Entrapment of α-Amylase in Agar Beads for Biocatalysis of Macromolecular Substrate.

Manu Sharma,Vinay Sharma,Dipak K Majumdar

doi:10.1155/2014/936129

Abstract

Attempts have been made to optimize immobilization parameters, catalytic property, and stability of immobilized α-amylase in agar. The work compares natural entrapment efficiency of agar with the ionotropically cross-linked agar hydrogel, with the advantage of easy scale-up and cost and time effectiveness. Beads prepared with 3% (w/v) agar and 75 mM calcium chloride and hardened for 20 minutes were selected for further studies on the basis of entrapment efficiency (80%) and physical stability. Following entrapment, pH and temperature optima of enzyme were shifted from 6 to 6.5 and 50 to 55°C, respectively. Michaelis constant (K m) for both free and entrapped enzymes remained the same (0.83%) suggesting no change in substrate affinity. However, V max⁡ of entrapped enzyme decreased ~37.5-fold. The midpoint of thermal inactivation for entrapped enzyme increased by 8 ± 1°C implying its higher thermal stability. The entrapped enzyme in calcium agar bead had an Ea value of 27.49 kcal/mol compared to 17.6 kcal/mol for free enzyme indicating increased stability on entrapment. Half-life of enzyme increased ~2.2 times after entrapment in calcium agar at 60°C indicating stabilization of enzyme. The reusability of beads was size dependent. Beads with diameter <710 μm were stable and could be reused for 6 cycles with ~22% loss in activity.

Highlights

Enzymes have an enormous potential as catalysts in chemical processes in wide range of industries [1]. α-Amylases are one of the largest selling industrial enzymes that find use in a wide variety of industrial applications such as production of ethanol, starch liquefaction, detergents, desizing of textile, modified starches, laundering, dye removal and feed preprocessing, and paper recycling [2]
On addition of agar solution to calcium chloride, instantaneous interfacial cross-linking takes place followed by a more gradual gelation of the interior and causes loss of enzyme from the beads, which was found to be proportional to the degree of cross-linking
Increase in viscosity with increase in agar concentration may retard the penetration of distilled water or calcium to the interior of the bead, resulting in decreased cross-linking and increased entrapment efficiency

Summary

Introduction

Enzymes have an enormous potential as catalysts in chemical processes in wide range of industries [1]. α-Amylases are one of the largest selling industrial enzymes that find use in a wide variety of industrial applications such as production of ethanol, starch liquefaction, detergents, desizing of textile, modified starches, laundering, dye removal and feed preprocessing, and paper recycling [2]. Α-Amylases are one of the largest selling industrial enzymes that find use in a wide variety of industrial applications such as production of ethanol, starch liquefaction, detergents, desizing of textile, modified starches, laundering, dye removal and feed preprocessing, and paper recycling [2]. Bacterial amylases used in industrial applications are highly thermostable with optimum temperatures greater than 90∘C [3, 6]. Such high temperature optima could lead to deleterious change in foods and result in burnt flavors. Fungal amylases (source Aspergillus niger and Aspergillus oryzae being mesophilic in nature) have the advantage of high catalytic rates at moderate temperatures of 50–60∘C without affecting the sensory appeal in starch liquefaction process leading to refined syrups and sweeteners widely used in the food industry. Various studies have been carried out on constraining the enzyme on to a solid support thereby immobilizing it and increasing its stability towards temperature [9,10,11]

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Results

Conclusion