Over the decades, many thermo-biocatalysts, viz. thermozymes resistant to irreversible inactivation and instability at elevated temperatures (50 to 125 °C), were purified from hyper/thermophiles [1]. Interestingly, other distinctive properties like pH resistance, chemical and substrate selectivity, and stability after chemical modifications are found to be reliable over their mesophilic counterparts [2]. For instance, α-amylases by Bacillus spp., Geobacillus stearothermophilus, Anoxybacillus, and other thermophiles hold potential for use in industrial processes [3]. However, α-amylases production requires a high starch substrate, which shows catabolite suppression or feedback inhibition by glucose and maltose, respectively. Regardless of production ability, these microbes produce low constitutive quantities of α-amylase [4]. Such enzymes manifest instability, poor recovery, and low reusability, driving up the process cost, especially for free enzymes [5]. In contrast, enzyme immobilization on the supporting materials by various means have subdued limitations in terms of stability, reusability, and selectivity in unfavorable condition by averting conformational changes that could be detrimental to catalytic capability and efficiency [6]. These includes; functionalized glass beads [7], Amberlite MB 150, chitosan beads [8], mesoporous silica [9], alginate [10], gelatin [11], agarose and agar matrices [12], polyaniline [13], silver nanoparticles, Acacia-gelatin-silica nanohybrid gum with doping [14], porous nitrocellulose [15], and carboxymethyl tamarind gum-silica nanohybrid are being used for this purpose. Notably, magnetic nanoparticles (MNPs) are also known for its exceptional mechanical strength, size, and biocompatibility. Due to its high extinction, super-paramagnetic, scattering coefficient, and for its Brownian motion in solution has robust practical applications [7]. Specifically, MNPs were used as imaging contrast agents [8], in magnetic resonance [9], in data storage, drug delivery, protein separation, enzyme immobilization [10], in biomedicine [11], and as a cation sensors [12].

In the current study, extracellular α-amylase from thermophilic Geobacillus thermoleovorans was purified, characterized and the immobilized onto MNPs. The sequential purification steps were carried out for the crude enzyme includes GEX, and DEAE-IEX and subsequently characterized by electrophoresis. Beforehand, the MNPs were synthesized by the co-precipitation method using Fe3+:Fe2+ (2:1). Owing to the benefits of excellent biocompatibility and high density of surface -NH2 functional group enables linking with other targeted biomolecules, aminopropyltriethoxysilane (APTES) was selected for the direct surface modification of Fe3O4 NPs with glutaraldehyde (GA) as a cross-linking agent. Effective α-amylase immobilization on to the amino-functionalized MNPs within a specific pH and temperature range was determined by SEM, XRD, and FTIR. Other factors on α-amylase activity and recovery were also performed.