A Three‐Enzyme Cascade Reaction through Positional Assembly of Enzymes in a Polymersome Nanoreactor

Abstract

Porous polymersomes based on block copolymers of isocyanopeptides and styrene have been used to anchor enzymes at three different locations, namely, in their lumen (glucose oxidase, GOx), in their bilayer membrane (Candida antarctica lipase B, CalB) and on their surface (horseradish peroxidase, HRP). The surface coupling was achieved by click chemistry between acetylene-functionalised anchors on the surface of the polymersomes and azido functions of HRP, which were introduced by using a direct diazo transfer reaction to lysine residues of the enzyme. To determine the encapsulation and conjugation efficiency of the enzymes, they were decorated with metal-ion labels and analysed by mass spectrometry. This revealed an almost quantitative immobilisation efficiency of HRP on the surface of the polymersomes and a more than statistical incorporation efficiency for CalB in the membrane and for GOx in the aqueous compartment. The enzyme-decorated polymersomes were studied as nanoreactors in which glucose acetate was converted by CalB to glucose, which was oxidised by GOx to gluconolactone in a second step. The hydrogen peroxide produced was used by HRP to oxidise 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to ABTS(.+). Kinetic analysis revealed that the reaction step catalysed by HRP is the fastest in the cascade reaction.

Functionalization of Block Copolymer Vesicle Surfaces

“…Polymersomes that have assembled from amphiphilic block copolymers have proven to be useful tools as drug delivery systems [8,[20][21][22], nanoreactors and sensors [13,17,[23][24][25][26][27][28]. The attachment of targeting ligands or enzymes to the polymersomes, as well as the immobilization of polymer vesicles on surfaces, is of crucial importance in most of the previously mentioned applications.…”

“…The state of the art in terms of common conjugation chemistry that fulfills all or some of these requirements is summarized in Table 1. [26,29] Horseradish peroxidase [26] and an azidofunctionalized Candida antarctica Lipase B [29] Azide Alkyne 1,4-disubstituted 1,2,3-triazole PBD-b-PEG [30,31], PS-b-PAA [32] Mannose functional polyester dendron [30,31], dansyl probe [32], biotin [32], eGFP [32] Biotin Streptavidin Biotin-Streptavidin interaction PBD-b-PEG and PEG-b-PEE [33], PMOXA-b-PDMS-b-PMOXA [17,20,34,35] Superavidin-coated microspheres [33], antibody [36], polyguanylic acid [20,34], streptavidin-modified glass surface [17], fluorescently labeled avidin [33,35] …”

Hybrid, Nanoscale Phospholipid/Block Copolymer Vesicles

“…Since the initial work by the groups of Discher and Eisenberg [1,2], sophisticated polymersome designs have been explored involving degradable or non-degradable systems [3], or those that are responsive towards external stimuli [4]. Potential applications of polymersomes include delivery of drugs [5] and other bioactive substances such as siRNA [6] and antibodies [7], and they have been used as carriers for probes in bioimaging [8,9] or for biocatalysts in the form of nanoreactors [10,11]. In general, the polymersome membrane displays enhanced stability as compared to liposomes [8,12] and they can, hence, more readily be modified.…”

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

“…After a pioneering study of Chang (1987) on cycling enzymatic routes inside non-lipid compartments (for example, the coupled system: urease, glutamate dehydrogenase, glucose dehydrogenase, glutamate-pyruvate transaminase), more recent approaches have been based on liposomes and polymersomes. Typical examples are glucose oxidase and horseradish peroxidase inside liposomes (Hill et al, 1997;Kaszuba & Jones, 1999) or polymersomes (Delaittre et al, 2009;van Dongen et al, 2009), possibly with the involvement of a third enzyme (a lipase). The other system that has received attention is the couple bacterhodopsin/ATP synthase (both membrane proteins) that have been reconstituted in liposomes at the aim of producing ATP after irradiation (Freisleben et al, 1995;Pitard et al, 1996;Choi and Montemagno, 2007).…”