Study on EcoRI And HinDIII Immobilization Using Sodium Alginate and Their Restriction Activity on Lambda DNA

In general, enzyme technology refers to the synthesis, separation, purification, and application of enzymes (either soluble or immobilized) for the benefit of humankind. Enzyme technology also includes the use of protein engineering and recombinant DNA technologies to produce more efficient and useful enzymes. One of the main components of the biotechnology industry is the commercial manufacture and use of enzymes ^[1]. The commercialization of enzymes is restricted to a certain degree based on their cost, as more expensive enzymes are not cost-effective due to their poor reusability factor. Notwithstanding their high cost, immobilization of beneficial enzymes with higher repeatability and functional efficiency is seen to be more promising for overcoming these constraints. Enzyme immobilization may be defined as confining the enzyme molecules to a solid matrix or support that is distinct from the one that contains the products or the substrate. The enzymes are attached to or contained within an appropriate support material in order to do this ^[2]. Enzymes are frequently immobilized, primarily to reduce the amount of money that goes toward the process by allowing for multiple uses of the enzyme. This involves physically confining the enzyme so that it cannot dissolve into solution, usually in the form of beads or membranes made of polymer matrix. Because an immobilized enzyme can be easily removed through sieving, it also typically makes downstream processing easier. In contrast, extracting a soluble enzyme from a reactor stream would require significant time, money, and effort ^[3]. 

History of enzyme immobilization:

When Chibata and colleagues created the immobilization of Aspergillus oryzae aminoacylase for the resolution of synthetic racemic D-L amino acids, it was the first time that immobilized enzymes were used in industry. This was published in 1966. The initial stage was the empirical industrial use of immobilized microbes at the start of the 19^th century. Enzyme immobilization's contemporary history began in the late 1940s. Only immobilized single enzymes were employed in the second stage, but more sophisticated systems, such as two-enzyme processes including cofactor regeneration and live cells, were created by the 1970s ^[1]. The first international conference on enzyme engineering was held in 1971, greatly aided by the first practical use of immobilized enzymes. "Immobilized enzymes" was the conference's main focus. At the conference, the term "immobilized enzyme" was suggested. Prior to that, a number of terminologies were in use, including "matrix supported enzyme," "water-insoluble enzyme," "trapped enzyme," and "fix enzyme"^[4].

Components of enzyme immobilization:

The major components for an enzyme immobilization include an enzyme, a support matrix and mode of attachment of a catalyst to the carrier (Figure 1).

Figure 1:  Basic components of enzyme immobilization

Types of Matrix:

Based on the chemical composition, a support matrix is generally two types. They are organic and inorganic carriers (Figure 2).

Figure 2. Classification of matrix

1. Organic matrix: It subdivides into natural and synthetic polymers.

a) Natural polymers: It exhibits favorable protein compatibility. Polysaccharides (Cellulose, dextran, agar, agarose, chitin, alginate etc.,), proteins (Collagen, albumin) and carbon are the natural polymers.

b) Synthetic polymers: It shows high chemical and mechanical stability. Polystyrene, polyacrylate, polyacrylamide, poly amides etc., are examples of synthetic polymers.

2. Inorganic matrix: It subdivides into natural and processed minerals.

1. Natural minerals: E.g. Bentonite, celite, centolite, silica, charcoal etc.,
2. Processed materials: E.g.Porous glass, metals and metal oxides ^[5].

3. Techniques:

BM Brena has divided enzyme immobilization techniques into two categories: reversible and irreversible. Irreversible immobilization of enzymes involves trapping and covalent binding. Adsorption, ionic binding, affinity binding, and metal binding are examples of reversible enzyme immobilization (Figure 3) ^[6]. Because immobilized enzymes may yield products in pure form with more selectivity than immobilized cells, they are often favored. The following methods are frequently used to immobilize enzymes: encapsulation, trapping, covalent binding, adsorption, and cross-linking. Physical adsorption: Enzymes are attached to a support material through weak physical interactions. Examples of support materials include activated carbon, silica, and metals ^[7].

Covalent binding: Enzymes are covalently attached to a support material through chemical bonds such as amide, ester, or thioester linkages. This method provides strong attachment and high stability, but it can also cause denaturation and loss of enzyme activity ^[8].

Entrapment: Enzymes are entrapped in a porous matrix, such as sodium alginate, Agarose, or polyacrylamide, which allows for the diffusion of substrates and products while preventing enzyme leaching ^[9].

Cross-linking: Enzymes are cross-linked with a support material using a chemical cross-linker, which forms covalent bonds between the enzyme and matrix. This method provides high stability and prevents enzyme leaching but can also cause loss of enzyme activity ^[10]. 

Encapsulation: Enzymes are encapsulated in a polymeric membrane, which allows for the diffusion of substrates and products while preventing enzyme leaching. This method provides high stability and enhances the enzyme's selectivity and specificity ^[11].

Figure 3: Various methods of enzyme immobilization

Sodium alginate:

It is a sulfated polysaccharide, obtained from brown algal cell wall. Increased enzyme activity and reusability are obtained by immobilizing enzymes with alginate salts, such as xanthan–alginate beads, alginate polyacrylamide gels, and calcium alginate beads. Alginate increases the stability of enzymes by forming crosslinks with glutaraldehyde and divalent ions ^[2].  One of the most significant classes of enzymes for DNA modification is restriction endonuclease. These are bacterial enzymes that have the ability to split and cleave DNA at particular locations in any source. They were initially found in E. coli, where they cleave the viral DNA to limit bacteriophage replication ^[12]. There are many restriction enzyme types utilized in molecular biology studies to manipulate DNA. There are various advantages to immobilizing restriction enzymes on a support substance such as sodium alginate. The enzyme's stability, activity, and resistance to harsh environmental conditions can all be increased through the immobilization procedure ^[13].  Sticky ends, are generated when the restriction enzyme cleaves the DNA at different positions on the two complementary strands, producing fragments with single-stranded overhangs or "sticky ends." These overhangs can anneal with complementary sequences in other DNA fragments, allowing for easy ligation. EcoRI and HindIII restriction enzymes that generate sticky ends.

1. EcoRI: recognizes the sequence GAATTC and produces sticky ends with a 5' overhang (AATT).
2. HindIII: recognizes the sequence AAGCTT and produces sticky ends with a 5' overhang (AGCT).

The type of ends generated by restriction enzymes can have significant implications for downstream DNA manipulation, as sticky ends can be easily ligated together, while blunt ends need additional enzymatic treatment to join together ^[14,15]. In this research the restriction enzyme activity of immobilized and free enzyme of EcoRI and Hind III were studied using lambda DNA.

METHODOLOGY

Immobilization of EcoRI and HindIII enzyme

A simple technique was adopted for immobilizing EcoRI and HindIII on sodium alginate is to combine the enzyme with the alginate solution, then extrude the mixture into a calcium ion solution to create beads. The restriction activity of the produced immobilized enzyme beads was assessed later.One gram each of potassium chloride and sodium alginate was dissolved in 50ml of distilled water to create 2% (w/v) solutions of each substance. A 0.2 M calcium chloride solution was prepared by dissolving 2.98 g of calcium chloride in 100 ml of distilled water ^[16].   4µl of EcoRI enzyme were pipetted into Eppendorf tubes, and then 4µl of a 2% (w/v) sodium alginate solution and 4µl of a 2% (w/v) potassium chloride solution were added. The range of 1:1:1 was maintained between the enzyme solution and the potassium chloride and sodium alginate solutions. The immobilized HindIII enzyme was prepared using the same procedure. Using a micro pipette, the resulting solution was added drop wise to 2% (w/v) calcium chloride. The EcoRI and HindIII enzymes are encapsulated in calcium alginate beads, which are created when the calcium ions in calcium chloride combine with sodium alginate. An hour was spent for the produced beads to cure in the calcium chloride solution. The prepared calcium alginate beads hardened during one-hour incubation. To get rid of any extra calcium chloride that might have been on the beads, they were thoroughly cleaned with distilled water. At 4°C, the prepared beads were kept in a buffer solution.

Evaluation of Immobilized Enzyme activity

Two similar reaction mixtures were created and evaluated for the free enzyme in order to compare the effectiveness of the immobilized enzymes. (Immobilized EcoRI digestion) reaction mixture Number one; 5.0 µl of lambda (λ) DNA; 2.5 µl of 10X Assay Buffer of EcoRI; 16.5 µl of molecular grade water; and immobilized EcoRI beads. Lambda (λ) DNA (5.0 µl), 10X Assay Buffer of HindIII (2.5 µl), molecular grade water (16.5 µl), and immobilized HindIII beads make up reaction mixture number two (immobilized HindIII digestion). Similar steps were taken to prepare reaction mixtures 3 and 4, substituting free enzyme for immobilized enzyme. The reaction mixtures were incubated at 37 °C for 3hrs and 1hr, respectively, to compare the activity of the free and immobilized enzymes.

Agarose Gel Electrophoresis:

Using 1X TAE buffer, 50 ml of 1% agarose solution were made. After cooling the solution to between 55°C, 0.5 µl of ethidium bromide was added. The gel was allowed to set in the gel tray at room temperature for approximately 30 minutes. After inserting the tray into the electrophoresis chamber and adding 1X TAE electrophoresis buffer, the comb was carefully taken out. 10 µl of DNA samples were mixed with 2 µl of 6X gel loading buffer to prepare the sample for electrophoresis. Carefully, the entire liquid was poured into the well. Electrophoresis at 90 mA and 120 volts until the dye markers moved away from the well in the proper amount of time ^[17]. When a gel was exposed to UV light (254–366 nm), DNA bands were able to be seen against a plain background. A gel documentation software (Alphadigidoc) was used to record the gel image. Table 1 provides information about the reaction mixtures and the enzyme activity.

Table 1:  Reaction mixture details and lane numbers from day 1 to 4

RESULTS AND DISCUSSION

In this study, two enzymes, EcoRI and HindIII, were immobilized utilizing natural material (sodium alginate) in mild conditions. Because alginates exchange monovalent ions (such sodium in sodium alginate) for divalent ions (particularly calcium in calcium chloride solution), the reaction proceeds relatively instantly, giving the low viscosity solution a gel-like structure. This is the main benefit of alginates. Using a micropipette, the enzyme solutions (EcoRI and HindIII) were poured into calcium chloride to immobilize them onto sodium alginate beads. For optimal activity, the ratio of enzyme solution to potassium chloride solution and sodium alginate solution was kept between1:1:1. From the results, we found that both free and immobilized EcoRI and HindIII digested the lambda DNA. The appearance of multiple lambda DNA bands on agarose gel electrophoresis served as confirmation of the enzyme activity. Table 2 presents the experimental findings. In order to give the enzyme adequate time to come into contact with lambda DNA, the immobilized enzyme was incubated for 3 hrs to assess its activity. For the free enzyme, 1hr contact duration was employed. For four days, the identical process was carried out daily on lambda DNA using an immobilized enzyme (Figure 4).

Table 2:  Activity of immobilized and free enzyme on lambda DNA

Figure 4: DNA bands appearance on agarose gel on first and fourth day