IONTOSORB - Bead Cellulose Derivatives
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Immobilization Enzymes by Covalent Bond

A Method Based on the Formation of Peptide Bond

To basic types of reactions using chemical binding of functional groups for enzymes immobilization a method based on the formation of peptide bond belongs too. This method is based on arising peptide bond between the protein part of an enzyme and water-insoluble support. Consequently there forms a peptide bond between free carboxylic groups or amino-groups of an enzyme and corresponding groups of a water-insoluble support.

The procedure used for the immobilization of the enzymes by covalent bond on supports IONTOSORB is described as follows:

To 250 mg support an adequate amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added (see following Table 1) in 5 ml distilled water and 5 ml enzyme solution. The mixture was shook for 24 hours at 4 oC. The conjugate was separated and rinsed 4 times with 80 ml NaCl solution (c = 1 mol/l). Immobilized enzymes were saved at 4 oC covered by phosphate buffer with adequate pH.

Table  1

Support Carbodiimide
[mg]		 Enzyme Enzyme amount
[mg]		 pH of buffer
   Iontosorb CM 100

71.9

 KAT 5 7.2
GOD 5 7.2
CHT 10 7.8
   Iontosorb DETA 100 43.1 KAT 5 7.2
GOD 5 7.2
CHT 10 7.8
   Iontosorb AV 100 67.3 KAT 5 7.2
GOD 5 7.2
CHT 10 7.8
   Iontosorb SALICYL 100 38.3 KAT 5 7.2
GOD 5 7.2
CHT 10 7.8

alpha-Chymotrypsine (CHT), Glucoseoxidase (GOD) and Catalase (KAT)

The results of immobilization on supports IONTOSORB by the method based on the peptide bond formation are given in the following table:

Iontosorb alpha-Chymotrypsine Glukoseoxidase Catalase
ukat/g MS ukat/g MS ukat/g MS ukat/g MS ukat/g MS ukat/g MS
  CM 0.632 0.905 0.022 0.030 0.84 1.15
  DETA 0.082 0.105 0.032 0.040 1.36 1.75
 SALICYL 0.544 0.666 0.037 0.050 7.58 9.13
  AV 0.141 0.165 0.028 0.032 5.76 6.82

MS = moist support                                 DS = dry support