Thermally Responsive Reversed Micelles for Immobilization of Enzymes

Abstract

In this study, thermally responsive alkyl end‐capped poly(N‐isopropylacrylamide‐co‐acrylic acid) amphiphilic copolymers were synthesized, and successfully utilized to fabricate reversed micelles and immobilize enzymes. Transmission electron microscopy (TEM) study showed that the reversed micelles were spherical in nature. The activity of Candida rugosa lipase immobilized in the reversed micelles towards catalyzing the esterification of lauric acid and 1‐propanol was analyzed in comparison with naked enzyme. Immobilized lipase provided much greater catalytic activity. In addition, the effects of pH, water content, polymer and enzyme concentration on the catalytic activity were investigated. The optimized fabrication conditions of lipase‐loaded reversed micelles, under which lipase gave the highest activity, were as follows: polymer concentration, 12 mg mL–1; enzyme concentration, 25 mg mL–1 phosphate buffered saline (PBS); pH, 7.4; W0 ([water]/[surfactant]), 83.3. Lipase immobilized in these micelles was much more stable than that in conventional sodium bis(2‐ethylhexyl) sulfosuccinate micelles. More importantly, the size of lipase‐immobilized micelles decreased, and the enzyme solution precipitated from the reaction mixture when the temperature increased to a value slightly higher than the lower critical solution temperature (LCST) of the polymer. The recovery rate of enzyme was about 75 %. The α‐helix structure of the recovered lipase remained intact. In addition, the enzymatic reaction was terminated after raising the environmental temperature slightly above the LCST. These thermally responsive micelles may make a promising system for enzyme immobilization.

Preparation strategies of thermo-sensitive P(NIPAM-co-AA) microspheres with narrow size distribution

“…Among them, poly (N-isopropyl acrylamide) (PNIPAM) is one of the most extensively investigated stimuli-responsive polymers, and has been widely used in biomedical fields including drug controlled release, and enzyme and protein immobilizations [5][6][7][8]. It undergoes a sharp volume transition near the lower critical solution temperature (LCST) from a highly hydrated swollen state below LCST to a dehydrated collapsed state above LCST [9], which can greatly influence the diffusion rate of drug molecules through PNIPAM [10][11][12]. The shrinking and swelling process of PNIPAM hydrogels is controlled by diffusion transport, thus the response rate is inversely proportional to the square of the smallest dimension of the gel [13,14].…”

Covalent immobilization of trypsin onto thermo‐sensitive poly(N‐isopropylacrylamide‐co‐acrylic acid) microspheres with high activity and stability

“…The decreased activity of enzyme could be considered as the result of the interaction between enzyme molecules. At a suitable enzyme concentration, trypsin could be immobilized onto microspheres in single layer status, which restrained the intermolecular reaction of immobilized trypsin molecules, and the immobilized trypsin could exhibit its maximal activity . However, a high enzyme concentration led to the increase of protein loading.…”

Effect of acrylic acid weight percentage on the pore size in poly(N-Isopropyl acrylamide-co-acrylic acid) microspheres

“…As one of the most extensively investigated thermally reversible polymers, poly(N-Isopropylacrylamide) (PNIPAM) has been widely used in drug delivery [1][2][3], enzyme immobilization [4][5][6], metal ion purification [7], fluorescence detection [8], and chromatography technique [9,10]. Particles of PNIPAM and its copolymers may be one of the most intensively studied subjects because of their small dimension for fast stimuli responses.…”