Evaluating enzyme stabilizations in calcium carbonate: Comparing in situ and crosslinking mediated immobilization.

Enzyme immobilization using inorganic materials has been shown to preserve enzyme activity improving and improve their practical applications in biocatalytic process designs. Proper immobilization methods have been used to obtain high recycling and storage stability. In this study, we compared the activity and stability of in situ or crosslink-immobilized enzymes in a CaCO3 biomineral carrier. More than 30% of the initial enzyme activity was preserved for both the systems after 180 days upon 15 activity measurements at room temperature, confirming the improved stability of these enzyme systems (100 mM phosphate buffer, pH 8.0); however, differences in enzyme loading, activity, and characteristics were observed for each of these methods. Each system exhibited efficacy of 80% and 20%, respectively. Based on the same amount of immobilized enzyme (0.2 mg), the specific activities of hydrolysis of p-nitrophenyl butyrate substrate at room temperature of in situ immobilized carboxyl esterase (CE) and crosslinked CE were 11.37 and 7.63 mM min-1 mg-1, respectively (100 mM phosphate buffer, pH 8.0). Moreover, based on the kinetic behavior, in situ immobilized CE exhibited improved catalytic efficiency (Vmax Km-1) of the enzyme, exhibiting 4-fold higher activity and efficiency values than those of the CE immobilized in CaCO3. This is the first study to describe the stabilization of enzymes in CaCO3 and compare the enzyme kinetics and efficiencies between in situ immobilization and crosslinking in CaCO3 carriers.

Immobilization and Stabilization of Enzyme in Biomineralized Calcium Carbonate Microspheres.

Biomineralized uniform and well-organized calcium carbonate microspheres were synthesized for enzyme immobilization, and the immobilized enzyme was successfully stabilized. The physicochemical parameters of calcium carbonate were studied using scanning electron microscopy with energy-dispersive X-ray spectroscopy, particle size analysis, X-ray diffraction analysis, Fourier-transform infrared spectroscopy, and surface area measurement. Additionally, Barrett-Joyner-Halenda adsorption/desorption analysis showed that the calcium carbonate microspheres provided efficient mesopore space for enzyme loading. As a model enzyme, carboxyl esterase (CE) was entrapped and then cross-linked to form an enzyme structure. In this aggregate, the cross-linked enzymes cannot leach out from mesopores, resulting in enzyme stability. The hydrolytic activities of the free and cross-linked enzymes were analyzed over broad temperature and pH ranges. The cross-linked enzyme displayed better activity than the free enzyme. Furthermore, the immobilized CE was found to be stable for more than 30 days, preserving 60% of its initial activity even after being reused more than 10 times. This report is expected to be the first demonstration of a stabilized cross-linked enzyme system in calcium carbonate microspheres, which can be applied in enzyme catalyzed reactions involved in bioprocessing, bioremediation, and bioconversion.

Improvement in modular scalability of polymeric thin-film photobioreactor for autotrophic culturing of Haematococcus pluvialis using industrial flue gas.

The aim of this study is investigate the effect of column diameter (D), height/diameter (H/D) ratio, and gas flow rate on microalgae cultivation, Haematococcus pluvialis. Bubble column reactors with various D and H/D ratio were tested to assess the hydrodynamic properties and biomass production performance. Then, H. pluvialis was cultured under outdoor autotrophic conditions using industrial flue gas. By optimizing the reactor module, reactor volume increased to 354% with minimized biomass loss. Compared to the control, developed module showed biomass and astaxanthin productivity of 0.052 versus 0.053 g/L/day, and 1.48 versus 1.47 mg/L/day, respectively. Consequently, biomass productivity was maintained with increased reactor scale, and the result is applicable to the scale up of overall microalgae cultivation process.

Expression and characterization of codon-optimized carbonic anhydrase from Dunaliella species for CO(2) sequestration application.

Carbonic anhydrases (CAs) have been given much attention as biocatalysts for CO(2) sequestration process because of their ability to convert CO(2) to bicarbonate. Here, we expressed codon-optimized sequence of α-type CA cloned from Dunaliella species (Dsp-aCAopt) and characterized its catalyzing properties to apply for CO(2) to calcite formation. The expressed amount of Dsp-aCAopt in Escherichia coli is about 50 mg/L via induction of 1.0 mM isopropyl-ß-D-thiogalactopyranoside at 20 °C (for the case of intact Dsp-aCA, negligible). Dsp-aCAopt enzyme shows 47 °C of half-denaturation temperature and show wide pH stability (optimum pH 7.6/10.0). Apparent values of K (m) and V (max) for p-nitrophenylacetate substrate are 0.91 mM and 3.303 × 10(-5) µM min(-1). The effects of metal ions and anions were investigated to find out which factors enhance or inhibit Dsp-aCAopt activity. Finally, we demonstrated that Dsp-aCAopt enzyme can catalyze well the conversion of CO(2) to CaCO(3), as the calcite form, in the Ca(2+) solution [8.9 mg/100 µg (172 U/mg enzyme) with 10 mM of Ca(2+)]. The obtained expression and characterization results of Dsp-aCAopt would be usefully employed for the development of efficient CA-based system for CO(2)-converting/capturing processes.

Analysis of expressed sequence tags from the antarctic psychrophilic green algae, Pyramimonas gelidicola.

Expressed sequence tags (ESTs) from the Antarctic green algae Pyramimonas gelidicola were analyzed to obtain molecular information on cold acclimation of psychrophilic microorganisms. A total of 2,112 EST clones were sequenced, generating 222 contigs and 219 singletons, and 200 contigs and 391 singletons from control (4 degrees C) and cold-shock conditions (-2 degrees C), respectively. The complete EST sequences were deposited to the DDBJ EST database (http:// www.ddbj.nig.ac.jp/index-e.html) and the nucleotide sequences reported in this study are available in the DDBJ/EMBL/ GenBank. These EST databases of Antarctic green algae can be used in a wide range of studies on psychrophilic genes expressed by polar microorganisms.

Expression and antioxidant enzymes in Chaetoceros neogracile, an Antarctic alga.

We examined low temperature-induced protein profile alterations in the Antarctic alga Chaetoceros neogracile using a proteomic approach. Chaetoceros neogracile was cultured at 4 degree C and then cooled to 0 degree C, and the resultant cold-induced alterations in protein expression patterns were analyzed by two-dimensional gel electrophoresis. Of the approximately 150 protein spots detected by Coomassie staining, we identified 15 with a greater than two-fold change in amount. Of these, ten proteins were up-regulated and five were down-regulated after cold exposure. Three cellular protein quality control proteins, such as chaperone protein DnaK, chaperone ClpB, and 26S protease regulatory subunit 6B homolog were prominently increased, whereas chaperone protein HtpG was decreased in response to cold stress. Moreover, changes in enzyme activity and isozyme profiles for superoxide dismutase, glutathione reductase, and glutathione S-transferase were also detected in the gel, using an enzyme activity staining method. These alterations in protein expression and antioxidant enzyme activity may be related to survival mechanisms of C. neogracile at low temperatures.

Purification and characterization of an anionic isoperoxidase from scented-geranium callus.

Secretory anionic isoperoxidase (EC 1.11.1.7), named PA1, was 68-fold purified from scented-geranium (Pelargonium graveolense) callus by using ion exchange chromatography and gel filtration. Isoperoxidase PA1 was a glycoprotein with an isoelectric point (pI) of 4.0. The molecular weight of PA1 was approximately 42.5 and 44 kDa, estimated by SDS-PAGE and Sephadex G-150 gel filtration, respectively. The optimum pH of the enzyme was 5.0 for guaiacol and H2O2, and the Km values for guaiacol and H2O2 were 1.96 and 8.5mM, respectively. Substrate studies in terms of optimum pHs and Km values with various synthetic and naturally occurring phenolic compounds were performed. In comparison with cationic isoperoxidase, PC3, which has been already characterized, anionic isoperoxidase PA1 had much lower Km values for synthetic phenolic compounds and much higher Km values for naturally occurring phenolic compounds than PC3. Moreover, anionic isoperoxidase PA1 could utilize ferulic acid as a substrate very well, while cationic isoperoxidase PC3 could not utilize ferulic acid as a substrate.