New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization

• Cloning of a gene encoding a protein related to alpha-amylase (AmyZK01).
• Expression and purification of a 55 kDa amylase enzyme by Ni2+ affinity chromatography.
• Proving the suitability of the B. subtilis spore as an organic matrix.
• Presentation of AmyZK01 on the spore surface by adsorption and covalent bonding.
• Comparison of properties of the immobilized enzyme with that of the free form.

Most of the studies in the field of enzyme immobilization have sought to develop a simple, efficient and cost-effective immobilization system. In this study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. Above all, their preparation is cost-effective. The alpha-amylase enzyme was immobilized on the spore surface by the covalent and adsorption methods. For the covalent method, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N hydroxysulfosuccinimide (NHS) were used. The maximum concentration of the alpha-amylase immobilized by the two methods onto the spore surface was 360 μg/1.2 × 1011 spore. However, maximum activity was achieved at an enzyme concentration of approximately 60 μg/.4 × 1010, corresponding to an estimated activity of 8 × 103 IU mg−1/1.2 × 1011 spore for covalent immobilization and 8.53 × 103 for the adsorption method. After washing the enzyme with 1 M NaCl and 0.5% Triton X-100, the enzyme immobilization yield was estimated to be 77% and 20.07% for the covalent and adsorption methods, respectively. The alpha-amylase immobilized by both methods, displayed improved activity in the basic pH range. The optimum pH for the free enzyme was 5 while it shifted to 8 for the immobilized enzyme. The optimum temperatures for the free and immobilized enzymes were 60 °C and 80 °C, respectively. The covalently-immobilized alpha-amylase retained 65% of its initial activity, even after 10 times of recycling. The Km and Vmax values were determined by the GraphPad Prism software, which showed that the Vmax value decreased moderately after immobilization. This is the first study which reports the covalent immobilization of an enzyme on the spore surface.