Immobilization of 2-Deoxy-d-ribose-5-phosphate Aldolase in Polymeric Thin Films via the Langmuir–Schaefer Technique

Abstract

A synthetic protocol for the fabrication of ultrathin polymeric films containing the enzyme 2-deoxy-d-ribose-5-phosphate aldolase from Escherichia coli (DERA) is presented. Ultrathin enzymatically active films are useful for applications in which only small quantities of active material are needed and at the same time quick response and contact times without diffusion limitation are wanted. We show how DERA as an exemplary enzyme can be immobilized in a thin polymer layer at the air-water interface and transferred to a suitable support by the Langmuir-Schaefer technique under full conservation of enzymatic activity. The polymer in use is a poly(N-isopropylacrylamide-co-N-2-thiolactone acrylamide) (P(NIPAAm-co-TlaAm)) statistical copolymer in which the thiolactone units serve a multitude of purposes including hydrophobization of the polymer, covalent binding of the enzyme and the support and finally cross-linking of the polymer matrix. The application of this type of polymer keeps the whole approach simple as additional cocomponents such as cross-linkers are avoided.

2-Deoxy-d-ribose-5-phosphate aldolase (DERA): applications and modifications

“…Three examples were described with different carriers but always utilizing the amino group of the surface lysines (Wang et al 2012; Fei et al 2014; Reinicke et al 2017). In all cases, the active immobilized DERA displayed improved, but not excellent activity or stability.…”

“…This type of protective covalent linking of the enzyme via a tether to the carrier material was also applied to stabilize DERA against high acetaldehyde concentrations. Three examples were described with different carriers but always utilizing the amino group of the surface lysines (Wang et al 2012 ; Fei et al 2014 ; Reinicke et al 2017 ). In all cases, the active immobilized DERA displayed improved, but not excellent activity or stability.…”

Self‐Assembled Polymeric Membranes and Nanoassemblies on Surfaces: Preparation, Characterization, and Current Applications

“…Lipoic acid functionalized p(butadiene)‐ b ‐p(ethyleneoxide) (LA‐ b ‐PB‐ b ‐PEO) triblocks were deposited via the LB technique to form a monolayer, and a highly ordered low defect bilayer was achieved by adding PB‐PEO‐OH via the LS technique . Blockcopolymers are also used for the formation of active surfaces, for which a poly( N ‐isopropylacrylamide‐ co ‐ N ‐2‐thiolactoneacrylamide) (PNIPAM‐ co ‐TlaAm) was first compressed to a homogeneous film and the subsequent horizontal dipping of an activated silica substrate lead to the transfer of it to the solid support . By mixing block copolymers and lipids, hybrid membranes become also accessible via layer deposition techniques, and permit the formation of domains or homogeneous membranes according to the miscibility of the lipid with the polymer .…”

“…Both copolymers and block polymers function as supporting membranes for proteins, and lipid/polymer membranes are also accessible via this technique. For instance, PNIPAM‐TlAa was deposited to promote aldolase insertion providing covalent bonding points without the use of further membrane crosslinking . Poly[9,9‐dioctylfluorene‐ co ‐thiophene] was used in combination with stearic acid for the immobilization of β‐galattosidase: all three components were deposited at the air water interface and compressed to a film, which was subsequently transferred to a solid support .…”

Bifunctionalized Redox-Responsive Layers Prepared from a Thiolactone Copolymer

“…34,35 However, polythiols are prone to oxidation under ambient atmosphere which considerably limits their manipulation. To overcome this drawback, Du Prez and co-workers [36][37][38][39][40][41][42][43] have recently developed copolymers incorporating thiolactone as a latent thiol functionality. By opening thiolactone cycles using a strong nucleophile such as an amine and/or thiol groups are liberated and are available to immobilize compounds of interest through a cleavable disulfide bond or via 'click chemistry'.…”