Abstract
Backgroundα-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.ResultsB. 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.ConclusionsStatistical methods are an important way to improve immobilization yield and efficiency. The ANOVA data confirmed the fitness of the model which possessed R2 value (0.975) and the adjusted R2 value (0.940). The results confirm the ability to reuse the immobilized enzyme in industrial processes.
Highlights
Enzymes have numerous characteristics, and they are more preferable over chemical catalysts in many areas of industry ranging from food to pharmaceuticals (Zdarta et al 2018). α-Amylase (EC 3.2.1.1) randomly hydrolyzes α-1,4-glycosidic bonds in the interior of starch molecule (Fig. 1) to produce branched and linear oligosaccharides of different chain length (Simair et al 2017; Frantz et al 2019)
One unit of enzyme activity (U) is defined as the Optimization of beads modifications using statistical design Response surface methodology (RSM) based on central composite design (CCD) was used to determine the optimum level of four important factors for beads modification of chitosan-magnetic nano-particles (Ch-MNP) beads. These factors include PEI percent (%) (W), PEI activation time (X), GA percent (%) (Y), and GA activation time (Z). These factors were tested at three levels as, low (− 1), central (0), and high (+ 1), resulting in experimental design of 25 experiments with respect to mean of Immobilization yield (IY) (%) of α-amylase on Ch-MNP beads as response
Immobilization yield (IY%) was calculated according to Wang et al (2015) as following: IY ð%Þ 1⁄4 I=ðA−BÞ Â 100 where I is the total activity of immobilized enzyme, A is the total activity offered for immobilization, and B is the total activity of unbounded enzyme
Summary
They are more preferable over chemical catalysts in many areas of industry ranging from food to pharmaceuticals (Zdarta et al 2018). α-Amylase (EC 3.2.1.1) randomly hydrolyzes α-1,4-glycosidic bonds in the interior of starch molecule (Fig. 1) to produce branched and linear oligosaccharides of different chain length (Simair et al 2017; Frantz et al 2019). High stability of enzyme under industrial conditions is considered an economic advantage due to low enzyme loss. Enzyme immobilization is the simplest way to solve the stability problem of protein and reduce the expensive cost of applying them on an industrial scale (Ahmed et al 2019a, 2019b). Immobilization improves enzyme properties as activity, reduces the inhibition, increases stability, specificity to substrates, and avoids contamination of product by enzyme (Ahmed et al 2019a, 2019b). Immobilization of enzymes can offer many benefits as reusability and recovery from their products enhance stability under both operational and storage conditions (Souza et al 2019). Magnetic nano-particles as Fe3O4, γ-Fe2O3, ZnO, and TiO2 are considered significant carriers that enhance immobilization efficiency because they have high stability and high electron conductivity
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