An Accessible Method to Improve the Stability and Reusability of Porcine Pancreatic α-Amylase via Immobilization in Gellan-Based Hydrogel Particles Obtained by Ionic Cross-Linking with Mg2+ Ions

Abstract

Amylase is an enzyme used to hydrolyze starch in order to obtain different products that are mainly used in the food industry. The results reported in this article refer to the immobilization of α-amylase in gellan hydrogel particles ionically cross-linked with Mg²⁺ ions. The obtained hydrogel particles were characterized physicochemically and morphologically. Their enzymatic activity was tested using starch as a substrate in several hydrolytic cycles. The results showed that the properties of the particles are influenced by the degree of cross-linking and the amount of immobilized α-amylase enzyme. The temperature and pH at which the immobilized enzyme activity is maximum were T = 60 °C and pH = 5.6. The enzymatic activity and affinity of the enzyme to the substrate depend on the particle type, and this decreases for particles with a higher cross-linking degree owing to the slow diffusion of the enzyme molecules inside the polymer's network. By immobilization, α-amylase is protected from environmental factors, and the obtained particles can be quickly recovered from the hydrolysis medium, thus being able to be reused in repeated hydrolytic cycles (at least 11 cycles) without a substantial decrease in enzymatic activity. Moreover, α-amylase immobilized in gellan particles can be reactivated via treatment with a more acidic medium.

Keywords: gellan hydrogel particles; hydrolysis; ionic cross-linking; magnesium acetate; α-amylase immobilized.

Figure 1

Schematic structure of α-amylase immobilized in ionically cross-linked gellan particles.

Figure 2

FT-IR spectra of gellan, α-amylase, and sample A4.

Figure 3

Scanning electron microscopy photographs of particles containing immobilized α-amylase cross-linked with (a) 1% and (b) 2% magnesium acetate in the cross-linking bath.

Figure 4

Variation of degree of swelling over time for three types of particles with immobilized α-amylase, obtained using different concentrations of magnesium acetate in the cross-linking bath (Q1%-A3, Q2%-A4, and Q3%-A5 magnesium acetate).

Figure 5

Michaelis–Menten kinetics (a) and Lineweaver–Burk plot (b) for free enzyme and enzyme immobilized in magnesium acetate cross-linked gellan particles A3 and A4.

Figure 6

Influence of pH (a) and temperature (b) on enzyme activity for free and immobilized enzyme.

Figure 7

The α-amylase inhibition percentage on NaCl for free and immobilized α-amylase (a) and enzyme activity in the presence of different salt concentrations (b).

Figure 8

Relative activity determinations in repeated hydrolytic cycles for particles with immobilized α-amylase (3 mg/mL), using starch as substrate at (a) pH 5.6 and (b) pH 5.2 and pH = 5.6. The temperature at which the catalytic activity of the particles was tested was 35 °C. The duration between hydrolytic cycles was 10 min.

Figure 9

Schematic presentation of the preparation of gellan particles with 3 mg of immobilized α-amylase.