Quantitative enzyme immobilization: Control of the carboxyl group density on support surface

In this paper, carboxyl groups were quantitatively introduced onto a support surface by casting a polystyrene (PS) solution with small molecular weight α,ω-dicarboxyl-terminated polystyrene (PS-(COOH)2) onto a glass plate. The support surface was examined using X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The surface density of the carboxyl groups was measured by UV spectrophotometry with methylene blue (MB) dye as an indicator. Lipase (from Candida rugosa) as a model enzyme was covalently immobilized on the support surface in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxylsuccinimide (NHS). It was found that with an increase of –COOH density from 0.42 × 10−6 mol/m2 to 1.21 × 10−6 mol/m2, the amount of immobilized protein increased linearly from 3.1 mg/m2 to 4.1 mg/m2, while the specific activity of lipase decreased from 7.79 U/mg to 5.14 U/mg with a breakpoint at the –COOH density of 1.08 × 10−6 mol/m2. As the trend of the specific activity of immobilized lipase was opposite to that of the amount of immobilized protein, the optimum carboxyl group density for obtaining the highest enzyme activity (26.4 U/m2) was 1.08 × 10−6 mol/m2 and the molar ratio of –COOH/lipase was 16.2. The decreasing rate of the specific activity of the immobilized enzyme accelerated when –COOH density was above 1.08 × 10−6 mol/m2, which may be regarded as a critical value of –COOH density, leading to the enzyme immobilization changing from single-site to multi-site immobilization. After immobilization, the optimum pH of enzyme was broader and the optimum temperature of enzyme was enhanced to 50 °C. This work is helpful for the design and preparation of supports for the immobilization of enzymes of higher activity and for increasing the understanding of the relationship between conformation variation of an immobilized enzyme and its corresponding catalytic activity.