Whey protein isolate for the preparation of covalent immobilization beads
Introduction
Enzymes are known to exhibit low stability and to be vulnerable to inhibition. Enzymes are also water soluble (dos Santos et al., 2015) and their solubility in their aqueous reaction mixtures causes their recovery and reusability to be difficult. In order for enzymes to be efficiently employed as industrial biocatalysts, the abovementioned drawbacks should be overcome. Immobilizing enzymes can actually help to overcome all of the abovementioned drawbacks. The immobilization carriers are insoluble; thus, they could be simply removed from reaction mixtures. This will allow the recovery and the reusability of the immobilized enzymes (Elnashar, 2010). The immobilization process could also serve to augment the stability of enzymes, whether monomeric or multimeric. In case of multimeric enzymes, the dissociation of their subunits could be avoided and their stability could be improved upon attaching these subunits to an immobilization carrier. This could be easily achieved in case of dimeric enzymes. However, if the multimeric enzyme possesses a more complex structure, it will not be likely that all of its subunits will be attached to the immobilization carrier as some of these subunits will be facing away from the carrier. This problem was previously addressed by chemically cross-linking the immobilized multimeric enzymes with poly-functional polymers. These polymers were capable of binding to the immobilized enzymes’ subunits which could not be attached to the immobilization carrier (Mateo et al., 2007). As for the monomeric enzymes, the multipoint covalent attachments formed between them and their immobilization carriers will rigidify their structure and stabilize them. It is worth pointing out that such enzymes’ structure rigidification was previously shown to reduce the immobilized enzymes’ allosteric inhibition. Decreasing the inhibition suffered by enzymes would lead to an increase in their observed activity. Other immobilization induced structural distortions could also alter the observed activity of immobilized enzymes as well as their specificity and selectivity. Immobilization could also serve to purify enzymes as the contaminants, which are present in the free enzymes’ preparations, might just fail to be immobilized onto the immobilization carrier (Rodrigues et al., 2013). Owing to all the possible outcomes from the enzymes’ immobilization process, it is always advantageous to develop novel immobilization carriers that are efficient, cost effective, and also safe to be involved in the industries of food and drugs.
ĸ-Carrageenan (Car) has been frequently employed as a carrier for enzymes’ immobilization (Elnashar and Yassin, 2009a, Elnashar and Yassin, 2009b, Elnashar et al., 2014, Wahba and Hassan, 2017). Car is a natural polysaccharide derived from red marine algae. It could be attained at a rational cost, and it is also accepted as a food adjunct. Nevertheless, Car does not possess the functional groups necessary for enzymes’ covalent immobilization (Delattre et al., 2011, Elnashar et al., 2014) which is the immobilization method that provides the strongest bonds between the immobilized enzymes and the carrier. Thus, it prevents the leakage of the immobilized enzymes (Elnashar, 2010). In order for Car to covalently immobilize enzymes, it was activated, and reactive functional groups were incorporated into its structure. The activation process comprised two steps. In the first step, an ionic exchange was allowed to occur between a polyamine compound and the Car beads. This ionic exchange involved the Car's anionic sulfate groups and the polyamine compound's cationic amino groups. This led to the binding of the polyamine compound to the Car beads. In the second step, glutaraldehyde (GA) was added. GA reacted with the nucleophilic unprotonated amino groups of the Car bound polyamine compound. During the enzymes’ immobilization process, an ionic exchange would occur between the charged groups of the enzymes and the charged groups of the Car bound polyamine compound which would cause the enzymes to be initially adsorbed onto the activated Car beads. Afterwards, the functional groups of GA would react with these adsorbed enzymes and covalently immobilize them. It should be noted that GA is a widely used protein cross-linker. The GA reacts with proteins via different mechanisms. For instance, at acidic and neutral pHs GA was debated to exist in both its linear and cyclic hemiacetal forms. The aldehyde groups of the linear GA moieties could react with the amino groups of proteins via Schiff's base (C˭N) formation. On the other hand, the cyclic hemiacetal GA forms, which offer hydroxyl groups rather than free aldehyde groups, could substitute their hydroxyl groups with amino groups from proteins via nucleophilic-substitution reactions and produce fairly stable cyclic derivatives (Barbosa et al., 2014). As regards to the polyamine compounds which were previously employed during the Car activation, they were chitosan and polyethyleneimine (PEI) (Elnashar and Yassin, 2009a, Elnashar and Yassin, 2009b, Elnashar et al., 2014, Wahba and Hassan, 2017). In this study, we activated Car beads through the utilization of another polyamine compound, the whey protein isolate (WPI) (Fig. 1). It should be noted that WPI was not employed before during the activation of any hydrogel for the covalent immobilization of enzymes.
WPI is considered a value-added product of whey. Whey is a liquid byproduct produced by cheese industries. The disposal of this waste whey imposes a big environmental issue. Whey is produced in huge amounts and its complex composition exerts too much load on any wastewater handling system. Thus, whey should be treated prior to its disposal. Whey could be converted into value-added goods, owing to its nutritious constituents, such as lactose and protein. These value-added goods include whey protein concentrate (WPC), WPI, and reduced lactose whey. The WPI is employed in food industries as a yogurt stabilizer, a texturizer in dips, and an egg alternative (Yadav et al., 2015). WPI was also investigated for a variety of pharmaceutical applications. For instance, a patented WPI named Immunocal was proven to increase the cytotoxic effect of anticancer drugs. WPI was also shown to inhibit the proliferation of melanoma cells (Castro et al., 2009). Moreover, WPI was employed to coat liposomes in order to enhance their stability (Frenzel and Steffen-Heins, 2015).
WPI is prepared through the elimination of the non-protein constituents of whey. Accordingly, its protein content is higher than 90%. The proteins present in the WPI are mainly β-lactoglobulins (β-LG) together with other whey proteins, such as α-lactalbumin and bovine serum albumin. The β-LG generally exists as a dimer under physiological conditions and at room temperature. Each monomer of this dimer is composed of 162 amino acids (Majhi et al., 2006, Yadav et al., 2015). These amino acids would provide the amino groups required for both the ionic exchange with the anionic Car beads and the covalent binding to the bifunctional GA. Thus, the WPI would be expected to efficiently activate Car according to the scheme presented in Fig. 1. Moreover, the amino acids of the WPI would also provide carboxylic groups which could also be utilized. Carboxylic groups could be aminated with ethylenediamine via the utilization of the carbodiimide chemistry (Rueda et al., 2016). This amination would increase the amino content of the WPI. Such increased amino content might improve the ionic exchange between this aminated WPI and the anionic Car beads. It might also enhance the covalent binding of GA to the aminated WPI treated Car beads, and this might eventually improve the enzyme loading capacity of these beads. It is worth mentioning that the carbodiimide chemistry could also be employed to covalently bind the carboxylic groups of immobilization supports to the amino groups of enzymes (Rueda et al., 2016). Thus, we could exploit the carboxylic groups in the WPI treated beads and covalently immobilize enzymes via the carbodiimide route without utilizing GA. This implies that the WPI treated beads could be activated for the covalent immobilization of enzymes via both the GA route, as in this paper, and the carbodiimide route, and this would increase their potential applications in the field of covalent immobilization.
It is worth pointing out that another covalent immobilization approach could also be adopted via utilizing the WPI treated beads. This approach relies on both the ionic groups of the WPI and the versatility of GA. The WPI treated beads would bear lots of ionizable carboxylic and amino groups. The presence of such large amounts of ionizable groups would enable these beads to immobilize enzymes via adsorption (Barbosa et al., 2014). Hence, enzymes could be first adsorbed onto the WPI treated beads. Afterwards, the adsorbed enzymes could be allowed to react with GA in order to covalently immobilize them. It should be noted that the GA treatment step should be accomplished under mild settings in order to assure that only one GA molecule would react with each reactive amino group of both the beads and the enzyme. This would provide amino-GA moieties on both the beads and the enzyme. The amino-GA moieties are reactive towards each other along a fairly wide pH range, and would covalently bind the enzyme to the beads. However, this approach subjects the enzyme to the GA mediated intra and inter-molecular alterations which could bring about positive or negative effects on the activity and stability of the immobilized enzymes (Barbosa et al., 2014, Zaak et al., 2017)
β-D galactosidase (β-gal) from Aspergillus oryzae was the enzyme selected to be immobilized onto the novel activated Car beads as it is a significant industrial enzyme. The A. oryzae β-gal is among the fungal β-gals which are active at acidic pH values. Accordingly, it could catalyze lactose hydrolysis in acid whey and whey permeate. The hydrolysis of whey lactose will help convert whey from an environmental polluting waste to sweet syrup which is a valuable product that has applications in the baking, sweets, and soft drinks industries. Moreover, if the β-gal reacts under high temperatures with elevated lactose concentrations, it will catalyze the trans-galactosylation of lactose and produce galacto-oligosaccharides (GOS). GOS are prebiotics that preferentially promote the growth of the beneficial colonic microflora (Panesar et al., 2010)
Section snippets
Materials
The Aspergillus oryzae β-D-galactosidase (EC 3.2.1.23) and the GA solution were acquired from Sigma-Aldrich (Germany). The WPI (BiPro®) was acquired from Davis Co. Foods International Inc. as a gift. All other employed chemicals exhibited Analar or equivalent quality.
Preparation and treatment of the Car beads
A 2% (w/v) Car solution was prepared via heating in a 70 °C water bath. This solution was then dropped via a thin needle of a disposable syringe into a 2% (w/v) KCl solution under stirring in order to form the Car beads. The formed
Box-Behnken Design
The results of the BBD (Table 1) were analyzed with the Design Expert statistical software, and a second order regression equation was obtained (Eq. (3)) which related the iβ-gal's activity recovery percent (response) to the influencing factors (A: WPI concentration, B: WPI pH, C:WPI soaking time). The analysis of variance (ANOVA) of the proposed statistical model and all of its participating
Conclusion
In this paper we reported, for the first time, on the utilization of WPI in the preparation of covalent immobilization supports. The Car activated with WPI and GA offered good activity recovery percents as compared to the other commonly used polyamino compounds. The novel activated polymer also caused the iβ-gal to exhibit superior operational stability where the immobilized enzyme maintained 93.84 ± 0.72% of its initial observed activity during the 13th reusability cycle.
Acknowledgements
This work was sponsored by National Research Centre, Egypt.
Conflict of interest
The authors declare that they have no conflict of interest.
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2020, International Journal of Biological MacromoleculesCitation Excerpt :This observation proved the merger of the PAMAM with the Carr beads. Similarly, the merger of other amino providing compounds, such as polyethyleneimine (PEI) and whey protein isolate (WPI) to the Carr gel was proven via the appearance of nitrogen in the beads' elemental structure [41,55,56–59]. However, the amount of nitrogen examined in the beads was higher, in case of PAMAM solution of pH 9.3 was employed to activate the beads, than the case at which PAMAM solution of pH 2.1 was employed.
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