Enzymes Immobilized on Columns
Solid Phase Enzyme Reactions.

Enzymes are immobilized on matrices and placed in small columns. Enzyme reactions occur when the substrate passes through and is recovered quantitatively by gravity flow or centrifugation. The enzyme remains bound to the column.

	Matrix Types F7m and K7m: Large pore sizes exclude >107Da molecules, i.e., most proteins/enzymes and substrates can enter the pores. High surface area, high enzyme activity. Elution volume is higherthan with G3m matrix. Eluted volume is diluted 3-5 fold.
	Matrix Type G3m: Small pore sizes exclude >103 Da molecules, i.e, all peptides, proteins and nucleic acid chains remain in void volume. Lower surface areas (outside surface only). Less enzyme activity. Low elution volume, e.g., it is possible to load 10 ”l and recover sample in 10 ”l.

Re-usability, Enzymes stay on the column: Compared with two other standard immobilization chemistries (CNBr, VA-Epoxy) the F7m immobilization method showed >10 fold less ÒbleedingÓ of the enzyme than the other methods. 1000 U of alkaline phosphatase were immobilized on the same volume of different activated resins. 200 ”l of Tris,HCl buffer pH 8 was applied to the columns for 20 minutes and eluted. The activity of the alkaline phosphatase in the eluate was assayed and the relative amounts are shown in the graph below. Also, the specific activity of the immobilized enzymes was compared and F7m showed 100%, CNBr 66%, and VA-Epoxy 66% (not shown, see ref 1). Thus the specific activity of the immobilized enzyme is considerably less for the CNBr and VA-epoxy compared to the F7m immobilization.

General Properties of Immobilized Enzymes:

Features:

• Immobilization with long linker to keep enzymes away from the matrix - no steric hindrance.
• Immobilized enzymes react efficiently and specifically with short reaction times.
• No bleeding of enzymes from columns.
• Columns can be washed clean between uses. After washing with 7.6 ml of buffer, the residual substrate has been measured at 104 molecules or 10-19M.
• Immobilized enzymes are especially stable. They can be stored for several months and used repeatedly.
• Columns can be centrifuged for fast, controlled elution behavior with negligible dilution of sample with G3m matrix.
• F7m matrix has effective working volume (and thus bound enzyme), 40 times that of G3m for very rapid reaction times.
• When the volume of the substrate is greater than that of the void volume of the column, use the F7m matrix.
• Can use G3m matrix when the volume of the substrate is less than the void volume of the column.

A few examples to illustrate the utility of Immobilized Enzymes:

• To eliminate a contaminating substrate from a solution without introducing another contaminant - the enzyme.
• Prior to sequencing a protein, it can be cut with an endoprotease, but to avoid leaving behind significant quantities of sequencable peptides from the enzyme, the reaction must be carried out over many hours or days with very low concentrations of enzyme. The reaction times can be short with no contaminating enzyme frag- ments if the enzyme is immobilized.
• Endoproteases are often their own substrate but if immobilized the reaction cannot take place.