Immobilization of Phenylalanine Ammonia-lyase via EDTA Based Metal Chelate Complexes – Optimization and Prospects

Immobilized metal ion affinity chromatography principles were applied for selective immobilization of recombinant polyhistidine tag fused phenylalanine ammonia-lyase from parsley (PcPAL) on porous polymeric support with aminoalkyl moieties modified with an EDTA dianhydride (EDTADa)-derived chelator and charged with transition metal ions. Out of the five investigated metal ions - Fe3+, Co2+, Ni2+, Cu2+, Zn2+ - the best biocatalytic activity of PcPAL was achieved when the enzyme was immobilized on the Co2+ ion-charged support (31.8 ± 1.2 U/g). To explore the features this PcPAL obtained by selective immobilization, the thermostability and reusability of this PAL biocatalyst were investigated. To maximize the activity of the immobilized PcPAL the surface functionalization of the aminoalkylated polymeric carrier was fine-tuned with using glycidol as a thinning group beside EDTADa. The maximal activity yield (YA=103 %) was earned when the EDTADa and glycidol were used in 1 to 24 ratio. The reversibility of the immobilization method allowed the development of a support regeneration protocol which enables easy reuse of the functionalized support in case of enzyme inactivation.

Geranyl Functionalized Materials for Site-Specific Co-Immobilization of Proteins

Different materials containing carboxylic groups have been functionalized with geranyl-amine molecules by using an EDC/NHS strategy. Chemical modification of the support was confirmed by XRD, UV-spectrophotometer, and FT-IR. This geranyl-functionalized material was successfully applied for four different strategies of site-selective immobilization of proteins at room temperature and aqueous media. A reversible hydrophobic immobilization of proteins (lipases, phosphoglucosidases, or tyrosinase) was performed in neutral pH in yields from 40 to >99%. An increase of the activity in the case of lipases was observed from a range of 2 to 4 times with respect to the initial activity in solution. When chemically or genetically functionalized cysteine enzymes were used, the covalent immobilization, via a selective thiol-alkene reaction, was observed in the presence of geranyl support at pH 8 in lipases in the presence of detergent (to avoid the previous hydrophobic interactions). Covalent attachment was confirmed with no release of protein after immobilization by incubation with hydrophobic molecules. In the case of a selenium-containing enzyme produced by the selenomethionine pathway, the selective immobilization was successfully yielded at acidic pH (pH 5) (89%) much better than at pH 8. In addition, when an azido-enzyme was produced by the azide–homoalanine pathway, the selective immobilization was successful at pH 6 and in the presence of CuI for the click chemistry reaction.

Selective Immobilization of Antimony Using Brucite-rich Precipitate Produced during In Situ Hypochlorous Acid Formation through Seawater Electrolysis in a Nuclear Power Plant

This study has investigated the selective immobilization of antimony using the brucite (magnesium hydroxide)-rich precipitate (BP) collected from a hypochlorous storage tank in a nuclear power plant of South Korea. The energy dispersive X-ray and X-ray diffraction analyses revealed that the BP mainly consisted of magnesium (72.5%) and its dominant mineral phase was brucite (Mg(OH)2). Therefore, brandholzite (Mg[Sb(OH)6]2·6H2O) was newly formed through the surface-induced precipitation during the adsorption of antimony using the BP. The adsorbed amount of antimony increased with decreasing pH values because of the increased positive surface charge of the BP (pHpzc = 9.6). The maximum adsorption capacity (Qmax) of BP, calculated by Langmuir adsorption isotherm, was 11.02 mg/g. The presence of competitive anions did not significantly affect the adsorption of antimony toward the BP due to its high selectivity. These results suggest that the facile utilization of the BP as a low-cost adsorbent seems to be a practical option for the selective removal of antimony from wastewater.

Bio-specific immobilization of enzymes on electrospun PHB nanofibers

Background Oriented immobilization of enzymes provides substantial advantages in terms of improving the efficiency of enzymatic process as well as enhancing the reusability of enzymes. Phasins have high binding affinity to PHA, and thus can potentially be used as a fusion partner to immobilize enzymes onto PHA supports. However, presently available PHA supports of granular configuration have low surface-area-to-volume ratio, which limits enzyme loading on the supports. In this study, we explored the use of electrospun PHB nanofibers as an alternative support for high density immobilization of a phasin-fused lipase in directed orientations.Results The electrospun PHB nanofibers were successfully used for selective immobilization of the phasin-fused enzyme, and could anchor 120-fold more enzyme than PHB granules of the same weight. The enzyme immobilized onto the PHB nanofibers exhibited exceptionally high stability. Furthermore, via oriented immobilization of the enzymes, PHB nanofibers loaded with the phasin-fused lipase provided 3- to 10-fold higher enzyme activity than the conventional immobilization supports.Conclusions Our approach combines the advantageous features of nanofibrous materials and the regio-specificity of biomolecular interactions for the efficient immobilization of enzymes, which can be widely adopted in the development of various enzymatic processes.