Elsevier

Food Chemistry

Volume 373, Part A, 30 March 2022, 131425
Food Chemistry

Efficient hydrolysis of starch by α-amylase immobilized on cloisite 30B and modified forms of cloisite 30B by adsorption and covalent methods

https://doi.org/10.1016/j.foodchem.2021.131425Get rights and content

Highlights

  • The cloisite 30B was activated by epichlorohydrin and tosyl chloride.

  • The cloisite 30B and its activated forms were used for immobilization of α-amylase.

  • Enzyme immobilization methods have been compared together.

  • Immobilization improved thermal resistance and storage stability of α-amylase.

Abstract

In this paper, α-amylase from Bacillus subtilis was successfully immobilized on three supports. First, α-amylase was immobilized on cloisite 30B via the adsorption method. Then cloisite 30B was activated with tosyl chloride and epichlorohydrin. These activated supports were used for covalent immobilization of α-amylase, and their enzymatic activities were effectively tested in the starch hydrolysis. The results demonstrated that the specific activity of α-amylase immobilized on cloisite 30B was 2.39 ± 0.03, for α-amylase immobilized on activated cloisite 30B with epichlorohydrin was 1.96 ± 0.05 and for α-amylase immobilized on activated cloisite 30B with tosyl chloride was 2.17 ± 0.05 U mg−1. The optimum pH for the activity of free α-amylase was 7, but for α-amylase immobilized on cloisite 30B was 8, and for α-amylase immobilized on activated supports was 7.5. The immobilized enzymes had better thermal resistance and storage stability than free α-amylase, and they also showed excellent reusability.

Introduction

Amylases are important industrial enzymes that belong to the hydrolase category and have achieved about a quarter of the market of industrial enzymes (Wang, Hu, Ma, Yan, Liu, & Jiang, 2020). Nineteen enzymes have been categorized in the amylase class (Mohanan & Satyanarayana, 2019). Among these nineteen enzymes, α-amylase (E.C.3.2.1.1) that catalyzes the hydrolysis of the internal α-1,4-glycosidic bond to glucose and maltose has greater importance due to its applications in biorefinery, textile, medicine, dairy, detergent, paper, and chemical industries (Fasim, More, & More, 2021). α-Amylase has many applications in the food industry and is used in alcohol production. Also, it is used as a starch hydrolysis agent in starch processing for the production of syrup, as a brewing agent in beverages, and as an antistaling agent in baking (Jin et al., 2020).

As mentioned above, one of the applications of α-amylase in the food industry is starch hydrolysis. Among carbohydrates, starch is an abundant source of energy, and global attention, especially in the food industry, has been drawn to using this economic source of carbon (Roy, Borah, Mahanta, & Mukherjee, 2013). The obtained starch from agricultural wastes and crops is a cheap source for the production of fructose, glucose, and maltose syrups that are used extensively in the food industry and ethanol production (Bai et al., 2012).

The large-scale industrial applications of amylases are limited by their high price, low operational stability, short life in the solution, and difficulties in their recovery (Cavalcante et al., 2021). The drawbacks mentioned above can be overcome by enzyme immobilization. This technique can be defined as the restriction of enzyme molecules on a support, physically or chemically or both, in such a way that it retains most of its activity (Aggarwal & Pundir, 2016). Enzyme immobilization is a powerful technique to tailor the catalytic features of enzymes such as recyclability over successive catalytic cycles, stability under various pH's and temperatures, activity, selectivity, specificity, and resistance to inhibitors (Taheri-Kafrani et al., 2020). Furthermore, simple separation of the immobilized biocatalyst from the reaction medium leads to products with higher purity (Galvão et al., 2018). The two most common ways to immobilize enzymes are physical methods, such as entrapment and adsorption, and chemical methods, such as cross-linking and covalent attachment. (Mortazavi & Aghaei, 2020). Among immobilization techniques, immobilization through adsorption and covalent bonding is more applicable than other methods. Adsorption is the most used strategy because of the simplicity and no need to support functionalization. Moreover, the activity is relatively high, and the conformation of the enzyme is retained since the enzyme is linked into the carrier through weak intramolecular forces such as ionic and hydrophobic interactions (Verma & Raghav, 2021). On the other hand, the covalent attachment of the enzyme has been superior to other methods, and it improved the storage and operational stability of the enzymes. The covalent immobilization offers a strong bond between the support and enzyme and significantly reduces the enzyme leaching (Dai, Kong, Wang, Zhu, Chen, & Zhou, 2018).

The support type also considerably affects the immobilization of enzymes (Nunes et al., 2021). Generally, ideal supports for enzyme immobilization should be non-toxic, cheap, and environmentally benign. They must not negatively affect the enzyme activity and should not lead to denaturation or deactivation of the enzyme (Monteiro et al., 2019). The carrier has to be insoluble in the solvent used for the immobilization and chemically, thermally, and mechanically stable (Rios, Neto, dos Santos, Fechine, Fernández-Lafuente, & Gonçalves, 2019).

As mentioned above, the large-scale industrial uses of amylases are limited by their high cost, problems in recovery, short solution life, and poor operational stability, and these disadvantages can be solved by enzyme immobilization. So, this work aimed to develop suitable carriers for α-amylase immobilization due to the numerous applications of this enzyme in the food industry. To reach this goal, cloisite 30B (CL) and its activated forms were used as supports. CL as a non-toxic and inexpensive carrier has high chemical and physical stabilities, and it is produced by Na-montmorillonite modification with N,N-bis(2-hydroxyethyl)-N-methyl-N-tallow ammonium chloride, where tallow is 65% C18; 30% C16; 5% C14). First, the adsorption method was employed for α-amylase immobilization on CL. Then CL was activated with epichlorohydrin and transformed into epoxy functionalized cloisite (ECL) by reaction of the CL hydroxyl groups and epichlorohydrin and converted to tosylated cloisite (TCL) by reaction of hydroxyl groups with tosyl chloride. These activated supports were also employed for covalent immobilization of α-amylase (Scheme 1). The biocatalytic activities of immobilized α-amylase on CL (CLA), immobilized α-amylase on ECL (ECLA), and immobilized α-amylase on TCL (TCLA) were effectively examined in the starch hydrolysis.

Section snippets

Materials

CL was purchased from Southern Clay Products (USA). α-Amylase from Bacillus subtilis (Product number: 10070, Lot # 0001419753, Activity: 50 U mg−1), maltose monohydrate (Product number: M9171, Purity ≥ 99%), Bradford reagent (Product number: B6916), and soluble potato starch (Product number: S2004, Lot # BCBM7676V) were bought from Sigma (USA). Epichlorohydrin (Product number: 803296, Purity ≥ 99%), p-toluenesulfonyl chloride (Product number: 808326, Purity ≥ 98%), triethylamine (Product

Characterization

The FT-IR spectra of CL, TCL, and ECL were previously reported in our papers (Aghaei et al., 2021, Khozeymeh Nezhad and Aghaei, 2021) and used here for comparison with the FT-IR spectra of CLA, TCLA, and ECLA (Fig. 1). The FT-IR of CL (Fig. 1a) demonstrated the stretching band of Si − O at 1046 cm−1. The hydroxyl (3630 cm−1) and methylene groups (2924 and 2852 cm−1) of the organic modifier are also marked on the CL spectrum. For ECL, the peaks of the epoxy group appeared around 1248, 914, and

Conclusions

The aim of this study was to develop suitable carriers for α-amylase immobilization. In order to achieve this goal, three supports, including CL, ECL, and TCL, were employed for the immobilization of α-amylase, and the enzymatic activities of the biocatalysts were effectively examined in starch hydrolysis. XRD, FT-IR, and SEM methods proved successful enzyme immobilization onto these carriers. The effects of α-amylase concentration, pH, thermal stability, storage stability, and reusability of

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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