A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes

Ghéczy, Nicolas; Sasaki, Kai; Yoshimoto, Makoto; Pour-Esmaeil, Sajad; Kröger, Martin; Stano, Pasquale; Walde, Peter

doi:10.1039/d0ra01204a

Cited by 9 publications

(66 citation statements)

References 71 publications

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“…Immobilized biocatalysts can give opportunities to continuously operate reactors for various purposes. − Support materials for enzyme immobilization can provide spatially or interfacially controlled reaction environments being potentially advantageous for multiple enzyme-catalyzed or bioelectrochemical reactions. − The alginate beads entrapped with BCALs were confined in a flow-through column and a TB2 solution containing 1.0 mM p -NA was passed continuously through the reactor. The absorbance at 405 nm was measured with respect to the fractions collected at the outlet of the reactor.…”

Section: Resultsmentioning

confidence: 99%

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions

Moriyama

Yoshimoto

2021

ACS Omega

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A versatile approach to entrap relatively small enzymes in hydrogels allows their diverse biotechnological applications. In the present work, bovine carbonic anhydrase (BCA) was efficiently entrapped in calcium alginate beads with the help of liposomes. A mixture of sodium alginate (3 wt %) and carbonic anhydrase–liposome conjugates (BCALs) was dripped into a Tris-HCl buffer solution (pH = 7.5) containing 0.4 M CaCl2 to induce the gelation and curing of the dispersed alginate-rich droplets. The entrapment efficiency of BCALs, which was defined as the amount of catalysts entrapped in alginate beads relative to that initially charged, was 98.7 ± 0.2% as determined through quantifying BCALs in the filtrate being separated from the beads. When free BCA was employed, on the other hand, a significantly lower entrapment efficiency of 27.2 ± 4.1% was obtained because free BCA could pass through alginate matrices. Because the volume of a cured alginate bead (10 μL) entrapped with BCALs was about 2.5 times smaller than that of an original droplet, BCALs were densely present in the beads to give the concentrations of lipids and BCA of 4.6–8.3 mM and 1.1–1.8 mg/mL, respectively. Alginate beads entrapped with BCALs were used to catalyze the hydrolysis of 1.0 mM p-nitrophenyl acetate (p-NA) at pH = 7.5 using the wells of a microplate or 10 mL glass beakers as batch reactors. Furthermore, the beads were confined in a column for continuous-flow hydrolysis of 1.0 mM p-NA for 1 h at a mean residence time of 8.5 or 4.3 min. The results obtained demonstrate that the conjugation of BCA to liposomes gave an opportunity to achieve efficient and stable entrapment of BCA in alginate hydrogels for applying to catalytic reactions in bioreactors.

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Section: Resultsmentioning

confidence: 99%

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions

Moriyama

Yoshimoto

2021

ACS Omega

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“…34,35,46 3.4 Co-immobilization of UGT1 and UGT2 Co-immobilized enzymes were established, as one of the onepot cascade enzymatic transformation system, to improve the productivity. [47][48][49] The UGT1 and UGT2 were co-immobilized onto the chitosan beads to form a dual-enzyme system for directly obtain Reb M from Reb A by the cascade reactions. Various ratios of UGT1 to UGT2 (1 : 2 to 1 : 10) were used for coimmobilization, the yield of Reb M was measured (Fig.…”

Section: Effect Of Temperature and Ph On Activity And Reusability Of The Immobilized Ugtsmentioning

confidence: 99%

Co-immobilized recombinant glycosyltransferases efficiently convert rebaudioside A to M in cascade

Wang

Liu

et al. 2021

RSC Adv.

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“…Enzyme molecules can be immobilized in various water-insoluble materials including phospholipids- or synthetic polymers-based vesicles, − inorganic substances, − hydrogels, , and DNA scaffolds. , Immobilized enzymes are different with respect to their stability, reaction efficiency, or practical applicability as compared to free enzymes homogeneously dispersed in an aqueous solution. − Cellular organelles are regarded as sophisticated immobilized enzyme systems which possess highly complex but organized networks of enzymes through being compartmentalized in the water phase or conjugated/adsorbed to a membranous interface. In this way, cell organelles achieve spatially and kinetically controlled mass transfer and multiple catalytic reactions.…”

Section: Introductionmentioning

confidence: 99%

Confinement of Metalloenzymes in PEGylated Liposomes to Formulate Colloidal Catalysts for Antioxidant Cascade

Iwasaki

Yoshimoto

2021

Langmuir

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Antioxidant cascade reactions detoxifying reactive oxygen species are of significance to control oxidative stresses-triggered diseases. In the present work, the antioxidant catalysts were prepared through the confinement of dual metalloenzymes in liposomes. The amino groups of superoxide dismutase (SOD) were conjugated to the carboxyl groups-bearing liposomes encapsulated with the catalase (CAT) to formulate a spatially organized antioxidant reaction network. The activity of SOD and CAT in the liposomal system was evaluated in detail on the basis of the prolonged xanthine oxidase/xanthine reaction producing superoxide anion radicals (O2̇–) and hydrogen peroxide (H2O2) coupled with redox reactions of cytochrome c. The liposome-confined SOD and CAT molecules were clearly demonstrated to catalyze the sequential disproportionation of O2̇– and H2O2 at 25 °C in a potassium phosphate buffer solution (pH = 7.8) under moderate transfer resistance with respect to the intermediate product (H2O2) within the liposomes. Furthermore, the liposomal catalysts were modified with the poly(ethylene glycol) (PEG)-conjugated lipids with the molecular mass of the PEG moiety of about 5000 through the post-PEGylation approach. The mean hydrodynamic diameter of the PEGylated liposomal catalysts was 140–150 nm. The dual enzyme activity in liposomes and the thermal stability of the encapsulated CAT were practically unaffected by the PEGylation. The above liposome-based antioxidant catalysts are highly biocompatible, PEG-modifiable, and reactive, thereby making the catalysts potentially applicable to therapeutic materials exhibiting functionality similar to cellular peroxisomes.

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A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes

Abstract: A cascade reaction catalysed by bovine carbonic anhydrase (BCA) and horseradish peroxidase (HRP) proceeds over two possible pathways, which explains differences in product formation for differently immobilised enzymes in flow-through reactions.

Cited by 9 publications

References 71 publications

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions

Co-immobilized recombinant glycosyltransferases efficiently convert rebaudioside A to M in cascade

Confinement of Metalloenzymes in PEGylated Liposomes to Formulate Colloidal Catalysts for Antioxidant Cascade

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