Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization

Abstract

Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein’s surface, resulting in a shift of the putative net charge from −1 to +21. This designed hCAII­(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII­(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn–hCAII­(+21) host–guest complex exhibits both greater activity and thermal stability when compared to hCAII­(+21). Upon immobilization on a solid support, AfFtn–hCAII­(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.

Multipoint Immobilization at Inert Center of Papain on Homo-Functional Diazo-Activated Silica Support: A Way of Restoring “Above Room-Temperature” Bio-Catalytic Sustainability

“…The elemental population of carbon (20.9%) in EDX was taken as standard to find out the numerical value (251) of "y" in inert−support−enzyme complex (Results and Discussion: Immobilization via Multi-Point Mode of Attachment). The molecular dimension of the inert−support− enzyme complex becomes {Si(OSi) 4 Though this elemental population (at. %) approves immobilization of the macromolecule, it does not convey the mode of attachment with the inert support.…”

“…The amount of immobilized papain (13.9 μM g −1 ; Experimental Section: Synthesis of {SiO 2 }@Papain) was nearly one-fourth of the diazo-groups (55 μM g −1 ). Thus, each enzyme molecule comprising 19 Tyr moieties can make only four diazo linkages with each unit cell as a multi-point mode of attachment, {Si(OSi) 4 Instrumental Analysis of the Immobilized Material. The SEM image of the SG immobilized papain (SG@papain) is shown in Figure 1a,b.…”

“…The immobilization, primarily via a multipoint attachment that hinders the unfolding of the protein, is an indispensable step in preventing enzyme thermo-inactivation. , The more rigid the enzyme globule is (e.g., the more rigidly fixed on a solid support), the more difficult it becomes to unfold. The well-developed immobilization methodologies that evolved with time include the adsorption on the surface of bulk solid via physical force, − the encapsulation employing multi-metal based inorganic–protein hybrid, , sol–gel or biomimetic mineralization approach, entrapment by physical process, the ion exchange, and the covalently linking onto a bulk solid surface.…”

“…11 After the addition is over, this reaction-admixture under constant stirring was kept for another 30 min. In this reaction course, papain employing its tyrosine residue (having phenolic −OH group) appears on the SiO 2 -surface through diazo coupling reaction as an identifiable entity of a definite dimension, {Si(OSi) 4 Estimation of Homo-Functional Diazo-Activating Surface Groups. At the first step, from 10 mM standard stock, a series of standard solutions viz., 0.078, 0.313, 0.625, 1.25, and 2.50 mmol L −1 of the aqueous orange II dye (Sigma-Aldrich) solution was prepared.…”

Protein encapsulation within the internal cavity of a bacterioferritin

“…Encapsulation of proteins within ferritin cages has been reported for horse spleen ferritin (employing a low pH method) with an artificial streptavidin-based transfer hydrogenase, 16 and for the Ftns from A. fulgidus and T. maritima. 15,17,32,52 For the latter, there is an apparent requirement for the encapsulated protein to carry a significant positive charge, suggesting that complementarity with the negatively charged inner surface of the protein is a key requirement. Here we demonstrate capture of hemin from solution by dimeric Bfr which can then be returned to the fully assembled form whilst retaining heme bound to the inner surface of the cage.…”