Covalent immobilization of enzymes on non-porous glass surfaces
Covalent immobilization of enzymes on non-porous glass surfaces
The resistance of glass to strong acids and solvents and its compact solid structure make it a valuable material for enzyme immobilization in technical processes. However, serious drawbacks are the inertness of the glass and the small acceptable surface. In order to introduce a reactive group to an inert glass surface, it must be silylated prior to the immobilization reaction. Source: Laboratory Manual of Enzymology
Operation method
basic program
Principle
Using 1 mm diameter glass beads, activation of the glass surface was achieved by reaction with the silane component to introduce the amino group. By further modification with p-nitrobenzoyl chloride, the nitro group was added to the amino group, which together with the nitroso group was transformed into a cationic heavy salt to which the enzyme was ligated via tyrosyl residues (Fig. 1). Fig. 1 Silanized glass surface and covalently immobilized enzyme. Ⅰ Si-OH groups on the silanized glass surface were modified with 3-aminopropyl-triethoxysilane; Ⅱ derivatization of the ammonia terminus by p-nitrophenyl chloride; Ⅲ reduction of the nitro group by sodium dithionite; Ⅳ formation of the cationic heavy salt by nitrite; Ⅴ the enzyme was ligated via a tyrosyl residue.
Materials and Instruments
Enzyme solutions Move 1. Activity of glass beads Glass beads (10 g) were heated in 40 ml of 5% nitric acid at 80-90 °C for 1 h. Thereafter the beads were washed three times with distilled water and were transferred to PE containers to avoid silanization and enzyme immobilization to the walls of long-necked bottles. Water (10 ml) and 3 g of 3-aminopropyl-triethoxysilane were added. The pH was first adjusted with concentrated HCl and finally adjusted to 3-4 with 2 mol/L HCl, which was detected by pH indicator paper (the pH electrode could be damaged). The reaction mixture was incubated at 65 °C for 12 h, after which the beads were rinsed five times with water and three times with acetone (20 ml each time) on a suction filter (porcelain, no glass powder). Finally, the beads were dried at 55 °C for 20 min. 2. Diazotization The latter procedure must be carried out in glass containers. For activation of the glass beads, 25 ml of dichloromethanol, 1.5 g of triethylamine and 5 g of p-nitrophenyl chloride are added and boiled at reflux for 4 h. Avoid humidity. The beads were rinsed three times with 10 ml of dichloromethanol and dried briefly at 55 °C. Afterwards, the beads were dried for free amino acid diazotization. The beads were then transferred to 100 ml of 2 mol/L HCl solution containing 1 g NaNO2 for free amino diazotization and allowed to stand at room temperature for 20 min. After vigorous rinsing of the beads with water, the beads were refluxed in 40 ml of 40 ml of aqueous solution containing 2 g of sodium hypodisulfite for lh. Aryl amine beads were formed, rinsed three times in 20 ml of water, and transferred to a long-necked flask with a round bottom with 88 ml of water. The arylamine beads were formed, rinsed three times with 20 ml of water, transferred to a round-bottomed, long-necked flask, and 88 ml of 2 mol/L HCl was added.The long-necked flask was mounted on a rotating dehydrator and rotated in an ice bath to ensure that the bath was at 0 °C. A portion of 1.12 g of NaNO2 was added slowly over a period of 20 min, during which time the flask was rotated in a non-vacuum state, and after the last portion was added, the flask had to be rotated for 10 min (approximately 20 mbar) with a weak water-jet vacuum thruster for the expulsion of nitrogenous gases. The resulting diazonium-based glass is rinsed 3 times with 20 ml of ice water and must be used for fixation immediately afterwards. It must be stored at 4°C for the duration of the period before being used for fixation. 3. Enzyme fixation Enzyme solution (10 mg dissolved in 10 ml of 0.1 mol/L pH 7.5 potassium phosphate) is added to the glass beads and the flasks are rotated slowly at 4°C overnight (12-16 h). The glass beads were then rinsed three times with 5 ml of 0.1 mol/L pH 7.5 potassium phosphate buffer. The buffer solution floating on the surface was collected and used to determine the activity of unimmobilized proteins and enzymes, and the efficiency of immobilization was assessed in this way Caveat Always be aware of toxic and aggressive reagents in your operations. Avoid the use of magnetic stirrers as it can damage the finishing of the glass surface. For more product details, please visit Aladdin Scientific website.
3-Aminopropyl-triethoxysilane P-nitrophenyl chloride Dichloromethanol Triethylamine Sodium nitrite Concentrated hydrochloric acid Hydrochloric acid Sodium dithionite Nitric acid Potassium phosphate
Glass beads