphate (Kiely et al., 1973). One unit of epimerase activity catalyzes oxidation of 1 ␮mol of NADH/min (Teige et al., 1998).

4.4. Assay of aldolase

4.8. Assay of PRK activity

Fructose bisphosphate (FBP) aldolase activity was measured in a coupled enzymatic assay with

Kinase activity was determined at 25 ◦ C by a spectrophotometric assay in which ADP, generated by the

4.3. Assay of triose phosphate isomerase

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Table 1 The physiocochemical properties of the enzymes Enzyme

MW (kDa)

pH

Temperature (◦ C)

Cofactor

Source

Reference

Phosphoglycerate kinase Glycerate-3-phosphate dehydrogenase Triose phosphate isomerase Aldolase (FBP) Phosphatase/FBPase Transketolase Aldolase (SuBP) Phosphatase/SBPase Transketolase Epimerase (RPEase) Phosphoribulokinase (PRK)

44 36

6.5 8.5

25 25

ATP ␤-NADH

Zymomonas mobilis ATCC29191 Yeast/rabbit muscle

32

7.4

60

B. stearothermophilus

Thomas and Scopes (1998) Worthington Enzyme Manual (1993) (pp. 201–206) Rentier-Delrue et al. (1993)

36.5 66 70 36.5 66 70 25 40

7.4 9.5 7.7 7.4 9 7.7 8 8

25 25 25 25 25 25 25 25

Streptomyces galbus DSM40480 Wheat (Triticum aestivum) Ascidia syndeiensis samea Streptomyces galbus DSM40480 Wheat (Triticum aestivum) Ascidia syndeiensis samea Rice (Oryza sativa) Cytosolic Spinach (S. oleracea) or yeast

Wehmeier (2001) Dunford et al. (1998) Ueki et al. (2000) Wehmeier (2001) Dunford et al. (1998) Ueki et al. (2000) Kopriva et al. (2000) Brandes et al. (1996)

␤-NADH ATP

kinase reaction, is coupled to NADH oxidation via phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase (Porter et al., 1986; Racker, 1957). The kinetic parameters for immobilized enzymes were calculated following published procedures (Pitcher, 1975; Bille et al., 1989). The physiochemical properties of the soluble enzymes are presented in Table 1.

Nylon beads having immobilized enzymes were soaked with 500 ml of 1 mg/ml respective substrates of the enzymes for 10–15 min before washing with buffer. After 15 min of incubation, the immobilized enzyme beads were washed with 4 × 5 ml of buffer (50 mM bicine pH 8.0, sodium EDTA 1 mM, ␤-mercaptoethanol 14 mM, glycerol 10%), containing 1 mg/ml of BSA and 0.1 mg/ml of trehelose in order to remove the residual free DCC.

5. Enzyme immobilization 5.1. Immobilization using DCC method

6. Modeling, design and construction of bioreactors

Immobilization of the enzymes was carried out on nylon beads using 1,3-dicyclohexylcarbodiimide (DCC) as described for other enzymatic proteins as described in previous reports (Shenoy et al., 1992; Agarwal and Bhattacharya, 1999; Chakrabarti et al., 2003a). The nylon beads used for immobilization had an average diameter of 0.1 cm. Immobilization was carried out in 50 mM potassium phosphate pH 8.0, sodium EDTA 1 mM, ␤-mercaptoethanol 14 mM, glycerol 10%, containing 1 mg/ml of BSA and 0.1 mg/ml of trehelose. Approximately 500 units of enzymes were applied onto the 1 ml beads in a final reaction volume of 2.5 ml. The immobilization was initiated by the addition of 20 ml of 5 mM 1,3-dicyclohexylcarbodiimide in the reaction mixture as described previously (Shenoy et al., 1992).

The conversion of 3-PGA to RuBP involves eight enzymes (Fig. 1). The product synthesized by one enzyme becomes the substrate for the next enzyme in the series with the exception of the epimerase that act in more than one reaction step in the penultimate step to produce ribulose-5-phosphate. Separate reactors each harboring single immobilized enzyme were employed in order to achieve good control on each conversion step and separation of byproducts in order to achieve increased overall efficiency of conversion 3-PGA to RuBP. The elaborate enzyme purification procedures also necessitate their reuse. Packed bed reactors were selected as the reactor type for this process (Fig. 2). In order to carry out the regeneration of RuBP from 3-PGA a cascade of eleven immobilized enzyme reactors (Fig. 3) in series were required (the penultimate


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Fig. 1. The schematic used for the conversion of 3-PGA into RuBP. The enzymes that catalyze particular steps have been identified. The abbreviations of the chemicals have been presented in parentheses. The carbon in the compounds has been indicated (italicized in square brackets).

three reactions catalyzed by the single immobilized epimerase reactor). The design and construction of laboratory-scale reactors were based on the results of a series of simulations carried out on simplified models of the reactors. The model was used to obtain gross dimensions of reactors, however, the final reactor construction was based on a series of trial and error

runs in addition to modeling. The simplified model was developed based on the following assumptions, that the Plug flow conditions prevail in the packed bed reactors; reaction is not diffusion-limited in the reactors and the available kinetic data describes the reaction rate; isothermal conditions are maintained and that the substrate(s) and product(s) are stable.

208


Fig. 2. A single prototype reactor. The definitions used in simulation have been schematically shown in this diagram. The reactors with varying lengths determined using simulation were constructed of glass material with fixed inner diameter.

Enzyme kinetics for soluble enzymes are described by the Michaelis–Menten equation v=

vmax s Km + s

(1)

where v is the initial reaction rate of the enzyme catalyzed reaction (␮mol/min mg), s is the substrate concentration (␮mol/cm3 ), vmax is the maximum reaction rate of the enzyme catalyzed reaction (␮mol/min mg) and Km is the Michaelis–Menten constant (␮mol/cm3 ). The kinetic parameters of the enzymes immobilized to solid supports are altered due to mass transfer limitations, particularly if the support is porous (Marrazzo et al., 1975). The mass transfer effects leads to additional resistances is substrate transfer to catalytic site within the enzyme and determination of the apparent kinetic parameters becomes necessary for accurate description of the system. For the reactors in series considered as ideal plug flow reactors, the enzymes are immobilized in beads that are assumed to be spherical. Considering a porosity ε for the bed and volume V for the reactor, the volume occupied by the

fluid is εV and the volume occupied by the beads containing the enzyme is (1 − ε)V. For a simple reaction of substrate to product, S → P the intrinsic reaction rate is represented by v(s, p). The overall product formation rate at z = z in the reactor is given by v|z = η(s∗ , p∗ )v(s∗ , p∗ )|z , where s∗ and p∗ are the substrate and product concentration at the exterior surface of the bead at the position z = z in the reactor. Performing a material balance of a differential section of the reactor (Fig. 3), the steady state model can be written as: ds 1−ε u =− η(s∗ , p∗ )v(s∗ , p∗ ) (2) dz ε where η(s∗ , p∗ ) is the effectiveness factor defined as the ratio of the observed rate to the rate with no concentration gradients existing within the bed of the beads. Both s∗ and p∗ can be expressed in terms of s, the bulk phase substrate concentration based on steady-state material balance on substrate over a spherical bead of radius R given by 4πR2 ks (s − s∗ ) = 43 πR3 η(s∗ , p∗ )v(s∗ , p∗ )

(3)


209

Fig. 3. The schematic diagram of cascade of eleven immobilized enzyme reactors in series used for the production of d-ribulose-1,5-bisphosphate from 3-PGA. The dashed arrows have been used to demonstrate recycling of the RuBP produced from 3-PGA (product of CO2 fixation) using this cascade of reactors. The cofactors NAD and ATP can be readily separated chromatographically and recycled using separate devices. The pH of the reactors was maintained using pH-stat. The predetermined amounts of DHAP (reactor numbers 6 and 8) and 3-PGAL (reactor number 5) based on preliminary measurements are added into reactors from a stock pool generated from 3-PGA by chemical catalysis steps (not shown in the scheme).

where ks is the mass transfer coefficient of the substrate. Assuming negligible intra-particle and external mass transfer resistances, η → 1 and s∗ → s, and considering this approximation, Eq. (2) reduces to ds 1 − ε vmax s u =− (4) dz ε Km + s which can be solved analytically to obtain the design equation of the reactor s s − s ε Km 0 0 t= + (5) ln 1 − ε vmax s vmax

In terms of the fractional substrate conversion δ = (s0 − s)/s0 , Eq. (5) simplifies to ε s0 δ Km t= (6) ln(1 − δ) + − 1−ε vmax vmax Under steady state conditions, the apparent kinetic parameters are usually calculated from a s0 δ versus ln (1 − δ) plot. For the initial simulation, the kinetics parameters reported in the literature (Table 2) were used assuming that the vmax values reported were reduced considerably (about 70%) due to immobilization. However, the apparent kinetic parameters were

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Table 2 Kinetic parameters of soluble and immobilized enzymes Enzyme

Soluble enzymes Km (␮M)

PGK (EC 2.7.2.3) pH 6.5 @ 25 ◦ C GAPDH (EC 1.2.1.12) pH 8.5 @ 25 ◦ C TIM (EC 5.3.1.1) pH 7.6 @ 25 ◦ C Aldolase (FBP) (EC 4.1.2.1.3) pH 7.4 @ 25 ◦ C PTASE/FP (EC 3.1.3.11) pH 9.5 @ 25 ◦ C TKL (EC 2.2.1.1) pH 7.7 @ 25 ◦ C Aldolase (SuBP) (EC 4.1.2.1.3) pH 7.7 @ 25 ◦ C PTASE/SBPase (EC 3.1.3.11) TKL (EC 2.2.1.1) pH 7.7 @ 25 ◦ C EMR (EC 5.1.3.1) pH 8.0 @ 25 ◦ C RPK (EC 2.7.1.19) pH 8.0 @ 25 ◦ C

Vmax (U/mg)

Immobilized enzymes

Reactor length

Km (␮M)

Designed (cm)

Vmax (U/mg)

Constructed (cm)

1000

415

1000

274

2.93

3

1000

25

720

21

47.39

48

3160, 10400 680 (G3P), 7270 (DHAP) 20

130

270

50

2500

13.5

1900, 7380 650 (G3P), 7010 (DHAP) 20

110

205

35

1670

Cadet and Meunier (1988)

1

Cadet and Meunier (1988) Schenk et al. (1998)

54000

520

1

Flechner et al. (1999)

560

110 (Ru5P), 60 (MgATP)

1

28

81000

200

Flechner et al. (1999)

27.44

600

17000

6

Schenk et al. (1998)

98

220

5.26

36

3810

510

1

35.57

140

13.5

1

Thomas and Scopes (1998) Worthington Enzyme Manual (1993) (pp. 201–206) Tang et al. (1999)

10.5

4000

510

Reference

10.5

1 48.11

48

11900

1

1.5

Chen et al. (1998)

380

1

1.5

Brandes et al. (1996)

90 (Ru5P), 55 (MgATP)

also experimentally determined for the final simulations (Table 2). Based on the physical restrictions of the final application of this process, the diameter of the reactor was taken to be 10 cm. The other process variables such as pH, temperature and reactor length have been provided in Table 2. Several flow rates ranging from 5 to 15 cm3 /min were investigated to determine the reactor length for a desired conversion. It was found that using a flow rate of 10 cm3 /min, the reactor lengths obtained was suitable for construction (Table 2). Simulations predicted that each of these reactors would achieve over 90% conversion of substrate. The bioreactor vessels all of which had a fixed internal diameter of 10 cm and lengths obtained from the simulation (Table 2) were constructed from glass. The reactors had cooling water jackets to maintain the temperature at the desired set point. Each of these

reactors were packed with the immobilized enzyme beads required for that stage, catalyzing a single reaction and the pH of the reactor was maintained constant at the optimum pH for that enzyme. The pH microelectrodes were employed for measurement of pH and were made following published procedures (de Beer and van den Heuvel, 1988). Briefly, a 100 ␮l glass pipette (Drummond Scientific) was stretched using a micropipette-tensioning device. A 10 ␮m tip was filled with silane solution (mixture of carbon tetrachloride and 20% (v/v) of trimethylchlorosilane) and then dried at 130 ◦ C. The membrane solution (an ion exchange solution) was prepared consisting of 10% (w/v) tridodecylamine and 7% (w/v) tetraphehylborate sodium salt dissolved with o-nitrophenyl octyl ether and was exposed in 100% CO2 gas for 16 h. The electrolyte solution was filled in the glass pipetter. A


0.3 mm diameter Ag/AgCl wire was inserted into the electrolyte solution. The pH measurements were fed to a pH stat, which had acid/base solution to control pH. For reactors that had small length, additional filler material was used to maintain structural integrity while the actual bead volume was maintained. The ATP and NADH solution (100 mM stock solutions) was fed from a reservoir, based on prior determinations, which pumped about 3–5-fold excess chemicals in the reactors. The mixing of chemicals was made in narrow openings just prior to entry in the reactor. 6.1. HPLC analyses The HPLC analysis of emanating solutions from each reactor were done on a Waters HPLC device model 510 using a 250 mm × 4.6 mm Econosphere column (5 ␮ Econosphere NH2 packing) at a flow rate of 4 ml/min, 75% acetonitrile, 25% water solution was used as mobile phase. The authentic sugar samples except sedoheptulose-1,7-bisphosphate were procured from Sigma Chemicals Co, St. Louis, MO, which served as standards during each HPLC analysis run. Sedoheptulose bisphosphate was a research gift from Dr. R.P. Sen. Detection was made using refractive index detector system connected to the HPLC.

211

perchloric acid (w/w), 10 ml N/1 hydrochloric acid and 25 ml 4% ammonium molybdate (w/v) made the volume 100 ml with water. The TLC plate is air dried and kept in an oven at 85 ◦ C for 5–7 min for development. The AgNO3 reagent detects total sugars, this reagent is composed of two parts: (I) 0.1 ml saturated aqueous silver nitrate plus 19 ml acetone and (II) 0.5 g NaOH dissolved in 5 ml water and diluted to 100 ml with ethanol. Part I was prepared immediately before use and the TLC plate dipped into it and allowed to air dry. The plate is then placed in Part II (ethanolic NaOH solution) and again allowed to air dry. After air drying the plate is soaked into dilute solution of sodium thiosulfate (5 g/l) for 1 min and rinsed in water and air dried.

7. Results A novel scheme for generation of RuBP from 3-PGA using a combination of different enzymes involved in sugar metabolism has been presented. This scheme employed in bioreactors using immobilized enzymes yields RuBP without any loss of carbon (Fig. 1).

6.2. TLC analyses

8. Purification of enzymes

The TLC analyses were performed on Plastic backed 20 cm × 20 cm Silica Gel 60 F254 plates with 0.2 mm layer thickness (Merck). After spotting the samples were air dried and placed in a TLC tank (27 cm ×24 cm ×7 cm) containing the solvent system, for first dimension solvent was isobutyric acid/1 N NH4 OH/0.1 M EDTA (100:60:1.6), run for 8 h and for second dimension n-butanol/propionic acid/water (375:180:245), run for 10 h (Tyszekiewicz, 1962). The two-dimensional TLC allowed greater resolution and accuracy in quantitation. The plates were dried and subjected to detection of sugars using ammonium molybdate reagent (Hanes and Isherwood, 1949) or with an AgNO3 reagent (Borders, 1972). Authentic sugars procured from Sigma Chemical Co, St. Louis, MO served as standards. The ammonium molybdate reagent detects sugar phosphate esters, each TLC plate after being air dried is sprayed with about 1 ml/100 cm2 with the following solution, 5 ml 60%

All enzymes used in this scheme with the exception of phosphoglycerate kinase, glycerate phosphate dehydrogenase and phosphoribulokinase has been produced as recombinant protein. Glycerate phosphate dehydrogenase was obtained commercially while PGK and PRK were purified from their native host. All proteins except GAPDH were either purified to homogeneity or near homogeneity or essentially free from any detectable interfering activity. The SDS–PAGE analyses of proteins have been presented (Fig. 4).

9. Enzyme immobilization and determination of kinetic parameters All enzymes used for conversion as depicted in Fig. 1 were immobilized using DCC protocol. The enzymes upon immobilization retained over 85% of the applied soluble activity. The apparent kinetic

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Fig. 4. The SDS–PAGE of analysis of purified proteins. The purified protein, phosphoglucokinase (PGK), glycerate phosphate dehydrogenase (GAPDH), triose-phosphate isomerase (TIM), aldolase, FBP/SBPase (fructose bisphosphatase/sedoheptulose bisphosphatase), ribulose-5-phosphate-3-epimerase (epimerase), phosphoribulokinase (PRK), were run on a 10% SDS–PAGE. The gel was stained with Coomassie blue R250.

parameters of the immobilized enzymes were determined using Lineweaver-Burk plot (Table 2) following published procedures (Pitcher, 1975; Bille et al., 1989). The immobilized rice epimerase was more

stable than the soluble epimerase. The rice epimerase has intermediate storage stability between spinach stable, which has least stability and yeast epimerase, which has most stability (Chen et al., 1998).

Fig. 5. Chromatographic profiles of the mixture of authentic sugars. (A) HPLC retention profile for sugars. The authentic sugars were used to determine the respective retention time with Econosphere column with a flow rate of 4 ml/min. The HPLC profile of mixture of sugars has been shown, the retention time of individual sugars were determined separately. 3-PGAL: 3-phosphoglyceraldehyde; 3-PGA: 3-phosphoglycerate; DHAP: dihydroxyacetone phosphate; G-1,3-BP: glycerate-1,3-bisphosphate; E4P: erythrose-4-phosphate; F6P and F1,6-BP: fructose-6-phosphate and fructose-1,6-bisphosphate; RuBP: d-ribulose-1,5-bisphosphate; Ru5P and R5P: ribulose and ribose-5-phosphate, respectively; X5P: xylulose-5-phosphate; SuBP: sedoheptulose-1,7-phosphate. The peaks of individual sugars determined using single authentic sugars have been identified. (B) TLC profile of sugars. F6P and F1,6-BP: fructose-6-phosphate and fructose-1,6-bisphosphate; 3-PGAL: 3-phosphoglyceraldehyde; RuBP: d-ribulose-1,5-bisphosphate; Ru5P and R5P: ribulose and ribose 5-phosphate, respectively. The peaks of individual sugars determined using single authentic sugars have been identified.


10. Bioreactor performance analyses The series of reactors as depicted in Fig. 3, enables conversion of 3-PGA, the product of biocatalytic carbon dioxide fixation (Bhattacharya, 2001; Chakrabarti et al., 2003a,b) into RuBP. At the onset of reactor operations the sampling was done at each reactor entry and exit port. Initial substrate and final product analyses were performed using chromatographic (HPLC and TLC) and enzymatic (the desired product of each reactor was determined using appropriate coupled enzymatic assays) methods. All substrates and products in entry and exit stream were sampled except, in reactors 4, 6, 7, 9, 10 and 11 where only DHAP, F6P, E4P, S7P, X5P, R5P in the entry and F6P, X5P, SuBP, X5P, R 5P, RuBP in the exit ports were subjected to measurement, respectively. The HPLC profile for a mixture of authentic sugars is presented in Fig. 5A and that for thin layer chromatography or TLC is

213

presented in Fig. 5B. The HPLC profile provides retention time of different sugars that are also products of enzyme-catalyzed reactions (Fig. 5A). The HPLC measurements closely resembled the measurements with serial dilution (enzymatic) analyses. The TLC enabled determining the position of sugars that are various reaction intermediates (data not shown). These initial measurements helped determine the amount of 3-phosphoglyceraldehyde (3-PGAL) fed into reactor 5 and dihydroxyacetone phosphate (DHAP) fed into reactors 6 and 8, respectively. The stock pool of 3-PGAL and DHAP were kept separately and generated using chemical conversion steps from 3-PGA (not shown). To evaluate the performance of bioreactors they were subjected to variation in flow rate (Fig. 6A and B) and variation in initial substrate concentration (Fig. 6C). The run time with varying flow rate deviates from linearity only at very low flow rates (data not shown). The profiles of product concentration as a function of

Fig. 6. Reactor performance analyses. The reactors were subjected to varying flow rate and measured for performance. (A) The variation in substrate concentration with run time. (B) The variation in product concentration with run time. (C) Product concentration with varying initial substrate concentration. The product, substrate concentration and run time are shown by dotted, solid and thick solid lines respectively. The symbols 1–11 show reactors 1–11, respectively. (D) The graph showing performance of individual reactors and the overall reactor assembly. The performance of each reactor was determined with respect to initial input taken as 100%. In the initial analysis, chromatographic and enzymatic method of determination was used. The final analyses were performed using HPLC as described in methods.

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varying flow rate (Fig. 6B) closely resembles the pattern of substrate concentration (Fig. 6A). The substrate and product profile with varying run time (a function of flow rate) divides the reactors in accordance to their performance roughly into two clusters, one with run times of up to 100 min and the other with run time between 200 and 1600 min (Fig. 6A and B). The product concentration with varying initial substrate concentration at a given flow rate shows a linear rise with increase in substrate concentration with the exception of first reactor, where two different regime of conversion appears to operate depending upon the applied initial substrate concentration (Fig. 6C). Based on performance of individual reactors attempts were made to determine the overall conversion by the serial cascade of eleven reactors. The performance of individual reactors during a single conversion cycle has been presented (Fig. 6D). The data presented in Fig. 6 is the average of 10 individual single conversion cycles and shows that all steps of enzymatic catalysis in individual reactors gives raise to overall degree of conversion. The maximum overall conversion achieved employing the series of reactors is about 56 ± 3% (Fig. 6D). The cofactors NAD and ATP can be readily separated chromatographically from the emanating solution. The recycling of cofactors (ATP/NAD) are achieved using separate devices as described elsewhere (Bhattacharya, 2001; Bhattacharya et al., 2002).

11. Discussion With the advancement of genetic engineering techniques the possibility of obtaining highly pure enzymes in large quantities has become feasible as well as economical. Many immobilization techniques have been established that successfully immobilize a wide range of enzymes. These developments have now made possible large-scale bioprocesses for complex enzymatic conversion suitable for industrial purposes. Several sugar molecules are used in different pharmaceutical and cosmetic industries. While d-glucose and d-fructose are produced in bulk from crop plants other sugars are prepared through chemical or biochemical syntheses. The reliable efficient bulk conversion of sugars has important industrial implication. A scheme enabling generation of RuBP from 3-PGA without loss of carbon can be achieved

using a cohort of enzymes (Fig. 1). In this scheme if the enzymes are used in a series of reactors, the product of every preceding reaction would be the substrate for the next reaction (Fig. 3). If the reactor series is truncated at a specific point, it will render generation of product up to that point. Therefore, this combination of reactors could also be used for deriving some specific sugars by truncation of bioreactor series at specific points. In this scheme the two enzymes transketolase and epimerase in soluble form deviates from Michealis–Menten kinetics with some but not all substrates. Using the substrates employed in reactors 6 and 9 (for transketolase) and that in reactor 10 (for epimerase) the immobilized transketolase and epimerase were subjected to detail kinetic analyses according to published procedures (Bhattacharya and Dubey, 1999), the kinetic data analysis according using FORTRAN program of Cleland employing statistical criteria (Cleland, 1979). All data were fit to PING-PONG, EQUARD and SEQUEN programs, however, the data showed best fit to PING-PONG and on further analysis is consistent with random rapid equilibrium random bi–bi mechanism (Cleland, 1979). For transketolase and epimerase based upon the reported kinetics mechanism (Kovina et al., 1997; Bykova et al., 2001; Samuel et al., 2001) a complex model was also explored, which provided very similar parameter values that has been used for reactor constructions using a simplistic model (Table 2). The current overall conversion of 3-PGA into RuBP employing the series of enzymatic reactors is about 56 ± 3%. Complete soluble carbon accounting using elemental analysis showed that input and output carbon id balanced and there is no carbon loss in the gas phase as expected from the scheme (data not shown). The yield of 56% RuBP in comparison to predicted 90% using this series of reactors therefore reflects incomplete enzymatic conversion. For prediction of conversion in reactors for the sake of simplicity, Michaelis–Menten kinetics was employed for our model, this was an approximation and actual kinetics occurring within the reactor could be more complicated. The kinetic properties of the enzymes used here remain amenable to modulation by the residual concentration of the metabolites from previous insufficient enzymatic conversions as well as residual cofactors such as NAD, NADH, ADP which may affect enzymatic conversions in subsequent steps. The use


of chemical engineering manipulations holds promise to improve individual conversion steps. For example, chemical engineering studies with varying operation variables may help improve erythrose-4-phosphate (E4P), one of the substrates involved in reactors 6 and 7 (that shows decreasing conversion levels; Fig. 6D) and thereby productivity. Erythrose-4-phosphate tends to form dimers at higher concentration levels, the back reaction to the monomer is much slower so decreasing the total concentration of E4P in reactors may favor greater conversion and improve productivity in reactors (Terada et al., 1985; Akowski and Bauerle, 1997). The use of chemical reaction engineering manipulations alone is not expected to enable in a better level of conversion. The genetic engineering of the enzyme holds the promise to enhance yield. The genetic engineering, however, would require substantial research into enzyme structure function for any particular enzyme. The other possibility to enhance yield is to use agents that binds the product and enables their removal from the emanating reaction mixture. This would help move the reaction in the forward direction and would result in increased productivity. The natural sugar binding lectins provides avenue to develop agents that bind products in a specific manner and can be made to reversibly bind the sugars with respect to applied physicochemical conditions. The chemical reagents that bind ketones and aldehydes, may also provide templates to develop into specific binders for sugars. Such reversible binding has potential for continuous operation in a series of reactors. The binder columns can be placed right after an enzyme reactor and the product can be trapped while the un-reacted product can be recycled. Alternately, the reagent can be eluted and subsequently fed to a downstream appropriate reactor. In vivo plants appear to employ a binding and release mechanism or a mechanism in which the entire conversion is handled within a complex where enzymes catalyzing successive steps reside in proximity, referred to as metabolic chanelling (Chakrabarti et al., 2003b). This is achieved by arrangement of enzymes as a complex where product of one enzyme acts as substrate for the next enzyme. A putative multienzyme complex of phosphoriboisomerase, phosphoribulokinase and RuBP carboxylase can be isolated from spinach leaves (Sainis and Jawali, 1994). The sequential reactions in a multienzyme complex of

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consecutive enzymes are essentially analogous to the coupled enzyme reactions where the product of one reaction is the substrate for the next reaction (Sainis and Jawali, 1994). In vivo, in plants, it has been possible to demonstrate that by manipulating RuBP regeneration and enhancing RuBP regeneration, the rate of carbon dioxide fixation and growth in the plant can be enhanced (Miyagawa et al., 2001; Perez et al., 2001) that is consistent with in vitro systems with respect to fixation process and indicates the importance of RuBP regeneration process. We envisage that specific product binding molecules as plug flow reactors with recycling of the substrates after product absorption would help increase in yield overall product yield and our work towards this direction in progress. Efficient recycling of 3-phospho-d-glycerate into RuBP is key to effective functioning of a continuous carbon dioxide fixation process. The continuous carbon dioxide fixation in contained manner results in abatement of pollution at least from stationary emissions, preliminary analyses showed beneficial ecological consequences and it is estimated to bring economic benefits as well (Bhattacharya et al., 2002). The efficient recycling would make the whole process feasible and economical. Acknowledgements We thank Professor Rainer Jaenicke for construct of triose phosphate isomerase, Dr. Udo Wehmeier for aldolase construct, Dr. Cristine Raines for construct of wheat SBPase/FBPase, Dr. Tatsuiya Ueki for construct of transketolase and Dr. S. Kopriva for rice epimerase construct and Dr. R.P. Sen for the sedoheptulose bisphosphate and Dr. W.W. Cleland for his useful comments on the manuscript. References Agarwal, P.K., Bhattacharya, S.K., 1999. Exploitation of mechanochemistry of restriction endonucleases: construction of a multi RE module. Biotech. Bioeng. 65, 233–239. Akowski, J.P., Bauerle, R., 1997. Steady-state kinetics and inhibitor binding of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (tryptophan sensitive) from Escherichia coli. Biochemistry 36, 15817–15822. Beaucamp, N., Ostendorp, R., Schurig, H., Jaenicke, R., 1995. Cloning, sequencing, expression and characterization of the

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