Immozymes are polymer beads that carry absorbed or covalently attached enzymes. The beads are designed to have a low diffusion limitation that even allows for the immobilization of enzymes with high specific activities. Although these beads have a very low water content the appearance is sometimes fluffy or sticky because of a minor static charge on the beads. This doesn’t affect the enzymes activity, however.
Adding Immozyme to reaction media
The Immozymes contain protein as active ingredient and should in principle be handled as any ordinary free protein. When putting the Immozyme in an aqueous buffer it is best to use cold solutions (<10 °C) after which it can be warmed up to the desired temperature.
Cosolvent - mono-phasic:
When using water-miscible organic solvents in combination with water, premix the two solvents prior to adding the immobilized enzyme. Make sure that the temperature has not increased because of mixing heat. Chill the mixture in case the temperature has exceeded 10 °C before Immozyme is added. When using a titrator do not use titrants that exceed a concentration of 1 mol base per liter.
Cosolvent - bi-phasic:
With non water-miscible solvents first add the Immozyme to the cold buffer solution and then add the cosolvent.
Water free:
Most organic solvent are compatible with the Immozymes’ carrier material. To preserve enzyme activity cold mixing is advised. Addition of substrate: Treat substrates in the same manner as the organic solvents. For optimal activity the water concentration in the organic solvent must be controlled.
Immozymes can be used in various media like MTBE, hexane, pentane, branched alkanes or more polar solvents like dioxane, methyl THF or alcohols like 2-propanol or higher alcohols (ethanol and 1-propanol are on the edge). The maximum temperature is dependent on the solvent but even at 60-70 °C high enzyme activity is found, although 30-40 °C is optimal for activity versus stability. The following solvents should be avoided: methanol, DMF and DMSO. For all solvents control of the water concentration is crucial, since a minor amount of water is needed for enzyme activity. Also it is best to exclude oxygen from the system if possible.
The Immozymes are delivered as dried material containing just a small amount of water to preserve enzyme activity (~ 2%). It is advised to determine the optimal water concentration in the reaction in a series of experiments. If water is consumed or produced in the reaction adequate compensation will be needed to preserve maximum reaction rates.
Stirring
After the Immozyme has been added to the reaction mixture stirring is best performed with a shaker or with a top stirrer to ensure minimal wearing and maximum filterability of the beads. For top stirrers, do not exceed 100 rpm (optimally 50-75 rpm). For small scale test reactions shaking of vials (either orbital or overhead) is recommended.
Do not use magnetic stirrer bars – this will grind the beads into powder
High rpm stirring (>100 rpm) or stirrer bars will grind any type of bead. Filtration and recycling will become more difficult. Special stirring devices are available (“Spinchem reactor”) to combine the benefits of batch and continuous process set-up.
Column
All beads can be applied in columns for continuous use. Because most of the beads swell, it is important to avoid overfilling. The beads will swell up to 50%, which will increase pore size. The stated bead diameters are always for fully rewetted beads. When the column is overfilled, beads will be crushed and the flow will be blocked.
Do not fill columns for more than 50% before swelling
It is possible to fill columns with dry material and swell them in situ or pre-swell and add the slurry to the column. When mixing dry immobilized enzyme with solvents, please read the “Adding Immozyme to reaction media” section carefully.
Filtration
Various ways of filtration can be used. For any of the Immozymes’ particle sizes metal sieves and polymer filter cloths with pore sizes of 50 to 100 µm work very well. It is not recommended to use filters with pore sizes below 50 µm since the flow rate will be decreased. P1 glass filters (100 – 160 µm pore size) work very well in combination with particle sizes from 150 µm and up.
Storage
Keep the Immozyme as dry as possible. When taken from the fridge, allow it to warm up to room temperature before opening. When the Immozyme has to be stored for recycling, wash away any remaining organic solvent or substrate with cold buffer. For prolonged storage after wetting, the use of 50% 1,2-propanediol (or 50% glycerol) solutions is recommended. Store at 4 °C. Do not freeze the Immozyme.
Safety
Avoid formation of dust. Avoid splashing and high-pressure washing. Ensure good ventilation of the room when handling this preparation. Like any other type of protein dust, enzyme dust can cause allergies if not managed correctly.
Make sure to take a representative sample for activity measurements. It must contain all particle sizes as specific activity may vary with particle size.
Download instructions for optimal handling of immobilized enzymes.