IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO(2) SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan-alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co(2+), Cu(2+), and Fe(3+), increased the enzyme activity, whereas CA activity was inhibited by Pb(2+), Hg(2+), ethylenediamine tetraacetic acid (EDTA), 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO(2) to CaCO(3). The maximum CO(2) sequestration potential was achieved with immobilized CA (480?mg CaCO(3)/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO(2) sequestration.     

Particle-tethered NADH for production of methanol from CO(2) catalyzed by coimmobilized enzymes

Efficient cofactor regeneration and reuse are highly desired for many important biotransformation applications. Here we show for the first time that cofactor NAD(H) covalently attached to micro particles, which can be easily recovered and reused, effectively mediated multistep reactions catalyzed by enzymes that were also immobilized with the micro particles. Such an immobilized enzyme-cofactor catalytic system was examined for the production of methanol from CO(2) with in situ cofactor regeneration. Four enzymes including formate, formaldehyde, alcohol, and glutamate dehydrogenases were coimmobilized using the same particles as that used for cofactor immobilization (enzymes and cofactor were immobilized separately). Reactions were performed by bubbling CO(2) in a suspension solution of the particle-attached enzymes and cofactor. It appeared that the collision among the particles afforded sufficient interactions between the cofactor and enzymes, and thus enabled the sequential transformation of CO(2) to methanol along with cofactor regeneration. For a 30-min batch reaction, a productivity of 0.02 micromol methanol/h/g-enzyme was achieved. That was lower than but comparable to the 0.04 micromol methanol/h/g-enzyme observed for free enzymes and cofactor at the same reaction conditions. The immobilized system showed fairly good stabilities in reusing. Over 80% of their original productivity was retained after 11 reusing cycles, with a cumulative methanol yield based on the amount of cofactor reached 127%. That was a promising enhancement in cofactor utilization as compared to the single-batch yield of 12% observed with free enzymes and free cofactor.     

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.     

CO 2 -fixing enzymes in a marine psychophile

A psychrophilic marine Pseudomonas was found to contain phosphoenolpyruvate (PEP) carboxylase and an adenosine triphosphate-linked PEP carboxykinase. Some properties of these CO(2)-fixing enzymes were compared with those homologous enzymes from the terrestrial mesophile Enterobacter cloacae. The PEP carboxylases from both organisms were activated by acetyl-coenzyme A (CoA) and inhibited by l-aspartate. The enzyme from Pseudomonas was less dependent on the presence of the activator, but maximal activation was attained at acetyl-CoA concentrations much lower (50 mum) than those required to saturate the enzyme from E. cloacae. In both cases the main effect of acetyl-CoA was to decrease the K(m) value for PEP. The activity of PEP carboxylase from Pseudomonas was only slightly inhibited by NaCl, KCl, or NH(4)Cl up to 100 mm, whereas the enzyme from E. cloacae was inhibited by about 70% under similar experimental conditions. Both PEP carboxylase and PEP carboxykinase from Pseudomonas showed considerably lower thermal stability than their counterparts from E. cloacae. Our results suggest that the CO(2)-fixing enzymes from a marine Pseudomonas and E. cloacae are similar in nature and regulation, but they differ in properties related to the peculiar conditions of the marine environment.     

Absorption characteristics and kinetics of CO2 capture into N-methyldiethanolamine aqueous solution catalyzed by the immobilized carbonic anhydrase

Abstract

Carbonic anhydrase (CA) is the most effective CO₂ hydratase catalyst, but the poor storage stability and repeatability of CA limit its development. Therefore, CA was immobilized on the epoxy magnetic composite microspheres to enhance the CO₂ absorption into N-methyldiethanolamine (MDEA) aqueous solution in this work. In the presence of immobilized CA, the CO₂ absorption rate of MDEA solution (10?wt%) (0.63?mmol·min^?1) was greatly improved by almost 40%, and their reaction equilibrium time was shortened from 150?min to 90?min compared with that into MDEA solution. The results indicated that the absorption of CO₂ into MDEA solution had been significantly enhanced by using CA. After the 7th reuse recycle, the activity of the immobilized CA was still closed to its initial value at 313.15?K. Moreover, enzyme catalytic kinetics of immobilized CA was investigated using the p-nitrophenyl acetate (p-NPA) as substrate. The values of Michaelis–Menten constant (K_m) and the maximum velocity (V_max) of the immobilized CA were calculated to be 27.61?mmol/L and 20.14?×?10^?3?mmol·min^?1·mL^?1, respectively. Besides, the kinetics of CO₂ reaction into MDEA with or without CA were also compared. The results showed that CO₂ absorption into CA/MDEA aqueous solution obeyed the pseudo first order regime and the second order kinetics rate constant (k₂) was calculated to be 929?m³·kmol^?1·s^?1, which was twice higher than that of MDEA aqueous solution without immobilized CA (k₂=414 m³·kmol^?1·s^?1) at 313.15?K.     

Topical inhibition of nasal carbonic anhydrase affects the CO2 detection threshold in rats

Previous studies indicate that Long-Evans rats can be operantly trained to discriminate inspired CO(2) concentrations as low as 0.5%. This ability has been proposed to be due to the presence of CO(2)-sensitive olfactory receptors that contain the enzyme carbonic anhydrase (CA). The objectives of the present study were as follows: 1) to determine whether Zucker rats could be operantly conditioned to discriminate low concentrations of CO(2) from control air and 2) to determine the rats' CO(2) detection thresholds before and after nasal perfusion of mammalian Ringers or methazolamide, a CA inhibitor. Rats were operantly trained to discriminate between 25% CO(2) and control air (0% CO(2)) and were then subjected to various CO(2) concentrations (0.5-12.5%) to determine their CO(2) detection thresholds. The average (+/-standard error of mean) baseline CO(2) detection threshold of 7 Zucker rats was 0.48 +/- 0.07% CO(2), whereas the average CO(2) detection thresholds after nasal perfusion of either mammalian Ringers or 10(-2) M methazolamide were 1.41 +/- 0.30% and 5.92 +/- 0.70% CO(2), respectively. The average CO(2) detection threshold after methazolamide was significantly greater (P<0.0001) than the baseline detection threshold. These findings demonstrate that like Long-Evans rats, Zucker rats can be trained to discriminate low concentrations of CO(2) and that inhibition of nasal CA reduces the ability of the rats to detect low concentrations (3.5% and below) but not higher concentrations of CO(2) (12.5%). These results add to the growing evidence that olfactory neurons exhibiting CA activity are CO(2) chemoreceptors sensitive to physiological concentrations of CO(2).     

Acclimation of wild-type cells and CO2-insensitive mutants of the green alga Chlorella ellipsoidea to elevated [CO2

CO(2)-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO(2). When air-grown, wild-type (WT) cells were transferred to 5% CO(2), an abrupt drop of P(max) to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO(2)-insensitive mutant, ENU16, exhibited a constant P(max) at maximum levels and a low K(1/2)[DIC] throughout the acclimation period. The rapid P(max) drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in P(max) in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO(2)], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14-0.4% [CO(2)], K(1/2)[DIC] values increased as [CO(2)] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the P(max) level in this intermediate [CO(2)] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO(2) conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO(2)].     

Immobilization of carbonic anhydrase in alginate and its influence on transformation of CO2 to calcite

Present investigation entails carbonic anhydrase (CA) immobilization and its influence on transformation of CO₂ to calcite. CA enzyme was immobilized in alginate beads, subsequently maintained its catalytic efficiency after sequential operational cycles. The immobilized beads showed better operational stability by retaining nearly 67% of its initial activity even after six cycles. Batch scale studies with free and immobilized enzyme revealed that the entrapped CA hydrates CO₂ to bicarbonate and/or carbonate which was then made to react with Ca²⁺ ions to transform into calcite. Calcite was characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The entrapped CA was employed for the performance evaluation with respect to several operational parameters including the influence of enzyme concentration in free and immobilized condition. It was concluded that immobilized CA in alginate beads would have the potential for CO₂ sequestration by biomimetic route.     

CO2 biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels

In this study, Scenedesmus obliquus SJTU-3 and Chlorella pyrenoidosa SJTU-2 were cultivated with 0.03%, 5%, 10%, 20%, 30%, 50% CO(2). The two microalgae could grow at 50% CO(2) (>0.69 g L(-1)) and grew well (>1.22 g L(-1)) under CO(2) concentrations ranging from 5% to 20%. Both of the two examined microalgae showed best growth potential at 10% CO(2). The maximum biomass concentration and CO(2) biofixation rate were 1.84 g L(-1) and 0.288 g L(-1) d(-1) for S. obliquus SJTU-3 and 1.55 g L(-1) and 0.260 g L(-1) d(-1) for C. pyrenoidosa SJTU-2, respectively. The main fatty acid compositions of the two examined microalgae were fatty acids with C(16)-C(18) (>94%) under different CO(2) levels. High CO(2) levels (30-50%) were favorable for the accumulation of total lipids and polyunsaturated fatty acids. The present results suggested that the two microalgae be appropriate for mitigating CO(2) in the flue gases and biodiesel production.     

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.     

CO2 and bicarbonate exchange in the rat liver

Several forms of carbonic anhydrase (CA) have been detected in hepatocytes. The distribution of these enzymes appears to be heterogeneous in the hepatic lobule, and the specific isoenzyme that predominates is influenced by sex steroid levels in the animal. In the present study, experiments were conducted in isolated male rat livers perfused with erythrocyte-free solutions, which were devoid of CA to see if there were sufficient tissue CA activity accessible to the plasma to ensure equilibration between labeled HCO3- and CO2 during a single passage from the portal vein to the hepatic vein. After injection of H14CO3- into the portal vein, emergence of the 14C label from the hepatic vein was slightly more rapid than after injections of 14CO2. After infusion of 5-250 microM of acetazolamide, an inhibitor of CA, H14CO3- was virtually confined to the extracellular space during a single transit through the organ, whereas the outflow of 14CO2 was very prolonged, suggesting that some of the 14C had been "trapped" within the hepatic cells as H14CO3-. Inhibition of CA activity in the intact organ with low doses of acetazolamide suggests the presence of a readily inhibitable isoenzyme of CA on the surface of the hepatocytes, which is directly accessible to both HCO3- and acetazolamide. The outflow patterns of 14CO2 and H14CO3- became the same after infusion of erythrocyte CA into the portal vein. On the basis of the pH of the perfusate and the cellular distribution of 14CO2 and H14CO3- in the presence of CA, an intracellular pH value of 7.26 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fate of photosynthetically-fixed carbon in Lolium perenne grassland as modified by elevated CO2 and sward management

Prediction of the impact of climate change requires the response of carbon (C) flow in plant-soil systems to increased CO(2) to be understood. A mechanism by which grassland C sequestration might be altered was investigated by pulse-labelling Lolium perenne swards, which had been subject to CO(2) enrichment and two levels of nitrogen (N) fertilization for 10 yr, with (14)CO(2). Over a 6-d period 40-80% of the (14)C pulse was exported from mature leaves, 1-2% remained in roots, 2-7% was lost as below-ground respiration, 0.1% was recovered in soil solution, and 0.2-1.5% in soil. Swards under elevated CO(2) with the lower N supply fixed more (14)C than swards grown in ambient CO(2), exported more fixed (14)C below ground and respired less than their high-N counterparts. Sward cutting reduced root (14)C, but plants in elevated CO(2) still retained 80% more (14)C below ground than those in ambient CO(2). The potential for below-ground C sequestration in grasslands is enhanced under elevated CO(2), but any increase is likely to be small and dependent upon grassland management.     

Uroplakins do not restrict CO2 transport through urothelium

Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) (～1.6 × 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited (～7 × 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold (～ 5.1 × 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.     

Physiological characteristics of the primitive CO2 concentrating mechanism in PEPC transgenic rice

C4 plants such as maize have CO2 concentrating mechanism and higher photosynthetic efficiency than C3 plants, especially under high light intensity, high temperature and drought conditions. In recent years, due to the rapid development of transgenic technique, different transgenic rice plants with high-level expression of C4 genes have been created by the successful introduction of genes encoding the key C4 photosynthetic path enzymes PEPC, PPDK and NADP-ME through agrobacteria-mediated…     

Proton transport by phosphate diffusion--a mechanism of facilitated CO2 transfer

We have measured CO2 fluxes across phosphate solutions at different carbonic anhydrase concentrations, bicarbonate concentration gradients, phosphate concentrations, and mobilities. Temperature was 22-25 degrees C, the pH of the phosphate solutions was 7.0-7.3. We found that under physiological conditions of pH and pCO2 a facilitated diffusion of CO2 occurs in addition to free diffusion when (a) sufficient carbonic anhydrase is present, and (b) a concentration gradient of HCO3- is established along with a pCO2 gradient, and (c) the phosphate buffer has a mobility comparable to that of bicarbonate. When the phosphate was immobilized by attaching 0.25-mm-long cellulose particles, no facilitation of CO2 diffusion was detectable. A mechanism of facilitated CO2 diffusion in phosphate solutions analogous to that in albumin solutions was proposed on the basis of these findings: bicarbonate diffusion together with a facilitated proton transport by phosphate diffusion. A mathematical model of this mechanism was formulated. The CO2 fluxed predicted by the model agree quantitatively with the experimentally determined fluxes. It is concluded that a highly effective proton transport mechanism acts in solutions of mobile phosphate buffers. By this mechanism; CO2 transfer may be increased up to fivefold and proton transfer may be increased to 10,000-fold.     

Evaluation of whole lysosomal enzymes directly immobilized on titanium (IV) oxide used in the development of antimicrobial agents

Lysosomal enzymes isolated from egg white were directly immobilized on titanium (IV) oxide (TiO₂) particles using shaking methods (150 rpm, room temperature, 10 min), and the immobilization efficiency, activity, and stability of lysosomal enzymes immobilized on TiO₂ were evaluated. Of the various mass ratios (w/w) of lysosomal enzymes to TiO₂ tested, we found that 100% immobilization efficiency was observed at a ratio of 1:20 (enzymes:TiO₂; w/w). Furthermore, the antimicrobial activities of the immobilized lysosomal enzymes were confirmed using viable cell counts against Escherichia coli. Our results showed that the antimicrobial activity of immobilized lysosomal enzymes is stable and can be maintained up to one month, but the antimicrobial activity of free enzymes without immobilization completely disappeared after five days in storage. In addition, enhanced immobilization efficiency was shown in TiO₂ pretreated with a divalent, positively charged ion, Ca²⁺, and the antimicrobial activity for E. coli increased as a function of increasing ratio of immobilized enzymes. However, K⁺, a monovalent, positively charged ion, did not have any positive effect on immobilization or antimicrobial activity. Finally, we suggest that activity and stability of immobilized lysosomal enzymes can be maintained for a longer time than those properties of free lysosomal enzymes.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

Proton-motive-force-driven formation of CO from CO2 and H2 in methanogenic bacteria

Cell suspensions of methanogenic bacteria (Methanosarcina barkeri, Methanospirillum hungatei, Methano-brevibacter arboriphilus, and Methanobacterium thermoautotrophicum) were found to form CO from CO2 and H2 according to the reaction: CO2 + H2----CO + H2O; delta G0 = +20 kJ/mol. Up to 15,000 ppm CO in the gas phase were reached which is significantly higher than the equilibrium concentration calculated from delta G0 (95 ppm under the experimental conditions). This indicated that CO2 reduction with H2 to CO is energy-driven and indeed the cells only generated CO when forming CH4. The coupling of the two reactions was studied in more detail with acetate-grown cells of M. barkeri using methanogenic substrates. The effects of the protonophore tetrachlorosalicylanilide (TCS) and of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide (cHxN)2C were determined. TCS completely inhibited CO formation from CO2 and H2 without affecting methanogenesis from CH3OH and H2. In the presence of the protonophore the proton motive force delta p and the intracellular ATP concentration were very low. (cHxN)2C, which partially inhibited methanogenesis from CH3OH and H2, had no effect on CO2 reduction to CO. In the presence of (cHxN)2C delta p was high and the intracellular ATP content was low. These findings suggest that the endergonic formation of CO from CO2 and H2 is coupled to the exergonic formation of CH4 from CH3OH and H2 via the proton motive force and not via ATP. CO formation was not stimulated by the addition of sodium ions.     

Biomimetic CO₂ sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials

The present study deals with immobilization of purified CA and whole cell of Pseudomonas fragi, Micrococcus lylae, and Micrococcus luteus 2 on different biopolymer matrices. Highest enzyme immobilization was achieved with P. fragi CA (89%) on chitosan-KOH beads, while maximum cell immobilization was achieved with M. lylae (75%) on chitosan-NH(4)OH beads. A maximum increase of 1.08-1.18 fold stability between 35 and 55°C was observed for M. lylae immobilized CA. The storage stability was improved by 2.02 folds after immobilization. FTIR spectra confirmed the adsorption of CA on chitosan-KOH beads following hydrophilic interactions. Calcium carbonate precipitation was achieved using chitosan-KOH immobilized P. fragi CA. More than 2 fold increase in sequestration potential was observed for immobilized system as compared to free enzyme. XRD spectra revealed calcite as the dominant phase in biomimetically produced calcium carbonate.     

Photosynthetic light and CO2 utilization and C4 traits of two novel super-rice hybrids

Characteristics of photosynthetic light and CO2 use efficiency from seedling to heading stage, and C4 pathway enzyme activities in both flag leaves and lemma were compared between two newly developed super-rice hybrids (Oryza sativa L.), Liangyoupeijiu and Hua-an 3, and a traditional rice hybrid, Shanyou 63. At seedling and tillering stages, Liangyoupeijiu and Hua-an 3 had higher net photosynthetic rates (Pn) and light saturated assimilation rates (Asat) than did Shanyou 63, at both normal (360 micromol mol(-1)) and doubled (720 micromol mol(-1)) CO2 concentrations. At the heading stage, the flag leaves of all three rice hybrids had similar Pn and Asat. However, the two super-rice hybrids had higher apparent quantum yield (AQY) and carboxylation efficiency (CE) during all three typical developmental stages, and higher quantum yield of CO2 fixation (PhiCO2) at the tillering and heading stages. In addition, Liangyoupeijiu showed significantly higher activities of the C(4) pathway enzymes in both flag leaves and lemmas than did Shanyou 63. As a result, flag leaves of the two super-rice hybrids had higher Pn at morning, noontime and late afternoon during the daily cycle. Since most of the grain yield of rice comes from the photosynthesis of flag leaves, the similar Asat and much higher AQY, CE and PhiCO2 at heading stage of the two super-rice hybrids indicates that higher photosynthetic efficiency rather than higher photosynthetic capacity may be the primary factor contributing to their higher grain yields.