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Background: α-Amylase randomly hydrolyzes starch molecule to produce oligosaccharides of different chain length. It is among the most significant hydrolytic enzymes used in industrial applications. Enzyme immobilization is the simplest way to solve the stability problem of protein under industrial harsh conditions. Magnetic nano-particles considered suitable for immobilization due to their unique characteristics. The polymer nanocarriers still the feature of modifiable surfaces of carriers for further conjugation with biomolecule. This study aims to promote the immobilization of Bacillus subtilis MK1 α-amylase using the statistical optimization of the chemical modification of the chitosan-magnetic nano-magnetic particle beads and their ability to apply. Results: B. subtilis MK1 α-amylase was successfully immobilized on chitosan-magnetic nano-particles using a method combining the advantages of both physical adsorption and covalent binding. The beads were chemically modified using polyethyleneimine (PEI) followed by glutaraldehyde (GA). Aminated beads by (PEI), activated beads by (GA), and immobilized enzyme on activated beads were characterized using FTIR. Morphological examinations of the beads surface before and after conjugation with the α-amylase enzyme were carried out using scanning electron microscope (SEM). Chemical modification parameters of the beads were optimized using response surface methodology based on central composite design. Statistical approach enhanced the immobilization yield (IY%) by 1.5-fold. The application of immobilized enzyme in the baking process enhanced dough-raising about 2.3-fold and can be reused for 5 cycles with 100% activity. Conclusions: Statistical methods are an important way to improve immobilization yield and efficiency. The ANOVA data confirmed the fitness of the model which possessed R 2 value (0.975) and the adjusted R 2 value (0.940). The results confirm the ability to reuse the immobilized enzyme in industrial processes.

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