Irreversible enzyme immobilization methods

Methods based on the inhibitory effect of the analyte and the use of an enzyme thermistor have primarily been applied to environmental samples and typically involve measuring the inhibitory effect of a pollutant on an enzyme or on the metabolism of appropriate cells [162]. The inhibiting effect of urease was used to develop methods for the determination of heavy metals such as Hg(II), Cu(II) and Ag(I) by use of the enzyme immobilized on CPG. For this purpose, the response obtained for a 0.5-mL standard pulse of urea in phosphate buffer at a flow-rate of 1 mL/min was recorded, after which 0.5 mL of sample was injected. A new 0.5-mL pulse of urea was injected 30 s after the sample pulse (accurate timing was essential) and the response compared with that of the non-inhibited peak. After a sample was run, the initial response could be restored by washing the column with 0.1-0.3 M Nal plus 50 mM EDTA for 3 min. Under these conditions, 50% inhibition (half the initial response) was obtained for a 0.5-mL pulse of 0.04-0.05 mM Hg(II) or Ag(I), or 0.3 mM Cu(II). In some cases, the enzyme was inhibited irreversibly. In this situation, a reversible enzyme immobilization technique... 

Most biosensors based on AChE have the enzymes immobilized on the surface of the sensor. The inhibition reaction being irreversible, the membrane with immobilized enzyme has to be replaced after several measurements or the biosensor can be use for only one determination. Due to this fact, the researchers tried to realize pesticide biosensors with a renewable surface or disposable biosensors based on screen-printed electrodes (SPE). The screen-printing technology provides a simple, fast and inexpensive method for mass production of disposable biosensors for different biomolecules starting with glucose, lactate and finishing with environmental contaminants as pesticides (Kulys et al., 1991) and herbicides (Skladal, 1992). 

Peptides consisting of residues from GroEL immobilized on agarose have proved effective minichaperones (Altamirano ef al., 1997). The procedure used both column chromatography and batch-wise methods to renature an insoluble protein from an inclusion body, refold apparently irreversibly denatured proteins, and to recondition enzymes that have lost activity on storage. Eragments were immobilized by two methods Ni-NTA resin and CNBr-activated Sepharose 4B. 

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