Chelation by collagen in the immobilization of Aspergillus oryzae β-galactosidase: A potential biocatalyst to hydrolyze lactose by batch processes

doi:10.1016/j.ijbiomac.2017.12.088

International Journal of Biological Macromolecules

Volume 109, 1 April 2018, Pages 303-310

https://doi.org/10.1016/j.ijbiomac.2017.12.088 Get rights and content

Abstract

This work is the first study of the immobilization of Aspergillus oryzae β-galactosidase (Gal) on powdered collagen (Col) that had formed a chelate with aluminum (Col-Al-Gal). Other collagen treatments, including those with acetic acid, glutaraldehyde, and a combination of aluminum and glutaraldehyde (Col-Al-Glu-Gal), were also tested. High-yield (superior to 80%) and high-efficiency (superior to 99%) immobilization was obtained for the derivatives Col-Al-Gal and Col-Al-Glu-Gal, even at high protein loads (500–1,000 mg g⁻¹ of support). The storage stability of Gal immobilized on Col-Al and Col-Al-Glu resulted in Gal retaining approximately 60% of its initial activity after 90 days at 4 °C. The half-life values of derivatives Col-Al-Gal and Col-Al-Glu-Gal were higher than those of soluble enzyme at 65, 68, 70, and 73 °C. The derivatives Col-Al-Gal and Col-Al-Glu-Gal retained high enzyme activity in batch hydrolysis of lactose in permeate and lactose solutions for 50 and 60 cycles, respectively. Our results suggest that powdered collagen treated with aluminum, a low-cost support, is a promising support for the immobilization of β-galactosidase.

Introduction

β-Galactosidase, most commonly known as lactase, is classified as a hydrolase and can transfer galactosyl groups, catalyzing the hydrolysis of the terminal residue from lactose to form a 1:1 mixture of glucose and galactose [1,2]. Lactase from Aspergillus oryzae is a monomeric enzyme having a molecular weight of 90 kDa, an optimal pH range from 4.0 to 5.0 and an optimal temperature of 55 °C. It is used to prepare lactose-free milk and other dairy products at the industrial level for lactose-intolerant individuals. It is also applied to reduce the problems of lactose insolubility and the lack of sweetness of dairy products [3,4].

The use of soluble enzymes in industrial processes has limitations such as their low stability, non-reusability and high cost. It would be advantageous to employ an immobilized enzyme since it allows product separation and the reuse of the biocatalyst [5,6]. An important stage in the enzyme immobilization process is the choice of the support, which requires the consideration of factors such as productivity, efficiency, availability and cost [7]. β-Galactosidase has been immobilized on different supports composed of inorganic and organic materials. However, the use of alternative supports that are of low cost, easily obtainable, and biodegradable could reduce the cost of enzyme immobilization and facilitate its implementation on the industrial scale [8].

Collagen is an insoluble organic protein found in skin, tendons, ligaments and bones. It is composed of three polypeptide chains with triple helical structures that aggregate through hydrogen bonds. This protein is obtainable as a fiber, as a powder and in the hydrolyzed form. The collagen structure has abundant functional groups such as single bond OH, COOH, CONH₂ and NH₂ and is thus ready to react with other chemicals [9,10]. Interest in the extraction of collagen and its derivatives has increased due to the trend of using this protein to replace synthetic agents in the diverse industrial processes [11].

Diverse forms of collagen have been employed as supports for enzyme immobilization, e.g., fibers have been used for lysozyme [12], catalase [13,14], and β-galactosidase [15]; powders for naringinase [16]; and membranes for β-galactosidase [17,18], and α-amylase [19]. The advantages of greater surface area and versatility for use in bioreactors are demonstrated when powdered collagen, compared to other forms, is used as a support for enzyme immobilization. Nevertheless, studies that use powdered collagen for β-galactosidase immobilization are lacking.

The Aspergillus oryzae β-galactosidase was immobilized in this work using powdered collagen as matrix. The support was treated by four different protocols, involving acetic acid, glutaraldehyde, aluminum sulfate or a combination of aluminum sulfate and glutaraldehyde (Fig. 1). The treated supports were tested by varying the protein loading. The collagen was characterized by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis. The immobilized derivatives were evaluated for their operational conditions, thermal and storage stabilities, kinetic parameters and reusability in batch processes.

Section snippets

Materials

β-Galactosidase from Aspergillus oryzae (Gal) was donated by the Prozyn Company (São Paulo, Brazil). Powdered skim milk and powdered cheese whey were purchased from Brazil Foods S.A. (Rio Grande do Sul, Brazil). Powdered whey permeate was provided by Arla Foods (Córdoba, Argentina). The enzymatic kit for glucose determination was acquired from Labtest (Minas Gerais, Brazil). Powdered collagen (type-I from bovine skin) was donated by the Luchebras Company (Cachoeirinha, Brazil).

Results and discussion

Collagens receiving one of four treatments, acetic acid (Col-HAc), glutaraldehyde (Col-Glu), aluminum sulfate (Col-Al), and a combination with aluminum sulfate and glutaraldehyde (Col-Al-Glu), were tested for their ability to immobilize Aspergillus oryzae β-galactosidase (Fig. 1). Table 1 shows the influence of the treatments in the yields and efficiencies of the β-galactosidase immobilized on the collagens.

Table 1 shows that the immobilization yields of Col-Al-Gal and Col-Al-Glu-Gal were

Conclusion

In this study, we immobilized Aspergillus oryzae β-galactosidase on collagen treated with four different methods, generating the derivatives Col-HAc-Gal, Col-Glu-Gal, Col-Al-Gal, and Col-Al-Glu-Gal. The best yields and efficiencies were obtained employing aluminum as a metal chelate. The efficiency of the processes was higher than 99%, with protein loads on Col-Al-Gal and Col-Al-Glu-Gal ranging from 500 to 1,200 mg.g⁻¹ of support. The temperature profiles of free and immobilized (Col-Al-Gal and

Acknowledgements

We acknowledge CNPq, CAPES, and FAPERGS for the scholarships and Univates for the financial support of this research paper. We also thank the Prozyn Company for the enzyme donation.

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Chelation by collagen in the immobilization of Aspergillus oryzae β-galactosidase: A potential biocatalyst to hydrolyze lactose by batch processes

Abstract

Introduction

Section snippets

Materials

Results and discussion

Conclusion

Acknowledgements

Catal. Commun.

Int. J. Biol. Macromol.

Biochem. Eng. J.

Biotechnol. Adv.

Enzyme Microb. Technol.

J. Mol. Catal. B Enzym.

J. Memb. Sci.

Trends Food Sci. Technol.

Food Hydrocoll.

Colloids Surf. A Physicochem. Eng. Asp.

Process Biochem.

Anal. Chim. Acta

Anal. Biochem.

Int. J. Biol. Macromol.

Biomaterials

Food Chem.

Carbohydr. Polym.

J. Chromatogr. A

Mol. Catal. B, Enzym.

Process Biochem.

Int. J. Biol. Macromol.