Glutaraldehyde-Activated Enzymes

Glutaraldehyde is a homobifunctional cross-linker containing an aldehyde residue at both ends of a 5 carbon chain. Its primary reactivity is toward amine groups, but the reaction may occur by more than one mechanism. As discussed in Chapter 4, Section 

Despite the obvious disadvantages of glutaraldehyde-mediated conjugation, the 

The one- and two-step procedures for enzyme activation and conjugation using glutaraldehyde can be found in Chapter 10, Section 1.2. 

Despite the obvious disadvantages of glutaraldehyde-mediated conjugation, the crosslinker continues to be used to form enzyme-antibody complexes and in other applications. Many diagnostic tests still utilize antibody-enzyme conjugates prepared through glutaraldehyde 

---

Purify the activated enzyme from excess glutaraldehyde by gel filtration using a desalting resin or by dialysis against PBS, pH 6.8. 

A colorimetric assay for lecithin and choline was described by Kotsira and Klonis (1998) using two enzymes (phospholipase and choline oxidase) and an indicator dye conjugate (bromothymol blue-glutathione) co-immobilised on a glutaraldehyde-activated polyacrylamide transparent gel. The change of the... 

Figure 1. pH-dependence of the half-life for loss of activity in native and glutaraldehyde-modified j9-D-glucosidase upon preincubation at the indicated pH values and at a range of temperatures from 55o to 70 oC, with subsequent assay at pH 5.0, 45 oC. A,B Linear-ordinate and logarithmic-ordinate plots, respectively, for the glutaraldehyde-modified enzyme. 

Branched-chain L-amino acids can be analyzed using leucine dehydrogenase immobilized on aminated PVA activated with glutaraldehyde. This enzyme catalyzes the deamination of L-Leu, L-Ile, and L-Val to 2-oxo acids in the presence of NAD. ... 

Treatment of -ABSC-HEMA with glutaraldehyde produced enzyme supports capable of binding up to 55 wt % trypsin. Incorporation of hydrophobic styrene units Into the support reduced the capacity to 2-. 4 wt X but enhanced the specific activity of the trypsin. The esterase activity of bound trypsin, assayed with TAME, was found to range from 11% to 45% of that exhibited by the free trypsin. Active-site titration of a PHEMA-trypsln conjugate with p-nltrophenyl-p -guanadlnobenzoate HCl Indicated the active species to be 31% of the total amount of protein bound. 

Principle a homobifunctional agent (glutaraldehyde = pentanedialdehyde) is allowed to react with amino groups on alkaline phosphatase and the activated enzyme is allowed to react with IgG or streptavidin. 

After overnight incubation at room temperature, remove excess free glutaraldehyde by gel filtration. To do this, use a gel matrix with an exclusion limit of 20,000"C50,000 for globular proteins. Use medium-sized beads (approx 100 pm in diameter). Prepare a column with 5 mL of bead volume according to the manufacturer s instructions. To make the column easier to load and run, first add 20 pL of glycerol and 20 pL of 1% xylene cylanol. The column should be prerun with a minimum of 10 column vol of 0.15 MNaCl. Allow the column to run until the buffer level drops just below the top of the bed resin. Stop the flow of the column. Carefully load the column with the glutaraldehyde-treated HRP. Release the flow and allow the HRP to run into the column. Just as the level of the HRP solution drops below the top of the column, carefully add 0.15 MNaCl. Run the column with 0.15 MNaCl. Pool the fractions that look brown. These contain the active enzyme. 

Reaction with agarose cyclic imido-carbonate or with an aminohexanoyl derivative of cross-linked agarose after modification of the enzyme with periodate and 1,2-diaminoethane or glycyl-L-tyrosine Co-immobilized with catalase on glutaraldehyde-activated controlled pore glass... 

Silanized and glutaraldehyde-activated sub-micron ferrite particles have been used to immobilize j3-D-galactosidase. The immobilized enzyme was used for the hydrolysis of lactose in whole milk, from which the enzyme is readily recovered magnetically. The immobilized j3-D-galactosidase is of potential industrial importance hydrolysis improves some processed dairy products and there are also nutritional products requiring modification for people with lactose intolerance. 

Lysozyme has been immobilized by reaction with a glutaraldehyde-activated silanized ferrite support to yield an active immobilized enzyme readily recoverable magnetically from reaction solutions. ... 

Controlled-pore glass with aminopropyl functional groups has proven to be the most successful support for enzyme immobilization. Most enzymes immobilized on this support, e.g., with glutaraldehyde, retain their activities. The goal is to achieve maximal loading of the active enzyme, resulting in optimal... 

Pahujani, S., S. S. Kanwar, G. Chauhan, and R. Gupta. 2008. Glutaraldehyde Activation of Polymer Nylon-6 for Lipase Immobilization Enzyme Characteristics and Stability. ... 

---